Mechanisms of angiogenesis and lymphangiogenesis in calcific aortic valve stenosis

WIHURI RESEARCH INSTITUTE

DIVISION OF CARDIOLOGY / DEPARTMENT OF MEDICINE

HELSINKI UNIVERSITY CENTRAL HOSPITAL

UNIVERSITY OF HELSINKI 2013

SUVI SYVÄRANTA

MECHANISMS OFANGIOGENESISANDLYMPHANGIOGENESISIN CALCIFIC AORTIC VALVE STENOSIS

Wihuri Research Institute, Helsinki, Finland &

Division of Cardiology, Department of Medicine, Helsinki University Central Hospital

University of Helsinki, Finland

ACADEMIC DISSERTATION

To be publicly discussed, with permission of the Medical Faculty

of the University of Helsinki, in lecture hall 3, Biomedicum Helsinki,

Haartmaninkatu 8, on September 6th, 2013 at 12 noon.

Helsinki 2013

SUVI SYVÄRANTA

MECHANISMS OFANGIOGENESISANDLYMPHANGIOGENESISIN CALCIFIC AORTIC VALVE STENOSIS

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Supervisors Satu Suihko, MD, PhD Wihuri Research Institute Helsinki, Finland Professor Petri T. Kovanen, MD, PhD Wihuri Research Institute Helsinki, Finland

Reviewers Docent Tuomas Rissanen, MD, PhD Department of Cardiology and Internal Medicine Central Hospital of North Karelia Joensuu, Finland Docent Jari Satta, MD, PhD Division of Cardiothoracic Surgery Oulu University Hospital Oulu, Finland

Opponent Professor Johanna Kuusisto, MD, PhD University of Tampere Tampere, Finland and Department of Medicine/ Centre for Medicine and Clinical Research University of Eastern Finland and Kuopio University Hospital Kuopio, Finland Cover art: Tessa Helle ISBN: 978-952-10-8848-3 (paperback) ISBN: 978-952-10-8849-0 (pdf) http://ethesis.helsinki.fi Unigrafia, Helsinki 2013

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“Joka ratkaisuun on monta ongelmaa.” -Äiti “There are many problems to each solution.” -Mom

To my extended family

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CONTENTS ORIGINAL PUBLICATIONS ............................................................................................................... 6 ABBREVIATIONS ............................................................................................................................. 7 ABSTRACT ...................................................................................................................................... 9 TIIVISTELMÄ ................................................................................................................................. 11 I INTRODUCTION .......................................................................................................................... 13 II LITERATURE REVIEW ................................................................................................................. 14

Anatomy of the aortic valve ........................................................................................... 14 1. Clinical course of aortic valve stenosis ........................................................................... 14 2.

2.1. Epidemiology ......................................................................................................... 14 2.2. Diagnosis and clinical course ................................................................................. 15 2.3. Treatment .............................................................................................................. 16

Pathobiology of AVS ....................................................................................................... 18 3.

3.1. Overview ................................................................................................................ 19 3.2. Lipids, lipoproteins, and scavenger receptors ....................................................... 19 3.3. Inflammation ......................................................................................................... 20 3.4. Calcification and bone formation .......................................................................... 21 3.5. Extracellular matrix remodeling ............................................................................. 22 3.6. Experimental animal models ................................................................................. 24

Angiogenesis and lymphangiogenesis ............................................................................ 25 4.

4.1. Normal vascular development ............................................................................... 25 4.2. Proteases in angiogenesis and lymphangiogenesis ............................................... 29 4.3. Angiogenesis and lymphangiogenesis in the aortic valve ...................................... 30 4.4. Local modulators of angiogenesis and lymphangiogenesis in aortic valves .......... 30 4.5. Angiogenic potential of mast cells ......................................................................... 32 4.6. Scavenger receptors and angiogenesis .................................................................. 33

III AIMS OF THE STUDY ................................................................................................................ 34 IV MATERIALS AND METHODS ..................................................................................................... 35 V RESULTS AND DISCUSSION ....................................................................................................... 41

Angiogenesis and lymphangiogenesis in AVS ................................................................. 41 1.

1.1. Morphological classification and localization of valvular neovessels and lymphatic vessels (II-III)........................................................................................................................ 41 1.2. Associations of valvular neovessels and lymphatic vessels with inflammation and calcification (II-III) ................................................................................................................ 43

Local regulation of valvular angiogenesis and lymphangiogenesis ................................ 46 2.

2.1. Extracellular matrix remodeling – cathepsins S, K, and V, and their inhibitor cystatin C in AVS (I) ............................................................................................................. 46 2.2. Valvular expression of VEGF-A, VEGF-C, VEGF-D, and their receptors VEGFR-2 and VEGFR-3 (II-III) ..................................................................................................................... 48 2.3. Oxidized LDL and scavenger receptors (IV) ............................................................ 53 2.4. Angiogenic and antilymphangiogenic actions of valvular mast cells (II-IV) ......... 57 2.5. Interplay between valvular mast cells and myofibroblasts (II-IV).......................... 58

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General discussion .......................................................................................................... 59 3.

3.1. Angiogenesis and lymphangiogenesis in AVS......................................................... 59 3.2. Clinical applications ................................................................................................ 61 3.3. Study limitations .................................................................................................... 63

VI SUMMARY AND CONCLUSIONS ............................................................................................... 64 VII ACKNOWLEDGEMENTS ........................................................................................................... 66 VIII REFERENCES ........................................................................................................................... 68

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ORIGINAL PUBLICATIONS This thesis is based on the following original publications, which are referred to in the text by their Roman numerals.

I. Helske S, Syväranta S, Lindstedt KA, Lappalainen J, Oörni K, Mäyränpää MI, Lommi J, Turto H, Werkkala K, Kupari M, Kovanen PT. Increased expression of elastolytic cathepsins S, K, and V and their inhibitor cystatin C in stenotic aortic valves. Arteriosclerosis, Thrombosis and Vascular Biology 2006;26:1791-8.

II. Syväranta S, Helske S, Laine M, Lappalainen J, Kupari M, Mäyränpää MI, Lindstedt KA, Kovanen PT. Vascular endothelial growth factor-secreting mast cells and myofibroblasts: a novel self-perpetuating angiogenic pathway in aortic valve stenosis. Arteriosclerosis, Thrombosis and Vascular Biology 2010;30:1220-7.

III. Syväranta S, Helske S, Lappalainen J, Kupari M, Kovanen PT. Lymphangiogenesis in

aortic valve stenosis--novel regulatory roles for valvular myofibroblasts and mast cells. Atherosclerosis 2012;221:366-74.

IV. Syväranta S, Alanne-Kinnunen M, Öörni K, Oksjoki R, Kupari M, Kovanen PT, and

Helske-Suihko S. Pathological role for oxidized low-density lipoprotein and scavenger receptors SR-A1, CD36, and LOX-1 in aortic valve stenosis. Submitted.

Publication I is also part of the doctoral thesis of Dr. Satu Suihko (née Helske). The original publications are reproduced with the permission of the copyright holders, and some previously unpublished data are also presented.

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ABBREVIATIONS

ACE-I Angiotensin-converting enzyme inhibitor

Ang Angiotensin

ARB Angiotensin receptor blocker

AVA Aortic valve area

AVR Aortic valve regurgitation

AVS Aortic valve stenosis

BMP Bone morphogenetic protein

CAVD Calcific aortic valve disease

Cbfa1 Core-binding factor subunit alpha-1

CRP C-reactive protein

ECM Extracellular matrix

ELISA Enzyme-linked immunoassay

HDL High-density lipoprotein

ICAM-1 Intercellular adhesion molecule-1

IL Interleukin

LDL Low-density lipoprotein

LOX-1 Lectin-like oxidized LDL receptor-1

Lp(a) Lipoprotein(a)

LRP5 Low-density lipoprotein receptor-related protein 5

LYVE-1 Lymphatic vascular endothelial hyaluronan receptor

MCP-1 Monocyte chemoattractant protein-1

M-CSF Macrophage colony-stimulating factor

MMP Matrix metalloproteinase

mRNA Messenger ribonucleic acid

NO Nitric oxide

OxLDL Oxidized low-density lipoprotein

(q)PCR (Quantitative) polymerase chain reaction

PlGF Placental growth factor

RANK Receptor activator of nuclear factor kappa β

RANKL Receptor activator of nuclear factor kappa β ligand

SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SR-A1 Scavenger receptor class A type 1

SR-B1 Scavenger receptor class B type 1

Statin 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor

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TGF-β(1) Transforming growth factor beta (1)

TIMP Tissue inhibitor of metalloproteinases

TNF-α Tumor necrosis factor-alpha

VCAM-1 Vascular cell adhesion molecule-1

VEGF Vascular endothelial growth factor

VEGFR Vascular endothelial growth factor receptor

vWF Von Willebrand factor

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ABSTRACT

Aortic valve stenosis (AVS) is the most common valvular disease in Western countries. Pharmacological prevention of AVS having proved unsuccessful, its current treatment is still valve replacement. The etiology of AVS is multifactorial, both genetic and external risk factors predisposing to the active pathological process eventually leading to clinically manifest stenosis. Histological features of the disease resemble those of atherosclerosis, including the accumulation and modification of lipoproteins, inflammation, extracellular matrix remodeling, and calcification. Furthermore, valvular interstitial cells undergo phenotypic differentiation into actively proliferating myofibroblasts, which contribute to the local inflammatory response as well as extracellular matrix remodeling in stenotic aortic valves. Blood vessels also grow into the normally avascular valve leaflets already in the early stages of the disease. This thesis aimed at elucidating the mechanisms behind the pathological neovascularization of the stenotic aortic valves. Furthermore, we characterized valvular lymphangiogenesis and investigated potential factors contributing to the balance between valvular angiogenesis and lymphangiogenesis, focusing on the role of valvular myofibroblasts and mast cells. For this purpose, we studied a total of 117 stenotic valves obtained at valve replacement surgery, and 49 control valves obtained at cardiac transplantations, at valve replacement surgery due to aortic valve regurgitation, or from deceased organ donors whose hearts were unsuitable for use as grafts. The valve leaflets were either used freshly for myofibroblast cell culture or frozen for e.g. PCR and immunohistochemical analyses. First, we assessed the adverse extracellular matrix remodeling of stenotic aortic valves, a process necessary for angiogenic sprouting to occur. We found that the mRNA expression levels of cathepsins S, K, and V, and their inhibitor cystatin C were higher in stenotic aortic valves than in control valves. Furthermore, the total activity of such cathepsins was increased in AVS. In immunohistochemical stainings, the expressions of cathepsin S, cathepsin V, and cystatin C localized to valvular macrophages, chondroblast-like cells, and endothelial cells lining both the valvular surface and the neovessels in the stenotic valves. Next, we characterized the neovessels and lymphatic vessels in stenotic aortic valves and control valves using immunohistochemistry. We found that in addition to immature microvessels, the stenotic aortic valves contained organized arterioles, indicating an advanced stage of angiogenesis. Lymphatic vessels correlated with valvular blood vessels, but were present in much fewer numbers. Valvular mast cells resided close to neovessels and secreted the angiogenic Vascular endothelial growth factor A (VEGF-A). Furthermore, we showed that the lymphangiogenic growth factors VEGF-C and VEGF-D are locally produced in the aortic valves, and that the receptors

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for all these VEGFs, VEGFR-2 and VEGFR-3, are upregulated in AVS. We also identified several factors that induce VEGF-A secretion in cultured valvular myofibroblasts. These include mast cell-derived components, the inflammatory cytokine TNF-α, hypoxia, and cigarette smoke. Myofibroblasts were also able to promote VEGF-A secretion by cultured human mast cells, suggesting potential angiogenic interplay between these two valvular cell types. Interestingly, mast cell-derived proteases also efficiently degraded the lymphangiogenic growth factor VEGF-C. Thus, by secreting VEGF-A, by urging myofibroblasts to produce VEGF-A, and by releasing VEGF-C-degrading proteases, mast cells may strongly influence the observed imbalance between valvular blood vessels and lymphatic vessels. Finally, we investigated the potential effects of oxidized low-density lipoprotein (oxLDL) on valvular angiogenesis. We found that oxLDL induces the expression of several inflammatory cytokines in cultured myofibroblasts. Moreover, we identified oxLDL-binding scavenger receptors to be locally expressed in the aortic valves. The mRNA expression levels of scavenger receptor class A type 1 (SR-A1) and Lectin-like oxidized LDL receptor-1 (LOX-1) were increased in AVS, whereas CD36 was downregulated in stenotic valves. Furthermore, the expression of LOX-1 in cultured valvular myofibroblasts increased in response to mast cell-derived components and TNF-α. The observed changes in valvular scavenger receptor expression particularly favor inflammation and angiogenesis. In conclusion, several angiogenic factors were found to be activated in stenotic aortic valves. Furthermore, valvular mast cells and myofibroblasts were identified as potential players promoting valvular angiogenesis and contributing to the pathological imbalance between valvular angiogenesis and lymphangiogenesis. This imbalance, in turn, could facilitate the harmful infiltration of inflammatory cells and lipoproteins into the stenotic aortic valves and ultimately contribute to the progression of the disease.

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TIIVISTELMÄ

Aorttaläpän ahtauma on väestön ikääntymisen myötä yleistynyt sairaus, johon ei ole lääkehoitoa. Aorttaläpän ahtautuminen johtaa sydämen vasemman kammion painekuormitukseen ja lopulta vajaatoimintaan, ja jo taudin lievään muotoon liittyy kohonnut sydänperäisen äkkikuoleman riski. Tauti havaitaan yleensä myöhään, sillä se on pitkään oireeton. Oireiden ilmaantuminen onkin aihe keinoläppätoimenpiteelle. Taudin riskitekijöitä ovat korkean iän lisäksi miessukupuoli, korkea verenpaine, tupakointi, diabetes ja veren suuri LDL-kolesterolin pitoisuus. Vaikka riskitekijät ovat osittain samat kuin sepelvaltimotaudissa, vain noin puolella läppäpotilaista on merkittävä sepelvaltimotauti. Kudostasolla läpän ahtautumiseen johtaa aktiivinen tapahtumaketju, jonka katsotaan alkavan läpän pintaa verhoavan endoteelisolukerroksen vaurioitumisesta. Sitä seuraa tulehdussolujen, kuten syöttösolujen, ja kolesterolin kertyminen läpän rakenteisiin. Lisäksi läpän sidekudossolut aktivoituvat sidekudostumista edistävään suuntaan myofibroblasteiksi ja läpissä tuotetaan runsaasti sidekudosta hajottavia proteaaseja. Normaalisti suonettomiin aorttaläppäliuskoihin kasvaa taudin myötä uudissuonia, jotka voivat edistää tautia kuljettamalla läppiin lisää tulehdussoluja ja rasvoja. Lisäksi verisuonien kuljettamat ravinteet ja happi mahdollistavat tulehdussolujen ja sidekudossolujen haitallisen toiminnan. Imusuonet puolestaan voisivat toimia verisuonien vastavaikuttajina kuljettamalla tulehdussoluja ja rasvoja kudoksista pois. Ahtautuneiden aorttaläppien uudissuonittumisen ja imusuonikasvun taustalla olevat mekanismit ovat kuitenkin tähän asti olleet pitkälti tuntemattomia. Tämän väitöskirjan osatyöt tähtäsivät ahtautuneiden aorttaläppien uudissuonittumisen luonnehdintaan sekä uudissuoni- ja imusuonikasvun taustalla olevien tekijöiden kartoittamiseen. Osatöissä on tutkittu 117 aorttaläpän ahtauman vuoksi leikatun potilaan aorttaläppää. 49 kontrolliläppää kerättiin aorttavuodon vuoksi leikatuilta potilailta, sydämensiirroissa poistetuista sydämistä ja käyttämättä jääneistä sydänsiirteistä. Ensimmäinen osatyö käsitteli katepsiineja S, K, ja V, sekä niiden estäjää kystatiini C:tä. Nämä proteaasit osallistuvat sidekudoshajotukseen, joka on välttämätöntä uusien verisuonien versoamiselle. Toisessa ja kolmannessa osatyössä selvitimme uudissuonten ja imusuonten määrää ja laatua ahtautuneissa ja terveissä aorttaläpissä sekä niiden yhteyksiä läpän kalkkiutumiseen ja tulehdusasteeseen. Lisäksi eristimme ihmisen aorttaläpistä myofibroblasteja ja tutkimme niiden vastetta erilaisiin ärsykkeisiin, kuten tulehdusvälittäjäaineisiin, hapenpuutteeseen ja tupakansavuun. Lisäksi tutkimme myofibroblastien ja syöttösolujen vaikutuksia toisiinsa soluviljelymallissa ja osoitimme, että niiden välisellä vuorovaikutuksella voi olla uudissuonittumista edistäviä vaikutuksia. Neljännessä osatyössä selvitimme aiemmin

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ahtautuneista läpistä löydettyjen hapettuneiden LDL-hiukkasten vaikutuksia uudissuonittumiseen. Havaitsimme, että näiden hiukkasten vaikutuksia välittävien jätereseptorien ilmentyminen on ahtautuneissa aorttaläpissä muuttunut tulehdusta ja uudissuonittumista edistävään suuntaan. Tässä väitöskirjassa on kuvattu useita ahtautuneiden aorttaläppien uudissuonittumisen mekanismeja. Havaitsimme, että uudissuonten ja imusuonten välinen tasapaino on ahtautuneissa aorttaläpissä häiriintynyt suosien mm. tulehdussolujen kertymistä läpän rakenteisiin. Näin ollen tämän epätasapainon taustatekijät, kuten läpän syöttösolujen ja myofibroblastien vuorovaikutukset, voivat myös edistää aorttaläpän ahtauman etenemistä.

INTRODUCTION │ 13 _____________________________________________________________________________

I INTRODUCTION Calcific aortic valve disease (CAVD) and its end-stage, aortic valve stenosis (AVS), have become the most common valvular heart diseases in Western countries due to the ageing of the population. In younger adults, AVS usually develops when the valve only has two leaflets instead of three, which occurs in 1-2 % of the population (Lindroos et al. 1993). Other risk factors resemble those of atherosclerosis, but only half of AVS patients manifest significant coronary artery disease (Otto et al. 1997), suggesting the existence of distinct pathobiological mechanisms. Histological changes of AVS develop over a long period of time and ultimately lead to thickening of the valve leaflets and symptomatic ventricular outflow obstruction. Because the symptoms appear late in the course of the disease, the diagnosis is commonly delayed, and the prognosis of a symptomatic patient without operation is extremely poor. Indeed, the onset of symptoms indicates the need for valve replacement surgery. Since the patients are often elderly and have several comorbidities, a pharmacological treatment option would be called for. The search for such pharmacotherapeutic targets has, however, been unsuccessful.

Active atheroinflammatory processes have been discovered in stenotic aortic valves. Early changes of the disease include endothelial damage, lipid accumulation, infiltration of inflammatory cells, neovascularization, fibrosis, and calcium deposition in the affected valve leaflets (Olsson et al. 1994a, Otto et al. 1994). Over time, these changes become more manifest, and lead to the pathological thickening and stiffening of the valve. Even active bone formation and remodeling occur in the normally translucid valves (Mohler et al. 2001, O'Brien et al. 1995). Angiogenic processes leading to valvular neovascularization could enable a number of the aforementioned pathological processes. More specifically, valvular blood vessels could e.g. facilitate the entry of lipids and inflammatory cells into the tissue, and provide nutrients and oxygen to the proliferating fibrotic cells in the deeper parts of the valves. Although angiogenesis is recognized as a prominent pathological feature in AVS (Soini et al. 2003), its underlying mechanisms in the aortic valve have remained unknown for the most part. The present thesis aimed at characterizing valvular neovascularization and at identifying some of the mechanisms behind the different stages of valvular angiogenesis. First, we studied the extracellular matrix remodeling initially required for vascular sprouting. We then investigated the valvular production of angiogenic and previously unreported lymphangiogenic growth factors as well as their receptors. Finally, we assessed the possible contribution of oxidized low-density lipoprotein to valvular angiogenesis. We focused on the role of valvular myofibroblasts and mast cells, and their interactions in these pathological processes.

14 │ LITERATURE REVIEW _____________________________________________________________________________

II LITERATURE REVIEW

Anatomy of the aortic valve 1. The aortic valve opens during ventricular systole, when blood flows from the left ventricle into the aorta. The valve closes during ventricular diastole, preventing blood from flowing back into the heart from the aorta. The opening surface area is normally 3-4 cm

2, which can be measured

by echocardiography. A normal aortic valve is tricuspid, i.e. it consists of three thin leaflets (Figure 1A). Each leaflet consists of strong elastic tissue covered by vascular endothelium. Normal valvular structure can be histologically divided into three layers: ventricularis, spongiosa, and fibrosa (Figure 1B). The ventricularis, located on the ventricular side of the leaflet, mostly consists of elastic fibers. Spongiosa is a loose layer of proteoglycan-rich connective tissue and valvular interstitial cells, whereas fibrosa forms a strong collagenous structure on the aortic side of the leaflet. In aortic valve stenosis, all these components are disrupted leading to a thick, calcified, non-elastic valve structure obstructing blood flow from the heart to systemic circulation (Mönckeberg 1904). Due to its central location in the circulatory system, the aortic valve is exposed to substantial biomechanical forces for several decades. Especially the turbulent blood flow on the aortic side of the valve may increase local shear stress, thus triggering endothelial dysfunction and other potentially AVS-initiating processes. When examined macroscopically, the calcific deposits of the diseased leaflets consistently localize to the aortic side of the valve.

