CYP26B1 as regulator of retinoic acid in vascular cells...

CYP26B1 as regulator of retinoic acid in vascular cells and atherosclerotic lesions

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Örebro Studies in Medicine 71

ALI ATEIA ELMABSOUT

CYP26B1 as regulator of retinoic acid in vascular cells and atherosclerotic lesions

© Ali Ateia Elmabsout, 2012

Title: CYP26B1 as regulator of retinoic acid in vascular cells and atherosclerotic lesions

Publisher: Örebro University 2012www.publications.oru.se

[email protected]

Print: Örebro University, Repro. 05/2012

ISSN 1652-4063ISBN 978-91-7668-877-9

Abstract Ali Ateia Elmabsout (2012): CYP26B1 as regulator of retinoic acid in vascular cells and atherosclerotic lesions. Örebro Studies in Medicine 71, 62 pp. Cardiovascular disease (CVD), currently the most common cause of morbidity and mor-tality worldwide, is caused mainly by atherosclerosis. Atherosclerosis is a chronic multi-focal, immunoinflammatory, fibroproliferative disease of medium and large arteries. Atherosclerotic lesions and vascular cells express different genes, among these are genes regulated by retinoic acid. Retinoids have pleiotropic effects and are able to modulate gene expression involved in growth, function and adaptation. During atherosclerosis development, there is endothelial perturbation, lipid accumulation, attraction of immune cells, smooth muscle cell migration and extracellular matrix remodeling and eventually fibrous cap formation which results in plaques. Retinoids have been demonstrated to either inhibit or modulate the above processes, resulting in amelioration of atherosclero-sis. So far, retinoids are known to have impact on cellular processes in SMC, vascular injury and atherosclerosis. However, little is known about catabolism of retinoids in vascular cells and lesions and the effects of alteration of retinoic catabolizing enzymes on retinoids’ status. Therefore, we investigated the expression of Cytochrome P450 26 (CYP26) which is thought to be dedicated to retinoid catabolism. In vascular SMCs and atherosclerotic lesions, we found that CYP26B1 was the only member of the CYP26 family expressed, and it was highly inducible by atRA. Our data revealed that blocking CYP26B1 by chemical inhibition, or by targeted siRNA knock-down, resulted in signifi-cantly increased cellular retinoid levels. This indicates that CYP26B1 is an important modulator of endogenous retinoic acid levels. Therefore, we studied the effect of the CYP26B1 nonsynonymous polymorphism rs224105 on retinoic acid availability and found that the minor allele was associated with an enhanced retinoic acid catabolism rate and also with a slightly larger area of atherosclerotic lesions. The expression of CYP26B1 in human atherosclerotic lesions was localized to macrophage rich areas, suggesting retinoic acid activity in macrophages. Furthermore, we demonstrated that a CYP26B1 splice variant, that lack exon two, is expressed in vascular cells and in vessels walls. It is functional, with a reduced catabolic activity to around 70%, inducible by atRA in vascular cells and expressed 4.5 times more in atherosclerotic lesions compared to normal arteries. Moreover, the statins simvastatin and rosuvastatin reduced CYP26B1 mediated atRA catabolism in a concentration-dependent manner, and in vascular cells increased the mRNA expression of the atRA-responsive genes CYP26B1 and RARβ. This could lead to statins indirectly augmenting retinoic acid action in vascular cells which mimic statins roles. In conclusion, CYP26B1 is a major retinoic acid modulator in vascular cells and athero-sclerotic lesions. Blocking of CYP26B1 could provide an advantageous therapeutic alter-native to exogenous retinoid administration for treatment of vascular disorders.

Keywords: CYP26B1, alternative splice, vascular cells, atherosclerosis, all-trans-retinoic acid, gene polymorphism, inflammation, statins Ali Ateia Elmabsout, School of Medical Sciences, Örebro University, SE-701 82 Örebro, Sweden, [email protected]

List of papers Ocaya PA, Elmabsout AA, Olofsson PS, Törmä H, Gidlöf AC, Sirsjö A. CYP26B1 plays a major role in the regulation of all-trans-retinoic acid metabolism and signaling in human aortic smooth muscle cells. J Vasc Res. 2011; 48(1):23-30 Krivospitskaya O, Elmabsout AA, Sundman E, Söderström LA, Ovchinni-kova O, Gidlöf AC, Scherbak N, Norata GD, Samnegård A, Törmä H, Abdel-Halim SM, Jansson JH, Eriksson P, Sirsjö A, Olofsson PS. A CYP26B1 polymorphism enhances retinoic acid catabolism which may aggravate atherosclerosis. Mol Med. 2012: In press. Elmabsout AA, Ashok Kumawat, Patricia Saenz-Méndez, Olesya Krivospitskaya, Helena Sävenstrand, Peder S Olofsson,Leif A. Eriksson, Åke Strid, Guro Valen , Hans Törmä, and Allan Sirsjö. Cloning and Func-tional Studies of a Splice Variant of CYP26B1 Expressed in Vascular Cells. Accepted for publication in PLoS ONE.

Elmabsout AA, Karin Franzén, Eva Sundman, George Karikas, Hans Törmä, Peder S Olofsson and Allan Sirsjö. Simvastatin and rosuvastatin inhibit CYP26B1-mediated retinoid catabolism. (Manuscript)

List of abbreviations ADH alcohol dehydrogenase AER apical ectoderm ridge AngII angiotesin II ApoE apolipoprotein E ARAT acyle-CoA:retinol acyltransferase atRA all-trans-retinoic acid BCMO1 beta carotene monooxygenase 1 BFGF basic fibroblast growth factor CEL carboxyl ester lipase CHD coronary heart disease CRABP cellular retinoic acid binding protein CRBP cellular retinol binding protein CVD cardiovascular disease CYP cytochrome P450 CYP26 cytochrome P 450 family 26 CYP26A1 cytochrome P 450 family 26 subfamily A polypeptide1 CYP26B1 cytochrome P 450 family 26 subfamily B polypeptide1 CYP26C1 cytochrome P 450 family 26 subfamily C polypeptide1 ECs endothelial cells ECM extra cellular matrix DCs dendritic cells DGAT1 diacylglycerol O-acyltransferase 1 DHCR7 7-dehydrocholesterol reductase FDFT1 farnesyl-diphosphate farnesyltransferase 1 FOXL2 forkhead box L2 FOXO1 forkhead box protein O1 HDL high density lipoprotein HOCL hypochlorous acid HUVECs human umbilical vein endothelial cells HSCs hepatic stellate cells ICAM-1 interacellaur adhesion molecules 1 IL1β interleukine 1 beta INFγ interferon gamma LDL low density lipoprotein LPS lipopolysaccharides LRAT lecithin: retinol acyle tranferase MAPK mitogen activator protein kinase MCP-1 monocytes chemotactic protein-1 MCSF macrophages colony stimulating factor MI myocardial infarction MMP matrix metalloproteinase MTP microsomal transfer protein

NFKB nuclear factor kappa-B DNA binding subunit NO nitric oxide NOS nitric oxide synthase PAX6 Paired box protein 6 PDGFb platelet derived growth factor b PLRP2 pancreatic lipase related protein 2 PPAR peroxisome proliferator-activated receptors PTL pancreatic triglyceride lipase RA retinoic acid RALDH retinal dehydrogenase RARE retinoic acid responsive gene RAR retinoic acid receptors RBP retinol binding protein RDH retinol dehydrogenase REH retinyl ester hydrolase RXR retinoid x receptor SEAP secreted alkalin Phosphatase SF1 steroidogenic factor 1 SHH sonic hedgehog homolog SMCs Smooth muscle cells SMMHC smooth muscle myosin heavy chain SNP single nucleotide polymorphism SR-B1 scavenger receptor B1 SRY sex determining region Y STRA6 stimulated by retinoic acid gene 6 TGFβ transforming growth factor beta TIMPs tissues inhibitor of metalloproteinase TNFα tumor necrosis factor alpha TLR toll like receptor TPa tissues activator plasminogen TRR transthyretin VCAM-1 vascular cellular adhesion molecules VEGF vascular endothelial growth factor VLDL very low density lipoprotein

Table of contents

1. ATHEROSCLEROSIS ........................................................................... 9 1.1. Development of atherosclerotic lesion ................................................. 9 1.2. Vascular cells in atherosclerosis ........................................................ 12

1.2.1. Smooth muscle cells in atherosclerosis ....................................... 12 1.2.2. Endothelial cells in atherosclerosis ............................................. 12

1.3. Inflammation in atherosclerosis ........................................................ 12 1.4. Lipids in atherosclerosis .................................................................... 13 1.5. Gene polymorphism in atherosclerosis .............................................. 14

2. VITAMIN A ........................................................................................ 14 2.1. Retinoid metabolism ......................................................................... 15 2.2. The retinoid signalling pathway ........................................................ 17 2.3. Catabolism of retinoic acid ............................................................... 18

2.3.1. The cytochrome P450 superfamily ............................................. 18 2.3.2. The CYP26 family ..................................................................... 18 2.3.2.1 Catabolic activity of CYP26A1, CYP26B1 and CYP26C1 ...... 19 2.3.2.2. CYP26A1 ................................................................................ 19 2.3.2.3. CYP26B1 ................................................................................ 20 2.3.2.4. CYP26C1 and CYP26C2 ........................................................ 22

2.4. Effects of single nucleotide polymorphisms ....................................... 23

3. RETINOIDS AND CARDIOVASCULAR DISEASES .......................... 23 3.1. Retinoids and cardiovascular development ....................................... 23 3.2. Perturbation of the Retinoid signalling pathway is associated with cardiovascular diseases ............................................................................. 24 3.3. Retinoids and atherosclerosis ............................................................ 25 3.4. Retinoids and lipids .......................................................................... 26 3.5. Retinoids and inflammation .............................................................. 28 3.6. Retinol/β-carotene and risk of cardiovascular diseases ...................... 29

4. AIMS OF THE STUDY ....................................................................... 31

5. MATERIALS AND METHODS .......................................................... 32

6. RESULT AND DISCUSSION .............................................................. 35 6.1. CYP26B1 plays a major role in the regulation of all-trans- retinoic acid metabolism and signalling in human aortic smooth muscle cells (AOSMCs): (Paper 1) ............................................................................... 35 6.2. A CYP26B1 polymorphism enhances retinoic acid catabolism which may aggravate atherosclerosis: (Paper 2) ....................................... 36

