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Angiogenesis Associated Mechanisms in Airway Inflammation and Remodelling
Cengiz KIRMAZ MD.
Associated Professor of Internal Medicine
Celal Bayar University
Department of Immunology and Allergy
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Angiogenesis (angio'gen'esis)
• The growth of new blood vessels…
• It is an important natural process occurring in the body,
• in health and • in disease…
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Control of Angiogenesis
• Angiogenesis occurs in the healthy body for healing wounds and for restoring blood flow to tissues after injury…
• The healthy body controls angiogenesis through a series of “ " and “ " switches:
• The main "ON" switches are known as angiogenesis-stimulating growth factors
• The main "OFF” switches are known as angiogenesis inhibitors…
ONON OFFOFF
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List of Known Angiogenic Growth Factors ON
OFF– Angiogenin – Angiopoietin-1 – Del-1 – Fibroblast growth factors: acidic (aFGF) and basic (bFGF) – Follistatin – Granulocyte colony-stimulating factor (G-CSF) – Hepatocyte growth factor (HGF) /scatter factor (SF) – Interleukin-8 (IL-8) – Leptin – Midkine – Placental growth factor – Platelet-derived endothelial cell growth factor (PD-ECGF) – Platelet-derived growth factor-BB (PDGF-BB) – Pleiotrophin (PTN) – Progranulin – Proliferin – Transforming growth factor-alpha (TGF-alpha) – Transforming growth factor-beta (TGF-beta) – Tumor necrosis factor-alpha (TNF-alpha) – Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF)
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List of Known Angiogenesis Inhibitors
– Angioarrestin – Angiostatin (plasminogen
fragment) – Antiangiogenic antithrombin III – Cartilage-derived inhibitor (CDI) – CD59 complement fragment – Endostatin (collagen XVIII
fragment) – Fibronectin fragment – Gro-beta – Heparinases – Heparin hexasaccharide fragment – Human chorionic gonadotropin
(hCG) – Interferon alpha/beta/gamma – Interferon inducible protein (IP-10)
– Interleukin-12 Interleukin-12 – Kringle 5 (plasminogen fragment) Kringle 5 (plasminogen fragment) – Metalloproteinase inhibitors (TIMPs) Metalloproteinase inhibitors (TIMPs) – 2-Methoxyestradiol 2-Methoxyestradiol – Placental ribonuclease inhibitor Placental ribonuclease inhibitor – Plasminogen activator inhibitor Plasminogen activator inhibitor – Platelet factor-4 (PF4) Platelet factor-4 (PF4) – Prolactin 16kD fragment Prolactin 16kD fragment – Proliferin-related protein (PRP) Proliferin-related protein (PRP) – Retinoids Retinoids – Tetrahydrocortisol-S Tetrahydrocortisol-S – Thrombospondin-1 (TSP-1) Thrombospondin-1 (TSP-1) – Transforming growth factor-beta Transforming growth factor-beta
(TGF-(TGF-ßß) ) – Vasculostatin Vasculostatin – Vasostatin (calreticulin fragment) Vasostatin (calreticulin fragment)
ON
OFF
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The Angiogenesis Process: How Do New Blood Vessels Grow?
Diseased or injured tissues produce and release Diseased or injured tissues produce and release angiogenic growth factors (proteins) that diffuse into the nearby tissues angiogenic growth factors (proteins) that diffuse into the nearby tissues The angiogenic growth factors bind to The angiogenic growth factors bind to specific receptors located on the endothelialspecific receptors located on the endothelialcells (EC) of nearby preexisting blood vessels cells (EC) of nearby preexisting blood vessels Once growth factors bind to their receptors, Once growth factors bind to their receptors, the endothelial cells become activated. the endothelial cells become activated. Signals are sent from the cell's surface to Signals are sent from the cell's surface to the nucleus. The endothelial cell's machinery the nucleus. The endothelial cell's machinery begins to produce new molecules including begins to produce new molecules including enzymes enzymes
Enzymes dissolve tiny holes in the sheath-like Enzymes dissolve tiny holes in the sheath-like covering (basement membrane) surrounding covering (basement membrane) surrounding all existing blood vessels all existing blood vessels The endothelial cells begin to divide (proliferate), The endothelial cells begin to divide (proliferate), and they migrate out through the dissolved holesand they migrate out through the dissolved holesof the existing vessel towards the diseased tissue of the existing vessel towards the diseased tissue
Specialized molecules called adhesion molecules, Specialized molecules called adhesion molecules, or integrins (avb3, avb5) serve as grappling hooksor integrins (avb3, avb5) serve as grappling hooksto help pull the sprouting new blood vessel sproutto help pull the sprouting new blood vessel sproutforward forward
Additional enzymes (matrix metalloproteinases, or Additional enzymes (matrix metalloproteinases, or MMP) are produced to dissolve the tissue in front ofMMP) are produced to dissolve the tissue in front ofthe sprouting vessel tip in order to accommodate it. the sprouting vessel tip in order to accommodate it. As the vessel extends, the tissue is remolded As the vessel extends, the tissue is remolded around the vesselaround the vessel
Sprouting endothelial cells roll up to form a blood Sprouting endothelial cells roll up to form a blood vessel tube vessel tube Individual blood vessel tubes connect to form blood Individual blood vessel tubes connect to form blood vessel loops that can circulate bloodvessel loops that can circulate bloodFinally, newly formed blood vessel tubes are Finally, newly formed blood vessel tubes are stabilized by specialized muscle cells (smooth stabilized by specialized muscle cells (smooth muscle cells, pericytes) that provide structural muscle cells, pericytes) that provide structural support. Blood flow then begins. support. Blood flow then begins.
