M echanotransduction in the Vascular System : Focus on Shear and Integrin -ECM

1. Mechanotransduction in the Vascular System : Focus on Shear and Integrin-ECM Summer Topic Review

2. Why do we care? • Mechanical forces sensed by the vasculature determine • blood vessel structure both acutely (through vasodilation) and chronically (though vascular remodeling) • gene expression and phenotypic status of endothelial and mural cells • Through these biological effects, mechanical forces • regulate susceptibility to inflammation and atherosclerotic plaque formation • affect endothelial cell junctions, migration, permeability and probably extravasation

3. Regulation of Vascular Structure by Flow • Chick Yolk Sac, Thoma 1893 • Vessels with high-flow enlarge • Those with low-flow regress John Hunter, 1728 - 1793

4. Shear Stress Frictional force of blood flow on the vessel lining (endothelium) Well defined role in: Vessel enlargement/regression Vasodilation (acute dilation of blood vessel) Atherosclerosis/inflammation Developmental vascular remodelling Arteriovenous specification And a suggested role in aortic hemogenesisin the embryo

5. Circumferential Stress Outward force of blood pressure on the vessel wall (endothelium and mural cells) Well defined role in: Switch from synthetic to contractile and proliferative to quiescent Myogenic tone (contraction of smooth muscle cells in response to lumen pressure) Supporting a likely role in vessel wall growth in hypertension

6. Discrete Molecular Responses to Shear and Circumferential Stress Yamamoto et al. Nat. Med. 2008

7. Discrete Molecular Responses to Shear and Circumferential Stress Yamamoto et al. Nat. Med. 2008

8. Shear not Circumferential Stress Promotes Vessel Growth Surgical Fistula Pressure shear = increase growth Rabbit hindlimb angiogram Eitenmüller et al. Circ. Res. 2006

9. Regulation of Arteriovenous Specification le Noble et al. Development 2004

10. Atherosclerotic Plaques Preferentially Develop in Areas of Chronic Low/Oscillatory Shear Stress the endothelium is the mediator of shear stress effects on the vasculature Dai et al. PNAS 2004

11. Chronic Low/Oscillatory Shear Stress Induces an “Activated” or “Atheroprone” Endothelial Phenotype Pre-treatment + IL-1 Dai et al. PNAS 2004

12. Klf2 is Induced by Chronic Laminar Shear and Mediates Atheroprotective Gene Expression Patterns Dekker et al. Blood 2002 Kush et al. JCI 2006

13. Acute Changes in Shear Regulate Permeability of the Endothelium Electrical resistance of porcine endothelial cells in culture is reduced by removing shear stress. Resistance is further decreased by calcium chelation.

14. Chronic Changes in Shear Regulate Permeability of the Endothelium 1200 70 Cucullo et al. J. Cereb. Blood Flow & Met. 2007

15. Chronic Shear Stress Inhibits Endothelial Apoptosis Shear inhibits endothelial cell death upon serum starvation. Dimmeler et al. FEBS 1996

16. Chronic Shear Inhibits Endothelial Proliferation Akimoto et al. Circ. Res. 2000

17. Acute Loss of Shear may Encourage Emboli/Cell Extravasation Lam et al Nature 2010 Kienast et al Nat. Med. 2009 Similar time-course of emboli and tumor cell extravasation in murinecerebrovasculature

19. Molecular Transducers of Endothelial Shear Stress

20. Time-line of Shear Induced Changes Vasodilation Cytoskeletal Reorganization Increased leukocyte recruitment Hahn & Schwartz, Nat. Rev. Mol. & Cell. Biol. 2009

21. Chronic Shear Stress Regulates Endothelial Cell-Cell Adhesion Noria et al. Circ Res. 1999 Porcine aortic endothelial cells upon application of FSS

22. Acute Shear Stress Induces Cytoskeletal Stress Confocal optical section of GFP-vimentin Before and after shear Helmke, Biophys. Journal 2003

23. Chronic Shear Causes Re-alignment of Focal Adhesions Davies et al. JCI 1993

24. Acute Shear-mediated IntegrinaV/b3 Activation Bovine aortic endothelial cells under shear for 5 minutes. Wow-1 binds activated IntegrinaV/b3. b3 shows total b3 integrin. Tzima et al. EMBO 2001