6.3. Cloning and functional study of a spliced variant of CYP26B1 expressed in vascular cells: (Paper 3) ........................................................ 37 6.4. Simvastatin and rosuvastatin inhibit CYP26B1-mediated retinoid catabolism: (Paper 4) ............................................................................... 38

CONCLUSION ........................................................................................ 40

FUTURE PERSPECTIVES ....................................................................... 41

ACKNOWLEDGEMENTS ...................................................................... 42

REFERENCES ......................................................................................... 43

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1. Atherosclerosis Cardiovascular disease (CVD) is currently the largest cause of morbid-ity and mortality worldwide (1, 2). CVD and cerebrovascular disease are caused mainly by atherosclerosis, a chronic inflammatory disease, characterized by accumulation of fat in the walls of large and medium sized arteries, infiltration of immunocytes and debris cells. The cells infiltrate from the surrounding milieu, which eventually may lead to fibrous cap formation (2, 3). The term “atherosclerosis” describes the relation of the degeneration of fatty acids and vessel stiffness (4, 5). The atherosclerotic lesions consist of three major parts; the cellular part composed of smooth muscle cells (SMC) and macrophages, the connective tissues and extracellular lip-ids, and the intracellular lipids accumulate within macrophages which may result in the formation of foam cells (3, 6). The development of atherosclerosis starts in teenagers and young adults with the formation of early lesions which slowly progress over decades. Affecting the vascular intima layer, they eventually restrict blood flow prior to the manifestation of the disease (7). There are sev-eral risk factors implicated in atherosclerosis including hypertension, diabetes, obesity, high levels of cholesterol and low density lipoproteins (LDL), age, male gender and smoking(8). Genetic polymorphisms may also play a role in the development of atherosclerosis. The risk factors can act alone or in combination (9).

1.1. Development of atherosclerotic lesion The development of atherosclerotic lesion is established mainly through the presence of high cholesterol levels in the blood. The process starts with endothelial leakage, followed by activation of endothelial cells resulting in a dysfunctional endothelium (10) (Figure 1). Endothelial cells express several adhesion molecules, e.g. VCAM-1, ICAM-1 and P-selectin, which play an important role in the development of the atherosclerotic lesion. The adhesion molecules capture leukocytes in the presence of irritative stimuli such as dyslipidemia, hypertension, smoking, pathogenic organisms, shear stress and cytokines (11). Blood borne cells, including neutrophils, monocytes and T-cells, adhere to the injured endothelium in the early atherosclerotic lesion (12). Simultane-ous changes in the endothelial permeability and the composition of the extracellular matrix (ECM) proteins in the subendothelial intima layer allows the entry of cholesterol and LDL (13). The accumulation of LDL in the intima layer of the arteries is caused by the adhesion of

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ApoB100, a component of the LDL molecule. ApoB100 adhere to pro-teoglycans in the ECM, which capture the LDL and cause the release of lipoxygenases and other enzymes. The lipoxygenases may in turn liber-ate free oxygen species, like hypochlorous acid (HOCL), which is gen-erated during the inflammatory process in the atherosclerotic lesion (13, 14). When the LDL molecules have entered the subendothelial layer, they may be oxidized by lipoxygenase, myeloperoxidase or through the production of nitric oxide (NO) by nitric oxide synthase (NOS) (15). The presence of oxidized LDL in the subendothelial layer activates the innate immunity through the binding of toll like receptors (TLR) (16). Atheromous lesions are rich in monocytes and macrophages, which produce high levels of cytokines like IL-1β and TNF-α that aggravate atherogenesis (17). Chemoattractant molecules like MCP-1, further attract monocytes and T-cells. Furthermore, mast cells and B-cells are also present in the lesions, but less abundant and mostly in the adventi-tia layer (18). The monocytes in the subendothelial intima may then differentiate into macrophages and take up LDL through the scavenger receptors CD36 and SR-a, which results in the accumulation of choles-terol esters. The cholesterol esters cause the development of foam cells (so called fatty streak), which may lead to apoptosis and necrosis of the macrophages. Endothelial cells also produce macrophages colony stimulating factor (MCSF) supporting the conversion of the monocytes into macrophages, which is essential for the development of atheroscle-rosis (19). Immunoinflammatory effects may heal and repair the injury over time with extended SMC migration and proliferation induced by platelet-derived growth factor (PDGF) (20). SMC in the intima layer produce ECM proteins, such as collagen and elastin, to cover the plaque and form fibrous caps and thus narrowing the lumen. This causes a limita-tion of blood flow, which may result in tissue ischemia or thrombosis. The atherosclerotic lesions prone to rupture are characterized by pro-found lipid cores, thinner fibrous caps with few SMCs, adventitial inflammation, plenty of macrophages and matrix remodelling (1).



Figure1. A-D. Atherosclerosis development. A) Endothelial dysfunc-tion, release of adhesion molecules and penetration of LDL. B) Re-leased adhesion molecules and LDL undergo oxidation. Monocytes infiltrate and differentiate into macrophages in the subendothelium. C) Foam cell formation through macrophages engulfing oxidized LDL. Plaque formation with fibrous caps and migration of SMC. D) Migration of SMC by action of growth factors. Rupture of vul-nerable plaques and release of thrombogenic materials into the blood stream



1.2. Vascular cells in atherosclerosis

1.2.1. Smooth muscle cells in atherosclerosis SMC are located in tunica media and tunica intima and play a role in the pathogenesis of atherosclerosis and restenosis. These cells have the ability of phenotype modulation and thereby the capability for prolif-eration and migration which implicated for cardiovascular develop-ment (21). Under normal physiological conditions, the SMCs of the tunica media exhibit a contractile phenotype with limited proliferative activities (22). SMCs in the intimae and medial layers differ significantly from each other and have unique atherogenic action in plaque initiation. Medial SMCs extensively express proteins with contractile function, including smooth muscle α-actin (SM α-actin) and myosin heavy chain (SM-MHC), while levels of these proteins are lower in intimal SMC (23, 24). Intimal SMCs instead have a synthetic phenotype with higher capability of migration and proliferation (25). The switching of SMCs from one phenotype to another, i.e. from contractile to synthetic phe-notype has been implicated in atherogenesis (25-27).

1.2.2. Endothelial cells in atherosclerosis Endothelial cells are mainly located in tunica intima, lining the inner surface of the blood vessels. This is a very strategic position in direct contact with the blood and also innermost contact with the vascular smooth muscle cells (28, 29). The endothelial cells play a vital role in tissue homeostasis, coagulation, and fibrinolysis, inhibition of SMC migration, proliferation and blood/tissues exchanges (30, 31). Endothe-lial dysfunction is substantially contributing to atherosclerotic proc-esses that eventually may lead to vascular occlusion and hypoperfusion (29). Furthermore, activation of endothelial cells through biomechani-cal stimuli may lead to phenotype alteration and thus the formation and progression of atherosclerotic lesions (32). Endothelial perturba-tion can be caused by high cholesterol levels, hypertension, diabetes, oestrogen deficiency and vascular aging (30).

1.3. Inflammation in atherosclerosis Inflammation plays primordial role in all stages, formation, progres-sion and perturbation of atherosclerosis and both stable and vulnerable plaques are characterized by inflammatory reactions (33). Several stud-ies have demonstrated that a subset of inflammatory hematopoietic cells including monocytes and macrophages, accumulate in atheroscle-



rotic lesions and release proinflammatory cytokines (34). This may activate the expression of adhesion molecules on the endothelial cell surface, attracting more monocytes that further aggravate inflamma-tory response (35). Furthermore, MCP-1 is also implicated in attracting macrophages in atherosclerotic lesion. T cells recruitment into athero-sclerotic plaque and activation results in the expression of the ligands that induce apoptosis of macrophages and accelerate lipid core forma-tion (36). Endothelial cells and SMCs interact with monocytes causing the mono-cytes to differentiate into macrophages (37). The interaction is mediated by adhesion molecules e.g. VCAM-1, ICAM-1 and CX3CL1 expressed on the surfaces of endothelia cells and SMCs (38, 39). VCAM and ICAM are induced by the proinflammatory cytokines IL-1β and IL-18 (40, 41). In addition, macrophages express TGF-β, which may enhance tissue fibrosis (42). The adaptive immune system’s T and B cells play significant roles in the development of atherosclerosis and it has been shown that defi-ciency of B and T cells significantly suppresses lesion development (43, 44). Recruitment and activation of T cells occur through dendritic cells (DCs) (antigen presenting cells) (45). The accumulation of DCs in the intimae of atherosclerotic lesions may further disrupt tissue homeosta-sis (45).

1.4. Lipids in atherosclerosis Lipids play central roles in the development of atherosclerosis but the mechanism for their action is not fully clear. Recent observations show a strong relationship between blood lipids and risk of cardiovascular events (46, 47). The presence of high plasma LDL has also been corre-lated to increased risk of atherosclerosis susceptibility (17). Hyperlipi-demia may be a result of lipids and/or cholesterol rich diet, obesity, genetic predisposition, pathological conditions, and therapeutic agents (48, 49). LDL penetrates the dysfunctional endothelium moving toward the intimal layer. After binding to proteoglycans, LDL is modified (50), stimulating the release of adhesion molecules and pro-inflammatory cytokines in endothelial cells and macrophages (51). Releasing of NO from the endothelium contributes to the inhibition of adherence of platelets and leukocytes as well as SMC proliferation and oxidation of LDL (52, 53). Presence of LDL has profound effects on endothelial cells through the inhibition of endothelium relaxation, accumulation of platelets and



thrombin and impaired activity of NOS (54, 55). Furthermore, as ob-served in hyperlipidemia and atherosclerotic vascular disease, LDL enhances the release of endothelin that may lead to disruption of vascu-lar homeostasis (56, 57).