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Angiogenesis and Remodelling
• Angiogenesis and microvascular remodelling are elements of the tissue remodelling in and tumors.
• Both types of change in the microvasculature result from endothelial cell proliferation and often occur together, but they represent different phenomena and responses to different stimuli.
• Angiogenesis is the growth of new blood vessels
• Microvascular remodelling involves structural alterations—usually enlargement—of arterioles, capillaries or venules, without the formation of new vessels.
chronic inflammatory disorderschronic inflammatory disorders
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Background of angiogenesis and microvascular remodelling in airway disease
• There are clues that changes in the airway microvascul-ature have long been recognized as a feature of asthma…
• it was recognized many years ago that the airway mucosa in fatal asthma is edematous and contains dilated, congested blood vessels…
Unger L. South Med J 1945;38:513–523.
Walzer I, Frost TT. J Allergy 1952;23:204–214.
Dunnill MS. J Clin Pathol 1960;13:27–33.
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• Early studies also showed that the airway wall of subjects with asthma is abnormally thick…
• The increased wall thickness would necessitate an expansion of the microvasculature to accommodate the extra tissue mass, and the abundant, enlarged, and congested mucosal blood vessels contribute to the wall thickness…
Macdonald IG. Ann Intern Med 1932;6:253–277.James AL, et al. Am Rev Respir Dis 1989;139:242–246.Carroll N, et al. Am Rev Respir Dis 1993;147: 405–410.
Background of angiogenesis and microvascular remodelling in airway disease
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WHAT IS
ASTHMA ???
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Definition of Asthma*Definition of Asthma*
A chronic inflammatory disorder of the airways
Many cells and cellular elements play a role
Chronic inflammation is associated with airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing
Widespread, variable, and often reversible airflow limitation
A chronic inflammatory disorder of the airways
Many cells and cellular elements play a role
Chronic inflammation is associated with airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing
Widespread, variable, and often reversible airflow limitation
** Captured directly from GINA 2006 slide set…
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Captured directly from GINA 2006 slide set…
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Inflammation in asthmaInflammation in asthma
Chronic inflammation
Structural changes
Superimposed acute
inflammation
Time
Airway
remodeling
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Remodeling
A critical aspect of wound repair in all organs representing a dynamic process that associates
• matrix production and degradation in reaction to an inflammatory insult,
• leading to a normal reconstruction process or a pathologic process.
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Remodelling in Airway
• Several years ago, Bousquet et al. proposed as definition of remodelling 'model again or differently, reconstruct‘…
Bousquet J, Chanez P, Lacoste JY, White R, Vic P, Godard P .
Asthma: a disease remodelling the airways?
Allergy 1992;47:3–11.
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Processes in Airway Remodelling
Angiogenesis
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The histologic hallmarks of remodeling are as follows
(1) infiltration by macrophages and lymphocytes;
(2) proliferation of fibroblasts that may take the form of myofibroblasts;
(3) angiogenesis;
(4) increased connective tissue (fibrosis);
(5) tissue destruction.
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Prototype Angiogenics
• VEGF
• Anjiyopoetin-1 (Ang-1)
• CXC Kemokinler…
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• VEGF is an interesting inducer of angio-genesis and lymphangiogenesis, becauseit is a highly specific mitogen for endothel-ial cells.
• Signal transduction involves binding to tyrosine kinase receptors and results in endothelial cell proliferation, migration, and new vessel formation.
• The VEGF family currently comprises seven members:
– VEGF-A, – VEGF-B, – VEGF-C, – VEGF-D, – VEGF-E,– VEGF-F, – PlGF.
• All members have a common VEGF homology domain.
VEGF
found in lung
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VEGF Receptors
• Three VEGF tyrosine kinase receptors have been identified:
– The fms-like tyrosine kinase Flt-1 (VEGFR-1/Flt-1),
– The kinase domain region, also referred to as fetal liver kinase (VEGFR-2/KDR/Flk-1),
– Flt-4 (VEGFR-3).