25. RGD Peptide Inhibits Acute Shear Induced Vasodilation Isolated coronary arterioles Muller et al. Circ. Res. 1997

26. Genetic Ablation of a1 Integrin Inhibits Acute Shear Induced Vasodilation Loufrani et al. Am. J. Phys. 2008

27. Bacteria-derived Peptide (pUR4/FUD) Blocks Fn Matrix Assembly and Interferes with Vascular Remodeling In Vivo Chiang et al.ATVB 2011

28. “Quiescent” vs “Activated” Endothelial ECM Vascular basement membranes contain laminins (predominantly laminin-8/ laminin411 and laminin-10/ laminin511) Type IV collagens perlecan nidogens collagen XVIII von Willebrand factor. In development, and during pathological remodeling additional ECM components include Fibronectin Fibrinogen

29. Acute NF-kappa B (p65 subunit) Activation by Shear is ECM Dependent BAE cells plated for 4 hrs were sheared for 60 min Orr et al. JCB 2005

30. Fn Is Deposited in Regions Chronically Exposed to Low/Oscillatory Shear Orr et al. JCB 2005

31. In vitro Fn Deposition is Increased by Chronic Low/Oscillatory Shear Feaver et al. Circ. Res. 2010

32. Modification of Fn Fibrils Which “Hide” EIIIA Domain Suppresses Shear-induced NF-kappa B Signaling by Increasing p38 Klein et al. JCS 2003 Orr et al. JCB 2005

33. Increased Expression of EIIIA Isoform in Pathological Vascular Response Protein Immunofluorescence RNA Dot Blot Hybridization Media EIIIA Fn isoform total Fn EIIIA Total Normal Rat Artery Intima EIIIA Total Human Atherosclerotic Lesion, 10ug RNA  2x dilutions EIIIA Fn isoform IgG control Wounded Rat Artery Glukhova et al. JCB 1989

34. Endothelial Cilia mediates Ca++ Release Ciliated mouse aortic endothelial cells are increase Ca++ release in response to increased shear when incubated in low or no-shear conditions, but not when incubated in high-shear. Nauli et al. Circulation 2008

35. Shear Activates Purified G proteins imbedded in Liposomes Cholesterol (which decreases membrane fluidity) reduces GTPase activation Gudi et al. PNAS 1998

36. A Mechano-sensory Complex of VE-cadherin/VEGFR2/PECAM-1 Regulates a Subset of Shear-induced Events Tzima et al Nature 2005

37. Endothelial IntegrinaV/b3 Activation by Shear Depends on the Mechano-sensory Complex Ve-cadherin/PECAM1/VEGFR2 VE-cadherin-/- or PECAM-/- cells Src kinase inhibitors PP2 or SU6656 PI3 kinase kinase inhibitors LY294002 or wortmannin aV/b3 activation (detected by WOW-1 antibody) Magnetic bead conjugated with PECAM-1 (but not VE-cadherin or CD44) induces WOW-1 signal upon mechanical pull on bovine aortic endothelial cells. Tzima et al Nature 2005

38. Take-away Points • Shear stress exerts acute and chronic effects on the vasculature with important implications in human development and disease • -Acute increase promotes vasodilation • -Acute and chronic decrease promotes permeability, proliferation, apoptosis • -Low/oscillatory shear promotes inflammation and atherosclerosis • Acutely responsive signaling pathways critical in these responses have been identified • PECAM/VE-cadherin/VEGFR2 complex • Integrin activation • Chronic changes have also been identified • -KLF2 • -Fibronectin (EIIIA and EIIIB)

39. Open Questions How do changes in ECM composition and integrin array induced by altered shear feed back into the in vivo consequences? - New integrin adhesions are required for shear-mediate cytoskeletal reorganization - Crude pharmacologic inhibition of FN matrix assembly inhibits inflammatory receptor expression, recruitment, and proliferative response to reduced FSS Can the different response to shear among vessels be attributed in part to differences in ECM composition? What portion of this is genetically controlled, and what portion is environmentally controlled?