1.5. Gene polymorphism in atherosclerosis Extensive studies have been carried out to investigate gene polymor-phisms as a risk for atherosclerosis development but results are not conclusive. Apolipoprotein E (ApoE) is essential for lipid transport and metabolism in vivo (58, 59) and plays a pivotal role in development of atherosclerosis (60). ApoE gene polymorphisms have been extensively studied and three risk alleles (E2, E3 and E4) resulting from missense alterations, have been identified. Allele E4 has in some studies been associated with increased risk for atherosclerosis (58, 61, 62), while other studies found a borderline (63, 64) or no significant association with cardiovascular disease (65). The Toll like receptor 4 (TLR4) has been the focus of several investiga-tions over the last few decades. TLR4 is a pattern recognizing receptor for LPS (lipopolysaccharide), LDL (16), heat-shock protein 60, fibrino-gen and fibronectin (66). Recent studies have found an association between the Asp299Gly polymorphism in TLR4 gene and decreased inflammatory response and diminished incidence of atherosclerosis (67, 68). In addition, the Asp299Gly polymorphism has also been associ-ated with myocardial infarction (69), which is a common result of atherosclerosis. However, Hommels (66) and Netea (70) could not verify these results.

2. Vitamin A Several nutritional studies have revealed the importance of vitamin A in biology (71). McCollum and Davis first found that rats fed with a mix-ture of ether-soluble compounds such as casein, butter and lard but not salt extract showed improved growth and survival (72). Later McCollum came to conclude that a fat soluble compound was essential for keeping animal healthy and named this fat soluble factor A (later is vitamin A) (73). The term of retinoids was introduced by Michael Sporn in 1976 and includes natural derivatives of vitamin A (retinol), both biologically active and non biologically active and synthetic ana-logs (74, 75). The term has recently been expanded to also include compounds structurally different from retinol but with retinoid activity (76).



RA (retinoic acid) is the active natural and physiological metabolite of vitamin A (77, 78) and a powerful regulator of gene transcription. There are three geometric isomers of RA (atRA, 9-cis-RA and 13-cis-RA), among which all-trans-retinoic acid (AtRA) seems to be most important for retinoid signalling (79).

2.1. Retinoid metabolism RA is produced in the body from dietary vitamin A, either ingested as retinol and retinyl esters from animal products or as carotenoids ( β-carotene) from plant sources. Some dairy products and cereal may also be fortified with retinyl esters. In the gut, retinyl esters are converted into retinol before entering the enterocytes. Carotenoids may enter enterocytes and chylomicrons unchanged or be cleaved to form retinal which is subsequently reduced to retinol. The majority of the entero-cytes’ retinol is then esterified to retinyl ester, secreted in chylomicrons into the lymphatic system and via the thoracic duct transferred to the circulation. The chylomicrons may be utilized by tissue directly, but the majority is taken up by hepatocytes in the liver. In the hepatocytes, retinyl ester is hydrolyzed to retinol, transferred to hepatic stellate cells (HSC) and re-esterified, in the retinoid-sufficient state, storage in lipid droplets in the HSC. When needed the stored retinyl esters may be mobilized and released to the circulation (Figure 2).

Figure 2. Enteral absorption of dietary vitamin A, metabolic steps for transportation from gut to liver for storage or resecretion into the cir-culation.



For transportation to target tissues, retinyl ester stored in HSC lipid droplets is hydrolyzed to retinol and released from the liver into the circulation bound to retinol binding proteins (RBP), also synthesized in the liver and adipose tissue. The concentration of retinol-RBP in plasma is strictly regulated at 2-3 μM in humans and 1μM in mice. To prevent filtration by kidney glomeruli, the retinol-RBP complex associ-ates with the thyroxine hormone carrier transthyretin (TTR). The reti-nol-RBP-TTR complex is taken up in target tissues by the transmem-brane spanning protein STRA6 which act as a RBP receptor mediating both uptake and release of retinol. Intracellularly, retinol is converted to retinyl ester by lecithin retinol acyltransferase (LRAT). Retinol may also be oxidized to retinal, catalyzed by membrane-bound short-chain dehydrogenase/reductases (SDR), retinol dehydrogenases (RDH) or cytosolic medium-chain alcohol dehydrogenases (ADH). Several tissue specific retinal dehydrogenases, RALDH1A1, RALDH1A2 and RALDH1A3, are then able to further oxidize retinal to retinoic acid (RA) (Figure 3). The retinoid metabolisms have been described in detail in several recent reviews (76, 80, 81).



2.2. The retinoid signalling pathway RA may act in a paracrine manner, affecting adjacent cells (82) or in an autocrine manner where remain in the cell it was produced (82, 83). The biological effects of retinoids are mediated by steroid hormone receptors, the retinoic acid receptors RARα, RARβ and RARγ and the retinoid X receptors RXRα, RXRβ and RXRγ (84). RARs and RXRs form heterodimers (one of the RARs combined with one of the RXRs) that bind to retinoid acid responsive element (RARE or RXRE) in the promoter region of the DNA sequence and act as ligand depend-

Figure 3. Retinoic acid metabolism and signaling pathways



ent transcription factors altering gene expression (85). In the absence of ligand, the RAR/RXR complex bound to DNA repress transcription by recruiting corepressors and histone deacetylase. When ligand binds to the RAR/RXR complex, a conformational change occurs, and tran-scription is facilitated by recruitment of coactivators and histone ace-tyltransfreases. Effects of RA on different cellular processes thus de-pend both on the present concentration of RA and the expression of RARs (86). Beside the function as classical transcription factors, RARs can promote epigenetic alterations such as modification of chromatin with resultant changes in DNA sequences (87).

2.3. Catabolism of retinoic acid

2.3.1. The cytochrome P450 superfamily Cytochrome P450 (CYP) was first identified in 1961 as a cellular chromophore which was referred to as P450 because the pigment (P) was found to have a 450-nm spectral peak when reduced and interact with carbon monoxide (88). Several microsomal CYP genes encode CYP enzymes that are involved in the metabolism of endogenous sub-strates, including vitamins, fatty acids and steroids. Furthermore, the CYP enzymes eliminate toxic exogenous compounds, such as pollut-ants, drugs and carcinogenic molecules (89). So far, more than 75 CYP proteins with different physiological roles have been identified in hu-man (90, 91).

2.3.2. The CYP26 family The CYP26 enzymes are heme-binding monooxygenases responsible for the catabolism of RA (89, 92), acting as a switch for RA signalling in vertebrate (87). The CYP26 family have three members; CYP26A1, B1 and C1, all known to metabolize RA (93). The CYP26 family is also essential for embryonic development, postnatal survival and germ cell development (94). CYP26A1 and B1 have 44% sequence similarity, CYP26A1 and C1 45% and CYP26B1 and C1 51%. Therefore, the highest conserved CYP26 is CYP26B1 and least conserved is CYP26C1 (95). The pre-dicted protein sequences of the three CYP26s are between 497 to 522 amino acids, the predicted molecular weight is between 50 and 60 kD with a chance for gylcosylation as observed in some CYPs. Both CYP26A1 and CYP26B1 are controlled by different nuclear receptors which give rise to different tissue expression patterns (94, 96).



2.3.2.1 Catabolic activity of CYP26A1, CYP26B1 and CYP26C1 CYP26A1 and CYP26B1 hydroxylase atRA into different polar me-tabolites (4-OH-RA, 4-OXO-RA and 18-OH-RA) (97, 98). While CYP26C1 also catabolize atRA into same polar metabolites as CYP26A1 and B1, its preferred substrate is 9-cis-RA (93, 97, 99). The metabolites from atRA catabolism can bind to RAR and activate gene transcription (100). There are conflicting evidence on differences be-tween CYP26A1 and CYP26B1 in atRA catabolizing activity and for-mation of metabolites (86, 99).

2.3.2.2. CYP26A1 CYP26A1 is a retinoic acid responsive gene, also affected by tissue dependent and cell type specific biological agents. It is the predominat-ing atRA catabolizing enzyme in the liver (101). The role of CYP26A1 was first reported in 1997 (92) and the DNA sequence was isolated and cloned in 1996 (102). The human CYP26A1 gene is located on chromosome 10q23–q24 and encodes 497 amino acids (103). CYP26A1 in rat has 95% of the amino acid sequence iden-tical to mouse and 91 % to human (104). CYP26A1 has four distinct RARE regions (1, 2, 3 and 4) in the promoter region which is located 2.2 kbp upstream of the transcription start site. Deletion of any of these regions results in loss of function and the responsiveness to RA (105-107). The importance of CYP26A1 is illustrated by its involve-ment in embryonic development (108). A null mutation of CYP26A1 is lethal because of severe defects in e.g. brain and spinal cord (109, 110). Over expression of CYP26A1 in xenopus results in a phenotype similar to vitamin A deficiency but may also rescue embryos from the terato-genic effects of RA (111). Several animal studies have shown that CYP26A1 is downregulated in the liver of rats fed with a vitamin A deficient diet. In contrast, rats fed with a vitamin A rich diet, have an increased expression of CYP26A1 (112). It has also been shown that CYP26A1 is induced by several different substances like organochlorine pesticides (113), the tumor suppressor gene product of adenomatous polyposis coli gene (94) and gestagens (114), while it is inhibited by clofibrate (94) and downregu-lated in patients with liver ischemia (95). In addition, it has been found that endogenous RA synthesized by RALDH does not alter the expres-sion of CYP26A1 in most embryonic tissues, whereas administration of RA increases CYP26A1 (115).



CYP26A1 response to RA treatment is dependent on the cell/tissue types (96, 106-107, 117). It has also been shown that activation of RARα, RARβ, RARγ and RXRα induce CYP26A1 in hepG2 cells, hepatocytes, F9 and NB4 cells (95, 118-120), while administration of lipopolysaccharide inhibit atRA mediated induction of CYP26A1 in rat liver (121). CYP26A1 is important during development which protects the embryo from teratogenic effect of RA and it could be altered by diseased and biological compounds which might result in retinoids imbalance.