Figure 1. A) A normal aortic valve viewed from the aortic side. B) The three layers of an aortic valve leaflet. Elastic van Gieson stain showing collagen fibers in pink and elastin in dark purple.

Clinical course of aortic valve stenosis 2.

2.1. Epidemiology Aortic valve stenosis (AVS) is the end-stage in a wide disease spectrum of calcific aortic valve disease (CAVD), characterized by prominent calcification and ventricular outflow obstruction (Rajamannan et al. 2011). Earlier stages are generally referred to as aortic sclerosis, the

LITERATURE REVIEW │ 15 _____________________________________________________________________________

pathological leaflet thickening prior to obstruction. The prevalence of CAVD increases with age. In Finland, 60 % of the people past 80 years of age have aortic sclerosis, and 4 % of them already manifest clinically significant stenosis (Lindroos et al. 1993). A recent Norwegian population-based cohort study on clinically significant AVS reported prevalences of 3.9 % in the 70-79 year cohort and 9.8 % in the 80-89 year cohort (Eveborn et al. 2013). Rheumatic fever no longer significantly contributes to valvular disease due to modern medical practices. The most distinct predisposing factor of AVS is a congenitally bicuspid valve, which occurs in 1-2 % of the population (Lindroos et al. 1993), but in up to 49% of AVS patients (Roberts and Ko 2005), varying between studies. The two-leaflet structure of the bicuspid valve has lower tolerance to shear stress, and stenosis usually develops at a younger age (Beppu et al. 1993). However, histopathological features seem to be similar in the both types of stenotic valves (Wallby et al. 2002). In addition to age and valve structure, several epidemiological risk factors have been connected with AVS. These include male gender, hypertension, smoking, elevated levels of serum low-density lipoprotein (LDL), and diabetes (Deutscher et al. 1984, Lindroos et al. 1994, Stewart et al. 1997). Metabolic syndrome is also an independent risk factor for aortic valve calcification (Katz et al. 2006). Even though all these factors are also associated with atherosclerosis, only half of AVS patients present with significant coronary artery disease at angiography (Otto et al. 1997). Uncommon risk factors of AVS comprise renal insufficiency, Paget’s disease, primary hyperparathyroidism, and alcaptonuric ochronosis (Freeman et al. 2004, Steger 2011). Genetics Several genetic factors have been suggested to predispose to CAVD. The identified genes include vitamin D receptor (Ortlepp et al. 2001), estrogen receptor alpha, and transforming growth factor beta (TGF-β) (Nordstrom et al. 2003), as well as apolipoprotein E (Avakian et al. 2001, Novaro et al. 2003), although the latter has generated contradictory reports (Ortlepp et al. 2006). In addition, mutations in NOTCH1 gene have been associated with the development of bicuspid aortic valves as well as valvular calcification in adulthood (Garg et al. 2005). Bicuspid valves predisposing to AVS may be an inherited trait, and it has also been suggested that calcific stenosis of the trileaflet aortic valve could show familial aggregation (Probst et al. 2006). Interestingly, angiogenesis has also been implicated: a variation in the angiogenic vascular endothelial growth factor A (VEGF-A) gene may associate with AVS (Zhao et al. 2012). Recently, a large genomewide study has identified one single-nucleotide polymorphism, or SNP, in the lipoprotein(a), or Lp(a), locus as a significant risk factor for aortic valve calcification and valve replacement surgery (Thanassoulis et al. 2013). The physiological function of Lp(a) is still unknown, but high serum Lp(a) levels are a risk factor for other cardiovascular diseases, and a causal effect to atherosclerosis has been proposed (Tsimikas and Hall 2012).

2.2. Diagnosis and clinical course A suspicion of AVS usually rises upon clinical examination when auscultation reveals a harsh systolic parasternal murmur on both sides of the sternum radiating to the neck. A stenotic aortic valve obstructs blood flow through the aortic opening, causing increased ventricular pressure and decreased aortic pressure, i.e. transaortic pressure gradient. Increased effort is


required to maintain sufficient blood circulation, leading to left ventricular hypertrophy sometimes detectable as increased voltage and a strain pattern in the ECG. A chest X-ray may reveal the calcification of the aortic valve, most apparent in the lateral view. The presence of symptoms, such as shortness of breath, chest pain, or collapses already indicates hemodynamically severe AVS, and the need for immediate echocardiographic evaluation and treatment. In indefinite cases, symptoms of AVS can be provoked with exercise testing. In severe stenosis, a narrow aortic opening area can cause acquired von Willebrand factor deficiency by shear stress-induced cleavage of high-molecular-weight multimers (Loscalzo 2012). The subsequent gastrointestinal angiodysplasia and bleeding, called Hayde’s syndrome, usually resolves upon valve replacement. Echocardiography reveals not only aortic valvular anatomy and calcification, but also the degree of stenosis, which is graded from mild (aortic valve area, or AVA, 1.5-2.0 cm

2, mean

transaortic pressure gradient <25 mmHg) to moderate (AVA 1.0-1.5 cm2, pressure gradient 25-

40 mmHg) to severe (AVA <1cm2, pressure gradient >40mmHg). Echocardiography also provides information on left ventricular function, which is important in evaluating the patient’s prognosis and thus, the timing of surgical treatment. The need for simultaneous coronary artery bypass surgery is detected by coronary angiography. AVS usually develops during a long period of time, but the prognosis rapidly deteriorates after the stenosis has become severe. If left untreated, AVS ultimately leads to heart failure and a significant risk of cardiac sudden death. The first-year survival rate of patients with hemodynamically severe AVS is 60 % (Turina et al. 1987), and the 5-year survival rate is only 18 %, mean 5-year survival rate being less than 2 years (Horstkotte and Loogen 1988). Interestingly, even mild AVS is associated with an increased risk of cardiovascular events. The underlying cause of this phenomenon remains obscure, but concurrent coronary artery disease and universal atherosclerosis have been suggested (Freeman and Otto 2005). In addition, aortic sclerosis is associated with impaired responsiveness of platelets to nitric oxide (NO), which might explain the increased thrombotic risk even in cases of aortic sclerosis and mild stenosis (Ngo et al. 2009).

2.3. Treatment

2.3.1. Surgical treatment The only curative treatment for aortic valve stenosis is surgical valve replacement. In young and middle-aged patients, the stenotic valve is replaced with a mechanical prosthesis, while biological prostheses are used mainly in patients over 70 years of age. The operative risk is balanced with the increased risk of sudden cardiac death, and the importance of correct timing in the treatment of AVS is highlighted in international guidelines (Bonow et al. 2008). B-type natriuretic peptide may be helpful in assessing the risk level of asymptomatic patients with severe AVS (Bergler-Klein et al. 2004). Operative mortality in a Finnish AVS study was 2,4 % within 30 days of aortic valve replacement surgery (Taskinen et al. 2008). In recent years, a new method has become available for replacing the aortic valve: Transcatheter aortic valve implantation can be performed either endovascularly via the


subclavian or the femoral artery or transapically through a small anterolateral minithoracotomy (Stortecky et al. 2012). Current recommended indication for this approach is a case of severe stenosis when major surgery is contraindicated, e.g. due to comorbidities (high risk, Euroscore >20%). The procedure involves risks that still need to be managed before the transcatheter method can compete with traditional replacement surgery in unselected patient material (Desai and Bonow 2012).

2.3.2. Pharmacotherapy Despite immense effort, no medical therapy has proven successful in preventing the progression of AVS. Many of the possible target processes of pharmacological prevention are early events in the development of AVS. Thus, the natural course of AVS with a prolonged asymptomatic period and a rapid end-stage advancement makes the timing and effectiveness of any pharmacological intervention extremely challenging. Common epidemiology and several histopathological similarities with atherosclerosis have also driven the search for common pharmacotherapy. The two forms of therapy that have advanced to clinical trials are hydroxymethylglutaryl coenzyme-A inhibitors (statins) and angiotensin-targeting medications, angiotensin-converting enzyme inhibitors (ACE-I) and angiotensin receptor blockers (ARB). Statins seemed to be promising candidates in retrospective and prospective observational analyses (Moura et al. 2007, Rosenhek et al. 2004). Regrettably, they proved ineffective in reducing AVS progression in randomized, controlled trials (Chan et al. 2010, Cowell et al. 2005, Rossebo et al. 2008). The result remained similar in a subpopulation of patients with only mild stenosis (Gerdts et al. 2010). In order to produce the desired effect, lipid-lowering possibly should have been initiated up to decades before detectable stenosis (Helske and Otto 2009). ACE-I therapy has been demonstrated to hinder AVS progression in a rabbit model (Ngo et al. 2011). Retrospective studies on humans, however, have been controversial (O'Brien et al. 2005, Rosenhek et al. 2004), and the link between overall improved survival rate of patients with aortic valve disease and the use of ACE-I/ARB medication is inconsistent (Ardehali et al. 2012, Nadir et al. 2011). A recent prospective follow-up study found a relationship between ACE-I/ARB therapy and a slower progression of tricuspid aortic valve sclerosis (Sverdlov et al. 2012a), but the results of ongoing prospective clinical trials on AVS patients remain to be seen. Nevertheless, adequate pharmacological treatment of cardiac risk factors is important in order to prevent concurrent coronary artery disease in patients with AVS (Bonow et al. 2008).


Pathobiology of AVS 3.

Figure 2. Schematic illustration presenting the cross sections of a normal and a stenotic aortic valve leaflet.


3.1. Overview

AVS was previously called “degenerative” aortic valve stenosis, separating it from the formerly common infective forms of the disease, such as rheumatic valve disease. Nowadays the metabolically active nature of AVS is acknowledged, and the term “degenerative” is no longer valid. The pathogenesis of AVS is characterized by endothelial injury and the subsequent infiltration of lipids and inflammatory cells into the valves (Olsson et al. 1994a, Otto et al. 1994). These early events result in the proliferation and transformation of valvular interstitial cells, extensive extracellular matrix remodeling, active calcification, and even bone formation (Mohler et al. 2001, O'Brien et al. 1995). The major pathological changes in AVS are introduced in Figure 2, and further discussed below. Valvular neovascularization is described in detail in Section 4.3 (Angiogenesis and lymphangiogenesis in the aortic valve and in vascular disease). Similarities and dissimilarities between AVS and atherosclerosis Investigations targeted at unraveling the pathobiology of AVS have mostly followed the footsteps of atherosclerosis research. Indeed, many of the key cell types and molecules regulating inflammation and extracellular matrix remodeling in arterial atherosclerotic plaques have also been identified in stenotic valve leaflets. However, the severity of aortic stenosis has been found to specifically associate with local calcification in the aortic valve rather than that in the coronary arteries or the aorta (Dweck et al. 2013). One of the culprits in both atherosclerosis and AVS is the myofibroblast. In atherosclerotic lesions, myofibroblasts initially derive from medial smooth muscle cells (Hinz et al. 2007, Libby and Theroux 2005), whereas in aortic valve leaflets they transdifferentiate from the local resident cell, the valvular interstitial cell, which is also able to assume mesenchymal and osteoblast phenotypes (Liu A. C. et al. 2007, Olsson et al. 1994b). More distinct dissimilarities also exist. For one, the strong causal effect of lipids demonstrated in coronary atherosclerosis has not been replicated in AVS (Helske and Otto 2009). Furthermore, there are differences in the clinical manifestations of these two diseases. Atherosclerotic changes can restrict blood flow in the affected artery either chronically due to the development of a fibrotic, calcified plaque or acutely by sudden rupture of an unstable plaque, even without a preceding arterial obstruction (Libby and Theroux 2005). In stenotic aortic valve leaflets, no acute event similar to plaque rupture occurs, but chronic fibrotic and calcified changes make the valve stiffer, thus advancing the outflow obstruction. Such fibrotic histology, in turn, is usually desirable in arterial atherosclerosis because of its stability and susceptibility to modern pharmaceutical and invasive therapies. Based on these findings and the emerging knowledge of other pathophysiological dissimilarities, it has been established that coronary atherosclerosis and valvular stenosis are, in fact, separate disease entities rather than manifestations of the same pathology.

3.2. Lipids, lipoproteins, and scavenger receptors The accumulation of lipids is considered one of the initiating events in valvular calcification (Demer 2001, Otto et al. 1994), supported by epidemiological data linking hyper-cholesterolemia to AVS (Deutscher et al. 1984). The association between lipid accumulation and AVS progression has also been demonstated in the hypercholesterolemic animal models


introduced below, as well as in histological studies. Indeed, protein components of the atherogenic lipoproteins LDL and Lp(a), specifically apolipoproteins B100 and (a), as well as apolipoprotein E, also accumulate in valvular lesions (O'Brien et al. 1996). In contrast, the amount of the potentially protective apolipoprotein A1, a component of high-density lipoprotein (HDL), is reduced in stenotic valves as compared to controls (Lommi et al. 2011). In atherosclerotic plaques, oxidatively modified accumulated lipoproteins may play a central role by triggering pathological immune responses (Tsimikas and Miller 2011). Oxidized low-density lipoprotein (oxLDL) has also been identified in stenotic valve leaflets, where it colocalizes with inflammatory cells and calcium deposits (Olsson et al. 1999). Moreover, circulating oxLDL levels associate with more advanced histopathological changes of AVS (Cote et al. 2008), and small dense LDL particles, which are potential sources of oxLDL, associate with the progression of AVS (Mohty et al. 2008). OxLDL and other modified forms of LDL bind to scavenger receptors, a diverse group of membrane proteins and soluble proteins implicated in the pathogenesis of atherosclerosis (Goldstein et al. 1979, Parthasarathy et al. 1987). Scavenger receptor class A member 1 (SR-A1) and CD36, a class B scavenger receptor, play a role in macrophage foam cell formation (Stephen et al. 2010). Foam cells form when macrophages take up excess lipids, and this key pathogenic process in atherosclerosis has also been identified in AVS (Otto et al. 1994). Interestingly, the plasma levels of CD36 decrease in AVS patients with increasing myocardial hypertrophy (Heather et al. 2011). Lectin-type oxidized LDL receptor 1 (LOX-1), in turn, is expressed mainly in endothelial cells and mediates their oxLDL uptake (Stephen et al. 2010). In contrast to the above-listed scavenger receptors, scavenger receptor class B member 1 (SR-B1) is considered to be atheroprotective, since it participates in reverse cholesterol transport by binding HDL (Stephen et al. 2010). In addition to proinflammatory actions, scavenger receptors participate in numerous processes potentially involved in valvular pathology, including apoptosis, collagen production, and vascular smooth muscle cell proliferation (Mitra et al. 2011, Stephen et al. 2010). The expression of these receptors in aortic valves, however, has not been previously demonstrated. OxLDL and scavenger receptors, especially CD36 and LOX-1, have also been implicated in angiogenesis, which is further described in Section 4.6 (Scavenger receptors and angiogenesis). The specific properties of scavenger receptors studied in this thesis are also summarized in Table 9 of the corresponding Results Section 2.3 (Oxidized LDL and scavenger receptors).

3.3. Inflammation Only sparsely scattered macrophages and mast cells can be found in healthy aortic valve leaflets, whereas extensive accumulation of inflammatory cells is observable in the stenotic valves (Helske et al. 2004b, Otto et al. 1994). Infiltrates of T and B lymphocytes, activated mast cells and macrophage foam cells characterize the diseased leaflets (Helske et al. 2004b, Mohler et al. 2001, Otto et al. 1994). The number of valvular mast cells has even been linked to the disease progression (Wypasek et al. 2012). The recruitment of inflammatory cells into valvular tissue may be facilitated by neovascularization. In fact, the endothelial cells lining valvular neovessels express vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) (Mazzone et al. 2004), that are known for their role in the transportation


of inflammatory cells into tissues. Lymphatic vessels, a possible exit route for these cells, have been identified close to inflammatory cell infiltrates in stenotic aortic valves (Steiner et al. 2010). Areas rich in inflammatory cells also express tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) (Kaden et al. 2005a, Kaden et al. 2003), both of which are powerful proinflammatory mediators. Interestingly, TNF-α is expressed by mast cells in coronary atherosclerotic plaques (Kaartinen et al. 1996b), and valvular mast cells may also contain TNF-α (Helske et al. 2004a). By stimulating the secretion of protein-degrading enzymes, TNF-α and IL-1β may contribute to the pathological extracellular matrix remodeling in stenotic valves (Kaden et al. 2005a, Kaden et al. 2003). Moreover, the expression of IL-1β along with the macrophage marker CD68 is increased in calcified areas of stenotic aortic valves as compared to macroscopically normal areas of the same valves (Nagy et al. 2011). Put together, these findings have led to the conclusion that inflammation is a central pathogenic process in AVS. In addition to inflammatory cells, cultured valvular interstitial cells secrete proinflammatory mediators, including monocyte chemotactic protein-1 (MCP-1), IL-6 and IL-8 (Helske et al. 2007, Helske et al. 2008, Meng et al. 2008). Several studies suggest that these cells actively participate in valvular inflammatory responses. First, the expression of bradykinin type-1 receptor, a potential inflammatory modulator, is increased in AVS and its expression in valvular myofibroblasts can be induced by TNF-α (Helske et al. 2007). Furthermore, the complement system, a part of the innate immune system comprising a complex protein activation cascade, is activated in AVS (Helske et al. 2008). Valvular myofibroblasts express C3a receptor (Helske et al. 2008), which mediates the effects of a complement activation product, C3a. Consequently, C3a induces the secretion of proinflammatory mediators by cultured valvular myofibroblasts (Helske et al. 2008). The involvement of the complement system was confirmed by a proteomic analysis showing increased expression of C3 along with five other complement components in diseased areas of the valves (Matsumoto K. et al. 2012). Finally, valvular interstitial cells express Toll-like receptors 2 and 4, as well as several leukotriene receptors, and the stimulation of these receptors results in the release of proinflammatory and pro-osteogenic cytokines by the interstitial cells (Meng et al. 2008, Nagy et al. 2011). As a marker for systemic inflammation, C-reactive protein (CRP) has also been of interest in AVS research. CRP is commonly present in the stenotic aortic valves and is frequently found in degenerative aortic valve bioprosthesis, potentially reflecting inflammatory processes within the valve (Skowasch et al. 2006). Epidemiological studies have failed to connect elevated levels of circulating CRP to AVS patients (Novaro et al. 2007, Sverdlov et al. 2012b). CRP could, however, serve as a marker for the development of aortic valve sclerosis (Sverdlov et al. 2012a), and disease progression as well as a prognosis once AVS has been diagnosed (Imai et al. 2008, Sanchez and Mazzone 2006).

3.4. Calcification and bone formation Extensive calcification is a hallmark of AVS and valvular calcification even correlates with the patients’ prognosis (Rosenhek et al. 2000). Besides calcification, active bone formation and remodeling occur in the diseased valves. In fact, lamellar bone with bone marrow elements can be found in 12% of the stenotic valves (Mazzone et al. 2004, Mohler et al. 2001). Endochondral bone formation and microfractures have been identified in stenotic valves, and


neoangiogenesis is associated with the calcified and ossified areas of the leaflets (Mohler et al. 2001). Targeted research has elucidated the underlying mechanisms and revealed active regulatory pathways of calcification and osteogenesis in the diseased aortic valves. Numerous osteogenic mediators have been identified in stenotic aortic valves, including osteopontin, osteocalcin, osteonectin, tenascin C, bone sialoprotein, core-binding factor subunit alpha-1 (Cbfa1), and bone morphogenetic proteins 2 and 4 (BMP 2/4) (Caira et al. 2006, Jian et al. 2001, Kaden et al. 2004b, Mohler et al. 2001, O'Brien et al. 1995, Rajamannan et al. 2003, Srivatsa et al. 1997). Another pathway implicated in the adverse valvular matrix calcification of stenotic aortic valves is the altered balance between receptor activator of nuclear factor kappa β ligand (RANKL), its decoy receptor osteoprotegerin, and actual receptor RANK (Kaden et al. 2004a, Nagy et al. 2013). The overall pro-osteogenic properties of calcific aortic valve tissue have also been demonstrated with mesenchymal stem cells in an in vitro co-culture study (Leskelä et al. 2006). Many of the abovementioned osteogenic factors associate with valvular inflammatory cells, implicating inflammation as a plausible trigger for aortic valve calcification (Mohler et al. 2001, O'Brien et al. 1995, Srivatsa et al. 1997). Further evidence for this notion has been provided by mechanistic studies on cultured valvular interstitial cells. TNF-α stimulates cultured valvular myofibroblasts to form calcific nodules and increases the expression of alkaline phosphatase and osteocalcin, both signs of a phenotypic change towards osteoblastic differentiation (Kaden et al. 2005c). Interestingly, the formation of such calcific nodules is also induced by oxLDL and BMP-2 (Mohler et al. 1999). Indeed, valvular interstitial cells are capable of osteoblastic differentiation through a mechanism potentially counteracted by statins (Osman et al. 2006). These cells also express the osteogenesis-associated factors BMP-2 and Cbfa1 in response to Toll-like receptor 2/4 stimulation (Meng et al. 2008). All in all, transformed osteoblastic valvular interstitial cells seem to play a central role in local osteogenesis (Liu A. C. et al. 2007).

