Positive & negative inflammatory signals in the vascular wall. The River of Life. Young-Guen Kwon

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1 Positive & negative inflammatory signals in the vascular wall The River of Life Young-Guen Kwon Vascular genomics Lab., Dept. of Biochemistry Yonsei University

2 Formation of a vascular network Two processes are responsible for the formation of new blood vessels: I. Vasculogenesis and Angiogenesis Vasculogenesis: in situ formation of blood vessels from progenitor endothelial cells or angioblasts Angiogenesis: formation of new blood vessels from preexisting blood vessles Structure of Blood Vessel Immune cell, Pericytes Smooth muscle cell Endothelial cell Basement membrane Collagen typeiv Heparan sulphate proteoglycan Entactin Von Willbrand factor Vitronectin Platelet Interstitial matrix Collagen types I, III, V and VI Fibronectin Other ECM components

3 Therapeutic angiogenesis Ways for treating myocardial ischemia and peripheral vascular disease Cell therapy for ischemia Gene or protein therapy EPC Endothelial progenitor cell bfgf Will any single growth factor or stem cell therapy be the magic bullet to cure vascular disease? >>> Combination therapy?

4 Therapeutic Angiogenesis (Gene or protein therapy) What to deliver? Clinical trials of angiogenic therapy Protein Trial type N Delivery FGF1 Phase I, OL 20 Safe 41 Capillary blush at injection site FGF2 Phase I, OL 30 Hypotension at high dosages Dilatation of epicardial coronaries FGF2 Phase II, DBR 337 Safe/ No effect on ETT or SPECT Short-term improvement in symptoms compared to placebo - A 165 Phase I, OL 15 Hypotension at low dosages Reduced SPECT defect size - A 165 Phase II, DBR 165 No improvement in ETT, symptoms, or SPECT compared to controls GM-CSF Phase I/II, DBR 21 Improved collateral flow index in the GM-CSF group What are the risks? Cardiovascular Research 2005 Aberrant Vascular Proliferation in Nontargeted Tissues Increased Vascular Permeability and Inflammation Induction of the Development of Functionally Abnormal Blood Vessels Triggering Growth of Neoplasms Increase in Atherosclerotic Plaque Mass and Instability Vasodilatation and Hypotension During Short-Term Administration Hazards Associated With Viral Vectors Hazards Associated With Direct Myocardial Delivery

5 Molecular link between angiogenesis and inflammation? >> Pro- and Anti-Inflammatory Signals Acting on Vascular Cells << Mediators Proinflammatory Signals Anti-Inflammatory Signals Cytokines TNF, IL-1, IL-8, IFN-, TGF-β, IL-10, IL-1ra, Oncostatin M, IL-4, IL-13 IL-13 Angiogenic factors, TRANCE Angiopoietin-1 Growth factors PDGF FGF, HGF Vasoactive agents Neuropeptides Nuclear receptors Angiotensin endothelin NO Substance P PPARs Mechanial forces Stretch Shear stress Other LPS, phobol esters, thrombin HDL, n3-fatty acids

6 ABIN-2 NF-κB; a key molecule of vacular inflammation Pro-inflammatory signals NBD Angiopoietin-1 HGF HDL Shear stress IL-10 Tie2 PRARα activators TGFβ IL-10Rα IL-10Rβ NEMO IKKα, β HDL NO SOD P p50 IκBα p65 p50 IκBα p65 PRARα P IκBα p50 p65 IκBα Smads Degradation CBP p50 p65 Target Genes Inflammatory cytokines Enzymes Adhesion molecules Receptors

7 Vascular Inflammation - a basic pathological mechanism that underlies atherosclerosis, ischemia/reperfusion, rheumatoid arthritis, psoriasis, restenosis, and bronchial asthma. > Inflammatory process > Potential makers of Vascular Inflammation The inflammatory process requires extravasation of leukocytes from the microvasculature at sites of inflammation or injury Increased endothelial permeability Up-regulation of leukocyte adhesion molecules Up-regulation of endothelial adhesion molecules (ICAM & ) Migration of leukocytes into the artery wall