2.3.2.3. CYP26B1 The CYP26B1 gene was identified shortly after CYP26A1, cloned from human retina (97, 121). It is highly conserved from human to mouse and zebra fish. It is located on chromosome 2p13.3, it covers about 18 000 base pairs (bp) divided into six exons and a large second intron of 8.57 kb (121) (Figure 4). The gene encodes a 512 amino acid protein, but also has a long 3´untranslated region of almost 3 kb (121). The importance of CYP26B1 expression can be illustrated by the fact that CYP26B1 knockout causes severe embryopathy and death after birth (122, 123). CYP26B1 is highly expressed in the central nervous system (hindbrain, spinal cord, and cerebellum), craniofacial area, arteries, testis, kidney, lung and spleen (97, 124, 125). The high expression of CYP26B1 in the nervous system, govern a tight regulation of RA levels. RA is a potent neuronal differentiating agent in cervical spinal cord cells and important for regeneration after spinal cord injury (126). CYP26B1 is also expressed, but to a lower extent, in liver cells, immune cells, lung and ileum (125, 127). In intimal smooth muscle cells, CYP26B1 is highly expressed, upregulated by atRA and has been shown to be the dominating atRA catabolizing enzyme (128). Unlike CYP26A1, CYP26B1 lack RARE in its promoter region (95, 97). During development, CYP26B1 is important for regulation of meiosis, apoptosis, gonad and germ cell development and postnatal survival (95, 129, 130). The activation of CYP26B1 blocks RA-induced meiosis in embryonic germ cells (131), while administration of the synthetic retinoid AM80 (which is not catabolized by CYP26B1) causes en-hanced meiosis and abnormal germ cells (123). Skeletal development is also dependent on CYP26B1. RA is required for limb bud morphogenesis (132) and chondroblast differentiation but



excessive levels of RA, as seen e.g. after mutation in CYP26B1, cause severe limb and craniofacial abnormalities (133-135). CYP26B1 is induced by atRA, but is also regulated by other signalling pathways including steroidogenic factor 1 (SF1) and sex determining region Y (SRY) box 9 gene (SOX9) (136). Knockout of Sf1 and Sox9 in mouse embryonic gonads decreased CYP26B1, while the increase in CYP26B1 caused by Sf1 and Sox9 was inhibited by forkhead box L2 (FOXL2) (136). In fatty liver patients, the CYP26B1 expression is in-creased, but its activity may be interrupted by lipopolysaccharids, TGFβ1 and IL4 (94, 120, 127). In human hepatocytes, Phenobarbital has been shown to induce CYP26B1 (94). RARα, RARβ agonist, RARα, RARβ, PPARα, PPARβ, PPARγ, TNFα, IL1β, IL10, IL6, IL17, FGF, sonic hedgehog homolog (SHH), apical ectoderm ridge (AER) and PAX6 may also affect the expression of CYP26B1(94, 127, 137-140).



2.3.2.4. CYP26C1 and CYP26C2 The human CYP26C1 is located on chromosome 10q23-q24 (103, 125, 141) and consists of 522 amino acids. It has been proposed that CYP26C1 is a gene duplication of CYP26A1, which is supported by the fact that they are both located on the same chromosome (125, 142). As a member of the CYP26 family, it is regulated by RA, al-

Figure 4. Human CYP26B1 nucleotides and corresponding amino acid sequences with highlighted exons



though it lacks a RARE promoter region (80). As opposed to the other members of the CYP26 family, the main substrate for CYP26C1 is 9-cis-RA. The role of CYP26C1 during embryonic development and adult life is less clear than for CYP26A1. It has low expression in adult tissues but implications for the pathogenesis of human optic nerve aplasia has been shown (143). In zebrafish, a CYP26C2 gene (initially named CYP26D1), with 99% similarity to CYP26C1, has been detected. CYP26C2 is active in RA catabolism similarly to other CYP26 members, but the gene has not yet been found in humans (95, 144).

2.4. Effects of single nucleotide polymorphisms A number of polymorphisms in genes encoding retinoid metabolizing or binding proteins have been identified. The majority of these poly-morphisms, for example RARα, RALDH2 and STRA6, cause various congenital malformations (145, 146). For STRA6, an association be-tween two common haplotypes and type II diabetes has also been found (147). Cellular retinoic acid binding proteins (CRAB) are specific carrier proteins for RA. CRABP1 deliver RA to CYP26 for degradation (148), whereas RA bound to CRABP2 is guided to the nucleus for binding to RARs (149). A polymorphism in CRABP2 was associated with an increase in total serum cholesterol and LDL cholesterol (150). A number of studies have investigated CYP26A1 or CYP26B1 poly-morphisms, but no links to disease have been found (145, 151-154) except an association between a CYP26B1 polymorphism and oral squamous cell carcinoma (155). Two CYP26A1 polymorphisms with a 40 to 80 % de-creased atRA catabolising activity has been found (156), as well as a short variant, missing 69 amino acid at the N terminal, with little or no activity (156), but none of them have yet been linked to disease.

3. Retinoids and cardiovascular diseases

3.1. Retinoids and cardiovascular development The heart is the first organ formed in the vertebrate embryo. The heart morphogenesis starts by determination of cardiomyocyte specification and heart tube formation, looping, development of chambers and the formation of valve and septal (157, 158). RA is required for normal cardiovascular morphogenesis and develop-ment and manipulation of RA receptors as well as Vitamin A defi-



ciency may alter the process (157). In species such as zebra fish, chick and quail, RA has been found to restricting cardiac progenitor cells (158), attenuate location of ventricular progenitors (159) and affect heart tube pattern formation (160). In the rodents vitamin A deficiency results in malformation of large arteries, ventricular septum and decreased out-growth capillaries (160) and decreased cardiomyocyte proliferation (161, 162). Cardiac mal-formations are also increasingly after local retinoids treatment in preg-nant woman (163). The cardiovascular malformations have been iden-tified in 2-8 weeks of gestation in human, whereas in mice in embry-onic days 7.7-8.25(day post coitus) (164). In patients with coronary artery disease (CAD) an altered gene expres-sion of circulating CD34+ cells has been demonstrated to be related to RA inducing differentiation program. Administration of RA reduced the CD34+ cells capacity to migrate to ischemic tissues (165). Further-more, administration of 13-cis-RA to patients caused marked heart remodelling including reductions in hear dimensions (166). In conclusion, RA plays critical role in cardiovascular development. Excess or deficiency of RA is associated with detrimental effects on both developmental and growth of the cardiovascular system.

3.2. Perturbation of the Retinoid signalling pathway is associ-ated with cardiovascular diseases Levels of RA need to be appropriate for proper heart development and, in line with this, changes in retinoids signalling pathway also disturb cardiovascular development. Deletion of RALDH2 in mouse result in severe consequences include second heart field defects, improper heart looping, reduction in heart ventricular compact zone outgrowth and anomalies in coronary vessel development, decreased myocardium cells differentiation and increased proliferation (167), and eventually em-bryonic death (168, 169). Deletion of RXRα in mice causes congestive heart failure and embryonic death (170). The availability of retinol and RA is determined by intra and extracellular retinoid binding proteins (CRBPs, CRABPs and RBP). Knockout of RBP in mice result in an increased number of myocytes, accumulation of fibronectin and prema-ture differentiation of myocytes (171). STRA6 play an important role in cellular retinol homeostasis (172) and a mutation in the human STRA6 gene has been associated with lethal cardiovascular anomalies (173). Individuals with a point mutation in STRA6 display atrial and ventricular septal defects as well as aortic arch defects (173). The con-



sequences of STRA6 deficiency in zebra fish are similar to those in humans and can be reduced by knockdown of RBP4 in the zebra fish embryo (174). Apparently RA signalling is critical for heart development, suggesting that defects in any enzymes considered critical in retinoid homeostasis could result in improper development.

3.3. Retinoids and atherosclerosis Retinoids are important for normal vascular development (161) but may also be involved in cardiovascular diseases, i.e. atherosclerosis and restenosis (175). Especially the ability of retinoids to reduce inflamma-tion and proliferation could be of importance for the development of cardiovascular diseases (176). Recent studies support the important role of retinoids in cell prolifera-tion, migration, apoptosis, tissue remodelling, coagulation, fibrinolysis and inflammation, all processes implicated in vascular diseases (176, 177). In a rat model for vascular injury used for stimulating effects of balloon angioplasty in humans, administration of retinoids limited neoinitma formation (176) and vascular occlusion (178) through de-creased intima to media ratio and increased lumen diameter. In the same model decreased intimae hyperplasia was observed after admini-stration of RA and a RARα agonist (179). Based on this observation, Lee et al extended their work and found that RA accelerated re-endothelialization and improved endothelial function (180). Extended to another species, a rabbit atherosclerotic model showed that RA limited initima thickness through inhibition cells proliferation and decreased intima to media ratio, and thus enhanced lumen flow (181). In a rabbit vein bypass graft model, RA decreased intima thickness and intima/media ratio and promoted cell apoptosis (182). Furthermore, retinoids inhibited neointima formation through induction of RARβ that in turn suppressed cells proliferation and promoted apoptosis in vascular SMCs in vivo (183). Herdeg et al showed that atRA admini-stration in an angioplasty model, stenosis was markedly decreased, neointima formation attenuated and vascular diameter augmented (184). Increases in the differentiation marker SM α-actin indicated that atRA increased SMCs differentiation (184). Furthermore, administra-tion of RA inhibits the development of atherosclerosis in apolipopro-tein E deficient mice (185). In vascular endothelial cells, RA has been found to enhance pros-taglandin 2 expressions and inhibit platelet aggregation (186) which could contribute to lowering the incidence of cardiovascular disease.



Retinoids may also be beneficial in increasing NO synthesis, suppress-ing endothelin-1 production in ECs (187, 188) and attenuating Ang II expression in vascular SMCs (VSMCs) (189). These effects induce cells differentiation and apoptosis in VSMCs which give rise to anti-atherosclerotic properties (190, 191). Taken together Retinoids suppress atherosclerosis development, de-creased intima proliferation in vascular injury models and, apart from potential usefulness as therapeutics in atherosclerosis intervention, are considered candidates for modulation of gene transcription in cardio-vascular disease (Figure 5).