3.5. Extracellular matrix remodeling Disruption of normal extracellular matrix (ECM) structure is evident in stenotic aortic valve tissue. Active ECM remodeling is, indeed, a part of the pathogenesis of AVS from its initial steps to the detrimental outcome. Some early pathological events that require ECM degradation are the recruitment of inflammatory cells into tissue through the endothelial basement membrane, migration of proliferating valvular myofibroblasts, and neovascular sprouting (Liu J. et al. 2004). Furthermore, the same mechanisms may be involved in active valvular osteogenesis and bone remodeling, as well as in the modulation of local inflammatory responses (Liu J. et al. 2004). In AVS, valvular interstitial cells undergo myofibroblast differentiation and assume an active, profibrotic phenotype (Olsson et al. 1994b, Otto et al. 1994). The myofibroblasts produce increased amounts of ECM components, such as collagen I (Eriksen et al. 2006). At the same time, extensive ECM degradation accounts for the disruption of normal laminar arrangement of the matrix protein and proteoglycan network. Increased overall matrix turnover has been detected in stenotic valves (Eriksen et al. 2006), and a recent proteomic analysis revealed a major decrease in ECM components in calcified areas of stenotic valves, when compared to macroscopically healthy areas of the same valves (Matsumoto K. et al. 2012). In fact, individual


extracellular proteases capable of degrading matrix proteins and proteoglycans have been identified in stenotic aortic valves. These include members of the metalloproteinase superfamily (such as matrix metalloproteinases, or MMPs) as well as serine proteases (tryptase, chymase, and cathepsin G) (Edep et al. 2000, Helske et al. 2004b, Helske et al. 2006). Macrophages, lymphocytes, and myofibroblasts in stenotic aortic valves show increased expression of MMPs -1, -2, -3, and -9, MMP-9 being expressed only in the diseased valves (Edep et al. 2000). Tissue inhibitor of metalloproteinases-1 (TIMP-1) has also been identified in myofibroblasts of the diseased valves (Soini et al. 2001). The expression of MMP-1 and MMP-2 by human aortic valve myofibroblasts increases in response to the inflammatory cytokine IL-1 and the osteogenic mediator RANKL (Kaden et al. 2003, Kaden et al. 2005b). Moreover, TNF-α stimulates the expression of MMP-1 but not TIMP-1 in the myofibroblasts (Kaden et al. 2005a). Taken together, the local inflammatory and osteogenic environment seems to promote ECM degradation by myofibroblast-derived MMPs. Information on the balance between MMPs and TIMPs in stenotic valvular tissue is, however, somewhat inconsistent (Fondard et al. 2005). Three serine proteases – chymase, tryptase, and cathepsin G – have been shown to be locally produced in the aortic valve, their only valvular source being the mast cell (Helske et al. 2004b, Helske et al. 2006). These powerful proteases may directly degrade ECM in the valves. Moreover, chymase may also promote valvular fibrosis by inducing mast cell migration and degranulation and by generating an active form of transforming growth factor beta 1 (TGF-β1) (Lindstedt et al. 2001). Importantly, chymase and the neutral protease cathepsin G, which also exerts elastolytic activity (Boudier et al. 1991), are capable of generating angiotensin II (Ang II) from Ang I (Genest et al. 1983). Ang II is part of the circulating renin-angiotensin-aldosterone hormone system and is also produced locally in tissues where it exerts autocrine and paracrine effects (Weber et al. 1999). This pathway is of particular interest because of the existing possibilities for pharmacological intervention. Ang II e.g. induces the production of TGF-β1 by macrophages (Weber et al. 1999). In stenotic aortic valves, areas rich in cathepsin G-positive mast cells show increased fragmentation of elastic fibers as an indication of their local elastolytic activity (Helske et al. 2006). Interestingly, mast cell-rich areas also accumulate TGF-β1 (Helske et al. 2006), potentially resulting from the generation of Ang II by the cathepsin G-secreting mast cells. TGF-β1, in turn, promotes the differentiation of valvular interstitial cells into the more profibrotic myofibroblasts, further contributing to adverse ECM remodeling (Weber et al. 1999). TGF-β1 has also been linked to valvular calcification, as it associates with calcific areas of the stenotic valves (Jian et al. 2003), and promotes calcification in cultured valvular interstitial cells (Mohler et al. 1999). Besides cathepsin G, other cathepsins have also been implicated in adverse ECM remodeling in vascular disease. Cathepsins S and K, lysosomal cysteine proteases also capable of extracellular degradation, are overexpressed in atherosclerotic lesions (Liu J. et al. 2004). The expression of their endogenous inhibitor cystatin C, in turn, is decreased in the same pathology (Shi et al. 1999). Accumulating evidence suggests a crucial role for the elastolytic and collagenolytic actions of these cathepsins in atherogenesis (Liu J. et al. 2004). The potent collagenolytic activity of cathepsin K enables its role in bone metabolism (Brömme et al. 1996), rendering a potential function for cathepsin K also in valvular calcification. Moreover, in 2009 Aikawa et al. demonstrated that cathepsin S-deficiency truly prevents aortic valve calcification in a mouse model of AVS, highlighting the importance of this particular cathepsin in valvular disease (Aikawa et al. 2009). Cathepsin V is an intracellular protease identified from activated


macrophages in atherosclerotic plaques (Yasuda et al. 2004). This potently elastolytic protease is also capable of degrading apolipoprotein B100, the protein component of LDL (Linke et al. 2006). These findings suggest a role for cathepsin V in valvular disease as well. The implications of proteases in angiogenic processes are introduced in Section 4.2.

3.6. Experimental animal models Several animal models of AVS have been developed to obtain valuable information on the underlying molecular mechanisms of valve stenosis and to connect them with clinical practice. Medical interventions demonstrated in these studies have, indeed, showed promise in the field of possible diagnostic assessment and pharmacotherapy for AVS in the future (Miller J. D. et al. 2011, Rajamannan 2009). Rabbit models Early AVS has been demonstrated in cholesterol-fed New Zealand White rabbits already in the 1980s, with changes in valvular surface endothelium (Sarphie 1985). Furthermore, changes in extracellular matrix production, foam cell formation and osteogenic markers have been identified in the same model, atorvastatin having been able to attenuate some of the pathological changes (Rajamannan et al. 2002). Atorvastatin has also been shown to inhibit calcification in Watanabe rabbits, which lack LDL receptors (LDLr

-/-) (Rajamannan et al. 2005).

Interestingly, valvular angiogenesis has been described in cholesterol-fed New Zealand White rabbits, with in vivo quantification using integrin-targeted nanoparticles and 19-fluoreine magnetic resonance imaging (Waters et al. 2008). In addition to histopathological findings, even hemodynamic changes of early AVS have been induced in these rabbits with a combined diet of cholesterol and excessive vitamin D (Drolet et al. 2003). Echocardiographically detectable changes have also been induced with vitamin D alone, and in this model ACE inhibitors were able to counteract most of the effect of vitamin D on valve thickening and calcification (Ngo et al. 2011). In some investigations, however, cholesterol and vitamin D diets induced calcification only in the ascending aorta rather than the aortic valve itself, questioning the ability of the diet models to mimic human disease (Hekimian et al. 2009). Mouse models Even hemodynamically severe stenosis has been demonstrated in mice lacking the receptor for LDL and expressing only human apolipoproteinB 100 (LDLr

-/-apoB

100/100) (Weiss et al. 2006). In

fact, several mechanistic studies on mice have strengthened the role of actively regulated osteogenic factors, hyperlipidemia, metabolic syndrome, and angiogenesis in the pathogenesis of AVS (Aikawa et al. 2007, Drolet et al. 2006, Miller J. D. et al. 2010, Yoshioka et al. 2006), and suggested a causal role for angiogenesis in the development of AVS (Yoshioka et al. 2006). Furthermore, the administration of angiotensin II induces aortic valve thickening independently of its effect on blood pressure in apolipoprotein E-deficient mice (Fujisaka et al. 2013). Some evidence of the potential benefits of ACE inhibitors has been presented in a uremic mouse model (Simolin et al. 2009). A major limitation in mice as an experimental model for AVS is their small size, which compromises the possibilities to reliably conduct extensive hemodynamic and histopathological analyses (Miller J. D. et al. 2011). Moreover, the effect of arterial atherosclerosis of the surrounding aorta and the sinuses of valsalva to the hemodynamic measurements of the aortic valve may not entirely be excluded.


Angiogenesis and lymphangiogenesis 4.

4.1. Normal vascular development

4.1.1. Overview Angiogenesis can occur by several mechanisms depending on the prevailing conditions. During early development, blood vessels form in a completely avascular tissue by the differentiation and proliferation of endothelial progenitor cells in a process called vasculogenesis. These newly formed blood vessels, i.e. neovessels, are then modified to meet the needs of their surroundings by angiogenic remodeling. Angiogenic remodeling affects all blood vessel components, including endothelial cells and their interactions, supporting cells, such as pericytes, and the surrounding extracellular matrix (Yancopoulos et al. 2000). Angiogenic sprouting, the prevalent type of vascular expansion, refers to a process in which new blood vessels sprout into tissue from existing vessels, generating a primary vascular network. This usually involves the destabilization of the pre-existing blood vessel, including extracellular matrix degradation to allow the endothelial cells to migrate and form the new vessel (Yancopoulos et al. 2000). New blood vessels can also split from a pre-existing vascular network through a process called intussusceptive angiogenesis or splitting angiogenesis (De Spiegelaere et al. 2012). The three mechanisms of angiogenesis are illustrated in Figure 3. In addition to these proliferative mechanisms, vascular regression and pruning are also vital for shaping a functioning vascular network. In the fully developed cardiovascular system, blood is pumped from the heart into the tissues via arteries and returned through the venous system.

Figure 3. Mechanisms of angiogenesis, as described in (De Spiegelaere et al. 2012). A) In vasculogenesis, endothelial progenitors differentiate and form rudimentary tube structures that eventually mature into functional blood vessels. B) Angiogenic sprouting from pre-existing blood vessels is an invasive process. The endothelial tip cell with its filopodia leads the sprout to the surrounding tissue by e.g. secreting matrix-degrading components. Proliferating stalk cells follow the tip cell, forming the vessel lumen. The remaining quiescent endothelial cells are called phalanx cells. C) Intussusceptive angiogenesis enables the expansion and remodeling of an existing vascular network by splitting pre-existing capillary tubes into parallel structures.


Lymphatic vessels collect extracellular fluid, macromolecules, and cells from the cellular interstitial space back into the circulating blood. Lymphatic capillaries are blind-ended, with loose lining endothelium, little or no basement membrane, and no supporting cells. From the capillaries, lymph flows to collecting lymphatic vessels that are surrounded by smooth muscle cells and also contain valves. On the way to the lymphatico-venous junctions, lymph goes through lymph nodes, where antigen-presenting cells may initiate immune responses. This is also a well-known route for cancer metastasis, and major advances in lymphatic research have, indeed, been made in cancer research (Alitalo et al. 2005).

4.1.2. Vascular endothelial growth factors and their receptors The best-known angiogenic growth factors are the vascular endothelial growth factors (VEGFs), which are also considered to be the most important factors in regulating both angiogenesis and lymphangiogenesis. Up to date, five members of this family have been identified in mammals: VEGF-A, -B, -C, and -D, and placental growth factor (PlGF) (Joukov et al. 1996, Leung et al. 1989, Maglione et al. 1991, Olofsson et al. 1996, Senger et al. 1986, Yamada et al. 1997). The cellular receptors of these growth factors are vascular endothelial growth factor receptors 1-3 (VEGFR-1, -2, and -3) (Aprelikova et al. 1992, de Vries et al. 1992, Terman et al. 1991). The binding pattern of the VEGFs to their receptors is illustrated in Figure 4. The affinity of the VEGFs to their specific receptors may also be affected by neuropilins 1 and 2, which act as their co-receptors (Lohela et al. 2009).

Figure 4. VEGFs and their receptors as described in (Lohela et al. 2009). The binding of VEGFs to their receptors causes receptor dimerization, autophosphorylation, and activation of corresponding cellular signaling cascades. VEGFR-2 is responsible for the


major angiogenic effects of the VEGFs, inducing endothelial cell proliferation and migration (Lohela et al. 2009). It also increases vasodilatation and vascular permeability by activating endothelial nitric oxide synthase (Shen et al. 1999). VEGFR-1, in turn, may act as a VEGF trap, thus scaling down the angiogenic effects of VEGFR-2 and guiding angiogenic sprouts (Saharinen et al. 2010). It may also modulate VEGFR-2 signaling and increase cell recruitment via VEGFR-2. These effects depend at least partly on receptor heterodimerization of VEGFR-1 with VEGFR-2 (Lohela et al. 2009). VEGFR-3 controls lymphangiogenesis during embryonic development and in pathological conditions later in life. In adults, the expression of VEGFR-3 mainly confines to lymphatic endothelium, but some angiogenic properties have also been described (Lohela et al. 2009). Indeed, VEGFR-3 seems to be involved at least in venous sprouting and tumor angiogenesis (Saharinen et al. 2010). VEGF-A is clearly required for both vasculogenesis and vascular homeostasis; lack of VEGF-A is lethal already at the embryonic stage, and endothelial cell survival depends on autocrine VEGF-A signaling (Lohela et al. 2009). VEGF-A binds to both VEGFR-2 and VEGFR-1, VEGFR-2 accounting for the angiogenic effects as described above. Several isoforms of VEGF-A have been identified, with differences in binding to ECM components and co-receptors of the VEGFRs (Lohela et al. 2009). Local hypoxia, shortage of oxygen resulting from increased cellular metabolic load, strongly induces the expression of VEGF-A in order to enhance the defective blood supply (Shweiki et al. 1992). The expression of VEGF-B and PlGF varies in different tissues. The highest levels of VEGF-B can be found in the heart and in skeletal muscle, whereas PlGF is expressed in the placenta, the heart, and the lungs. Both growth factors only activate VEGFR-1 (Lohela et al. 2009), resulting in corresponding modulatory effects. VEGF-C and VEGF-D act through VEGFR-3, and are, thus, lymphangiogenic (Lohela et al. 2009). VEGF-C is first synthesized as a precursor, and the generation of active VEGF-C from a dimerized propeptide requires both intracellular and extracellular proteolytic modification (Joukov et al. 1997). These modifications increase the affinity of VEGF-C to VEGFR-3 and enable the binding of VEGF-C to VEGFR-2. The most processed form, in which the disulfide bonds of both N- and C- terminal peptides are cleaved (VEGF-CΔNΔC), activates VEGFR-2 equally to VEGF-A at 4- to 5-fold concentrations (Joukov et al. 1997). Correspondingly, the activation of VEGFR-2 by VEGF-A or VEGF-CΔNΔC stimulates endothelial cell invasion and tube formation in vitro (Tille et al. 2003). The posttranslational processing of VEGF-D is similar to that of VEGF-C, as the mature form of VEGF-D is able to activate VEGFR-2 in addition to VEGFR-3 (Achen et al. 1998). In fact, the angiogenic activity of the processed form of VEGF-D may even surplus that of VEGF-A at high expression levels, as demonstrated in an adenoviral gene transfer model in rabbits (Rissanen et al. 2003). Other angiogenic growth factors with established roles in angiogenic sprouting and remodeling are the angiopoietins and ephrins, and their respective receptors Ties and Ephs. Angiopoietin-1 participates in maintaining the stability of mature blood vessels, whereas angiopoietin-2 may act as a de-stabilizing signal initiating angiogenic remodeling (Yancopoulos et al. 2000). Ephrins, in turn, are linked to the interactions of arterial smooth muscle and endothelial cells (Yancopoulos et al. 2000). Nevertheless, the VEGFs remain the most critical initiating factors for both vasculogenesis and angiogenic sprouting.


4.1.3. Angiogenic remodeling and vascular sprouting Following vasculogenesis, the maturing of primary vasculature into functioning capillaries involves several changes in the vessel wall. The blood vessels enlarge, endothelial cells integrate, assume a quiescent phenotype, and become covered with supporting cells, such as pericytes in capillaries and postcapillary venules, and smooth muscle cells in arterioles and larger arteries (Allt and Lawrenson 2001, Yancopoulos et al. 2000). The supporting cells provide stability to the blood vessel by regulating endothelial cell function and by secreting reinforcing ECM structures. Also, by secreting TGF-β, pericytes inhibit endothelial cell growth and migration, thus inhibiting angiogenic sprouting. Accordingly, the loss of pericytes leads to uncontrolled capillary proliferation and microaneurysms (Allt and Lawrenson 2001). Arterioles and larger arteries are even more organized, consisting of three distinct layers: a thin luminal intima, a medial layer of contractile smooth muscle cells and elastic ECM, and an outer adventitial layer of loose connective tissue. In addition to structural support, the contractile smooth muscle cell layer enables the local and hormonal control of blood flow, adjusting the delivery of oxygen and nutrients into tissues in demand. The organized structures described above need to be disrupted for angiogenic sprouting to occur. Importantly, the local sustained inflammation observed already in early sclerotic aortic valve lesions could serve as a trigger for an angiogenic response (Arroyo and Iruela-Arispe 2010). Inflammatory cytokines, including TNF-α and MCP-1, enhance vascular permeability, which, in turn, allows for both inflammatory and endothelial cell migration (Arroyo and Iruela-Arispe 2010). Endothelial tip cell activation is the first step of angiogenic sprouting, and VEGF-A ia a crucial promoting factor in this process. In fact, VEGF-A and -C induce endothelial cell migration via VEGFR-2 and -3 expressed by the tip cells (Lohela et al. 2009). Furthermore, the balance between VEGFs, angiopoietins, and ephrins seems to be central in regulating vascular stability and permeability, as well as in inhibiting excessive angiogenesis (Yancopoulos et al. 2000). In addition to VEGF signaling, regulation of the periendothelial ECM also plays a role in the angiogenic activation of endothelial tip cells, collagen I being a potent angiogenic component and laminin 1 its counteractor (Davis and Senger 2005). To invade the surrounding tissue, endothelial tip cells secrete ECM-degrading proteases, including MMPs -2, -3, and -9 also found in stenotic aortic valves (Arroyo and Iruela-Arispe 2010, Edep et al. 2000). Furthermore, inflammatory cells participate in the angiogenic invasion by secreting proteases that both degrade ECM and cleave the VEGFs into their active forms (Arroyo and Iruela-Arispe 2010). Proliferating stalk cells follow the tip cell, forming the vessel lumen. The commitment of stalk cells to tip cell phenotype is inhibited by the tip cell via NOTCH1-mediated downregulation of VEGFR- and -3 (Lohela et al. 2009). In addition to the locally proliferating cells, circulating endothelial progenitors may also contribute to vascular sprouting (Urbich and Dimmeler 2004). Again, VEGF may play a role in mobilizing these cells from the bone marrow to areas of active neovascularization (Costa et al. 2007). Regression of the newly formed capillaries follows the resolution of the inflammatory response (Arroyo and Iruela-Arispe 2010). In many pathological conditions, however, chronic inflammation maintains the vascular network, and the primary sprouts may mature to organized vessels (Costa et al. 2007).


4.1.4. Lymphatic vessel development and sprouting Lymphatic vessels arise from the venous system during early embryonic development. Transformed vascular endothelial cells expressing VEGFR-3, the homeobox transcription factor PROX-1 and Lymphatic vascular endothelial hyaluronan receptor (LYVE-1) emerge in the cardinal vein. This lymphatic commitment results in the formation of primary lymph sacs that continue maturing into lymphatic capillaries and collecting structures (Karpanen and Alitalo 2008). The predominant pathway in lymphatic vessel growth is the activation of VEGFR-3 by VEGF-C and -D (Alitalo et al. 2005). In addition, several co-receptors as well as interplay between angiogenic growth factors, such as VEGF-A and angiopoietin-1, have been identified to be involved in lymphangiogenesis (Alitalo et al. 2005). In adults, lymphatic vessel growth follows the angiogenic response in physiological and pathological conditions, such as wound healing, tumor growth and inflammation, but the exact origin of lymphatic sprouts in these conditions remains unclear (Karpanen and Alitalo 2008). Nevertheless, the proinflammatory cytokines IL-1β and TNF-α may play a role in inflammation-induced lymphangiogenesis, as they are able to induce the production of VEGF-C by human lung fibroblasts (Ristimaki et al. 1998). Lack of lymphatic vasculature leads to interstitial fluid and protein accumulation. The chronic stage, lymphedema, results in dermal fibrosis, an increase in adipose and connective tissue, and impaired wound healing and immune defense in the affected area (Karpanen and Alitalo 2008). Remarkably, compensating lymphatic vasculature has been successfully grown in experimental models using a VEGFR-3-specific form of VEGF-C (Karpanen and Alitalo 2008), highlighting the importance of this potent lymphangiogenic growth factor.

