Hypothesis: Targeting Plaque Angiogenesis in Atherosclerosis

Transcription

1 Hypothesis: Targeting Plaque Angiogenesis in Atherosclerosis Karen Moulton, M.D. Dept. of Medicine, Cardiology University of Colorado Anschutz Medical Campus Learning Objectives: 1. Progress of investigations on neovascularization in atherosclerosis 2. Potential therapeutic rationales for selective regulation plaque neovascularization. 3. Translational goals: Tools for imaging. Clinical evidence. Disclosure: Sponsored research grant to test effects of VEGF164 aptamer on atherosclerosis in mice. Use of approved drug for alternative application.

2 Functions of Plaque Neovascularization Promote atheroma growth: Werber, Atherosclerosis (1987). Moulton K, Circulation (1999). Khurana R, Circulation (2004). Vessel wall perfusion: Geiringer, J Path Bact. (1951). Heistad DD, Circulation Res. (1981). Williams JK, Circulation Res. (1988) Conduits for inflammatory cell entry: O Brien K, Circulation (1996). Moulton K, PNAS (2003). Eriksson E. Circulation (2011) Intravital microscopy on atherosclerosis in ApoE-/- mice Plaque hemorrhage and vulnerability for rupture: Patterson, Arch. Pathology (1938). RBCs-atherogenic stimulus Kolodgie, NEJM (2003). Neovascularization-rupture Virmani, ATVB (2005);Moreno, Circulation (2004). Adventitia vs intimal neovascularization. Hemorrhage secondary effects on macrophage phenotypes. Finn, JACC (2011) PNV correlates with increased risk for CV events Hellings et al. Circ (2010)

3 Neovascularization in advanced atheromas of atherosclerosis-prone mice CD31 vwf In Situ CD31 MicroCT WT Ae -/- Moulton et al (1999) Circ 99: ApoE;ApoB100: Celleti, Nat Med (2001). ApoE: Heeschen, Nat Med (2001). LDLR-/-: Petrovan, ATVB (2007) ApoE;LDLR KO, 80 wks Langhenrich. ATVB (2006) LDLR-ApoB100 Drinane. Circ Res. (2009)

4 Angiogenesis and Plaque Growth Angiogenesis inhibitors delay growth of advanced plaques Endostatin EC Moulton, Circulation (1999) TNP-470 fumagillin EC Moulton, Circulation (1999) VEGFR1-Ab EC, immune Luttun, Nat Med (2002). Angiostatin EC Moulton, PNAS (2003) sol. Flt1 (rabbit, mice) EC, immune Khurana, Circ (2004); Zhou, ATVB 2004 FGFR-dn EC, SMC Khurana, Circulation (2004) αvβ3-tnp NP (rabbit) EC Winter, ATVB (2006) VEGFR2-vaccine EC Petrovan, ATVB (2007) rpai-1(23) EC, SMC Drinane, Circ. Res. (2009) pravastatin (monkey) EC, Mac Williams, JACC (1998) Angiogenesis stimulators promote plaque growth VEGF (iv) EC, immune Lazarous Circulation (1996) VEGF (iv) EC, immune Celletti, Nat Med (2001) Nicotine EC, Heeschen, Nat Med (2001) PlGF EC, immune Khurana, Circ (2005) VEGF-A,B,C,D (rabbit) EC, immune Leppanen, Circ (2005) bfgf afgf (rabbit, pig) EC, Mac, SMC Edelman, JCI 1992; Nable Nature Angiogenesis independent and dependent phases of plaque growth.

5 Intravital Microscopy: seeing is believing Eriksson E. Circulation (2011)

6 Angiogenesis and Chronic Inflammation Mononuclear cells in plaque neovascularization. mice (Moulton, Circ (1999) Human (Daemen JACC (2009). ApoE Permeability: limited pericytes. abnormal EC junctions basement membranes and membrane blebs. (Sluimer et al JACC (2009). EC and leukocytes share receptors for various angiogenesis factors Human

7 Natural History of Atheromas +/- NV: Can we convert the vascularized phenotype? Avascular Plaques Composition: fewer macs, T cells. Matrix: collagen, Ca++ less lipid pool Non-ACS sx Plaques with NV Composition: more immune cells. Proteoglycans lipid pool ACS culprit intraplaque blood +effect on Macs, +CV outcomes-hellings Circ 2010? excess VEGF, FGF (animal models) Statin, ACE, AT 1 R

8 Translation: gaps in knowledge What are active molecular pathways? VEGF, replacement of endogenous inhibitors, hypoxia, ROS, inflammatory cell phenotypes. Anti-angiogenesis agents are standard treatments for cancer and macular degeneration. Effects on patients with existing atherosclerosis (known and unknown) could be assessed, but have not been well studied. controls or adjustments for confounding variables. Systemic VEGF antagonists are not specific to pathological angiogenesis and do not spare normal arteries. Different goals for therapy in CAD vs cancer: permeability, maturation, means to target specific vascular beds. spare normal arteries Modification Regression

9 Endogenous Regulators of Plaque Angiogenesis I. Angiogenesis stimulators in atheromas. VEGF-A (VEGF164) is major angiogenesis activity in atheromas VEGF164 and its receptors. (ATVB 2009) II. III. IV. Endogenous inhibitors in normal arteries that are depleted by atherosclerosis and vascular inflammation. Collagen XVIII (and proteolytic endostatin domain). Moulton et al. Circ (2004) and Zeng et al. JLR (2005). Other candidates: Collagen IV, fibulin-5, elastin and perlecan have cryptic anti-angiogenesis molecules that are depleted by atherosclerosis. Replacement therapy to restore a normal artery component. Hypertension, Diabetes, smoking. Diabetes: Moulton (unpublished 2010) Plaque angiogenesis is induced by T1D and accelerates atherosclerosis. Hypertension: Ritman & Lehrman CVR (2007) Hypoxia-related transcription factors or metabolic stress. Sluimer & Daemen J Path (2009); Detection of hypoxia by [18f]ef5 in atheromas in mice. ATVB ( 2011).