• Each receptor has • seven immunoglobulin like domains in the extracellular
domain, • a single transmembrane region, • a consensus tyrosine kinase sequence interrupted by a
kinase insert domain
Most important receptorin lung
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VEGF and Receptors in Airway
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Signal Transduction
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Regulation of VEGF Gene Regulation of VEGF Gene ExpressionExpression
• Hipoxia• Cytokines and growth factors
• TNF-, • TGF-, • EGF, PDGF-BB, • IGF-1, • COX-2
• PGE2
• Hormons• Estrogen• Testosterone
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VEGF and Angiopoietins in Angiogenesis
• The endothelial cell-specific growth factors, VEGF and angiopoietin 1 (Ang1), have potent and clinically relevant actions on the microvasculature.
Yancopoulos GD. Nature 2000;407:242–248.
• Both of these growth factors play essential but separate roles in vascular development in the embryo.
Ferrara N, et al. Nature 1996;380:439–442.
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VEGF and Angiopoietins in Angiogenesis
• VEGF is key to the formation of the initial vascular plexus in early development.
• VEGF expression increases in the airways of subjects with asthma and correlates with mucosal vascularity…
Hoshino M, et al. J Allergy Clin Immunol 2001;107:295–301.Hoshino M, et al. J Allergy Clin Immunol 2001;107:1034-1038.
• Ang1 appears to be essential for maturation of the vasculature from primitive tubes into a hierarchical network of vessels composed of endothelial cells and pericytes or smooth muscle cells.
Suri C, et al. Cell 1996;87:1171–1180.
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VEGF and Angiopoietins in Angiogenesis
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VEGF and Angiopoietins in Angiogenesis
• Ang2 antagonizes the effects of Ang1 on Tie2 receptors and in some contexts acts as a natural inhibitor of Ang1.
• Whereas Ang1 is widely expressed in normal adult tissues, Ang2 is expressed mainly at sites of vascular remodeling such as the ovary, placenta, uterus, and tumors.
• Ang2 expression in the presence of VEGF is accompanied by angiogenesis, but Ang2 expression in the absence of VEGF is associated with vascular regression.
Maisonpierre PC, et al. Science 1997;277:55–60.Holash J,et al. Science 1999;284:1994–1998.Zagzag D, et al. Lab Invest 2000;80:837–849.
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CXC Chemokines and Angiogenesis
• Chemo-attractive cyto-kines• On a structural level, they have four highly
conserved cysteine amino acid residues.• The NH2 terminus of the majority of the CXC
chemokines contain a three-amino-acid motif (Glu-Leu-Arg: the ELR motif).
• The family members that contain the ELR motif (ELR+) are potent promoters of angiogenesis,
• ELR- members are potent inhibitors of angiogenesis…
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Angiogenic and Angiostatic CXC Chemokines
Angiogenic CXC chemokines containing the ELR motif (ELR+)Interleukin-8 (IL-8)Epithelial neutrophil activating protein-78 (ENA-78)Growth-related gene alpha (GRO-)Growth-related gene beta (GRO-)Growth-related gene gamma (GRO-)Granulocyte chemotactic protein-2 (GCP-2)Platelet basic protein (PBP)Connective tissue activating protein-III (CTAP-III)Beta-thromboglobulin ( -TG)Neutrophil activating protein-2 (NAP-2)
Angiostatic CXC chemokines that lack the ELR motif (ELR-)Platelet factor-4 (PF4)Interferon- -inducible protein (IP-10)Monokine induced by interferon- (MIG)
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CXC Chemokine Receptors
Interleukin-8 (IL-8)Granulocyte chemotactic protein-2 (GCP-2)
Epithelial neutrophil activating protein-78 (ENA-78)Growth-related gene alpha (GRO-)Growth-related gene beta (GRO-)Growth-related gene gamma (GRO-)Platelet basic protein (PBP)Connective tissue activating protein-III (CTAP-III)Beta-thromboglobulin ( -TG)Neutrophil activating protein-2 (NAP-2)
CXCR1
CXCR2
End
othe
lial C
ell
Ang
ioge
nic
activ
atio
n
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Angiogenesis and Treatment (Inhaled Steroide)
• Chetta A, et al. Vascular endothelial growth factor up-regulation and bronchial wall remodelling in asthma. Clin Exp Allergy
2005;35:1437-42. • They showed that
» The counts of VEGF(+) cells, mast cells and EG2(+) cells were higher in asthmatics than in controls.
» The number of vessels, the vascular area in the lamina propria, and the basement membrane thickness were significantly higher in asthmatics than in healthy volunteers.
» In asthmatic patients, the number of VEGF(+) cells was significantly related to the number of vessels, to mast cells and to basement membrane thickness.