4.2. Proteases in angiogenesis and lymphangiogenesis

A variety of proteases participate in the modulation of periendothelial and extracellular matrix involved in angiogenic sprouting and vascular remodeling. The proteolytic degradation of ECM not only clears the way for the emerging angiogenic sprouts but also modulates angiogenesis by releasing fragments from ECM components and by inducing conformational changes in some ECM proteins (Bellon et al. 2004). Among the generated fragments, called matrikines, are plasminogen-derived angiostatin, endostatin cleaved from collagen XVIII, and several non-collagenous domains of type IV collagen, all of which are considered anti-angiogenic (Bellon et al. 2004). Remarkably, excess endostatin has also been shown to hinder tumor lymphangiogenesis in mice (Brideau et al. 2007). Angiostatin can be converted from plasminogen by elastase and by MMP-3, -7, and -9 (Bellon et al. 2004), whereas endostatin is generated by elastase, several MMPs, and cathepsins, including MMP-3, MMP-9, and cathepsin K (Ferreras et al. 2000). Some MMPs, such as MMP-1 and -10, even seem to be involved in vascular regression rather than mere angiogenic modulation (Arroyo and Iruela-Arispe 2010). In contrast to the abovementioned antiangiogenic actions, MMP-3 and -9 also promote angiogenesis by modulating VEGF-A to its more angiogenic forms (van Hinsbergh et al. 2006). Furthermore, proteolytically cleaved fragments of collagen I, collagen IV, and elastin attract inflammatory cells and are subsequently able to induce the expression of various proteases and cytokines (Arroyo and Iruela-Arispe 2010), thus indirectly promoting both angiogenesis and lymphangiogenesis. More specifically, neutral elastolytic proteases, including MMP-2, MMP-9,


and cathepsin G, have been speculated to promote angiogenesis by generating such fragments from intact elastin (Bellon et al. 2004). Some cathepsins even seem to be crucial for angiogenesis. This has been demonstrated in cathepsin S-deficient mice that showed decreased endothelial cell invasion capacity, as well as defective microvessel growth, during wound repair (Shi et al. 2003). Indeed, loss of cathepsin S activity in mice lacking LDL receptor resulted in reduced neovascularization in atherosclerotic lesions as well (Liu J. et al. 2004). Lymphatic capillary endothelium lacks a proper basement membrane and is attached to its surroundings by loose anchoring filaments composed mainly of elastic fibers. Nevertheless, the proteolytic processes involved in lymphatic endothelial cell migration resemble those in angiogenic sprouting (Ji 2006). Importantly, the intracellular and extracellular proteolytic processing of VEGF-C and -D is essential for both their lymphangiogenic and angiogenic actions (Achen et al. 1998, Joukov et al. 1997).

4.3. Angiogenesis and lymphangiogenesis in the aortic valve In studies using injection techniques to identify vascular structures, blood vessels have been infrequently identified in healthy human aortic valve leaflets, while lymphatic vessels have been completely absent (Johnson and Blake 1966). In stenotic valve leaflets, neovessels have been consistently detected, and angiogenesis has been recognized as a potential pathological process contributing to the progression of AVS (Mazzone et al. 2004, Soini et al. 2003). Neoangiogenesis has even been suggested to be the initiating trigger of AVS (Hakuno et al. 2009). Neovessels can be seen in up to 85 % of the stenotic valve samples, and they correlate with valvular inflammatory cells and calcium content (Mazzone et al. 2004). In fact, blood vessels particularly manifest themselves adjacent to cell-rich and calcific areas, but also close to valve surface (Charest et al. 2006). Abundant neovessels in cellular islets together with the presence of endothelial progenitor cells implicate that vasculogenesis occurs in the stenotic aortic valves (Charest et al. 2006). Valvular neovessels have also been speculated to originate from existing arteries of the aorta, i.e. the vasa vasorum, and by budding from valvular surface endothelium (Charest et al. 2006, Steiner et al. 2010). Like angiogenesis, lymphangiogenesis has been linked to the pathogenesis of atherosclerosis (Nakano et al. 2005). Even though the role of lymphatics in valvular disease was speculated already in the 1980s (Miller A. J. 1982), lymphatic vessels were immunohistochemically detected from stenotic aortic valves only recently, and their role in valvular pathology is unclear (Kholova et al. 2011, Steiner et al. 2010).

4.4. Local modulators of angiogenesis and lymphangiogenesis in aortic valves

Inflammation being an important trigger for angiogenic sprouting (Arroyo and Iruela-Arispe 2010), the infiltration of inflammatory cells and the ensuing hypoxia from the rapidly increasing cellular metabolic load could launch an angiogenic response in the normally avascular valve


leaflets. Furthermore, the identified extensive mechanisms of ECM remodeling provide a favorable environment for angiogenic sprouting. In addition to these pathological events, several specific local mechanisms potentially contribute to valvular angiogenesis. Table 1 summarizes potential antiangiogenic and proangiogenic players identified in aortic valves. Detailed descriptions and corresponding references are provided in the text below and in Sections 3.5 (Extracellular matrix remodeling), and 4.2 (Proteases in angiogenesis and lymphangiogenesis). Table 1. Factors potentially involved in valvular angiogenesis. These mediators may contribute to valvular angiogenesis by different mechanisms: VEGFs strongly affect endothelial cell (EC) migration and survival, whereas MMPs and cathepsins may exert both anti- and proangiogenic effects by ECM degradation and proteolytic processing. In addition, some literature-based potential contributors of valvular angiogenesis are shown in grey. Links to corresponding thesis substudies are also denoted. Arrows indicate observed changes in AVS.

Mechanism Antiangiogenic Angiogenic

EC migration Endostatin (↑) (II) VEGF-A (via VEGFR-2 ↑) (II)

and/or survival Chondromodulin I ↓ Inflammation (e.g. TNF-α, MCP-1 ↑) (II-IV)

TGF-β ↑ Elastin (proteolytically processed)

Thrombospondin-1 ↔ VEGFs -C and -D (III)

Angiopoietin-1 Angiopoietin-2

Laminins Collagen I

Cathepsin S (I)

ECM degradation TIMP-1 ↔ MMP-2, -3, and -9 (macrophages, myofibroblasts) ↑

(expressing cells) Thrombospondin-2 ↑ Tryptase, chymase, Cathepsin G (mast cells) ↑

Other cathepsins (I)

Proteolytic MMP-3 and -9 ↑ (endostatin) MMP-3 and -9 ↑ (VEGF-A)

processing MMP-3 ↑ (osteonectin) MMP-3 ↑ (osteonectin)

(target molecule) Cathepsin K (endostatin) (I) MMP-2 and -9, cathepsin G ↑ (elastin)

Other Osteonectin ↑ Osteonectin ↑

Angiotensin II ↑

LRP5 ↑

OxLDL ↑ (scavenger receptors) (IV)

VEGF-A has been detected in stenotic aortic valves, and its receptors VEGFR-1 and VEGFR-2 are expressed by valvular endothelial and interstitial cells (Soini et al. 2003). In addition, the VEGFR-2 positivity of valvular interstitial cells correlates positively with valvular blood vessel density (Soini et al. 2003). Osteonectin (aka SPARC; secreted protein, acidic and rich in cysteine) is also associated with blood vessels in stenotic aortic valves, being altogether absent from healthy leaflets (Charest et al. 2006). Traditionally, osteonectin is a mineralization-promoting glycoprotein secreted by osteoblasts (Termine et al. 1981), but it also has pro- and antiangiogenic effects (Bellon et al. 2004). Notably, serine proteases and cathepsins can cleave angiogenic fragments from the intact protein (Iruela-Arispe et al. 1995). One of the proteases involved in generating these modulatory peptides is MMP-3 (Sage et al. 2003), which is upregulated in AVS (Edep et al. 2000). Osteonectin can also induce the expression of MMP-9


(Bradshaw 2012), and indeed, osteonectin and MMP-9 colocalize in stenotic valves, mainly in the areas of neovascularization (Charest et al. 2006). In addition, other potentially angiogenic factors with less evident association to valvular neovascularization have been detected in stenotic aortic valves. Upregulation of the low-density lipoprotein receptor-related protein 5 (LRP5) pathway may contribute to bone formation in stenotic aortic valves (Caira et al. 2006), but the same protein has also been linked to capillary maturation (Xia et al. 2008). Furthermore, angiotensin II-producing systems are upregulated in AVS (Helske et al. 2004b). Since Ang II is able to induce VEGF-A expression in cardiac microvascular endothelial cells (Fujiyama et al. 2001), we may surmise that the locally generated Ang II also possesses some angiogenic potential in the diseased valves. The proangiogenic environment in stenotic aortic valves may also depend on the suppression of local antiangiogenic factors. Convincing evidence from an aging mouse model as well as human samples and cell culture experiments suggests that chondromodulin-I prevents angiogenesis in healthy valve leaflets and that its downregulation is involved in valvular angiogenesis (Yoshioka et al. 2006). Indeed, VEGF-A-expressing areas of human diseased leaflets show increased vascular densities and diminished chondromodulin-I positivity (Yoshioka et al. 2006). Another antiangiogenic molecule detected from stenotic valve tissue is the collagen XVIII-derived matrikine endostatin, which inhibits angiogenesis by e.g. counteracting VEGF-A-induced endothelial cell migration (Bellon et al. 2004, Chalajour et al. 2004). In contrast to the upregulation of proangiogenic and downregulation of antiangiogenic factors described above, certain antiangiogenic mechanisms seem to be induced in AVS. The expression of thrombospondin-2, an intracellular protein involved in MMP degradation and inhibition of angiogenesis, is increased in AVS (Pohjolainen et al. 2012). As several MMPs are also upregulated in AVS, the compensatory actions of thrombospondin-2 may be insufficient in controlling the proangiogenic actions of the MMPs (Edep et al. 2000, Pohjolainen et al. 2012). In contrast to the identified mechanisms of angiogenic regulation described above, the list of specific factors potentially promoting lymphangiogenesis in stenotic aortic valves is still short. In addition to the general ECM destruction and remodeling, the only pro-lymphangiogenic factor detected from stenotic aortic valves is macrophage-derived TNF-α (Kaden et al. 2005a). However, potential candidates can be found in atherosclerotic plaques, where e.g. VEGF-D expression decreases in complicated lesions, and the remaining VEGF-D localizes to areas of neovascularization (Rutanen et al. 2003). Thus, examining the expression of lymphangiogenic growth factors in AVS could uncover previously unrecognized disease-altering mechanisms.

4.5. Angiogenic potential of mast cells Mast cells are inflammatory cells that store histamine, neutral proteases and inflammatory cytokines in heparin-rich granules that are released into the surrounding tissue upon mast cell activation. Mast cells are consistently found close to small blood vessels and lymphatic vessels, the former having been demonstrated in atherosclerotic plaques as well (Kaartinen et al. 1996a). In fact, a role for mast cells has been proposed in the pathological angiogenesis associated with atherosclerosis, abdominal aortic aneurysms, obesity, and cancer (Ribatti and Crivellato 2011, Xu and Shi 2012). Mast cells also contribute to lymphangiogenesis in cancer, and cultured mast cells secrete lymphangiogenic growth factors (Detoraki et al. 2009, Ribatti


and Crivellato 2011). However, the link between mast cells and lymphangiogenesis has remained unestablished in atherosclerotic diseases. In addition to the proteolytic ECM degradation required in angiogenic sprouting, mast cells can participate in angiogenesis by secreting VEGF-A and a wide variety of other angiogenic mediators including growth factors, agents increasing vascular permeability, proteases that release growth factors from ECM, and chemokines attracting macrophages and lymphocytes (Grützkau et al. 1998, Norrby 2002). In stenotic aortic valves, activated mast cells secrete ECM-degrading tryptase, and also chymase and cathepsin G, which, in addition to being capable of degrading ECM, are also able to generate the angiogenic effector angiotensin II (Helske et al. 2004b, Helske et al. 2006). Even though VEGF-A has been detected in endothelial cells and myofibroblasts in stenotic aortic valves (Soini et al. 2003), and mast cells in other tissues have been found to contain VEGF-A (Grützkau et al. 1998), the ability of valvular mast cells to produce this potent angiogenic factor is unknown.

4.6. Scavenger receptors and angiogenesis Both antiangiogenic and proangiogenic actions of oxLDL on vascular endothelial cells have been described; oxLDL may inhibit endothelial cell proliferation but it also induces capillary tube formation (Chen et al. 2000, Dandapat et al. 2007, Yu et al. 2011). The inhibitory effects seem to result from downregulation of another angiogenic molecule, basic fibroblast growth factor (Chen et al. 2000). The increase in capillary tube formation, in turn, was first linked to LOX-1-mediated VEGF-A upregulation (Jiang et al. 2011). More recently, a vast range of genes involved in cell adhesion, migration, and angiogenesis were found to be regulated by oxLDL via LOX-1 (Khaidakov et al. 2012), consolidating the role of this scavenger receptor in the angiogenic actions of oxLDL. Intriguingly, one of the proangiogenic properties of angiotensin II is its ability to induce LOX-1 expression (Hu et al. 2007). The class B scavenger receptor CD36 has a variety of functions in the immune system, and it is centrally involved in macrophage foam cell formation in atherosclerosis (Stephen et al. 2010). In contrast to LOX-1, the angiogenic actions of CD36 seem to be independent of oxLDL. In endothelial cells, CD36 mediates apoptotic signals of thrombospondins-1 and -2. Furthermore, thrombospondin-1 inhibits endothelial cell migration via CD36, thus inhibiting angiogenesis (Lawler and Lawler 2012). Notably, thrombospondin-1 has been detected from stenotic aortic valves, but unlike thrombospondin-2, its expression levels remain unchanged in AVS (Pohjolainen et al. 2012). CD36 is also involved in the thrombospondin-induced inhibition of endothelial cell nitric oxide synthase pathway, thus counteracting the effects of VEGF-A on vascular permeability (Lawler and Lawler 2012). Moreover, CD36 has been linked to thrombospondin-1-mediated decrease in VEGF-C and inhibition of lymphangiogenesis in mice (Cursiefen et al. 2011), suggesting that CD36 may also mediate antilymphangiogenic signals. Ligands for both LOX-1 and CD36 have been identified in stenotic aortic valves (Olsson et al. 1999, Pohjolainen et al. 2012), as well as factors potentially inducing their expression, including angiotensin II and endothelin-1 (see Results, Table 9) (O'Brien et al. 2002, Peltonen et al. 2009b, Stephen et al. 2010). The expression of these scavenger receptors capable of modulating valvular angiogenesis has remained, however, an unstudied area of AVS research.


III AIMS OF THE STUDY The aim of this thesis was to investigate angiogenesis and lymphangiogenesis in aortic valve stenosis. The substudies focused on different steps of vascular remodeling, as well as the possible role of valvular mast cells and myofibroblasts along with their interactions. The following specific questions were addressed:

1) Are the elastolytic cathepsins S, K, and V, and their inhibitor cystatin C, expressed in stenotic aortic valves, and could they be involved in valvular neovascularization? (I)

2) Are mast cells involved in valvular neoangiogenesis and lymphangiogenesis, and could the interplay between mast cells and valvular myofibroblasts contribute to these processes? (II-IV)

3) Are lymphangiogenic growth factors expressed in the aortic valve, and could changes

in their expression play a role in AVS? (III)

4) Are scavenger receptors SR-A1, SR-B1, CD36, and LOX-1 expressed in the aortic valve, and may changes in their expression affect valvular neovascularization? (IV)

MATERIALS AND METHODS │ 35 _____________________________________________________________________________

IV MATERIALS AND METHODS The methods used in this thesis are summarized in Table 2. The methods are briefly described here, and detailed descriptions of all the methods including supplementary references are provided in the original publications. Table 2. Methods used in Studies I-IV.

Method Study Reference

Collection and preparation of tissue material I-IV

Histological stainings (Elastic van Gieson stain) I

Immunohistochemistry and double immunofluorescence I-IV (Helske et al. 2004b)

Confocal microscopy II

Computer-assisted morphometry I-III (Helske et al. 2006)

Fluorometric detection of cathepsin activity I (Werle et al. 1999)

Polymerase chain reaction (PCR) and semi-quantitative PCR I-II (Helske et al. 2004b)

Quantitative real-time PCR (qPCR) I, III-IV (Livak and Schmittgen 2001)

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to detect endostatin degradation

II (Laemmli 1970)

Isolation, culture and stimulation of human aortic valve myofibroblasts

II-IV (Helske et al. 2007)

LAD2 cell culture and activation II (Kirshenbaum et al. 2003)

Human peripheral blood-derived mast cell culture and activation

II-IV (Lappalainen J. et al. 2007)

Enzyme immunoassays II-IV

Isolation and oxidation of LDL IV (Havel et al. 1955)

Protein quantitation with Lowry IV (Lowry et al. 1951)

Measurement of thiobarbituric acid-reactive substances (TBARS) as a means to determine the degree of LDL oxidation

IV (Hessler et al. 1983)

Cellulose acetate electrophoresis (CAE) of lipoproteins as a means to determine the degree of LDL oxidation

IV

Statistical analyses I-IV

Clinical material (I-IV) The study population used in this thesis is characterized in Tables 3 and 5. Different subpopulations were used in each study, and they are further described in the original articles. The number of valves used in each analysis is denoted in the text. A total of 95 stenotic aortic valves were obtained at valve replacement surgery due to symptomatic, isolated AVS. We excluded patients with moderate to severe aortic regurgitation, mitral valve disease, a history of myocardial infarction, significant (>50 %) proximal coronary artery stenosis, complicated diabetes, renal insufficiency (serum creatinine >170 µmol/l), endocarditis, or malignancy.

36 │ MATERIALS AND METHODS _____________________________________________________________________________

Control material was collected from patients undergoing cardiac transplantation, valve replacement surgery due to aortic valve regurgitation, or from deceased organ donors whose hearts were unsuitable for use as grafts due to advanced age or suspected ischemia. Only macroscopically and microscopically healthy valves were accepted as controls, and samples with mild calcification were considered sclerotic. Typical causes of death in the organ donor group were brain trauma (n=9) and spontaneous intracerebral hemorrhage. Diagnoses leading to heart transplantation were dilated cardiomyopathy (n=11) and ischemic cardiomyopathy (n=4). Aortic valve regurgitation (AVR) was evaluated as moderate to severe at the time of surgery. The study complies with the declaration of Helsinki, and the protocol was approved by the Ethics committee of Helsinki University Central Hospital (approval ref. nr. 557/E5/02, clause nrs. 88/1999, 549/2002, 382/2005, and 141/13/03/01/10 for AVS and AVR material; approval ref. nr. 264/E06/05 for transplant and organ donor material). All the patients signed an informed consent, and the use of organ donor tissue was approved by The National Authority for Medicolegal Affairs of Finland (approval ref. nr. 4444/32/300/05). Table 3. Characteristics of all the patients and control subjects included in the immunohistochemical analyses and PCR analyses in Studies I-IV. Data are presented as mean ± SD (range) or number of patients (%).

AVS patients (n=95)

Organ donors (n=23)

Transplants (n=15)

AVR (n=6)

Age (years) 67 ± 10 (39-86)

57 ± 7 (43-68)

52 ± 11 (27-62)

66 ± 9 (55-79)

Sex, male/female 46/49 12/8 13/2 5/1

NYHA class, 1/2/3/4 4/58/28/2 1/3/2/0

Aortic valve area (cm2) 0.67 ± 0.18

(0.3-1.1)

Mean pressure gradient (mmHg) 49 ± 16 (14-95)

Number of cusps, 2/3/4 13/75/1* 1/22/0 0/15/0 0/6/0

Smoking, No/Ex-smoker/Current 60/20/15 ?/?/8 6/4/0 4/2/0

Diabetes 5 (5%) 2 (33%)

Hypertension 32 (34%) 5 (83%)

LDL cholesterol (mmol/l) 3.1 ± 0.9 (1.3-5.0)

Medication ACE-I/ARB 24 (25%) 1 (13%)** 15 (100%) 3 (50%) β-blocker 58 (61%) 2 (25%)** 12 (80%) 3 (50%) Diuretic 32 (34%) 0 (0%)** 15 (100%) 4 (67%) Statin 22 (23%) 0 (0%)** 4 (27%) 0 (0%) Digitalis 6 (6%) 0 (0%)** 11 (73%) 0 (0%)

*Information available for 89 subjects **Information available for 8 subjects

Fluorometric detection of cathepsin activity (I) To detect the total activity of cathepsins S, K, and V in the aortic valves, cryostat sections of stenotic and control valves were diluted in 50µl of 20 mM MES buffer (pH 6.0; containing


2.5mM EDTA, 150 mM NaCl, 0.035% Brij, and 2.8 mM DTT) on 96-well microtiter plates. Enzymatic activity was kinetically measured by detecting the degradation of the synthetic substrate Z-Phe-Arg-7-amido-4-methylcoumarin (AMC) by fluorometry. A specific inhibitor of cysteine proteases, trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E64; 20 µM), was used in the inhibition experiments. Human recombinant cathepsins S and V were used as positive controls. Table 5. Primary antibodies used in Studies I-IV.