10 Clinical effects of chronic systemic VEGF antagonists: not selective for pathologic functions of VEGF Agent Mice Rabbit Pig Human Sol Flt-1, + + BP, proteinuria, preeclampsia VEGFRinhibitors BP, proteinuria Pro-thrombotic Vegf-ab + + BP, proteinuria Pro-thrombotic Target microvascular plaque NV and spare normal arteries.

11 Differential effects of VEGF isoforms in atherosclerosis Ae-/-VEGF-wt Ae-/-VEGF 164/164 Ae-/- VEGF 188/188 VEGF isoform mice: Same total VEGF transcripts, Isoform pattern is restricted 0.15% chol. diet. Early stage,10 wks * 24 wks angiogenic stage * * *

12 Aptamer binding heparin-binding domain of VEGF164 inhibited early and late-stage atherosclerosis. LDLR -/- ;ApoB100 tm/tm mice on 0.15% chol. Diet for 12, 24, and 32 wks. Matrigel Implants Control Tx mice Macugen: Macular Degeneration NEJM (2004)

13 Nrp2 is VEGF164 receptor expressed on PNV, but less abundant on arterial endothelium. ApoE -/- and ApoE -/- ;NRP2/LacZ littermates, 0.15% chol 28 wks Nrp2 promoter LacZ activity (blue)

14 Endogenous inhibitor Collagen XVIII in aorta WT KO Measurement Fold-WT P VV count VV density Int.Neov. (%) ES-IgG + C18 in basement membrane of NV and artery wall in ApoE-/- mouse. Moulton et al. Circ. (2004

15 VV and PNV: plaque hypoxia and wall perfusion. Anatomy: correlations with plaque size, or lamellar units. Geiringer, J Path Bact. (1951). Wolinsky, Circ Res (1967) Vessel wall perfusion: microsphere measures of flow, vasoactive changes in flow, compressive forces of artery wall, flow may vary based on cardiac cycle.: Heistad DD, Circulation Res. (1981). Williams JK, Circulation Res. (1988); Ritman and Lerman, Cardiovasc. Res (2007) End-vessels: implications for drainage and vascular targeting. Gossl et al, Am J Phys Heart (2003). Hypoxia and oxidative stress markers in vessel wall: Gossl et al, Am J Phys Heart (2003 Bjornheden (Wiglund) ATVB (1999): Hypoxic areas in vessel wall in vivo. Heterogeneous distributions of vasa vasorum and hypoxia. Levin ATVB (2003). Mapping glucose, ATP, glycogen and lactate in vessel wall. Detection of hypoxia by [18f]ef5 in atheromas in mice ATVB. (2011) Pleotropic effects of hypoxia, HIF mac phenotype, apoptosis, lipid metabolism.

16 Translation: methods to investigate outcomes Imaging methods for research and outcomes: OCT (intimal vs adv) ACS culprit vs non-acs lesions MRI +/- molecular agents CT contrast US Goals of NV therapy other than stimulation or inhibition: permeability maturation of PNV -- choroid and retinal vessel and visual response to VEGF antagonists precede vascular regression. leukocyte recruitment Outcome on plaque growth. On plaque vulnerability? vulnerable plaque- multiple others are present. Preemptive PCI for controlled rupture. Rationale unclear. No reduced MI risk in PCI + med. tx versus med tx groups. Vascular zipcode targeting.

17 Targeting Plaque Neovascularization to deliver therapy to plaques Monitor plaque angiogenesis-related biomarkers. Neovascular imaging to assess risk and treatment response. Specific delivery mechanisms (improve efficacy and safety). Challenges: approval as device and drug. Integrin-targeted fumagillin or contrast-loaded nanoparticles to plaque neovascularization Winter et, Circulation 2003.

18 Conclusion: Future Directions in Neovascular Therapy I. Cancer and Macular Degeneration: chronic treatments Targets beyond growth factors, receptors, downstream signaling. Combined agents for synergy: tumor vessels transiently normalize vasomotor function to improve XRT and chemo efficacy. Imaging as biomarker and monitor of therapy. II. Plaque Angiogenesis: chronic or periodic tx. Target beyond growth factor and receptors. Imaging as biomarker and monitor of therapy. Treatment duration? Tx effect after withdrawal? Microvascular versus large vessel endothelium. CV goals: reduce permeability, leukocyte recruitment. Selective targeting with lower doses in circulation.

19 Acknowledgements Moulton Lab Ian McKittrick Laura Allmon Weston Truman Ashley Johns Dmitry Belchencko Joslin Diabetes Center Giulio R. Romeo Vascular Biology (Children s Boston) Marsha Moses Diane Bielenberg Rick Rogers UC Denver (Diabetes Biomarker) Kristen Nadeau (TCH, Denver) Janet Snell-Bergeon (Barbara Davis) Talia Brown (Barbara Davis) Moshe Levi (Renal Division) Yolanda Bogaert (Renal) Animal Imaging Core Schepens Eye Institute (VEGF) Patricia D Amore