» High doses of inhaled fluticasone propionate significantly reduced VEGF(+) cells, vessel number, vascular area and basement membrane thickness in a subgroup of asthmatic patients
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Angiogenesis and Treatment(Inhaled 2-agonist)
• Bernadette E, et al. Effect of a long-acting 2-agonist over three months on airway wall vascular remodeling in asthma. Am J Respir Crit Care Med 2001;164:117-121.
• They showed that» airways of subjects with asthma had a
significant increase in the number of vessels/mm2 of lamina propria compared with airways of normal subjects
» a decrease in the density of vessels of lamina propria after treatment only in the salmeterol group compared with baseline…
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Angiogenesis and Treatment (Leukotriene antagonists)
• Lee KS, et al. Cysteinyl leukotriene receptor antagonist regulates vascular permeability by reducing vascular endothelial growth factor expression. J Allergy Clin Immunol 2004, 114:1093-1099.
• They showed that» asthmatic lungs have increased numbers of inflammatory cells of
the airways, airway hyperresponsiveness, increased vascular permeability, and increased levels of VEGF.
» Administration of cysLT receptor antagonists markedly reduced plasma extravasation and VEGF levels in allergen-induced asthmatic lungs.
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Angiogenesis and Treatment (VEGFR antagonists)
• Lee YC, et al. Contribution of vascular endothelial growth factor to airway hyperresponsiveness and inflammation in a murine model of toluene diisocyanate-induced asthma. J Immunol 2002;168:3595-600.
• They used» Inhibitors of VEGFR tyrosine kinase, SU5614 and SU1498 for
treatment of toluene diisocyanate (TDI) sensitized and challenged mice…
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Angiogenesis and Treatment (VEGFR antagonists)
H&E-stained sections of the lungs
VEGF-positive cells in the bronchioles
VEGF-positive cells in the BAL fluids
Placebo
TDI
TDI +
VEGFR inhibitors Lee YC, et al.Lee YC, et al.
J ImmunolJ Immunol
2002;168:3595-600. 2002;168:3595-600.
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What about us???
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Increased expression of angiogenic markersin patients with seasonal allergic rhinitis
• We aimed to investigate the presence of increased angiogenesis and its relation to angiogenic molecules,
• VEGF, • CD34,• FvW, in endothelial cells of nasal mucosa in patients with seasonal
allergic rhinitis (SAR)
• Twenty patients with symptomatic SAR, who were not under treatment, were enrolled in the study.
• Ten patients with nasal septal deviation NSD, who needed surgical therapy, served as the non-allergic control group.
• Three different immunohistochemical analysis methods, • HSCORE, • microvessel density (MVD),• vascular surface density (VSD) were used.
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Results of Our Study
• Especially;– HSCORE showed statistically significant
differences among the two groups;
• p=0.002 for VEGF; • p=0.045 for CD34;• p=0.016 for FvW...
125
150
175
200
225
250
275
300
325
350
VEGF*
FvW*
CD34* a
patients control patients control patients control
HS
CO
RE
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Conclusion
• Although we have not evaluated other markers of remodelling;– Our observation suggests that
– The presence of angiogenesis and related remodelling cannot be denied in patients with SAR despite a certain amount of controversy.
– Proving the presence of increased angiogenesis, an important part of remodelling and other chronic inflammatory changes in the nasal mucosa, may improve treatment choices, and follow-up in subjects with SAR.
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Our Another Research
• Icreased expression of tissue VEGF and Flk-1 receptor in seasonal allergic rhinitis and relevance to asthma component (Yuksel H, Kose C, Yilmaz O, Ozbilgin K, Kirmaz C)…
• Final revised form underreview in Clinical and Experimental Allergy
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Aim of this study
• Compare VEGF and its receptor Flk-1 expression – between SAR patients and non-allergic
controls and – between SAR patients with and without
asthma.
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Results• Mean HSCORE and VSD for VEGF
was significantly higher in the patient group,
• Mean HSCORE and VSD for Flk-1 in the patient group was significantly higher than those in the control group,
• The difference between mean VEGF HSCORE and VSD scores in patients with pure AR and AR with asthma was insignificant ,
• Mean HSCORE and VSD scores for Flk-1 in patients with pure AR were significantly lower than those in AR patients with asthma…
Flk-1
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Conclusion• Angiogenic factor VEGF and its receptor Flk-1 is increased in AR
patients when compared to the non-allergic controls,
• VEGF is increased similarly in AR patients with and without asthma. • However, Flk-1 that is the main receptor of VEGF is significantly
lower in pure AR patients than that in patients with AR and asthma.
• This may be a possible explanation to the excessive presence of angiogenesis in airway wall in patients with asthma but not in the ones with AR not associated with asthma.
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Imagination is better than knowledge… Einstein
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