Antigen Clone / Cat number Host Isotype Conc. Source Study

Cathepsin S 1E3 / ab11059 Mouse IgG1 1.0 µg/ml Abcam Ltd, Cambridge, UK I

Cathepsin V AF1080 Goat IgG 1.0 µg/ml R&D Systems, MN, USA I

CD-31 (EC)* JC70A / M0823 Mouse IgG1k 10.0 µg/ml DAKO, Clostrup, Denmark I-IV

CD-34 (EC)* QBEnd/10 / NCL-L-END

Mouse IgG1k 0.5 µg/ml Novocastra, Newcastle upon Tyne, UK

I-IV

CD-36 FA6-152 Mouse IgG1 2.0 µg/ml Beckman Coulter, CA, USA IV

CD-68 (MØ) PG-M1/M0876 Mouse IgG3 0.4 µg/ml DAKO I, III, IV

CD-163 (MØ) 5C6-FAT / BM4041

Mouse IgG1 1.0 µg/ml Acris Antibodies, Hiddenhausen, Germany

I, III, IV

Cystatin C AF1196 Goat IgG 4.0 µg/ml R&D Systems I

LOX-1 MAB1798 Mouse IgG2b 5-10 µg/ml R&D Systems, MN, USA IV

LYVE-1 AF2089 Goat IgG 1.0 µg/ml R&D Systems III

MCSP (aka NG2, P)

LHM2 / ab20156 Mouse IgG1k 2.4 µg/ml Abcam plc, Cambridge, UK II

Osteocalcin (O)

MAB1419 Mouse IgG1 2.0 µg/ml R&D Systems II

S-100 Z0311 Rabbit 9.0 µg/ml DAKO I

SMC Actin (MFB)

1A4 / M0851 Mouse IgG2a 1.4 µg/ml DAKO II-IV

SR-A1 MAB27081 Mouse IgG1 1.0 µg/ml R&D Systems IV

SR-B1 ab369 Rabbit 5 µg/ml Abcam IV

Tryptase (MC) AA1 / M7052 Mouse IgG1k 2.4 µg/ml DAKO II-IV

Tryptase (MC) Rabbit 0.6 µg/ml A kind gift from I. Harvima, Kuopio, Finland

II, IV

VEGF A-20 / sc-152 Rabbit 4.0 µg/ml Santa Cruz Biotechnology, CA, USA

II

VEGF-C AF752 Goat IgG 5.0 µg/ml R&D Systems III

VEGF-D AF286 Goat IgG 10.0 µg/ml R&D Systems III

VEGF-R3 AF349 Goat IgG 6.7 µg/ml R&D Systems III

Vimentin (MFB)

VIM 3B4 / NCL-VIM

Mouse IgG2a 0.13 µg/ml Novocastra II, III

EC = endothelial cells, MØ = Macrophages, P = Pericytes, O = Osteoblasts, MFB = Myofibroblasts, MC = Mast cells *Used as a mixture in order to detect neovessels (Jeziorska and Woolley 1999)


Histological, immunohistochemical, and double immunofluorescence stainings (I-IV) Cryostat sections of snap-frozen aortic valve leaflets were used for histological and immunohistochemical analyses in Studies I-IV. Myofibroblasts were cultured on coverslips and fixed with paraformaldehyde for the immunohistochemical stainings in Study II. In the histological staining used to detect elastin degradation in Study I, elastin fibers were stained dark purple with a hematoxyline-iodine-ferric chloride solution. Excess staining was then differentiated with a dilute ferric chloride solution, and the samples were counterstained with van Gieson solution to visualize collagen fibers (Accustain Elastic stain kit, Sigma-Aldrich). Immunohistochemical stainings were performed with standard avidin-biotin complex (ABC) (I-III) and horseradish peroxidase (HRP) (IV) techniques using commercial kits according to the manufacturers’ instructions (Vectastain Elite ABC kit, Vector laboratories; Dako Envision system-HRP, Dako) with minor modifications. All the primary antibodies used in Studies I-IV are presented in table 5. Cathepsin S signal was amplified with a commercial tyramide signal amplification (TSA) Kit (#22, Molecular probes). In order to detect possible colocalization of the studied antigens with valvular cell types, primary antibodies were used as mixtures and detected with isotype-specific Alexa Fluor® secondary antibodies (Invitrogen). In all the stainings, host and isotype-specific nonimmune immunoglobulins were used as negative controls. The stainings were analyzed using light or epifluorescent microscopy, and the subcellular distribution of tryptase and VEGF-A in mast cells (II) was also visualized with confocal microscopy. The sample area, the size of calcific nodules, and valvular elastin and myofibroblast contents were calculated with computer-assisted morphometry using SPOT Advanced software. The grade of valvular calcification was determined by assessing the number of minor (cross section <5 mm

2) and major (cross section >5 mm

2) calcific nodules as

follows: 1 = none; 2 = one minor; 3 = several minor; 4 = one or more minor and major, or several major nodules. Human aortic valve myofibroblast cell culture (II-IV) Myofibroblasts were isolated from human aortic valves freshly obtained at valve replacement surgery due to AVS or AVR (Table 5) using a previously described outgrowth method (Helske et al. 2007). The myofibroblast phenotype was confirmed with immunohistochemical stainings against α-actin and vimentin (II, Supplemental Figure II). To investigate the effect of various stimuli on the myofibroblasts, the cells were incubated in serum-free culture media with or without the studied substances for 24 and/or 48 hours at 37 °C. All experiments were performed with subconfluent myofibroblasts at passages four to five and in triplicates with cells from multiple donors. The secretion of VEGF-A and inflammatory cytokines into the cell culture media was detected with commercial enzyme-linked immunoassays (ELISA) according to instructions provided by the manufacturer (R&D Systems). Changes in cellular messenger RNA (mRNA) expression were detected from cell lysates with PCR methods as described below. In order to investigate the possible effects of cigarette smoke on the myofibroblasts, cigarette smoke-treated culture medium was prepared as follows. A burning cigarette was connected to a sterile 50 ml syringe with plastic tubing. Cigarette smoke was suctioned into the syringe containing 10 ml of serum-free culture medium with vigorous mixing between cycles. The medium containing the water-soluble components of cigarette smoke was then diluted and used in culture experiments.


Table 5. The number and origin of aortic valves used in cell culture experiments. A total of 22 stenotic and 5 control valves were used in Studies II-IV.

Study Aortic valve stenosis Aortic valve regurgitation

II 7 2

III 11 0

IV 12 4

Polymerase chain reaction (PCR), competitive PCR, and quantitative PCR (I-IV) Conventional (Cathepsins S and K, and cystatin C), semi-quantitative competitive (VEGF-A), and quantitative (Cathepsin V, VEGF-A, VEGF-C, VEGF-D, VEGFR-2, VEGFR-3, SR-A1, SR-B1, CD36, and LOX-1) PCR were performed using standard PCR methods further described in the original publications. The used primers and probes are also described in the original articles. In conventional and competitive PCR, the PCR products were quantified with a Gel Doc 2000 gel documentation system (Bio-Rad), and in competitive PCR the competitor-to-target ratio was plotted against the logarithm of the competitor DNA molecules. In the qPCR analyses, the data were normalized to an endogenous control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and relative units were calculated with a comparative Ct method using the formula 2

-∆∆CT (Livak and Schmittgen 2001).

LAD2 and human mast cell culture (II-IV) Human LAD2 mast cells, described in (Kirshenbaum et al. 2003), were only used to generate mast cell-conditioned medium (referred to as releasate in the text) in Study II. To prepare the releasate, the LAD2 cells were stimulated with calcium ionophore, and the media containing mast cell-derived substances was collected after a 24-hour incubation at 37 °C. All other experiments on mast cells were performed with human peripheral blood-derived mast cells generated and activated according to Lappalainen and coworkers (Lappalainen J. et al. 2007). In order to study the effect of myofibroblasts on the VEGF-A production by the mast cells, the mast cells were stimulated with serum-free myofibroblast-conditioned medium collected from valvular myofibroblast cell culture after a 24-hour incubation at 37 °C. VEGF-A secretion into the mast cell culture media was detected with a commercial ELISA kit (R&D Systems). Endostatin and VEGF-C degradation experiments (II-III) So as to study the effect of mast cell tryptase on endostatin, human recombinant endostatin (ProSpec-Tany TechnoGene Ltd) was incubated with or without human skin beta tryptase (Promega) in 50 mM Tris buffer (pH 7.4) for 0 min, 15 min, and 1,3,6, and 24 hours at 37 °C. Protein fragmentation was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by analyzing the gels with GelDoc 200 (Bio-Rad) after Simply blue staining (Invitrogen). To investigate the effects of mast cells on VEGF-A and VEGF-C secreted by valvular myofibroblasts, myofibroblast-conditioned media was incubated with or without mast cell releasate (prepared as above). The effect of specific proteases was tested by incubating recombinant human VEGF-C (R&D Systems) with or without mast cell releasate, human skin beta tryptase (Promega), recombinant human cathepsin G (Calbiochem), or recombinant human chymase (Sigma-Aldrich) for 24 hours at 37 °C. VEGF-A and VEGF-C were quantified with commercial ELISA kits (R&D Systems)


Isolation and oxidation of low-density lipoprotein (LDL) (IV) Human LDL was isolated from the plasma of healthy volunteers (Finnish Red Cross) by sequential ultracentrifugation as described by Havel and coworkers (Havel et al. 1955), and protein concentrations were measured according to the Lowry method (Lowry et al. 1951). For oxidation, the lipoproteins were dialyzed overnight at +4 °C against phosphate-buffered saline (PBS) and incubated in the presence of 10 µM CuSO4 for 18 hours at 37 °C. LDL oxidation was verified with Cellulose Acetate Electrophoresis (CAE) of Lipoproteins, and the degree of oxidation was determined by measuring thiobarbituric acid-reactive substances (TBARS). Sterile-filtered oxLDL was then used in cell culture experiments. Statistical analyses (I-IV) Normality of distributions was tested with the Kolmogorov-Smirnov method. In group comparisons, Student’s T-test was used for normally distributed data, and the nonparametric Mann-Whitney U test for skewed data and also for a small number of cases (n≤10/group). Respectively, Pearson’s product-moment or Spearman’s rank correlation coefficients were calculated for correlations. Statistical significance was set at p≤0.05. The current versions of SPSS for Windows software were used in all statistical analyses.

RESULTS AND DISCUSSION │ 41 _____________________________________________________________________________

V RESULTS AND DISCUSSION

Angiogenesis and lymphangiogenesis in AVS 1.

1.1. Morphological classification and localization of valvular neovessels and lymphatic vessels (II-III)

First, we characterized valvular neovascularization using immunohistochemical stainings against the endothelial cell markers CD31 and CD34, and the pericyte marker NG2. We found that blood vessels were present in 80 of the studied 85 stenotic aortic valves (94 %), and in only 2 of the studied 20 control valves (10%). The density of blood vessels was significantly higher in the stenotic valves as compared to the controls (II, Figure 2A). In line with previous reports (Charest et al. 2006), neovessels in the stenotic aortic valves localized close to calcific nodules, amidst inflammatory cell infiltrates, and close to valvular surface. Some of the typical localizations of valvular neovessels are demonstrated in Figure 5 (unpublished results). The findings were similar in bicuspid (n=13) and tricuspid (n=65) valves. In the two control valves, neovessels were detected primarily at the leaflet base, i.e. close to its aortic interface.

Figure 5. CD31/CD34 stainings of stenotic aortic valves showing cross-sectional neovessels as reddish-brown circles. Previously unpublished results. A) Neovessels are abundant in a cell-rich area (right), but distinctly absent from an acellular, fibrotic area (left). B) Neovessels localizing to CD34-positive progenitor cell islets as described in (Charest et al. 2006). C) Neovessels close to calcifying nodules (blue). Scale bars = 100 µm.

42 │ RESULTS AND DISCUSSION _____________________________________________________________________________

Cross-sectional neovessels were classified into three categories according to their size: small microvessels with one to two endothelial cells forming a small lumen, medium microvessels with more than two endothelial cells forming a larger lumen, and mature arterioles with an organized, α-actin-positive vessel wall (II, Figure 1, A-C). All three types of blood vessels were found in the stenotic aortic valves, and only small microvessels were detectable in the control valves. In the NG2 stainings, all arterioles and some of the microvessels were surrounded by pericytes (II, Figure 1, D and E). The presence of lymphatic vessels in advancing AVS was assessed with LYVE-1 and VEGFR-3 stainings of stenotic, sclerotic, and control aortic valves (III, Figure 1, A-D). Like blood vessels, lymphatic vessels typically resided in areas with abundant inflammatory cell infiltration (not shown), and the density of lymphatic vessels correlated positively with the density of neovessels (III, Figure 1F). In contrast to the high occurrence of valvular blood vessels, cross sections of lymphatic vessels were found in only 15 (38%) of the studied 40 stenotic valves, in two (40%) of the studied 5 sclerotic valves, and in one (5 %) of the studied 20 control valves (III, Figure 1E). The number of investigated sclerotic valves was small, rendering it infeasible to compare lymphatic vessel densities between the sclerotic and the stenotic aortic valve samples. Nevertheless, the highest lymphatic vessel densities were observed in the stenotic valves (III, Figure 1E). Importantly, the finding that even early sclerotic valves contained several lymphatic vessels suggests that, besides angiogenesis, lymphangiogenesis represents an early phenomenon in the pathogenesis of AVS. The one control sample staining positive for lymphatic vessels contained two cross sections of lymphatic vessels and also presented with the highest density of blood vessels (8 cross sections of blood vessels, data not shown). There were noticeably fewer lymphatic vessels than blood vessels in the stenotic valves (Figure 6A): In the 15 valve samples containing lymphatic vessels, the average proportion of lymphatic vessels compared to the number of blood vessels was 5 % (calculated from data in Figure 6A). Similar relative rarity of lymphatic vessels as compared to blood vessels has also been observed in atherosclerotic plaques (Nakano et al. 2005), specific percentages being incomparable due to differences in the methodological assessment. Most blood vessels observed in the stenotic aortic valves were small or medium-sized microvessels, while arterioles were more sparse (Figure 6B). The proportion of vascular pericyte coverage was not specifically assessed in Study II, but pericytes mostly colocalized with organized blood vessels, and the vast majority of valvular microvessels were pericyte-negative. Interestingly, pericytes integrate with vascular endothelial cells during blood vessel maturation, and decreased pericyte coverage is thought to correlate with vascular instability and endothelial cell proliferation (Allt and Lawrenson 2001, Yancopoulos et al. 2000). Thus, immature microvessels lacking pericytes are more susceptible to angiogenic sprouting. In addition to providing structural support, pericytes may also modulate microvessel permeability (Allt and Lawrenson 2001). Whether the pericytes covering valvular blood vessels act in favor of vascular permeability or stability remains unclear. Either way, the possible stabilizing effects of valvular pericytes fail to hinder the observed angiogenic response and the infiltration of inflammatory cells and lipids into the stenotic valves.


Figure 6 (Redrawn from data presented in II, Figure 3; and in III, Figure 1E). A) The numbers of blood vessels and lymphatic vessels in the same subpopulation of 40 stenotic aortic valves. Neovessels were present in two and lymphatic vessels in one out of the 20 control samples (not shown). Individual data points are shown, lines indicate medians. B) The average numbers of different types of blood vessels found in the studied 85 stenotic aortic valves (classification: II, Figure 1, A-C). Data are presented as mean + SD.

1.2. Associations of valvular neovessels and lymphatic vessels with inflammation and calcification (II-III)

1.2.1. Blood vessels, lymphatic vessels, and inflammation

As stated above, neovessels and lymphatic vessels in the stenotic aortic valves typically localized in areas abundant with inflammatory cells. Moreover, we found that the density of valvular blood vessels correlated positively with the density of mast cells in the same samples (II, Figure 2B). The correlation remained significant for all three vessel types individually (r=0.29, p=0.007; r=0.39, p<0.001; and r=0.36, p<0.001 for small microvessels, medium microvessels, and arterioles, respectively). Furthermore, in double immunofluorescence stainings of a subpopulation of 10 stenotic aortic valves, 83% of valvular mast cells resided in the close proximity of a neovessel (<50µm) (II, Figure 4, A-C). Similar to previous findings (Helske et al. 2004b), almost all the mast cells in the stenotic valves were activated, as determined by a degranulated histological appearance. The density of lymphatic vessels also correlated positively with valvular mast cell density (III, Figure 1G), which corresponds to the evident association between valvular lymphatic vessels and blood vessels described above. The spatial proximity of valvular mast cells and blood vessels together with the overall correlation of valvular mast cells with blood vessels and lymphatic vessels suggests that mast cells may influence valvular angiogenesis and lymphangiogenesis. Such close localization of mast cells and neovessels has also been observed in abdominal aortic aneurysms (Mäyränpää et al. 2009) and in coronary atherosclerotic plaques (Kaartinen et al. 1996a), where mast cells secrete the proangiogenic basic fibroblast growth factor (Lappalainen H. et al. 2004). In


addition to the secretion of specifically proangiogenic molecules, inflammatory cells may promote angiogenesis by increasing vascular permeability and by promoting angiogenic processes in ECM remodeling (Arroyo and Iruela-Arispe 2010). Furthermore, activated mast cells accumulate subendothelially in coronary artery plaques, in which they potentially contribute to endothelial erosion by secreting proteases capable of degrading VE-cadherin and fibronectin, both of which are necessary for endothelial cell adhesion (Mäyränpää et al. 2006). Overall, mast cells could act in concert with valvular macrophages and other inflammatory cells in forming the inflammatory environment that potentially triggers and sustains valvular angiogenesis.

1.2.2. Blood vessels associated with valvular calcification Valvular blood vessel density also correlated with valvular calcification grade (II, Figure 3), which was defined by the presence and extent of cross-sectional calcific nodules. Out of the three vessel types, this correlation was evident for the density of medium microvessels and arterioles, but not for small microvessels (r=0.29, p=0.007; r=0.37, p<0.001; r=0.26, p=0.02 for medium microvessels, arterioles, and all neovessels, respectively). Importantly, as the strongest correlation was found with mature arterioles, advanced angiogenic remodeling seems to parallel other histological hallmarks of AVS. As the small microvessels, in turn, are likely to be the most newly formed vessels, their equal presence at all stages of valvular calcification would indicate active angiogenesis even at a histologically advanced stage of the disease. Moreover, in group comparisons, the densities of all types of blood vessels were higher in the most severe calcification group as compared to moderately calcified valves. In contrast to our findings, the highest density of blood vessels has previously been reported in moderately calcified valves and hemodynamically moderate stenosis rather than at the end-stage of the disease (Charest et al. 2006, Soini et al. 2003). In these investigations, neovessels were detected with stainings against von Willebrand factor (vWF) and Factor VIII, which occur as a complex in endothelial cells. In our studies, we used a combination of CD31 and CD34 antibodies, which has been shown to be significantly more effective than targeting vWF in detecting small microcapillaries in atherosclerotic plaques (Jeziorska and Woolley 1999). Considering that 46 % of all the blood vessels in Study II were such small microvessels (calculated from data in Figure 6B), the chosen antibodies form a major source of error in the past investigations. In addition, we used unprocessed frozen tissue, whereas other studies assessing valvular blood vessel density have used paraffin-embedded (Soini et al. 2001) or partially decalcified (Charest et al. 2006) tissue material. In unprocessed frozen tissue, calcified areas often disintegrate upon cryostat sectioning, whereas e.g. paraffin-embedding facilitates the sectioning of such calcified tissue, providing better morphology at the cost of losing some antigen reactivity. To avoid methodological bias from the altered morphology in our cryostat sections, we calculated the section area from the remaining connective tissue. Thus, massive calcification of severely stenotic samples was excluded in our calculations, resulting in relatively higher vessel densities in the stenotic valves as compared to the previous investigations. The average number of at least larger vessels has likely been similar in all the studies. This would also explain why we found a positive correlation with valvular calcification grade, while the previous results have shown the opposite (Charest et al. 2006) or being insignificant (Soini et al. 2003), possibly due


to the increase in cross-sectional area in the density equations in valves with massive calcific nodules. Considering these methodological differences, a feasible conclusion from our findings would be that noncalcified areas of otherwise heavily calcified valves have higher vessel densities than those of less calcified valves. Furthermore, the observed decrease in vascular densities in some studies seems to result from an increase in the calcific masses as the stenosis progresses rather than from an actual decrease in valvular blood vessel content. Thus, angiogenic activity may contribute to the histological progression of AVS even at the end-stage of the disease.