8 TRANCE, TNF-related activation-induced cytokine also called ODF, OPGL, and RANKL exists either in a cell-bound form or a truncated ectodomain variant cleaved by TACE The biological function from the phenotype of RANKL-deficient mice > osteoclast functions and bone remodeling > immune cell cross-talks, dendritic cell survival, and lymph node organogenesis. > Mammary gland development RANK, Receptor activator of NF-κB OPG, Osteoprotegerin Extracellular domain 1 Cysteine-rich domains Cysteine-rich domains Death domain Homologous regions TRANCE in the vascular system Prominent expression of TRANCE in the vascular cells in vitro and in vivo Expression of RANK in endothelial cells >> up-regulation of RANK by in endothelial cells (JBC 2003) A potential risk factor for progressive atherosclerosis and cardiovascular disease >> mice deficient in OPG exhibit arterial calcification in addition to early onset osteoporosis TRANCE in angiogenesis and vascular inflammation >> induced angiogenesis in vitro and in vivo (JBC 2002)

9 Soluble TRANCE Induces Angiogensis in Vivo Chicken Chorioallantoic Membrane Assay CON PMA TRANCE % of Positive Disc /22 21/25 10/12 0 CON PMA TRANCE Mouse Matrigel plug assay CON TRANCE 80 Neovessel Area (x10 3 ) ( mm 2 ) CON TRANCE bfgf JBC 2002

10 TRANCE fails to promote angiogenesis in enos-knock out mice in vivo A Control TRANCE enos +/+ enos +/+ B 10 C enos -/- Control Control enos +/+ enos -/- Control enos -/- TRANCE TRANCE enos +/+ enos -/- TRANCE Hb Contents (g/dl) TRANCE (10 ug) D Number of neovessel TRANCE (1 ug) enos +/+ enos -/ enos +/+ enos -/-

11 TRANCE increases monocyte adhesiveness and transendothelial migration of leukocyte in vivo A Control TRANCE (1 µg/ml) B 4h 8h 12h 16h TRANCE ICAM-1 Actin TRANCE (3 µg/ml) TRANCE (5 µg/ml) (20 ng/ml) ICAM-1 C Untreated TRANCE Counts Counts EC Lu Fluorescence Intensity

12 TRANCE induces Vascular permeability in vivo ; impairment in enos-deficient mice A Control TRANCE B enos +/+ 0.8 Absorbance 620 nm enos -/- Control TRANCE 0 TRANCE (10 ug) enos +/+ enos -/- C Control TRANCE Control TRANCE enos +/+ enos -/-

13 Schematic illustration of TRANCE-induced Angiogenesis & Inflammation TRANCE TRANCE/RANK PI3K KDR/Flk Src PLC RANK TRAF2 PLC TRAF6 PI3K AKT EC PKC DG Ca 2+ enos Raf MEK Raf MEK PKC- α ROS PKC- ζ FAK Migration NO Permeability JBC2002 JBC2003 JI2005 In preparation RANK mrna ERK1/2 NF-κB Morphogenesis Proliferation CAMs Angiogenesis Inflammation Bone, Atherosclerosis, RA, Tumor

14 Circulation Research. 2004;95:1046 Schematic diagram of potential expression, regulation, and function of RANKL, RANK, and OPG in atherosclerotic vascular calcification. Elevated level of TRANCE in the vasculature may be importantly involved in the pathogenesis of atherosclerosis.

15 External regulation of inflammatory signals HGF suppresses inflammatory responses in EC HGF NF-κB NF-κB Tubulogenesis by epithelial cells Angiogenesis by endothelial cells Scattering Invasion and metastasis HGF; hepatocyte growth factor

16 HGF inhibits -stimulated leukocyte adhesion & CAM expression in HUVECs A Control HGF B - 4h 6h H V HV H V HV ICAM-1 Actin +HGF C Counts Control ICAM-1 HGF + HGF D ICAM-1 2h 4h - H V HV H V HV Actin Fluorescence Intensity

17 HGF suppresses -induced transcriptional activity through inhibition of NF-κB activation A B ICAM-1-luc bp AP-1 ARE NF-κB TATA NF-κB TATA AP-1 STAT Luc ICAM-1-luc bp TATA NF-κB TATA AP-1 STAT Luc -luc bp Oct AP-1 GATA TATA TRE NF-κB Luc -luc -353 bp TATA NF-κB Luc Promoter Activity (% of control ) ICAM-1 ### Promoter Activity (% of control ) ICAM-1 ### 0 - V HV H 0 - V HV H

18 HGF inhibits -induced NF-κB activation through inhibition of IκB-α phosphorylation and degradation A 5 min 10 min 20 min 45 min H - V H V V H H V V H H V H V H V B - H 5 min V HV IκB-α P-IκB-α IP :α- IKKγ KA : GST- IκBα (1-54) Actin IB : α- IKK γ IKKγ C D Control HGF - 60 min 90 min H H H V V H V V C p65 p65/p50 +HGF