3.4. Retinoids and lipids Elevated plasma cholesterol is a risk factor for CVD and in itself suffi-cient for the development of atherosclerosis (18). The impact of RA on lipid metabolism seems to be very complex and is still unclear. RA has been shown to enhanced transcription of a wide array of target genes (192). RA decreased hepatic ApoA-1 expression both in vivo and in vitro in one study (193), while in other studies in hepatoma cells and

Figure 5. Summary of the influence of retinoids on various bio-logical aspects in atherosclerosis development



cynomolgus monkey cells, RA stimulated apoA-1 expression (194, 195). It has been found that prolonged RA treatment induced apoA-IV and apoA-I which prevented the development of early atherosclerosis in transgenic mice (196). RA regulates adipose tissue function, and treatment with RA may re-duce body weight, lipid metabolism and adiposity. An in vivo study showed that, treatment with RA resulted in increased expression of RXRα and PPAR, and simultaneously lowering levels of fatty acid synthase, VLDL and liver triacylglycerol. Administration of RA to mice decreased lipogenesis and increased lipid metabolism (197, 198) which was associated with a decreased in body fat and weight (197). However, in the other studies, retinoids have been found to trigger dyslipidemia and to lower HDL which is established as a risk factor for CVD (199-201). Retinoids increased hepatic synthesis of lipids (202), decrease degradation of lipoproteins (203) and increased endogenous cholesterol synthesis through effect on FDFT1 and DHCR7 (199). Furthermore, RA induced hyperlipidemia through induction of tran-scription factors FOXO1 (204), which activate MTP (microsomal transfer protein) and the ApoC3 gene that eventually stimulate VLDL and TG synthesis (205, 206). Retinoids have also been found induce hyperlipidemia through inhibition CYP7A1, an enzyme that converts cholesterol to bile acid (207). Deficiency of vitamin A in mice resulted in lipid accumulation in liver and elevation of TG levels (208), and single dose of atRA to these mice enhanced hepatic fatty acid oxidation (208). Mice fed with high cholesterol diet displayed increased tissue levels of RA and RAR, and cholesterol induced expression of Raldh2 which is the last limiting step in retinoid biosynthesis (209). The same study found that a high cholesterol diet had impact on retinoids catabolizing genes causing induction of CYP26A1 and inhibition CYP26B1, con-tributing to retinoids imbalance (209). The molecular mechanism behind the effects of retinoids on lipid me-tabolism remains unclear and complex (Figure 6).



3.5. Retinoids and inflammation Inflammatory processes are crucially important in development and progression of atherosclerosis, affecting both vascular cells and im-mune cells and their interaction. Retinoids have powerful effects on immune systems and the inflammatory milieu (210). The significance of retinoids in the vascular inflammation is illustrated by vitamin A deficiency in rats, by which the rats provoked inflammation reaction and infectious diseases (211, 212), and also by patients suffering from POEMS syndrome which is associated with augmented cytokines such as IL6, TNF and IL1, cytokines that were decreased after atRA admini-stration (213). In human lung fibroblasts, RA was observed to disable IL1β-enhancing expression of IL6 (214) and in human monocytes for enhance IL8 (215). In vitro experiments on macrophages and periph-eral blood cells have shown that RA downregulated TNFα and IL1β secretion (216, 217). In other experiments, RA treatment of human monocytes and macrophages led to increased expression of IL1β and IL8 and suppress IL1 receptor antagonist IL1RN (218-219). However, the result of experiments may be dependent on the experimental setup

Figure 6. Possible mechanism for retinoic influence on lipid ho-meostasis



because in yet another study, treatment of monocytes with IL1β re-sulted in marked increased of IL8, IL1β and IL6 which drastically in-hibited by addition of RA (220). Though the exact mechanisms may be uncertain, it is clear that reti-noids have immunomodulatory actions (221). In a study carried out by Beibei et al RA treatment of rats dramatically inhibit NFKB and IL1β (222), and thus LPS induced inflammation. An in vitro study showed that RA may inhibit LPS -TLR4 protein and NFkB activity, indicating that RA attenuates the inflammatory response to LPS through suppres-sion of the TLR4/NFkB pathway (222). Additionally, LPS induced expression of IL12 and IFNγ in the macrophages was suppressed by the addition of RA (223), and synthetic retinoids treatment in mice suppressed the LPS stimulated IL6 expression (224). Furthermore, reduced inflammation and ameliorated atherosclerosis index were observed in ApoE deficient mice treated with RXR agonist (225). RA also inhibited neutrophil migration and reduced target in-flammation (226) CRP is an inflammation marker which shows an inverse relationship with retinol (227), suggesting that retinoids could inhibit provoked inflammatory processes. In conclusion, retinoids modulate inflammatory processes via several mechanisms which may be targeted to ameliorate vascular disease.

3.6. Retinol/β-carotene and risk of cardiovascular diseases As motioned earlier, retinol (vitamin A) is the precursor of biologically active RA (176) and has a hormone like activity that regulates reactiv-ity of endothelial and smooth muscle cells, modifies cellular interaction in inflammation (176) and also participates in angiogenesis and inhibits arterial restenosis (175). Vitamin A deficiency has been associated with deregulation of cellular differentiation, increased vulnerability to infec-tion and to sometimes death (176). The effect of vitamin A on cardio-vascular diseases is debated and an inverse relationship was observed in myocardial infarction patients and low β-carotene levels in blood (228). Furthermore supplementation of carotenoids changed the inci-dence of MI (229) and increasing plasma β-carotene was associated with decreased risk of MI (230). A clinical trial studied subjects treated with retinyl palmitate and β-carotene, but was interrupted after 21 months because of 26% increase in cardiovascular deaths and 28% increase in lung cancer incidence (231, 232). These detrimental effects have been associated with in-creased serum cholesterol and TG levels in the patients receiving vita-



min A (233, 234). Similarly, in a study by Rapola et al administration of β-carotene to numbers of male smokers increased risk of coronary heart diseases (235). Nicotine has been proposed one of the compo-nents of cigarettes that may enhance vitamin A catabolism, however, the mechanisms is unclear (236) and Lee and colleagues could not ver-ify the results (237). Low plasma retinol has been associated with an increased risk of CHD in middle aged men (238). Gey et al found a strong correlation between retinol deficiency and increased IL-6 and decreased HDL and HDL/cholesterol ratio, which could contribute to an increasing risk CHD (238). In LDLr deficient mice a diet containing β-carotene was found to pre-vent hypercholesterolemia and atherosclerosis promoted by high fat intake (239). Collectively, the available observations outline a role of vitamin A in cardiovascular disease. Furthermore, the effect may depend on the duration and nature of vitamin A exposure.



4. Aims of the study The aim of the present study was to investigate the role of CYP26B1 in retinoid metabolism in vascular cells and atherosclerotic lesion.

1. Investigate the effect and importance of CYP26B1 for the regu-lation of retinoic acid metabolism in human aortic smooth muscle cells (paper 1).

2. Investigate the expression of CYP26B1 in atherosclerosis and the role of non-synonymous CYP26B1 polymorphism in atRA metabolism. (paper2).

3. Characterization, expression and function of a CYP26B1 splice variant in vascular cells and atherosclerotic lesions (paper 3).

4. Effect of statins on retinoic acid metabolism by CYP26B1 and investigate whether statins could be metabolized by CYP26B1 (paper 4)



5. Materials and methods Cell culture Human aortic smooth muscle cells (AOSMCs) passages 4-10 and Hu-man umbilical cord endothelial cells (HUVECs) (Invitrogen, Stock-holm, Sweden) passages 4-7, were used (Papers 1 ,3 and 4). Human biopsies Fifteen patients scheduled for carotid endarterectomy were included in the study and one lesion from each patient was utilized. Nine of the fifteen lesions were snap frozen and furthered used for qPCR. The six additional lesions were divided in two and incubated in DMEM/F12 medium enriched with 30 mg/mL of human albumin (Biovitrum AB, Stockholm, Sweden) for 6 h with 1 μM atRA (Papers 2 and 3). Murine biopsies Female ApoE-/- mice on C57BL/6 background (n=11, local breeding) and C57BL/6 mice (n = 6, Taconic, USA) were used, and fed standard mouse chow and euthanized at 18 weeks of age. RNA was extracted from atherosclerotic aortas from ApoE -/ - mice and atherosclero-sis free aortas from C57BL/6 mice and analyzed for CYP26B1 expres-sion. (Paper 2) Total RNA extraction and cDNA synthesis Total RNA from cells and biopsies were isolated using the E.Z.N.A Total RNA kit (Omega Bio-Tek, Doraville, GA, USA) according to the manufacturer’s protocol. RNA was reverse-transcribed to cDNA using random hexamers and Superscript II reverse transcriptase (Invitrogen, Stockholm, Sweden. (Papers1-4) QRT-PCR The mRNA levels were determined by quantitative real-time poly-merase chain reaction (qRT-PCR) by using TaqMan universal PCR master mix (Applied Biosystems, Foster City, CA, USA) and Assay on demand primers and probes for each particular gene (Papers 1-4). The PCR analysis was performed using a 7900HT Fast Real-Time PCR System according to the manufacturer’s instructions (Papers 1-4). Construction of expression vectors for CYP26B1 Constructs of full-length CYP26B1 with the major (wild-type) variant of the rs2241057 polymorphism was commercially obtained as an



Ultimate ORF Clone ID IOH37861 (Invitrogen, Stockholm, Sweden). The minor (mutated) variant of the rs2241057 polymorphism was obtained from the major variant by site-directed mutagenesis using mutagenic primers (Invitrogen, Stockholm, Sweden; Paper 2).

Transient transfect of AOSMCs, COS-1 and THP-1 AOSMCs (Paper 1), COS-1 and THP-1 cells were transfected with each particular plasmid by lipofectamin 2000 (Invitrogen, Stockholm, Swe-den). The transfection were performed by incubate the mixture of li-pofectamin 2000 with plasmids for 20 minutes and later added to the cells. (Paper 2-4). Purification of CYP26B1 variants, measurement of CYP26B1 activity and western blot CYP26B1 protein was purified from transfected COS-1 cells using monoclonal antibodies (Abnova, Taipei City, Taiwan ) and the Pierce IP Kit and stored in buffer (50mM KH2PO4, 50mM K2HPO4, 0.5 EDTA mM and 20% of glycerol, pH 7.4) at -80°C for further use (Pa-pers 2-4) Genotyping The NCBI ENTREZ SNP database (http://www.ncbi.nlm.nih.gov/snp) was used to identify SNPs and prevalence determined in a cohort of Swedish individuals consisting of 387 survivors of a first myocardial infarction and 387 controls. Genotyping was performed using a TaqMan SNP assay (Paper 2). Angiography Quantitative coronary angiography was performed QCA-CMS system (Medis medical imaging systems, Leiden, The Netherlands) (Paper 2) Quantification of [3H] atRA by HPLC Cell-associated radioactivity was separated by high performance liquid chromatography following sample hydrolysis and extraction (Papers1-4). In all experiments the [3H] atRA levels was measured. Immunohistochemistry Formalin fixed 10 μm cryosections of arterial biopsies were incubated with mouse-anti-CYP26B1 (Abnova, Taipei City, Taiwan) anti-CD68



or anti-alpha-actin (Dakopatts, Glostrup, Denmark) monoclonal anti-bodies or a relevant isotype (Paper 2). CYP26B1enzymatic assay CYP26B1 enzymatic assay were performed by using followed reagents CYP26B1, oxidoreductase (Electra-Box Diagnostica AB, Tyresö, Swe-den), [3H] atRA/statins (paper 4) and NADPH were mixed. The total volume of the reaction was adjusted to 100μl with 100 mM pH 7.4 potassium phosphate buffers (KPi buffer). The mixtures were incubated at 37°C for different times. The reactions were stopped by addition of 99.5% ethanol (Paper 2-4) Western blot Western blot was performed by using mouse anti-human CYP26B1 monoclonal antibodies (Abnova, Taipei City, Taiwan) followed by horseradish-peroxidase-conjugated secondary antibodies anti-mouse, both were dissolved in 3% non-fat dry milk (GE Healthcare, Uppsala, Sweden) in TBS. Peroxidase activity was detected using the ECL detec-tion kit (GE Healthcare) and recorded on HyperFilmTMMP (GE Healthcare) (Paper 2 and 3).