Local regulation of valvular angiogenesis and 2.lymphangiogenesis

2.1. Extracellular matrix remodeling – cathepsins S, K, and V, and their

inhibitor cystatin C in AVS (I) Disruption of normal valvular connective tissue structure is involved in the development of AVS (Otto et al. 1994). In addition, a number of proteolytic enzymes, including matrix metallo-proteinases and mast cell-derived tryptase, chymase, and cathepsin G, have been implicated in the pathological ECM remodeling that leads to thickening and stiffening of the stenotic valves (Edep et al. 2000, Helske et al. 2004b, Helske et al. 2006). These proteases may also be involved in valvular angiogenesis, as summarized in the Introduction, Table 1. In Study I, we aimed at identifying additional ECM-degrading proteases, cathepsins S, K, and V, in the aortic valves and at clarifying their role in AVS and valvular angiogenesis. We also assessed their endogenous tissue inhibitor cystatin C, the downregulation of which has been implicated in the pathological ECM remodeling of atherosclerotic plaques (Shi et al. 1999). The results of Study I are summarized in Table 6. Measuring total cathepsin activity from cryostat sections of 7 stenotic and 7 control valves using a fluorogenic substrate revealed the presence of enzymatically active cathepsins in the aortic valves. Furthermore, total cathepsin activity was significantly higher in the stenotic valves as compared to the controls (I, Figure 6B). In order to investigate the selected cathepsins individually, the mRNA expression of cathepsins S and K, along with cystatin C were studied from 32 stenotic and 13 control valves with semi-quantitative PCR, and the mRNA expression of cathepsin V was quantified with qPCR (Table 6). No differences were observed between the mRNA levels in bicuspid and tricuspid aortic valves. To find out which cells were responsible for the increased cathepsin and cystatin C production in the stenotic valves, we performed a series of immunohistochemical and double immunofluorescence stainings against macrophage, myofibroblast, endothelial cell, and chondrocyte markers (see Methods, Table 5). Due to the lack of a reliable primary antibody, we were unable to investigate the cellular expression profile of cathepsin K. The expression patterns of the cathepsins and cystatin C in AVS are summarized in Table 6. Cathepsins S and V were present in the valvular surface endothelium of both stenotic and control valves. Remarkably, cathepsin V was also produced by neovascular endothelial cells of the stenotic valves and was present in some surrounding pericytes. As the staining of cathepsins S and V, cystatin C-positive staining was also more abundant in the stenotic valves. Importantly, cystatin C was absent from vascular endothelial cells of neovessels in the stenotic valves. Cystatin C also colocalized with preserved elastin fibers in the stenotic valves, suggesting a role for cystatin C in maintaining valvular elasticity. This was further investigated by incubating cryostat sections of healthy aortic valve leaflets with recombinant human cathepsins S, K, and V in the absence or presence of cystatin C. The sections were then stained for elastin and collagen, and analyzed by computer-assisted morphometry. The average elastin content after a 24-hour incubation with control buffer was 45 ± 16 %. Incubation with the cathepsins resulted in statistically significant decreases of the valvular elastin content to 25 ± 12 % for cathepsin S,


33 ± 13 % for cathepsin K, and 34 ± 13 % for cathepsin V (I, Figure 6A). Cystatin C was, indeed, able to counteract the elastolytic effects of all three cathepsins (I, Supplemental Figure III). Table 6. The expression of cathepsins S, K, and V, and cystatin C in AVS (I). Detailed descriptions and corresponding references are provided in the text.

Substrates Particular actions

mRNA expression in AVS (I, Figure 1)

Staining profile in the aortic valves (I, Figures 2-4)

Cathepsin S Multiple ECM components

ECM degradation in capillary sprouting, AVS progression

Increased

Increased in AVS; Some macrophages, chondroblast-like cells, endothelial cells lining valvular surface

Cathepsin K Elastin, collagen I Bone remodeling Increased -

Cathepsin V Elastin, Plasminogen

ECM degradation, anti-angiogenic?

Increased

Increased in AVS; Endothelial cells lining valvular surface and neovessels, some pericytes

Cystatin C Cathepsin inhibitor

Counteracting the cathepsins

Increased

Increased in AVS; Macrophages, endothelial cells lining valvular surface, chondroblast-like cells

Altogether, the results from Study I show that cathepsin activity is increased in stenotic aortic valves, and that the local valvular production of cathepsins S, K, and V, as well as their inhibitor cystatin C, is induced in AVS. Moreover, their localization suggests that they have distinct roles in valvular ECM degradation, bone remodeling, and angiogenesis. Remarkably, only cathepsin V was expressed in the endothelial cells of valvular neovessels, while its inhibitor, cystatin C, was absent from these cells. Cathepsin V possesses potent elastolytic properties (Yasuda et al. 2004), which could contribute to matrix degradation required for angiogenic sprouting. In contrast to this potentially angiogenic feature, cathepsin V has been proposed to have an inhibitory effect on corneal angiogenesis by generating angiostatin-like molecules from plasminogen (Puzer et al. 2008). Confusingly, this action would also provide an indirect angiogenic function for cystatin C, which has traditionally been considered a protective factor in atherosclerotic diseases. Although the presence of angiostatin in aortic valves is currently unknown, the neovascular localization of cathepsin V suggests that it may possess a function, that for the time being remains unfocused, in the regulation of valvular angiogenesis. Unlike cathepsin V, cathepsin S has a clearly established role in angiogenesis. Cathepsin S seems to be crucial for the formation of neovessels even in atherosclerotic plaques (Liu J. et al. 2004). Subsequent investigations have confirmed the pathological nature of cathepsin S in AVS by revealing that its actions may actually be crucial for valvular calcification. In a mouse model of AVS, cathepsin S deficiency led to a significant decrease in elastin degradation, and reduced


calcification in the valve leaflets (Aikawa et al. 2009). Interestingly, serum levels of cystatin C were significantly lower in the mice lacking cathepsin S than in the control group (Aikawa et al. 2009), parallel to our findings which state that cathepsin S and cystatin C expressions were simultaneously increased. These results are inconsistent with findings in atherosclerotic lesions, where cystatin C is, in fact, downregulated (Shi et al. 1999). Neovascularized areas of the plaques, however, contain more cystatin C than the surrounding lesion (Shi et al. 1999), possibly in effort to control pathological angiogenesis. In fact, the observed parallel overexpression of the cathepsins and cystatin C, as well as their multiple functions, complicate the interpretation of their net effect in the aortic valve. Our observation on cathepsin S being present in the chondroblast-like cells in calcifying human valve tissue suggests that the role of cathepsin S in valvular calcification reported by Aikawa and coworkers could extend to humans as well (Aikawa et al. 2007). Cathepsin K being predominantly expressed by osteoclasts (Brömme et al. 1996), increased valvular cathepsin K expression could also contribute to bone formation in the stenotic valves. In atherosclerotic plaques, cathepsin K participates in plaque development and remodeling, and disruption of the cathepsin K gene slows down the progression of atherosclerosis and promotes plaque fibrosis in an animal model (Lutgens et al. 2006). Cathepsin K is also present in atherosclerotic plaques of human coronary arteries, where it may serve macrophage foam cells in degrading plaque ECM components (Barascuk et al. 2010). Controversially, cathepsin K downregulation in the mouse model augmented scavenger receptor-mediated uptake of lipids by macrophages, increased CD36 expression being particularly implicated in this process (Lutgens et al. 2006). Interestingly we found CD36 mRNA expression to be decreased in AVS in Study IV (see Section 2.3.1. below). As cathepsin K mRNA expression, in turn, was increased in AVS, a regulatory connection between CD36 and cathepsin K, as proposed by Lutgens and coworkers, could also be operative in the aortic valves. Unfortunately, the immunohistochemical staining of cathepsin K in our study of AVS (I) was unsuccessful, leaving several unanswered questions regarding the stature of cathepsin K in this disease. Therefore, more information on the valvular localization of cathepsin K, as well as the functional role of cathepsin V, is needed to demonstrate their relevance in the pathogenesis of AVS.

2.2. Valvular expression of VEGF-A, VEGF-C, VEGF-D, and their receptors VEGFR-2 and VEGFR-3 (II-III)

2.2.1. Local production Angiotensin II, which is capable of inducing VEGF-A expression (Williams et al. 1995), is present and locally produced in stenotic aortic valves (Helske et al. 2004b, O'Brien et al. 2002). Both VEGF-A and VEGFR-2 have been previously reported in stenotic aortic valves, VEGF-A having been detected only in valvular endothelial cells and myofibroblasts (Soini et al. 2003). In studies II and III, VEGF-A, VEGF-C, VEGF-D, and VEGFR-3 were detected from the aortic valves using immunohistochemistry. VEGF-C, VEGF-D, VEGFR-2, and VEGFR-3 were also studied with qPCR (III, 10 stenotic and 10 control valves). The expression profiles of the studied VEGFs and VEGFRs in aortic valve tissue are summarized in Table 7. Detailed descriptions of their properties and function along with corresponding references can be found in the Introduction (Section 4.1.2), as well as in the discussion below.


Table 7. The expression of VEGF-A, VEGF-C, VEGF-D, and their receptors VEGFR-2 and VEGFR-3 in aortic valve tissue (II and III).

Receptor / Function

Expression in the aortic valve (mRNA levels) (III, Figure 3)

Staining profile in the aortic valve (II, Figure 4; III, Figures 1-2)

VEGF-A (II)

VEGFR-1/2 - Mast cells, myofibroblasts, endothelial cells lining valvular surface and neovessels

VEGF-C (III)

VEGFR-2/3 High, similar in AVS and controls

Myofibroblasts, endothelial cells lining valvular surface and neovessels

VEGF-D (III)

VEGFR-2/3 Low, increased in AVS Myofibroblasts, endothelial cells lining valvular surface

VEGFR-2 (III)

Angiogenesis, vascular permeability, osteogenesis

High, increased in AVS -

VEGFR-3 (III)

Lymphangiogenesis, osteoblast differentation?

Low, increased in AVS Lymphatic endothelial cells, endothelial cells lining valvular surface

In Study II, we found that 62% of valvular mast cells stained positive for VEGF-A. The presence of VEGF-A-positive extracellular granules was confirmed by confocal microscopy, indicating that mast cells in the stenotic aortic valves produce VEGF-A and release it upon activation. VEGF-A was also found in the previously described cell types. In the control valves, surface endothelium and some subendothelial, resting mast cells were VEGF-A-positive. VEGF-D was only patchily detected in the surface endothelium, which concurs with previous work indicating endothelial cells as a source of VEGF-D in normal arteries (Rutanen et al. 2003). In the normal arteries, VEGF-D was also expressed by smooth muscle cells, whereas in atherosclerotic plaques it was expressed mainly by macrophages (Rutanen et al. 2003). In our stainings, however, valvular macrophages remained VEGF-C- and VEGF-D-negative (not shown). Furthermore, mast cells have been shown to express VEGF-C in mouse tumors (Brideau et al. 2007). Thus, they were considered a potential source of VEGF-C in our study as well, but remained negative for both VEGF-C and -D in the immunohistochemical stainings (not shown). VEGF-C and VEGF-D were not double-stained with lymphatic vessels due to overlapping primary antibody immunoglobulin classes. Because CD34 may also be expressed by lymphatic endothelial cells (Cueni and Detmar 2006), it is conceivable, that some of the observed positive staining of VEGF-C in the neovascular sprouts actually localized to lymphatic vessel endothelium. Interestingly, VEGFR-2 mRNA levels were significantly higher in patients with a history of smoking, and correlated positively with the peak pressure gradient at echocardiography (r=0.744). Although the sample size was small, it is tempting to hypothesize that the soluble components of cigarette smoke may promote valvular neovascularization by affecting local VEGFR-2 expression. In fact, in the series of experiments on valvular myofibroblasts performed


in Study III (presented in III, Figure 4D, discussed in Section 2.2.2), stimulation of the myofibroblasts with cigarette smoke also resulted in an increase in VEGFR-2 expression by these cells (Figure 7, unpublished data).

Figure 7. The VEGFR-2 mRNA expression of cultured human aortic valve myofibroblasts in response to cigarette smoke in the same three independent experiments presented in III, Figure 4D. Previously unpublished data, presented as medians and interquartile ranges.

The identification of mast cells as a novel valvular source of VEGF-A is in line with previous work demonstrating VEGF-A production by mast cells (Grützkau et al. 1998). Furthermore, the observed release of VEGF-A-containing granules close to valvular neovessels suggests a substantial role for these cells in valvular angiogenesis. The increase in valvular VEGFR-2 mRNA levels, in turn, is likely to portray the angiogenic activation of valvular endothelial cells, which have been shown to express VEGFR-2 (Soini et al. 2003). VEGFR-2 has also been immunohistochemically detected in valvular myofibroblasts both in valve tissue and in cultured cells (Chalajour et al. 2007, Soini et al. 2003). The investigation by Chalajour and coworkers derived from their earlier demonstration of an ex vivo 3D collagen model of capillary tube formation by valvular endothelial cells (Chalajour et al. 2004). In this earlier investigation, however, only morphological evidence of endothelial outgrowth was presented without immunohistochemical confirmation. In subsequent work, the same group found that most of the migrated cells were, in fact, α-actin-positive, indicating a myofibroblast rather than an endothelial cell phenotype (Chalajour et al. 2007). In addition to VEGFR-2, the myofibroblasts also stained positive for the angiopoietin receptor Tie-2 and formed capillary sprout-like structures (Chalajour et al. 2007). The specific consequences of VEGFR-2 and Tie-2 activation in valvular myofibroblasts, however, remain to be determined. The observed VEGFR-3-positive vascular structures were also LYVE-1-positive, denoting lymphatic endothelium. Interestingly, VEGFR-3 was also expressed by some of the endothelial cells lining the valve surface. As VEGFR-3 is expressed by tip cells of angiogenic sprouts (Lohela et al. 2009), VEGFR-3-positivity of surface endothelial cells could indicate angiogenic activation of these cells. In fact, based on the findings that neovascular clusters concentrate close to valvular surface and on the morphological observation that some sprouts seem to originate directly from the valve surface, it has been suggested that some of the valvular neovessels may form by budding from valvular surface endothelium (Steiner et al. 2010). We also observed areas of endothelial migration from the valve surface into the tissue in our immuno-histochemical stainings (Figure 8, unpublished). Such vascular sprouting could account for some of the observed VEGFR-3 upregulation in AVS.


Figure 8. CD31/CD34 staining of the surface endothelium of a stenotic aortic valve (unpublished). Some of the endothelial cells staining brownish-red in the valve tissue seem to directly connect with the valvular surface, supporting the hypothesis that endothelial sprouts may originate from the endothelium lining the valvular surface. Scale bar = 100µm. In addition to promoting lymphangiogenesis, the activation of VEGFR-3 by VEGF-D has been shown to induce osteoblastic differentiation in mice (Orlandini et al. 2006). The expression of VEGFR-3 mRNA, however, was extremely low at least in our experiments with cultured valvular myofibroblasts (data not shown), which are a potential source of osteoblastic cells in stenotic aortic valves (Liu A. C. et al. 2007). Nevertheless, the increased VEGF-D expression in AVS could contribute to the pathological osteogenic environment of the diseased valves. Although VEGF-C mRNA expression remained unchanged in AVS, the abundance of VEGF-C mRNA as compared to that of VEGF-D, as well as the upregulation of VEGFR-2 and VEGFR-3, supports the potential role of VEGF-C in valvular lymphangiogenesis. Furthermore, as the receptor-binding properties of both VEGF-C and VEGF-D may be posttranslationally modified (Achen et al. 1998, Joukov et al. 1997), the correlation between their mRNA expression and physiological function may not be linear. Unlike in the expression of VEGF-C, there was a significant increase in the valvular mRNA expression of VEGF-D in AVS. As discussed previously, VEGF-D may, in addition to being lymphangiogenic, also exert angiogenic effects. Because angiogenesis is overrepresented as compared to lymphangiogenesis in the stenotic valves, it is conceivable that VEGF-D exerts mostly angiogenic effects in these valves. On the other hand, if the main effects of VEGF-D in the valves are lymphangiogenic, these actions are evidently insufficient to induce valvular lymphangiogenesis in proportion to angiogenesis.

2.2.2. The role of valvular myofibroblasts Studies on cultured valvular myofibroblasts Since VEGF-A and VEGF-C were detectable in part of the myofibroblasts in stenotic aortic valves, we wanted to further explore the production of these growth factors by cultured human myofibroblasts derived from stenotic aortic valves. Both growth factors were constitutively secreted into the cell culture media by the myofibroblasts. We also explored the possibility of the myofibroblasts producing VEGF-D, but it remained undetectable in the cell culture media (data not shown). Next, to study the local conditions potentially regulating the production of VEGF-A and VEGF-C in the myofibroblasts, we stimulated these cells with factors


implicated in the pathogenesis of AVS. We studied the effects of mast cell-derived components, the inflammatory cytokine TNF-α, hypoxic conditions, and osteogenic stress. Smoking being a risk factor for AVS (Deutscher et al. 1984), we also investigated the influence of soluble components of cigarette smoke on the myofibroblasts. Importantly, cigarette smoke has previously been shown to activate mast cells, and it has been linked to the pathological inflammation and ECM remodeling in AVS (Helske et al. 2008, Helske et al. 2006). The results of these experiments are summarized in Table 8. Table 8. The effects of external stimuli on the VEGF-A/C production by valvular myofibroblasts derived from stenotic aortic valves. Arrows indicate an increase, decrease, or no response. VEGF-A secretion was also studied in control myofibroblasts, the results being similar to those observed in myofibroblasts from stenotic valves (not presented).

Mast cell releasate

TNF-α Hypoxia Cigarette

smoke Osteogenic

stress

VEGF-A (II) ↑ ↑ ↑ ↑ ↓

VEGF-C (III) ↔/↓* ↑ ↔ ↔/↓** ↔/↓**

* No effect at mRNA level, protein degradation ** Only one time point statistically significant

Both of the inflammatory stimuli, i.e. the mast cell-derived components and TNF-α, significantly induced the VEGF-A secretion of the myofibroblasts (II, Figure 5, A and B). Furthermore, incubating the myofibroblasts in the presence of soluble components of cigarette smoke or in hypoxic culture conditions also resulted in increased VEGF-A production (II, Figure 5, C and D). The effect of cigarette smoke and TNF-α was similar at the mRNA level (II, Figure 5F). Thus, in addition to the association between cigarette smoke and increased VEGFR-2 described above, smoking could contribute to the harmful angiogenic response in stenotic aortic valves by inducing VEGF-A production. These findings provide a possible mechanism for this known risk factor of AVS in the pathogenesis of the disease. Finally, introducing the myofibroblasts to osteogenic stress decreased the amount of secreted VEGF-A (II, Figure 5E), a phenomenon also occurring during early osteogenic differentiation (Mayer et al. 2005). The VEGF-A production by valvular myofibroblasts in response to the above stimuli was similar between cells derived from stenotic and non-stenotic aortic valves, the latter ones being obtained at valve replacement surgery due to aortic regurgitation (II, Supplemental Figure III). In addition to VEGF-A, TNF-α also stimulated the VEGF-C expression of valvular myofibroblasts, both at protein level and at mRNA level (III, Figures 4B and 5B). Hypoxic culture conditions, in turn, showed no effect on the VEGF-C secretion of the myofibroblasts (III, Figure 4C). These findings are concurrent with previous work on human fibroblasts demonstrating that hypoxia significantly induces the expression of VEGF-A, but not VEGF-B or VEGF-C (Enholm et al. 1997). TNF-α was also a plausible inducer of VEGF-C expression based on literature, having been shown to increase VEGF-C production in human fibroblasts (Ristimaki et al. 1998). In contrast to VEGF-A, cigarette smoke had a minor decreasing effect on VEGF-C production by the myofibroblasts evident only at the 24-hour time point (III, Figure 4D). Finally, the VEGF-C


response to osteogenic stress was similar to that of VEGF-A, being significant only at the 48-hour time point (III, Figure 4E). The effects of hypoxia, cigarette smoke, and osteogenic stress at protein level were concurrent with the mRNA findings by qPCR (not shown). Myofibroblasts and mast cells Remarkably, the effect of mast cell-derived components on cellular VEGF-C secretion seemed to be opposite to the effect it had on VEGF-A production. In fact, adding mast cell releasate to the myofibroblasts derived from stenotic valves resulted in a decrease of detectable VEGF-C in the culture media (III, Figure 4A). At the mRNA level, however, the production of VEGF-C remained constant (III, Figure 5A). These results raised questions regarding possible VEGF-C degradation, addressed below in Section 2.4 (Angiogenic and antilymphangiogenic actions of valvular mast cells). We also studied the potential effects of myofibroblast-derived factors on the VEGF-A production of cultured human mast cells. The mast cells were first generated from circulating progenitors by a previously established protocol (Lappalainen J. et al. 2007). We then incubated the mast cells with cell culture medium obtained from cultured myofibroblasts derived from stenotic aortic valves. Interestingly, the myofibroblasts had produced substances that were able to induce the VEGF-A secretion of cultured human mast cells (II, Figure 5G). The effect was more pronounced at the 24-hour time point than at the 1-hour time point, suggesting a more sustained state of active VEGF-A production rather than an acute release of stored VEGF-A in mast cell degranulation process. The interactions between valvular myofibroblasts and mast cells are discussed in Section 2.5 (Interplay between valvular mast cells and myofibroblasts).