19 HGF inhibits -induced leukocyte infiltration in vivo A Control HGF B 500 +HGF Leukocyte Adhesion (% of control) V HV H C Control HGF +HGF CD11a (x200)

20 Co-treatment of HGF with shows the synergistic effect on neovessel formation in the mouse skin CTL HGF +HGF PECAM-1 (x200)

21 and HGF synergistically stimulates angiogenesis A CTL B CTL HGF +HGF HGF +HGF 1.Xin X, Yang S, Ingle G, Zlot C, Rangell L, Kowalski J, Schwall R, Ferrara N, Gerritsen ME. Hepatocyte growth factor enhances vascular endothelial growth factor-induced angiogenesis in vitro and in vivo. Am J Pathol. 2001; 158: Mary E. Gerritsen, HGF and : A Dynamic Duo, Circ. Res : Therapeutic angiogenesis using & HGF co-administration may be more clinically applicable Reduce inflammation & potentiate angiogenesis Min et al Circ. Res. 2005

22 Internal regulation of inflammatory signals ERK suppresses inflammatory responses in EC - - TRANCE U73122 BAPTA PDTC PD98059 Wortmannin NAC (-) U0126(-) (+) U0126(-) Actin (+) U0126(+) (-) U0126(+) ERK-I U0126 NF-κB NF-κB

23 ERK negatively regulates -induced expression in ECs A U B U0126 PD Actin p-erk ERK Actin p-erk ERK C Mock DN-MEK D CON ERK1 ERK p-erk P-ERK1/2 ERK Actin

24 ERK negatively regulates -Induced transcriptional activity of A PD98059 Actin 3h 6h 12h B -luc -1.7Kb TRE GATA NF-κB GATA NF-κB TATA luc luc ty Relative promoter activ i U DN-MEK Relative promoter activity Relative promoter activity MOCK ERK1 ERK2

25 Inhibition of ERK increases -induced IκB-α phosphorylation and NF- κb activation A (+) U0126(-) (+) U0126(+) E F p-ikb-α (min) PD (-) PD98059(-) (+) PD98059(-) IkB-α p65/50 p-erk ERK NS C P-IκB-α P-ERK1/2 CON ERK1 ERK D U IP : α-ikkγ KA : GST- IκB-α (1-54) IκB-α IKKγ (+) PD98059(+) (-) PD98059(+) IB : α- IKKγ

26 PKC mediates both ERK and NF- κb activation in response to A Chelerythrine GF109203X U0126 P-IκB-α P-ERK1/2 ERK1/ FlK-1/KDR PLC-γ PKC MEK-1 B Chelerythrine GF109203X U ERK NF-κB IKK Actin

27 ERKs inhibit IL-1β- and TNF-a-induced CAM expression via inhibition of the NF-kB pathway in EC A TNF-α P-IκB-α CON - + ERK1 ERK2 CON IL-1β - + P-IκB-α ERK1 ERK C TNF-α CON - + ERK1 ERK IL-1β CON ERK1 ERK P-ERK1/2 P-ERK1/2 ICAM-1 ICAM-1 Actin Actin B - IL-1β TNF-α con ERK1/2 D Relative promoter activity IL-1β Relative promoter activity TNF-α MOCK ERK1 ERK2 MOCK ERK1 ERK2 7

28 ERK inhibits inflammatory cytokine-induced leukocyte adhesion to ECs A (-) U0126(-) (+) U0126(-) B - IL-1β TNF-α con ERK2 (+) U0126(+) e Fold-Increas U (-) U0126(+) - + P-ERK1/2 con ERK IL-1β - TNF-α - ERK2 - e Fold-Increas

29 Schematic pathway for the negative regulation of -induced expression by ERK TNF-α, IL-1β HGF Shear stress? FlK-1/KDR PLC-γ PKC MEK-1 MEK-1 ERK IKK ERK NF-κB Inflammatory responses

30 Balanced Regulation of Angiogenesis & Inflammation in the vascular wall Shear Stress HGF, FGF etc, TNF-α, IL-1β, TRANCE etc ERK ERK ERK ERK A I A & I A & ai Angiogenesis Inflammation

31 Acknowledgement Vascular genomics laboratory Yonsei University Jeong-Ki Min, Ph. D. Yong-Sun Maeng Kangwon National University Young-Myeong Kim, Ph. D. Young-Mi Kim, Ph.D Ewha Women s University Goo-Taeg Oh, Ph. D.