or anti-alpha-actin (Dakopatts, Glostrup, Denmark) monoclonal anti-bodies or a relevant isotype (Paper 2). CYP26B1enzymatic assay CYP26B1 enzymatic assay were performed by using followed reagents CYP26B1, oxidoreductase (Electra-Box Diagnostica AB, Tyresö, Swe-den), [3H] atRA/statins (paper 4) and NADPH were mixed. The total volume of the reaction was adjusted to 100μl with 100 mM pH 7.4 potassium phosphate buffers (KPi buffer). The mixtures were incubated at 37°C for different times. The reactions were stopped by addition of 99.5% ethanol (Paper 2-4) Western blot Western blot was performed by using mouse anti-human CYP26B1 monoclonal antibodies (Abnova, Taipei City, Taiwan) followed by horseradish-peroxidase-conjugated secondary antibodies anti-mouse, both were dissolved in 3% non-fat dry milk (GE Healthcare, Uppsala, Sweden) in TBS. Peroxidase activity was detected using the ECL detec-tion kit (GE Healthcare) and recorded on HyperFilmTMMP (GE Healthcare) (Paper 2 and 3).


6. Result and discussion

6.1. CYP26B1 plays a major role in the regulation of all-trans- retinoic acid metabolism and signalling in human aortic smooth muscle cells (AOSMCs): (Paper 1) Vascular smooth muscle cells (SMCs) play a role in the pathogenesis of atherosclerosis and restenosis. These cells have the ability for pheno-type modulation and thereby carry the capability for proliferation and migration which is hallmark for cardiovascular development (22). Retinoids have vast biological effects on vascular SMC, including pro-liferation, migration, differentiation and apoptosis (176). In this study, we show that CYP26B1 is the predominantly expressed CYP26 enzyme in human AOSMCs and that inhibition of the CYP26 family enzymes or CYP26B1 alone results in increased retinoid signalling. Using a po-tent inhibitor of CYP26, R115866, we demonstrated that inhibition of this family of enzymes decreased retinoid clearance in AOSMCs, which result in retinoidal effects, i.e. induction of gene expression and inhibi-tion of cellular proliferation. Furthermore, CYP26B1 mRNA in AOSMCs was induced by atRA. This induction suggests a negative feedback loop in which atRA in-duces its inactivation and by which these cells regulate their retinoid levels, a notion supported also by data from cell lines (118, 240). We have also found that the CYP26 inhibitor (R115866) induced expres-sion of atRA-responsive genes (CYP26B1, RARβ). Reduction of CYP26B1 enzymatic activity by using the synthetic inhibitor R115866 or by silencing of the CYP26B1 gene expression with siRNA respec-tively, decreased retinoid clearance in AOSMCs and increased atRA-mediated signalling which resulted in decreased SMCs cell prolifera-tion. The therapeutic potential of retinoids in vascular diseases has been investigated in vivo in atherosclerotic mouse and rabbit model (178, 181). Retinoids have also been shown to limit neoinitma forma-tion (181) and vascular occlusive (178) through decreased intimae and media ratio, increased lumen diameter, and decreased intimae hyper-plasia. However, the ability of atRA to accelerate its inactivation by inducing cytochrome P450 activity, as has been implicated in acquired retinoid resistance (241). This could limit the effectiveness of retinoids, therefore, our results showing that inhibition of CYP26B1 increases retinoid signalling in human vascular cells adds new insight into the role of CYP26 in vascular cells and suggest a potential novel way to overcome the development of retinoid resistance. Furthermore, our



result also demonstrated that atRA-induced CYP26 expression in AOSMCs, could give rise to an atRA-CYP26B1 feedback loop. Inhibi-tion of such loop with a CYP26 inhibitor or siRNA increased retinoid signalling, which indicate the essential role of CYP26B1 for maintain-ing cellular RA in AOSMCs.

6.2. A CYP26B1 polymorphism enhances retinoic acid ca-tabolism which may aggravate atherosclerosis: (Paper 2) Retinoids are important for normal vascular development (161) but may also be involved in cardiovascular diseases, i.e. atherosclerosis and restenosis (175) and especially their ability of retinoids to reduce in-flammation and proliferation could be of importance for the develop-ment of cardiovascular diseases (176). In this study we investigated CYP26B1 expression in human arterial biopsies and in ApoE deficient mice. In addition, we treated human atherosclerotic tissue with atRA in vitro. Our study demonstrated that CYP26B1 mRNA is over expressed in murine and human atherosclerotic lesions and that CYP26B1 is induced by atRA. Moreover, the CYP26B1 expression was localized in macrophage rich areas in the atherosclerotic lesions. This suggests that the regulation of atRA levels in the lesions by CYP26B1 may affect the ligand availability for the inflammation cells. Our findings imply that atRA levels are regulated locally in the vessel wall and thereby possibly may influence the development of atherosclerosis via the expression of CYP26B1. By searching the NCBI/SNPs database for non-synonymous SNPs in CYP26B1 and comparing with the prevalence in Swedish population, we investigated the rs2241057 SNP in the CYP26B1 gene. The genetic variation is a T to C shift in exon 4, which encodes a Leu to Ser amino acid shift in position 264 of the protein, which potentially may affect the function of the CYP26B1 protein. Therefore, we studied the catabolic activity of the two different variants. Vector of the CYP26B1 major (T) and minor (C) variants were constructed in cell free experiments followed by transfection of COS-1 (African green monkey kidney epithelial cells line) and THP-1 (Human acute mono-cytic leukemia cell line) cells. The result showed that the minore allele of the CYP26B1 variants catabolised atRA faster compared to the ma-jor CYP26B1 variants in vitro, which implies that carriers of the minor CYP26B1 variants may have reduced availability of atRA that thereby may result in an increased inflammation level in the lesion. However,



result also demonstrated that atRA-induced CYP26 expression in AOSMCs, could give rise to an atRA-CYP26B1 feedback loop. Inhibi-tion of such loop with a CYP26 inhibitor or siRNA increased retinoid signalling, which indicate the essential role of CYP26B1 for maintain-ing cellular RA in AOSMCs.

6.2. A CYP26B1 polymorphism enhances retinoic acid ca-tabolism which may aggravate atherosclerosis: (Paper 2) Retinoids are important for normal vascular development (161) but may also be involved in cardiovascular diseases, i.e. atherosclerosis and restenosis (175) and especially their ability of retinoids to reduce in-flammation and proliferation could be of importance for the develop-ment of cardiovascular diseases (176). In this study we investigated CYP26B1 expression in human arterial biopsies and in ApoE deficient mice. In addition, we treated human atherosclerotic tissue with atRA in vitro. Our study demonstrated that CYP26B1 mRNA is over expressed in murine and human atherosclerotic lesions and that CYP26B1 is induced by atRA. Moreover, the CYP26B1 expression was localized in macrophage rich areas in the atherosclerotic lesions. This suggests that the regulation of atRA levels in the lesions by CYP26B1 may affect the ligand availability for the inflammation cells. Our findings imply that atRA levels are regulated locally in the vessel wall and thereby possibly may influence the development of atherosclerosis via the expression of CYP26B1. By searching the NCBI/SNPs database for non-synonymous SNPs in CYP26B1 and comparing with the prevalence in Swedish population, we investigated the rs2241057 SNP in the CYP26B1 gene. The genetic variation is a T to C shift in exon 4, which encodes a Leu to Ser amino acid shift in position 264 of the protein, which potentially may affect the function of the CYP26B1 protein. Therefore, we studied the catabolic activity of the two different variants. Vector of the CYP26B1 major (T) and minor (C) variants were constructed in cell free experiments followed by transfection of COS-1 (African green monkey kidney epithelial cells line) and THP-1 (Human acute mono-cytic leukemia cell line) cells. The result showed that the minore allele of the CYP26B1 variants catabolised atRA faster compared to the ma-jor CYP26B1 variants in vitro, which implies that carriers of the minor CYP26B1 variants may have reduced availability of atRA that thereby may result in an increased inflammation level in the lesion. However,


the actual catabolic rates in vivo in patients will depend on several factors, including the local concentration of atRA. As described above, the highest expression of CYP26B1 was found in macrophage-rich inflammatory areas of the lesions. We showed that carriers of the minor allele, which catabolises atRA faster, indeed had significantly larger atherosclerotic lesions as determined by angiogra-phy. The difference in lesion size of the carriers of the minor and major alleles was small, and carriers of the minor allele of the CYP26B1 gene have a larger lesion size depending on decreased levels of atRA. How-ever, since we could not show the causality in the present investigation by direct measurement of the atRA levels in the atherosclerotic lesions and due to limited cohort size, these interpretations should therefore be done with caution. Collectively, these data suggest that the genotype of the CYP26B1 gene may affect the level of retinoids and thereby possibly also the develop-ment of atherosclerosis, but further studies are needed to determine the mechanistic pathway of retinoid action in atherosclerotic lesions.