2.3. Oxidized LDL and scavenger receptors (IV)

2.3.1. Local expression of scavenger receptors SR-A1, SR-B1, CD36, and LOX-1

The presence of oxLDL and its increased deposition in stenotic aortic valves have been reported previously (Olsson et al. 1999), but its potential actions in the aortic valves have remained unclear, and the presence of oxLDL-binding scavenger receptors in aortic valves has not been previously reported. The properties of the studied scavenger receptors, as well as the results from all the following analyses, are summarized in Table 9. First, we examined the expression of scavenger receptors SR-A1, CD36, and LOX-1, and the atheroprotective SR-B1 in 10 stenotic valves and 11 control valves with qPCR. Notably, the relatively highest expression was observed in the mRNA levels of SR-A1. We then analyzed the potential expression of these scavenger receptors in cultured valvular myofibroblasts derived from both stenotic and control valves (n=4 in each group). Based on previous findings reporting that inflammatory cytokines may induce the expression of several scavenger receptors (Stephen et al. 2010), we tested the effect of two selected inflammatory agents, mast cell releasate and TNF-α, on the cultured valvular myofibroblasts. Both the mast cell releasate and TNF-α were able to induce LOX-1 expression in the myofibroblasts. In order to examine which cells may be responsible for the observed changes in valvular scavenger receptor expression in AVS, we conducted immunohistochemical and double immunofluorescence stainings of both stenotic (n=24) and control (n=7) aortic valves (Table 9).


By performing these stainings, we also wanted to address the apparent discrepancies in SR-A1 and CD36 expression between valvular tissue and cultured aortic valve myofibroblasts. In the qPCR analyses, SR-A1 mRNA expression was increased in stenotic valve tissue but not in the myofibroblasts derived from stenotic aortic valves as compared to controls. In the immunohistochemical stainings, SR-A1 was more abundant in the stenotic valves than in the control valves, and localized to macrophages in both stenotic and control valves. Thus, valvular macrophages rather than myofibroblasts are likely to account for the observed increase in SR-A1 mRNA expression in stenotic valve tissue. Table 9. The expression of scavenger receptors (SR) SR-A1, SR-B1, CD36 and LOX-1 in AVS. The first five columns are modified from (Stephen et al. 2010).

Class SR Examples of ligands

Role in atherosclerosis

Potential expression-altering factors in AVS

Expression in the aortic valve (mRNA levels) (IV, Figure 1-2)

Staining profile in the aortic valve (IV, Figure 3-4)

A SR-A1

AcLDL, OxLDL, ECM, apoptotic cells, activated b-cells, bacteria

OxLDL uptake by macrophages leading to foam cell formation

Increased by oxidative stress, OxLDL, M-CSF

Upregulated in stenotic valves, no change in cultured myofibroblasts in AVS

Macrophages in both control and stenotic valves

B SR-B1 HDL, LDL, OxLDL, apoptotic cells

Reduces atherosclerosis through reverse cholesterol transport of HDL

Decreased by OxLDL, TNF-α, IL-1, LPS

Expression unchanged in AVS

-

B CD36 AcLDL, OxLDL, HDL, LDL, VLDL, apoptotic cells

OxLDL uptake by macrophages leading to foam cell formation, inhibition of angiogenesis*

Increased by IL-4, OxLDL*; Decreased by IL-10, statins*

Downregulated in stenotic valves, upregulated in cultured myofibroblasts in AVS

In endothelial cells lining the valve in both control and stenotic valves, in neovessels of stenotic valves, in some myofibroblasts

E LOX-1

OxLDL, ECM, apoptotic cells, activated platelets, bacteria

OxLDL uptake in endothelial cells leading to endothelial dysfunction, pro-angiogenic**

Increased by Ox-LDL, TNF-α, shear stress, oxidative stress, endothelin-1, angiotensin II

Upregulated in stenotic valves and in cultured myofibroblasts in AVS, mast cell releasate and TNF-α induce expression

In some endothelial cells lining the stenotic valves

*(Febbraio and Silverstein 2007) **(Jiang et al. 2011)


Surprisingly, CD36 was not present in valvular macrophages, but localized to endothelial cells lining both valvular surface and neovessels of the stenotic aortic valves. In double immunofluorescence stainings, CD36 was also found in some sparse valvular myofibroblasts. In the qPCR analyses, CD36 mRNA expression in AVS was decreased in valvular tissue but increased in cultured myofibroblasts. The predominant protein expression of CD36 by valvular endothelial cells could indeed explain the difference in CD36 mRNA expression between valve tissue and cultured myofibroblasts. More specifically, if most of the mRNA expression in the qPCR analyses had been derived from valvular endothelial cells, the decrease in their CD36 expression would have been reflected in the tissue mRNA analyses. Correspondingly, if only few valvular myofibroblasts had expressed CD36, their increased CD36 mRNA expression would have remained undetected in the tissue analyses. In any case, the possibility remains that the changes observed in valvular cells cultured over several passages in vitro do not fully represent the in vivo expression profile of these cells in the valvular tissue. Finally, LOX-1 was immunohistochemically detectable only in the stenotic aortic valves, where its expression localized to the valvular surface endothelial cells and the pericytes surrounding neovascular endothelial cells. In contrast to the cell culture analyses, LOX-1 expression could not be seen in valvular myofibroblasts in the immunohistochemical stainings. As LOX-1 is mainly an endothelial receptor, it is conceivable that the lower expression in valvular myofibroblasts may have remained below the immunohistochemical detection limit. Our findings on SR-A1 expression increasing in AVS and localizing to valvular macrophages propose a similar role for SR-A1 in AVS than in atherosclerosis, where it significantly contributes to macrophage foam cell formation (Stephen et al. 2010). In addition to SR-A1, CD36 is also a crucial contributor in atherogenesis, where its inhibitory effects on angiogenesis are clearly overcome by its proatherogenic role in macrophage foam cell formation (Febbraio and Silverstein 2007). In our analyses, CD36 mainly localized to valvular endothelial cells, however, suggesting that it could be a player in the local regulation of valvular angiogenesis in addition to its role in foam cell formation. In addition, the expression of this antiangiogenic mediator was decreased in AVS, possibly contributing to the local proangiogenic environment. The expression of LOX-1 was increased in AVS in all the analyses in Study IV. First, the mRNA levels of LOX-1 were elevated in the stenotic aortic valves. Second, cultured myofibroblasts derived from stenotic valves expressed higher levels of LOX-1 mRNA than those derived from control valves. Finally, LOX-1 protein was present in endothelial cells lining the stenotic valves but not in those lining the control valves. Notably, its patchy expression may portray differences in local blood flow shear stress inducing LOX-1 expression, a mechanism implicated in the localized formation of atherosclerotic lesions (Mitra et al. 2011). In line with previous findings (Stephen et al. 2010), we found that LOX-1 expression was also induced by TNF-α in aortic valve myofibroblasts. Furthermore, we found that mast cell-derived components were able to induce LOX-1 mRNA expression in these cells. Our results link LOX-1 to the pathogenesis of AVS and indicate that local inflammation could induce its expression in the stenotic aortic valves.


2.3.2. A role for oxLDL in valvular inflammation and angiogenesis The angiogenic potential of small concentrations of oxLDL has been demonstrated in human coronary artery endothelial cells, in which oxLDL induces VEGF-A expression and capillary tube formation via LOX-1 (Dandapat et al. 2007). Furthermore, oxLDL has been shown to induce the expression of a large variety of genes involved in e.g. endothelial cell migration and adhesion (Khaidakov et al. 2012). Remarkably, these effects were also counteracted by a LOX-1-blocking antibody, suggesting a major role for LOX-1 in all of the angiogenic effects of oxLDL on endothelial cells. OxLDL also increases LOX-1 expression (Khaidakov et al. 2012), potentially further enhancing the angiogenic effect. Therefore the observed increase in LOX-1 mRNA expression in AVS may also result from local deposition of oxLDL in addition to the surrounding inflammatory environment as suggested above. More importantly, the eminent role of LOX-1 in angiogenesis suggests that its increased expression may be one of the factors behind the pathological neovascularization of stenotic aortic valves. The possibility of increased LOX-1-mediated angiogenesis has also been highlighted in atherosclerosis (Mitra et al. 2011). Besides its effects on angiogenesis, oxLDL has an established role in the pathological inflammation of the arterial wall in atherosclerotic lesions (Tsimikas and Miller 2011). Thus, we aimed at further examining the connection between oxLDL and valvular inflammation. For this purpose, we stimulated myofibroblasts derived from stenotic aortic valves with 10, 25, or 50 µg/ml oxLDL, and measured the responses in inflammatory cytokine production from the cell culture media by ELISA. Indeed, 25 µg/ml of oxLDL was able to induce the expression of four proinflammatory cytokines, MCP-1, IL-6, IL-8, and macrophage colony-stimulating factor (M-CSF), in the myofibroblasts (IV, Figure 5, A-D). Furthermore, the effect of oxLDL on interleukin production increased in a dose-dependent manner (IV, Figure 5, B and D). Considering the increased expression of LOX-1 and CD36 in valvular myofibroblasts, these receptors are potential contributors to the observed inflammatory response. In addition to the increased production of inflammatory cytokines, the activation of scavenger receptors by oxLDL may enhance monocyte infiltration and differentiation into foam cell-forming macrophages (Stephen et al. 2010), a process implicated in the pathogenesis of AVS as well (Otto et al. 1994). Notably, the overall proinflammatory effects of oxLDL may indirectly promote local angiogenesis in addition to the more specific mechanisms mediated by LOX-1. Scavenger receptor activation may also result in a number of other effects potentially involved in valvular pathology. Such effects include apoptosis, the expression of adhesion molecule genes, increased collagen production, and vascular smooth muscle cell proliferation (Mitra et al. 2011, Stephen et al. 2010). Interestingly, LOX-1 is upregulated by angiotensin II (Wang et al. 2011), which is actively generated and abundantly present in stenotic aortic valves (Helske et al. 2004b, O'Brien et al. 2002). Furthermore, oxLDL induces angiotensin type 1 receptor expression parallel to LOX-1 expression (Li et al. 2000), suggesting that the previously observed upregulation of this angiotensin receptor in stenotic aortic valves (Helske et al. 2004b) may be linked to the increase in LOX-1 expression detected in Study IV and may partly derive from the actions of oxLDL in the diseased leaflets. Thus, the above novel findings demonstrating scavenger receptor expression in the aortic valves could be central to the comprehensive understanding of the local pathological mechanisms leading to AVS.


2.4. Angiogenic and antilymphangiogenic actions of valvular mast cells (II-IV)

Our initial immunohistochemical findings that valvular mast cells produce VEGF-A, but not VEGF-C, already indicated an angiogenic but not a lymphangiogenic role for this potent inflammatory cell. This impression was further consolidated by experiments on valvular myofibroblasts demonstrating that mast cell-derived components induced the expression of VEGF-A but not VEGF-C in the myofibroblasts. In fact, the amount of detectable VEGF-C decreased as a result of adding mast cell releasate to the myofibroblast culture, suggesting that mast cells actually possess antilymphangiogenic potential. As described above, mast cell releasate decreased the amount of VEGF-C protein while VEGF-C mRNA expression remained constant. We therefore hypothesized that some proteases produced by the mast cells could degrade VEGF-C, and acknowledged the possibility of similar effects on VEGF-A. In fact, incubating recombinant VEGF-A and VEGF-C proteins with mast cell releasate revealed that VEGF-C, but not VEGF-A, was degraded by mast cell-derived components (III, Figure 5C). Next, we selected tryptase, chymase, and cathepsin G as potential effector proteases, as all three are produced by activated mast cells in stenotic aortic valves (Helske et al. 2004b, Helske et al. 2006). Notably, all three proteases were also able to degrade intact VEGF-C in vitro (III, Figure 5D). We also studied the potential effects of valvular mast cells on endostatin, an antiangiogenic molecule detected from stenotic aortic valves (Chalajour et al. 2004). Incubating purified human endostatin with recombinant mast cell tryptase indeed resulted in endostatin degradation in a time-dependent manner. Proteolytic loss of endostatin was detected by SDS-PAGE and was visible as a decrease in the intensity of the intact (20 kDa) endostatin band (II, Figure 6). All in all, mast cells in stenotic aortic valves seem to be angiogenic but antilymphangiogenic. They produce and secrete the angiogenic VEGF-A but degrade the lymphangiogenic VEGF-C, not VEGF-A. Mast cells are also capable of degrading the antiangiogenic endostatin, which would further consolidate the role of valvular mast cells as angiogenic effectors. Finally, mast cells may indirectly increase the angiogenic effects of oxLDL in the stenotic aortic valves by inducing LOX-1 expression in valvular myofibroblasts. Yet another angiogenic action of mast cells arises from a recent study in which human mast cells secreted and processed the proteoglycan perlecan into fragments containing functional endorepellin, which, in turn, regulates angiogenesis and matrix turnover (Jung et al. 2013). Even though the expression of perlecan in aortic valves remains obscure, it is obvious that valvular mast cells possess intensive angiogenic potential. The proposed antilymphangiogenic role of mast cells is somewhat shaken by the above-noted ability of mast cells to degrade endostatin, because endostatin also has antilymphangiogenic effects (Brideau et al. 2007). This would provide the mast cells with indirect lymphangiogenic activity as well. Considering that induced angiogenesis is strongly associated with AVS (Mazzone et al. 2004, Mohler et al. 2001, Soini et al. 2003), the possible increase in valvular endostatin observed by Chalajour and coworkers seems to be insufficient in controlling the prominent angiogenic response (Chalajour et al. 2004). Moreover, endostatin overexpression has been shown to decrease mast cell infiltration (Brideau et al. 2007), a phenomenon clearly


absent from stenotic aortic valves. In fact, the opposite is true: activated mast cells are abundantly present in the diseased leaflets, and their number is increased in comparison to non-diseased leaflets (Helske et al. 2004b). The increased amounts of valvular mast cells, in turn, would significantly contribute to endostatin degradation, thus counteracting the production of endostatin. Thus, the net effect of mast cells in stenotic aortic valves would conceivably be antilymphangiogenic, even when the prolymphangiogenic effect of endostatin degradation by the mast cells is taken into account. In the VEGF-C degradation experiments, the amount of intact protein was measured with a commercial enzyme-linked immunoassay. According to the information provided by the manufacturer (R&D Systems, e-mail communication, 2010), this assay should also detect the fully processed, angiogenic form of VEGF-C. Nevertheless, a possibility remains that at least some mast cell-derived proteases could cleave VEGF-C or other VEGFs into effective, modulated forms with distinct receptor-binding patterns. Future investigations on the potential capacity of mast cells to generate these modulated VEGFs would certainly elucidate the complex role of mast cell proteases on angiogenesis and lymphangiogenesis.

2.5. Interplay between valvular mast cells and myofibroblasts (II-IV) AVS is characterized both by the abundant accumulation of activated mast cells and by the proliferation and activation of valvular myofibroblasts (Helske et al. 2004b, Olsson et al. 1994b). These cells act together on several pathological valvular processes including the sustained inflammatory reaction. Myofibroblasts in the stenotic valves also produce increased amounts of collagen (Eriksen et al. 2006), thus contributing to the thickening of the valve leaflets. Valvular mast cells, in turn, participate in this adverse remodeling by producing ECM-degrading proteases, e.g. tryptase, chymase, and cathepsin G (Helske et al. 2004b, Helske et al. 2006). Mast cell-derived tryptase and chymase also possess profibrotic potential, and pharmaceutical inhibition of chymase has been shown to prevent myocardial collagen deposition and fibrosis in an experimental animal model (Matsumoto T. et al. 2003, Wygrecka et al. 2013). Mast cell-derived cathepsin G, in turn, induces the expression of the profibrotic TGF-β1 in human fibroblasts (Helske et al. 2006). Moreover, the colocalization of valvular mast cells and TGF- β1 indicates a potential contribution of this mechanism to valvular fibrosis as well (Helske et al. 2006). Thus, myofibroblasts and mast cells act in concert in the pathological modulation of valvular ECM in stenotic aortic valves. The joint contributions of mast cells and myofibroblasts extend beyond ECM remodeling. In this thesis, we identified several novel mechanisms coordinated by valvular mast cells and myofibroblasts that affect angiogenesis and lymphangiogenesis in stenotic aortic valves. First, both cell types express VEGF-A in the aortic valve, and both are also able to induce the expression of VEGF-A in the other cell type (II). Furthermore, mast cell-derived proteases are able to degrade VEGF-C produced by the myofibroblasts (III). Finally, mast cells induce LOX-1 expression in valvular myofibroblasts (IV), rendering them more susceptible to the effects of oxLDL. These mechanisms together with other major findings in this thesis are summarized in Figure 9. All in all, the interactions of valvular mast cells and myofibroblasts may promote angiogenesis in the aortic valve. In addition, changes in the balance between VEGF-C production by the myofibroblasts and its degradation by the mast cells may regulate valvular lymphangiogenesis.


General discussion 3.

3.1. Angiogenesis and lymphangiogenesis in AVS

3.1.1. Pathological angiogenic switch in AVS In normal tissue repair, the newly formed capillary network regresses with the resolution of the inflammatory stimulus. In the absence of inhibitory signals, however, the positive feedback between the inflammatory cells and the immature, leaky vasculature leads to chronic inflammation and increased ECM turnover (Arroyo and Iruela-Arispe 2010). This pathological angiogenic loop is present in e.g. atherosclerotic lesions (Costa et al. 2007), and many histological features indicate that the signals involved in normal vascular regression are also impaired in AVS. The presence of organized arterioles in heavily calcified valves implicates that these valves have already reached an advanced stage of angiogenic remodeling (II). At the same time, both mildly and severely calcified valves contain plenty of newly formed small microvessels, indicating active neoangiogenesis (II). Furthermore, the expression of the potent angiogenic factor VEGF-A by multiple valvular cells, including mast cells (II), points to a proangiogenic switch in the valvular microenvironment. In Study II, we also observed increased VEGF-A secretion by valvular myofibroblasts in response to hypoxia. Thus, the increased cellularity in stenotic valve tissue and the subsequent increase in local oxygen demand comprise one possible mechanism behind the pathological angiogenic activation in AVS. Moreover, the extensive connective tissue remodeling in the stenotic aortic valves, including the increased expression of cysteine cathepsins (I), enables angiogenic sprouting in the presence of abundant VEGF-A (Yancopoulos et al. 2000).

3.1.2. The function of valvular blood vessels and lymphatic vessels In addition to their response to hypoxia, valvular myofibroblasts may have an angiogenic (II) and lymphangiogenic (III) response to the local production of proinflammatory cytokines such as TNF-α. Thus, inflammation may be a trigger for angiogenesis and lymphangiogenesis in AVS, as it is in other pathological conditions (Arroyo and Iruela-Arispe 2010). Valvular blood vessels and lymphatic vessels, in turn, may also affect the course of events leading to chronic, sustained inflammation in AVS. Angiogenesis has a distinct role in chronic inflammation, where it e.g. facilitates inflammatory cell infiltration into tissue (Griffioen and Molema 2000). Because valvular neovascular endothelial cells express cell adhesion molecules involved in the recruitment of inflammatory cells from systemic circulation (Mazzone et al. 2004), neovessels are also likely to facilitate inflammatory cell infiltration into the aortic valves, thereby feeding the proinflammatory territory of the stenotic leaflets. Lymphatic vessels, in turn, are known to participate in the initiation of antigen-specific immune responses involved in e.g. transplant rejection (Cursiefen et al. 2004, Kerjaschki et al. 2004). Their role in other types of inflammatory responses is, however, less established. In contrast to the pathological role of lymphatic vessels in alloimmunization, lymphangiogenesis has been proposed to have an anti-inflammatory role in peripheral tissues (Alitalo 2011). In fact, genetically modified mice with increased lymphatic network demonstrate enhanced capability to limit acute inflammatory reactions (Huggenberger et al. 2011). Thus, sufficient lymph drainage may indeed be crucial to resolving


acute inflammation in an attempt to prevent the development of chronic inflammatory conditions. Importantly, the imbalance between angiogenesis and lymphangiogenesis has been suggested to contribute to the sustained inflammation of atherosclerotic lesions (Nakano et al. 2005). Furthermore, lymphatic vessels in atherosclerotic plaques may be dysfunctional, which could add to this pathological imbalance (Alitalo 2011). Blood vessels also transport lipoproteins into tissue, whereas lymphatic vessels serve as an exit route for both lipids and inflammatory cells. Thus, blood vessels and lymphatic vessels counteract each other in several pathological processes during the pathogenesis of atherosclerosis (Alitalo 2011). All in all, as the amount of lymphatic vessels was relatively small as compared to blood vessels in the stenotic aortic valves (III), their imbalance may promote lipid accumulation and maintain chronic inflammation in AVS as well. The role of intraleaflet hemorrhage In atherosclerotic plaques, neovascularization may contribute to the accumulation of lipids and inflammatory cells into the lesions also via intraplaque hemorrhage, i.e. the rupture of the newly-formed, fragile blood vessels (Kolodgie et al. 2003). Notably, such enhanced accumulation of lipid and inflammatory cells may also occur via less prominent leakage through the immature vasculature as implied above. In our material (II), prominent intraleaflet hemorrhage, as evaluated by neovascular rupture and the presence of microscopic thrombotic masses, was extremely rare. However, the search for such changes was unsystematic, rendering it possible that the incidence of hemorrhagic changes was underestimated. A recent study does in fact describe abundant intraleaflet hemorrhage in the diseased valves, suggesting a parallel pathology with atherosclerosis in this respect (Akahori et al. 2011). Moreover, the presence of erythrocyte-positive extraluminal staining indicating intraleaflet hemorrhage associated with the rapid progression of clinical stenosis (Akahori et al. 2011). Angiogenesis and calcification Angiogenesis and osteogenesis are closely associated during bone development and remodeling. Interestingly, VEGF-A could be the factor connecting these parallel processes (Yang et al. 2012). VEGF-A-mediated capillary invasion is also required for normal growth plate function (Gerber et al. 1999), and VEGF-A may promote both endochondral ossification and intramembranous ossification by directly affecting osteoblasts and osteoclasts (Yang et al. 2012). As discussed above (Results and discussion, 2.2.1), VEGF-D may also promote the differentiation of osteoblasts (Orlandini et al. 2006). Furthermore, pericytes may promote calcification by acting as progenitors for calcifying cells (Collett and Canfield 2005). Interestingly, this pathway has already been recognized as one of the factors behind pathological calcification of the arterial wall in atherosclerosis (Boström et al. 1993). Based on these findings, there is a plausible connection between angiogenesis and calcification in AVS. Other, weaker connections also exist. For instance, the formation of sprouting endothelial tip cells is regulated by NOTCH1 (Hellstrom et al. 2007), which has also been linked to aortic valve calcification (Garg et al. 2005). Without specific assessment, however, the relevance of these findings to AVS pathogenesis remains obscure.