6.3. Cloning and functional study of a spliced variant of CYP26B1 expressed in vascular cells: (Paper 3) In the previous experiments (Papers 1 and 2), we demonstrated that CYP26B1 is highly expressed and induced by atRA in vascular cells. During the cloning and screening of CYP26B1 in paper 2, we noticed the existence of a CYP26B1 splice variant missing exon 2. Using prim-ers targeting exons 1 and 3, the predicted size of the PCR product us-ing cDNA as a template was 438 bp in the presence of exon 2, com-pared to 213 bp in the absence of exon 2. In addition, we found that both the full length and the spliced CYP26B1 variants were expressed in AOSMCs, HUVECs and in normal kidney arteries. We also investi-gated if the spliced variant of CYP26B1 was expressed in atheroscle-rotic lesions by quantitative RT-PCR. Interestingly, the transcript levels of the full length and the spliced variant of CYP26B1 was 2.5 times and 4.5 higher, respectively, in atherosclerotic lesion compared to normal arteries. Next, we investigated if atRA could induce the mRNA and protein expression of the spliced variant in atherosclerotic lesions; we therefore treated atherosclerotic lesions in vitro with 1μM atRA and found that atRA induced the spliced and full length of CYP26B1 by 20 and 7 fold respectively. In addition, atRA also induced CYP26B1 protein expres-



sion in COS-1 cells. Our results therefore show that both CYP26B1 variants were induced by atRA, but that the mRNA expression of the spliced variant was higher compared to the full-length variant. Next, we investigated the activity of CYP26B1 splice variants by trans-fection of either the CYP26B1 full length or the spliced variant only or in combination with each other, or with the corresponding empty vec-tor pcDNA3.2/V5-DEST in COS-1 cells. The results showed that the full length CYP26B1 reduced the cellular levels of atRA compared with cells transfected with the empty vector. Interestingly, cells transfected with the spliced variant of CYP26B1 showed an increase in cell-associated [3H]atRA compared to cells transfected with full length CYP26B1 and empty vector. This indicates that the spliced variant interferes with endogenous enzymes that catabolise atRA in COS-1 cells, reducing the rate of atRA catabolism. To further explore whether this is a general phenomenon, THP-1 cells that catabolise atRA at a lower rate than COS-1 cells were transfected with the different plasmid constructs. In contrast to COS-1 cells, an increased degradation of atRA by the CYP26B1 splice variant, compared with empty vector alone, was seen. The above data indicated that CYP26B1 splice variant could increase or decrease the catabolism of atRA depending on the levels of other atRA catabolizing enzymes in the cells. The function of CYP26B1 splice variant was further investigated by affinity purified CYP26B1 and incubated with either 25 or 100 nM of [3H atRA] for different time points. The assay shows that the spliced variant of CYP26B1 retained the capability of atRA degradation but with less activity by around one third of full length CYP26B1. Our result showed that the spliced variant of the CYP26B1 gene, which is atRA-inducible in vascular cells, is capable to degrade atRA.

6.4. Simvastatin and rosuvastatin inhibit CYP26B1-mediated retinoid catabolism: (Paper 4) Statins belong to a group of antihyperlipidemia drugs widely used for lowering the levels of cholesterol and LDL in the blood (242). Statins have vast biological effects that overlap with the effects of atRA includ-ing anticancer (243) and inhibit cells proliferation (243) and anti-inflammatory capability (244, 245) but the detail mechanisms behind these effects have not been resolved. In this study we investigated if



sion in COS-1 cells. Our results therefore show that both CYP26B1 variants were induced by atRA, but that the mRNA expression of the spliced variant was higher compared to the full-length variant. Next, we investigated the activity of CYP26B1 splice variants by trans-fection of either the CYP26B1 full length or the spliced variant only or in combination with each other, or with the corresponding empty vec-tor pcDNA3.2/V5-DEST in COS-1 cells. The results showed that the full length CYP26B1 reduced the cellular levels of atRA compared with cells transfected with the empty vector. Interestingly, cells transfected with the spliced variant of CYP26B1 showed an increase in cell-associated [3H]atRA compared to cells transfected with full length CYP26B1 and empty vector. This indicates that the spliced variant interferes with endogenous enzymes that catabolise atRA in COS-1 cells, reducing the rate of atRA catabolism. To further explore whether this is a general phenomenon, THP-1 cells that catabolise atRA at a lower rate than COS-1 cells were transfected with the different plasmid constructs. In contrast to COS-1 cells, an increased degradation of atRA by the CYP26B1 splice variant, compared with empty vector alone, was seen. The above data indicated that CYP26B1 splice variant could increase or decrease the catabolism of atRA depending on the levels of other atRA catabolizing enzymes in the cells. The function of CYP26B1 splice variant was further investigated by affinity purified CYP26B1 and incubated with either 25 or 100 nM of [3H atRA] for different time points. The assay shows that the spliced variant of CYP26B1 retained the capability of atRA degradation but with less activity by around one third of full length CYP26B1. Our result showed that the spliced variant of the CYP26B1 gene, which is atRA-inducible in vascular cells, is capable to degrade atRA.

6.4. Simvastatin and rosuvastatin inhibit CYP26B1-mediated retinoid catabolism: (Paper 4) Statins belong to a group of antihyperlipidemia drugs widely used for lowering the levels of cholesterol and LDL in the blood (242). Statins have vast biological effects that overlap with the effects of atRA includ-ing anticancer (243) and inhibit cells proliferation (243) and anti-inflammatory capability (244, 245) but the detail mechanisms behind these effects have not been resolved. In this study we investigated if


statins could interact and inhibit CYP26B1-mediated atRA catabolism. Our study show that both simvastatin and rosuvastatin, in clinically relevant concentrations, increase atRA levels in human aortic smooth muscle cells. We also investigated the effect of simvastatin and rosuvas-tatin on the expression of retinoic acid responsive genes like CYP26B1, RAR , which were previously shown to response to retinoic acid in vascular aortic smooth muscle cells (Paper 1). We found that both concentrations of 1 and 5 ng/ml of Simvastatin and rosuvastatin sig-nificantly induced CYP26B1 and RAR in AOSMCs. The ability of the statins to increase intracellular atRA levels in the vascular wall, make way for possible explanations to some of the beneficial effects of statins not related to lipid lowering. An increase in atRA levels in vascular cells could potentially reduce atherosclerotic inflammatory activity, and thus at least partially explain the anti inflammatory effects of statins and for example why statin treatment is more beneficial in individuals with high levels of circulating C-reactive protein (CRP). Therefore the induction of CYP26B1 and the inhibition of retinoic acid degradation by statins could result in increased RA levels which are a potential mechanism for the anti-inflammatory effects of statin-treatment. We therefore speculate that the physiological concentrations of statin administered to AOSMC in this study mimic the situation in the vascular wall in vivo, where statins exert their anti-inflammatory effects by the inhibition of CYP26B1-mediated atRA catabolism.



Conclusion 1. CYP26B1 is an important regulator of retinoid levels in human AOSMCs and our results suggest that CYP26 inhibitors may provide an alternative way to exogenous retinoid administration. 2. CYP26B1 polymorphism accelerates retinoic acid clearance and is induced by atRA. CYP26B1 mRNA was increased in murine athero-sclerosis and was co-localized to macrophage-rich areas of human atherosclerotic lesions. This suggests that the genotype may have an impact on CYP26B1-regulated levels of retinoids and possibly athero-sclerosis development. 3. A CYP26B1 spliced variant is missing exon 2 is atRA-inducible in vascular cells and vessels wall and shows a slower and reduced degra-dation of atRA as compared to the full length CYP26B1. 4. Simvastatin and rosuvastatin increased intracellular atRA-levels by reducing CYP26B1-mediated atRA catabolism and simultaneously inducing transcript levels of CYP26B1 and RAR . This mechanism may contribute to the anti-inflammatory effects of these statins. Collectively, the findings in the present thesis contribute to increased knowledge about the role of CYP26B1 in the regulation of retinoic acid metabolism in vascular cells and atherosclerotic lesion.



Conclusion 1. CYP26B1 is an important regulator of retinoid levels in human AOSMCs and our results suggest that CYP26 inhibitors may provide an alternative way to exogenous retinoid administration. 2. CYP26B1 polymorphism accelerates retinoic acid clearance and is induced by atRA. CYP26B1 mRNA was increased in murine athero-sclerosis and was co-localized to macrophage-rich areas of human atherosclerotic lesions. This suggests that the genotype may have an impact on CYP26B1-regulated levels of retinoids and possibly athero-sclerosis development. 3. A CYP26B1 spliced variant is missing exon 2 is atRA-inducible in vascular cells and vessels wall and shows a slower and reduced degra-dation of atRA as compared to the full length CYP26B1. 4. Simvastatin and rosuvastatin increased intracellular atRA-levels by reducing CYP26B1-mediated atRA catabolism and simultaneously inducing transcript levels of CYP26B1 and RAR . This mechanism may contribute to the anti-inflammatory effects of these statins. Collectively, the findings in the present thesis contribute to increased knowledge about the role of CYP26B1 in the regulation of retinoic acid metabolism in vascular cells and atherosclerotic lesion.


Future perspectives This thesis demonstrated that CYP26B1 is a major retinoic acid cata-bolises enzyme modulator express in vascular cells and atherosclerotic lesion. Therefore, genetic alterations and the expression levels in such enzymes results in imbalance in retinoic acid levels. Retinoids are con-sidered to be a potential candidate for the modulation of gene tran-scription in cardiovascular diseases and the present thesis opens up for additional investigations:

- In the present thesis, we investigated a polymorphism in CYP26B1 and we found an association of the minor allele of the rs2241057 polymorphism in the CYP26B1 gene with in-creased lesion size, but a larger cohort will be needed to vali-date these results.

- Additional SNPs were identified in the CYP26B1 gene, which could be of interest to further investigations.

- Synthetic retinoids are potential alternative candidate drugs, which may alleviate the side effect of retinoids. Blocking CYP26B1 may increase the endogenous RA levels. Therefore, the use of RAMBA alone or in combination with RA may pro-vide a useful tool in the treatment of atherosclerosis. In addi-tion, it could also be of interest to further investigate the speci-ficity of new RAMBAs on CYP26B1 in vivo for the inhibition of atherosclerosis development in a mouse model.

- Since we found that statins inhibit retinoic acid catabolism in vitro, it would be of great value to investigate the effects of statins in vivo on atRA levels or to investigate if statins could be catabolized by CYP26B1.



Acknowledgements This study was performed at the School of Health and Medical Sci-ences (Division of Clinical Medicine, Cardiovascular research unit), the department of Biomedicine, Örebro University under supervision of Professor Allan Sirsjö. First of all, I would like to express my sincere gratitude to my supervi-sor Professor Allan Sirsjö for giving me opportunity to join his research group as a PhD student. Thanks for being patient, giving me unlimited time for reading, your suggestions, corrections and support in writing this thesis. My gratitude extends to my co-supervisors Karin Fransén for her time and energy spent on reading and correcting this thesis. Thanks a lot for helpful suggestions and improvements. Unforgettable and enormous thank to my co-authors Eva Sundman and her husband Peder Olofsson for helping me a lot to bring my thesis into its final version. My acknowledgments also go to Örebro University and administrative staff and for providing good research facilities, place for the work and for administrative support. I would like to express my acknowledgment to all Libyan students in Sweden, MENA coordinator and Libyan embassy members in Sweden. My grateful thanks go to all my former and present colleagues and friends that I met at the Örebro University and the Örebro University Hospital. Special thanks to my parents, brothers and sisters for their non-selfish help and support. Finally, I would like to express my great sincere thanks to my family for solid support and enormous patience during my study time.



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221. Vertuani S, Dubrovska E, Levitsky V, Jager M, Kiessling R, Levitskaya J. Retinoic acid elicits cytostatic, cytotoxic and im-munomodulatory effects on uveal melanoma cells. Cancer Im-munol Immunother. 2007;56:193-204.

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Publications in the series

Örebro Studies in Medicine

1. Bergemalm, Per-Olof (2004). Audiologic and cognitive long-term sequelae from closed head injury.

2. Jansson, Kjell (2004). Intraperitoneal Microdialysis. Technique and Results.

3. Windahl, Torgny (2004). Clinical aspects of laser treatment of lichen sclerosus and squamous cell carcinoma of the penis.

4. Carlsson, Per-Inge (2004). Hearing impairment and deafness. Genetic and environmental factors – interactions – consequences. A clinical audiological approach.

5. Wågsäter, Dick (2005). CXCL16 and CD137 in Atherosclerosis.

6. Jatta, Ken (2006). Inflammation in Atherosclerosis.

7. Dreifaldt, Ann Charlotte (2006). Epidemiological Aspects on Malignant Diseases in Childhood.

8. Jurstrand, Margaretha (2006). Detection of Chlamydia trachomatis and Mycoplasma genitalium by genetic and serological methods.

9. Norén, Torbjörn (2006). Clostridium difficile, epidemiology and antibiotic resistance.

10. Anderzén Carlsson, Agneta (2007). Children with Cancer – Focusing on their Fear and on how their Fear is Handled.

11. Ocaya, Pauline (2007). Retinoid metabolism and signalling in vascular smooth muscle cells.

12. Nilsson, Andreas (2008). Physical activity assessed by accelerometry in children.

13. Eliasson, Henrik (2008). Tularemia – epidemiological, clinical and diagnostic aspects.

14. Walldén, Jakob (2008). The influence of opioids on gastric function: experimental and clinical studies.

15. Andrén, Ove (2008). Natural history and prognostic factors in localized prostate cancer.

16. Svantesson, Mia (2008). Postpone death? Nurse-physician perspectives and ethics rounds.

17. Björk, Tabita (2008). Measuring Eating Disorder Outcome – Definitions, dropouts and patients’ perspectives.

18. Ahlsson, Anders (2008). Atrial Fibrillation in Cardiac Surgery.

19. Parihar, Vishal Singh (2008). Human Listeriosis – Sources and Routes.

20. Berglund, Carolina (2008). Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus. Epidemiological aspects of MRSA and the dissemination in the community and in hospitals.

21. Nilsagård, Ylva (2008). Walking ability, balance and accidental falls in persons with Multiple Sclerosis.

22. Johansson, Ann-Christin (2008). Psychosocial factors in patients with lumbar disc herniation: Enhancing postoperative outcome by the identification of predictive factors and optimised physiotherapy.

23. Larsson, Matz (2008). Secondary exposure to inhaled tobacco products.

24. Hahn-Strömberg, Victoria (2008). Cell adhesion proteins in different invasive patterns of colon carcinoma: A morphometric and molecular genetic study.

25. Böttiger, Anna (2008). Genetic Variation in the Folate Receptor-α and Methylenetetrahydrofolate Reductase Genes as Determinants of Plasma Homocysteine Concentrations.

26. Andersson, Gunnel (2009). Urinary incontinence. Prevalence, treatment seeking behaviour, experiences and perceptions among persons with and without urinary leakage.

27. Elfström, Peter (2009). Associated disorders in celiac disease.

28. Skårberg, Kurt (2009). Anabolic-androgenic steroid users in treatment: Social background, drug use patterns and criminality.

29. de Man Lapidoth, Joakim (2009). Binge Eating and Obesity Treatment – Prevalence, Measurement and Long-term Outcome.

30. Vumma, Ravi (2009). Functional Characterization of Tyrosine and Tryptophan Transport in Fibroblasts from Healthy Controls, Patients with Schizophrenia and Bipolar Disorder.

31. Jacobsson, Susanne (2009). Characterisation of Neisseria meningitidis from a virulence and immunogenic perspective that includes variations in novel vaccine antigens.

32. Allvin, Renée (2009). Postoperative Recovery. Development of a Multi-Dimensional Questionnaire for Assessment of Recovery.

33. Hagnelius, Nils-Olof (2009). Vascular Mechanisms in Dementia with Special Reference to Folate and Fibrinolysis.

34. Duberg, Ann-Sofi (2009). Hepatitis C virus infection. A nationwide study of assiciated morbidity and mortality.

35. Söderqvist, Fredrik (2009). Health symptoms and potential effects on the blood-brain and blood-cerebrospinal fluid barriers associated with use of wireless telephones.

36. Neander, Kerstin (2009). Indispensable Interaction. Parents’ perspectives on parent–child interaction interventions and beneficial meetings.

37. Ekwall, Eva (2009). Women’s Experiences of Gynecological Cancer and Interaction with the Health Care System through Different Phases of the Disease.

38. Thulin Hedberg, Sara (2009). Antibiotic susceptibility and resistance in Neisseria meningitidis – phenotypic and genotypic characteristics.

39. Hammer, Ann (2010). Forced use on arm function after stroke. Clinically rated and self-reported outcome and measurement during the sub-acute phase.

40. Westman, Anders (2010). Musculoskeletal pain in primary health care: A biopsychosocial perspective for assessment and treatment.

41. Gustafsson, Sanna Aila (2010). The importance of being thin – Perceived expectations from self and others and the effect on self-evaluation in girls with disordered eating.

42. Johansson, Bengt (2010). Long-term outcome research on PDR brachytherapy with focus on breast, base of tongue and lip cancer.

43. Tina, Elisabet (2010). Biological markers in breast cancer and acute leukaemia with focus on drug resistance.

44. Overmeer, Thomas (2010). Implementing psychosocial factors in physical therapy treatment for patients with musculoskeletal pain in primary care.

45. Prenkert, Malin (2010). On mechanisms of drug resistance in acute myloid leukemia.

46. de Leon, Alex (2010). Effects of Anesthesia on Esophageal Sphincters in Obese Patients.

47. Josefson, Anna (2010). Nickel allergy and hand eczema – epidemiological aspects.

48. Almon, Ricardo (2010). Lactase Persistence and Lactase Non-Persistence. Prevalence, influence on body fat, body height, and relation to the metabolic syndrome.

49. Ohlin, Andreas (2010). Aspects on early diagnosis of neonatal sepsis.

50. Oliynyk, Igor (2010). Advances in Pharmacological Treatment of Cystic Fibrosis.

51. Franzén, Karin (2011). Interventions for Urinary Incontinence in Women. Survey and effects on population and patient level.

52. Loiske, Karin (2011). Echocardiographic measurements of the heart. With focus on the right ventricle.

53. Hellmark, Bengt (2011). Genotypic and phenotypic characterisation of Staphylococcus epidermidis isolated from prosthetic joint infections.

54. Eriksson Crommert, Martin (2011). On the role of transversus abdominis in trunk motor control.

55. Ahlstrand, Rebecca (2011). Effects of Anesthesia on Esophageal Sphincters.

56. Holländare, Fredrik (2011). Managing Depression via the Internet – self-report measures, treatment & relapse prevention.

57. Johansson, Jessica (2011). Amino Acid Transport and Receptor Binding Properties in Neuropsychiatric Disorders using the Fibroblast Cell Model.

58. Vidlund, Mårten (2011). Glutamate for Metabolic Intervention in Coronary Surgery with special reference to the GLUTAMICS-trial.

59. Zakrisson, Ann-Britt (2011). Management of patients with Chronic Obstructive Pulmonary Disease in Primary Health Care. A study of a nurse-led multidisciplinary programme of pulmonary rehabilitation.

60. Lindgren, Rickard (2011). Aspects of anastomotic leakage, anorectal function and defunctioning stoma in Low Anterior Resection of the rectum for cancer.

61. Karlsson, Christina (2011). Biomarkers in non-small cell lung carcinoma. Methodological aspects and influence of gender, histology and smoking habits on estrogen receptor and epidermal growth factor family receptor signalling.

62. Varelogianni, Georgia (2011). Chloride Transport and Inflammation in Cystic Fibrosis Airways.

63. Makdoumi, Karim (2011). Ultraviolet Light A (UVA) Photoactivation of Riboflavin as a Potential Therapy for Infectious Keratitis.

64. Nordin Olsson, Inger (2012). Rational drug treatment in the elderly: ”To treat or not to treat”.

65. Fadl, Helena (2012). Gestational diabetes mellitus in Sweden: screening, outcomes, and consequences.

66. Essving, Per (2012). Local Infiltration Analgesia in Knee Arthroplasty.

67. Thuresson, Marie (2012). The Initial Phase of an Acute Coronary Syndrome. Symptoms, patients’ response to symptoms and opportunity to reduce time to seek care and to increase ambulance use.

68. Mårild, Karl (2012). Risk Factors and Associated Disorders of Celiac Disease.

69. Fant, Federica (2012). Optimization of the Perioperative Anaesthetic Care for Prostate Cancer Surgery. Clinical studies on Pain, Stress Response and Immunomodulation.

70. Almroth, Henrik (2012). Atrial Fibrillation: Inflammatory and pharmacological studies.

71. Elmabsout, Ali Ateia (2012). CYP26B1 as regulator of retinoic acid in vascular cells and atherosclerotic lesions.

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