3.1.3. Cause or consequence VEGF-A has been shown to induce the progression of atherosclerotic plaques in several animal models (Celletti et al. 2001). Moreover, inhibiting angiogenesis has resulted in reduced histological atherosclerosis in mice (Moulton et al. 2003). The role of angiogenesis in AVS, however, is mainly based on ex vivo histological evidence. Therefore the neovascularization of the stenotic aortic valves could also be a consequence rather than the cause of valvular thickening and inflammation. So far, only one in vivo study has assessed the significance of valvular angiogenesis in the development of AVS. Yoshioka and coworkers investigated the inhibitory effect of chondromodulin-I to angiogenesis in cardiac valves (Yoshioka et al. 2006). Remarkably, they observed increased lipid deposition and calcification in the aortic valves of mice with increased valvular angiogenesis due to chondromodulin-I downregulation. This finding indicates that valvular angiogenesis may, indeed, have a causal role in the development of AVS. Furthermore, the finding of Soini et al. indicating that even aortic valves with only mild stenotic changes contain neovessels suggests that valvular angiogenesis occurs in the early course of this disease (Soini et al. 2003). In any case, neovascularization may potentiate other pathological valvular processes by providing an entry route as well as nutrients and oxygen to the various cells sustaining valvular ECM remodeling and inflammation. Further investigations are required, however, to establish the exact role of neovascularization in this disease. As lymphatic vessels have remained unreported in animal models of AVS, assessing the balance between valvular angiogenesis and lymphangiogenesis in vivo remains an important long-term goal in the field of AVS research.

3.2. Clinical applications

3.2.1. Detecting valvular angiogenesis Multiple tools are already available for the detection of angiogenesis and inflammation in atherosclerotic plaques in research settings even in humans (Zagorchev and Mulligan-Kehoe 2011). One group has also measured angiogenesis in the sclerotic aortic valves of cholesterol-fed New Zealand White rabbits using integrin-targeted nanoparticles and 19-fluorine MRI/MRS (magnetic resonance imaging/spectroscopy) (Waters et al. 2008). They were able to demonstrate the development of detectable angiogenesis in this model, but possible correlations between the extent of angiogenic signals and histological findings were not included in the study design. Furthermore, the measurements were performed ex vivo, limiting the applications of this technique in humans. Disease progression rate was not assessed in the substudies of this thesis. In any case, the actual question would be how the in vivo measurements correlate with disease progression. Furthermore, in measuring the overall disease activity in a patient, all three leaflets should be accounted for in a three-dimensional manner instead of a cross-sectional assessment of one leaflet used in histological approaches. After addressing these initial questions, imaging disease activity in AVS could provide important clinical applications in the early detection of valvular disease, in recognizing patients with high risk of disease progression, and in targeting individual pharmacotherapies.


3.2.2. Pharmacotherapeutic possibilities A number of valvular processes leading to AVS, including those identified in this thesis, may be modulated even with existing pharmacotherapies (Cawley and Otto 2009, Parolari et al. 2009). The question remains if these therapies are still effective locally in the aortic valve, especially when the diagnosis is usually delayed to end-stage disease. Even if effective, restricting the effects of a drug therapy to the relatively small mass of aortic valve tissue is problematic. For instance, the systemic effects of a therapy aiming at the resolution of calcific masses in the valves could lead to a detrimental outcome in the bones. Finally, since the roles of many of the pathological processes remain unestablished in vivo, the ultimate consequences of successful therapy may be unpredictable even in valvular tissue. If these concerns could be addressed, a variety of possible candidates for pharmacological intervention stand in line to be tested in the treatment of AVS, even in the field of preventing valvular angiogenesis. Currently, the most promising drugs in AVS treatment are ACE-I/ARB medications. Two ongoing randomized clinical trials listed at clinicaltrials.gov assess the potential effects of ARB therapy on the progression of AVS, namely ROCK-AS (Helsinki, Finland; NCT00699452; Candesartan vs. placebo) and the ALFA Trial (Seoul, South Korea; NCT01589380; Fimasartan vs. placebo). Interestingly, there seems to be a regulative connection between LOX-1 and angiotensin II receptors; The activation of angiotensin 1 receptor induces the expression of LOX-1 and vice versa, the effects being counteracted by their respective receptor blockers (Wang et al. 2011). Thus, combining ARB medication with LOX-1-targeted therapy could further reduce the adverse effects of angiotensin II in the aortic valve. More importantly, ARB therapy could also act by reducing the adverse effects of oxLDL by means of downregulating LOX-1. Regarding the adverse effects of oxLDL, it may play a role in AVS pathogenesis even though the lipid-lowering statins seem to be ineffective in AVS (Rossebo et al. 2008). In fact, several investigations have indicated that the proangiogenic effect of oxLDL is more pronounced particularly at low concentrations (Dandapat et al. 2007, Khaidakov et al. 2012, Yu et al. 2011). Furthermore, the relative proportions of oxidatively and otherwise modified lipid particles are significantly increased in stenotic aortic valve leaflets as compared to healthy ones (Lehti et al. 2013). Thus, the pathological role of lipoproteins in AVS could be a question of quality rather than quantity, and the fact that statins produce a mass effect could at least partially explain the unsuccessfulness of lipid-lowering in the treatment of AVS. Both antiangiogenic and proangiogenic therapies targeting the VEGF family have been developed for the treatment of coronary atherosclerosis (Vuorio et al. 2012), since angiogenesis is mostly pathogenic in the arterial wall but desirable in the ischemic heart muscle after infarction. Other antiangiogenic therapies have been suggested as possible counteractors of valvular angiogenesis, including chemically modified tetracyclines and medications targeting the apelin pathway (Peltonen et al. 2009a, Salo et al. 2006). Some of these medications may, indeed, be useful in the future treatment of AVS in addition to other atheroinflammatory conditions. However, as stated above, the ultimate outcome of the inhibition of valvular angiogenesis should first be clearly established in adequate in vivo animal models before advancing to clinical settings with any of the antiangiogenic pharmacological agents.


3.3. Study limitations

Immunohistochemical detection of valvular blood vessels with antibodies against CD31 and CD34 does not differentiate blood vessels from lymphatic vessels. Thus, lymphatic vessels may be included in valvular small and medium-sized microvessel counts. As the number of lymphatic vessels was relatively small (Figure 6A), and there is a positive correlation between lymphatic and blood vessels, a significant bias from this cross-detection is unlikely. VEGF-A has previously been detected only from cells co-expressing its receptors VEGFR-1 and -2 (Soini et al. 2003). In the study in question, only protein, not mRNA, was analyzed at tissue level, leaving the unlikely possibility of ligand-receptor binding rather than actual local production. We found VEGFR-3 in valvular lymphatic vessels and surface endothelium, and processed forms of VEGF-C and -D may also bind to VEGFR-2 (Achen et al. 1998, Joukov et al. 1997). Thus, ligand-receptor binding cannot be ruled out regarding our observations of immunohistochemical colocalization of VEGF-A (II), -C, and –D (III) with valvular myofibroblasts and endothelial cells. Nevertheless, VEGF-A (II) and VEGF-C (III) were also produced by cultured valvular myofibroblasts, and VEGF-A was detected in valvular mast cells lacking both VEGFR-2 and -3 (II), indicating local valvular production of these VEGFs. The substudies in this thesis include data from tissue analyses and cell culture experiments from both AVS patients and control subjects. The lack of animal models limits the possibilities of drawing causal conclusions based on these data. The results may also be affected by the heterogeneity of the control material, which is a common problem in this field of research mainly due to availability issues. In addition to patients undergoing cardiac transplantation due to cardiomyopathies and organ donors without any diagnosed heart disease, control valves were also obtained from patients undergoing valve replacement surgery due to aortic valve regurgitation (AVR). Even though only macroscopically and microscopically healthy valves were included in Studies I-IV, the valves from AVR patients potentially represent another valve pathology. The differences between the three control groups were analyzed in each substudy, however, and the results of AVR patients deviated from those of organ donor tissue only in the scavenger receptor qPCR analyses in Study IV. Consequently, AVR samples were excluded from Study IV, except for some of the cell culture experiments, where other control tissue was unavailable.

64 │ SUMMARY AND CONCLUSIONS _____________________________________________________________________________

VI SUMMARY AND CONCLUSIONS This thesis aimed at characterizing neoangiogenesis and lymphangiogenesis in aortic valve stenosis. We identified several factors involved in these processes, summarized in Figure 9. Based on our findings, the following conclusions can be presented.

1) Cathepsins S, K, and V, and their inhibitor, cystatin C, are locally produced in aortic valves, and their activity is increased in AVS. In addition to promoting the stiffening of the valves by fractioning elastin fibers, these cathepsins may contribute to valvular angiogenesis by participating in the local ECM degradation, which enables endothelial cell migration and vascular sprouting.

2) Stenotic aortic valves contain blood vessels and lymphatic vessels, and the balance of these vessels favors the accumulation of lipids and inflammatory cells into the valves rather than their clearance. Furthermore, mature arterioles, an advanced stage of angiogenesis, correlate positively with valvular calcification.

3) Angiogenic and lymphangiogenic vascular endothelial growth factors and their

receptors are locally expressed in the aortic valves. In addition, the expression of angiogenic and lymphangiogenic VEGF receptors is increased in stenotic aortic valves, suggesting that the VEGFs are involved in the pathogenesis of AVS.

4) Activated mast cells in stenotic aortic valves secrete VEGF-A, and the VEGF-A secretion of mast cells is augmented by valvular myofibroblasts and vice versa. Mast cells are also capable of degrading the antiangiogenic endostatin and lymphangiogenic VEGF-C. Thus, valvular mast cells appear to possess angiogenic and antilymphangiogenic properties.

5) The oxLDL-binding scavenger receptors SR-A1, CD36, and LOX-1 are expressed in the

aortic valve. Furthermore, the expression of proinflammatory SR-A1 and proangiogenic LOX-1 are increased in AVS, whereas the potentially antiangiogenic CD36 is downregulated. Thus, changes in the expression of scavenger receptors in AVS may favor valvular inflammation and angiogenesis. Furthermore, oxLDL may even contribute to valvular angiogenesis indirectly by inducing the production of proinflammatory cytokines in aortic valve myofibroblasts.

General conclusions and future prospects Valvular myofibroblasts, mast cells, and their interactions may contribute to angiogenesis in stenotic aortic valves. In AVS, the balance between blood vessels and lymphatic vessels is disturbed, which could facilitate the infiltration of lipids and inflammatory cells into the stenotic valve leaflets. Even if the complex mechanisms behind pathological valvular angiogenesis and lymphangiogenesis could be altered, pharmacological treatment targeting valvular angiogenesis is unlikely to reverse the pathological changes of end-stage stenotic valves. Rather than a pharmacotherapeutic target of AVS, angiogenesis could potentially serve as a target for valvular imaging. Conceivably, valvular angiogenesis could not only portray the

SUMMARY AND CONCLUSIONS │ 65 _____________________________________________________________________________

presence of valvular disease but also associate with disease progression. If disease activity could be detected at earlier stages, targeted follow-up and future pharmacological treatment could perhaps alter the currently inevitable consequences of this detrimental disease.

Figure 9, modified from (III). Schematic illustration summarizing the main findings in this thesis and the proposed regulatory mechanisms of angiogenesis and lymphangiogenesis in AVS. (1) Cathepsins S and V are produced by valvular endothelial cells (I). These cathepsins may facilitate endothelial cell migration and vascular sprouting (I) (Shi et al. 2003). (2) Valvular myofibroblasts secrete VEGF-A (II), VEGF-C (III), and VEGF-D (III), and the production of VEGF-A by the myofibroblasts is increased by mast cell-derived components, hypoxic conditions, and cigarette smoke (II). Short-term osteogenic stress, in turn, decreases the secretion of VEGF-A (II) and VEGF-C (III) by the myofibroblasts. (3) Cigarette smoke can activate mast cells (Helske et al. 2006), and activated mast cells secrete VEGF-A (II). Furthermore, myofibroblast-derived factors promote VEGF-A secretion by the mast cells (II). Mast cells are also able to degrade antiangiogenic endostatin (II) and lymphangiogenic VEGF-C (III). (4) VEGF-A and VEGF-C are also produced by endothelial cells lining the valve as well as valvular neovessels, the former producing VEGF-D as well. (5) Valvular neovessels express VEGFR-2 (Soini et al. 2003) and lymphatic vessels express VEGFR-3. Both VEGFR-2 and -3 are upregulated in AVS (III). Valvular neovessels also express VEGF-A and -C, and the amount of pericyte-covered mature vessels increases with valvular calcification (II). (6) LOX-1 expression is increased in the endothelial cells of stenotic aortic valves (IV). OxLDL may thus increase VEGF-A secretion by the endothelial cells via LOX-1 (Dandapat et al. 2007). Oxidative modulation of LDL in the valves may also ensue from the actions of external risk factors such as cigarette smoke (Valkonen and Kuusi 1998). (7) Valvular neovessels may be harmful in AVS because they provide nutrients and oxygen to the proliferating myofibroblasts as well as form an entry route for inflammatory cells and lipids. Lymphatic vessels, in turn, could serve as an exit route. The imbalance between valvular angiogenesis and lymphangiogenesis may promote AVS progression by favoring lipid accumulation, inflammation, and thickening of the diseased valve leaflets.

66 │ ACKNOWLEDGEMENTS _____________________________________________________________________________

VII ACKNOWLEDGEMENTS This work was carried out at the Wihuri Research Institute during the years 2004-2013, in co-operation with the Division of Cardiology, Department of Medicine / Heart and Lung Center of Helsinki University Central Hospital. I wish to thank my supervisor Professor Petri Kovanen as the former director of the institute for having provided the excellent research facilities, a stimulating working environment, and the flexible arrangements that made it possible to work during my medical studies and with a growing family. I also wish to sincerely thank the current director of Wihuri Research Institute, Professor Kari Alitalo, for warmly welcoming us to Biomedicum as well as for his scientific insight that has proven valuable in the writing process. My warmest gratitude goes to my supervisor Satu Suihko for guiding me through this whole process and for her unconditional support in all the important moments of my life. I couldn’t have hoped for a better mentor. I am also most grateful to my other supervisor, Professor Petri Kovanen, for always appreciating my work and special points of view, as well as for sharing his vast knowledge on atherosclerosis in numerous enthusiastic conversations over the years. I express my sincerest thanks to the reviewers of this thesis, Docents Jari Satta and Tuomas Rissanen, for their expert opinions and constructive criticism that helped me improve this thesis. I wish to acknowledge my collaborators who, based on their expertise, have made this work possible by supplying the critically important human samples, teaching methods, and providing data: Mervi Kinnunen, Jani Lappalainen, Katariina Öörni, Mikko Mäyränpää, Riina Oksjoki, Markku Kupari, Ken Lindstedt, Mika Laine, Jyri Lommi, Heikki Turto, and Kalervo Werkkala. Professor Markku Kupari initiated this research project on aortic valve stenosis and provided a well-characterized group of patients for the study. Docent Ken Lindstedt had a significant role in my early years at Wihuri, and I am grateful for the ideas he threw around as well as for his general advice in the lab. Mikko M. and Riina, thank you for the insightful introduction to the research as well as the medical world. The late-night calls to transplantation surgery already seem so distant. Kati, especially this past year would have been so much more difficult without your kind, yet straightforward guidance. I would also like to warmly thank Liisa Blubaum, who has had a substantial role in collecting both the original material and subsequent patient and control samples for cell culture. Her kind and enthusiastic attitude has made many matters easier to handle. The heart transplantation coordinators and the cardiac transplantation team of Helsinki University Central Hospital are also sincerely thanked for their help in obtaining the control samples. I have had the privilege of working with two young gentlemen, Jaakko Lommi and the late Mikko Simolin (Pikku-Mikko), with whom I have shared numerous both hilarious and serious moments. Pikku-Mikko passing away ahead of his time taught me to appreciate what I have, and I will always remember him. I am ever so thankful to Jaana Tuomikangas, Suvi Sokolnicki, Mari Jokinen, and Maija Atuegwu for teaching me how to work and how not to work in the lab. Their help has been substantial in completing this work, and I am also quite happy with my seemingly everlasting nickname Pikku-Suvi (at least compared to the other alternatives).

ACKNOWLEDGEMENTS │ 67 _____________________________________________________________________________

I also wish to express my warmest thanks to all my other colleagues at Wihuri Research Institute. Special thanks go to Laura Fellman for her patience and all her help with practical matters. I would also like to acknowledge all my roommates over the years. The latest of them, Mervi, is warmly thanked for all the interesting conversations over so many matters, big and small, and for the apple jam. The Biomedicum crew, Ilona, Satu L, Krisse, Kata N, Kata M, Reija, Su, and Miriam – thank you for your patience during these final months and for all the fun times we have had over the years. Especially Ilona and Satu L, your friendship has saved me from a lot of worry and sorrow. Working at Kaivopuisto would not have been the same without Anna, Andrea, Steffi, Artturi, Hanna H, Hanna L, Riia, Kata L, Elina, Inka, Julia, Marja, Mia, Laura N, Marru, Jukka, Tuula, Julio, Maria, Jarmo, Katri, Pia, Lennu, and Terttu, who are warmly acknowledged. I would also like acknowledge my clinical colleagues in Porvoo and in Martinlaakso, thank you for providing other aspects into a researcher’s life. I would also like to thank all my friends, both in and outside the research world, for being there for me. My dearest thanks go to my beloved academic knitting squad, Taru, Jenni, and Sari, for all the tea and fluffiness. I would also like to thank Mervi and Aino for their support in both work and family matters. My old friends Liina, Liisa, and Hanne also had a role in this even if they didn’t know it, so thank you for teaching me how much candy is too much, that it’s fun to argue just for the sake of argument, and that organic milk is not lactose-free. My newer friends from Metsoforte, thank you for the music! One of you even had to read this thesis through – thank you Sanni for that. I owe special thanks my family, without whom none of this would have been possible: My parents Jatta and Ari for always believing in me, and my sister Tessa and her husband Petteri, “the Kamppi tech support”, also for the practical help with graphic design. My grandmother Helmi, who was so proud of all her grandchildren but regrettably isn’t here to see this day. My parents-in-law Hilve and Seppo for always appreciating me and for their interest towards my work, and my sisters and brothers-in-law Elina, Jan, Perttu, and Nita for their support and general advice in life. Thank you all for the numerous enjoyable brunches, lunches, and dinner parties as well. I would also like to acknowledge my cousins Jari, Janne and Kalle, who have always been a part of my life, near or far. Elsa’s cousins Arttu, Elias, Elmo, Ella, Iida, and Rosa are warmly thanked for all the joy they have brought into our lives and also for making time fly. Finally, I express my deepest gratitude to my husband Lauri, for encouraging me to apply to medical school and into this research group in the first place, and for all his love and patience over the years. I also wish to thank him, our dearest daughter Elsa, and her yet unborn baby brother for all the memorable moments we have had so far, and for helping me sort out my priorities. This work was financially supported by the Jenny and Antti Wihuri Foundation, the Finnish Cultural Foundation, the Aarne Koskelo Foundation, the Finnish Foundation for Cardiovascular Research, the Finnish Medical Foundation, and the Finnish Medical Society Duodecim. Helsinki, August 2013 Suvi Syväranta

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