1. Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University

2. Sensors for shear stress on vascular endothelial cells Kimiko Yamamoto. Circulation Journal. 2009

3. Endothelial cells isolated from mouse express CD31 and have primary cilia. Nauli, et al. Circulation 2008 Do vascular endothelial cells isolated from mouse express primary cilia?

4. Colocalization of polycystin-1 (PC1) and polaris. Nauli, et al. Circulation 2008 Polycystin-1 is localized in aortic endothelial cilia of embryonic mouse aorta. Does polycystin-1 (TRPP1, PKD1) express and localize in endothelial cilia of embryonic mouse aorta?

5. Fluid shear–induced calcium and transient NO require polaris and polycystin-1. Nauli, et al. Circulation 2008 Does TRPP1 or cilium play a role in regulation of endothelial function?

6. Is TRPP2 localized in the cilia of vascular endothelial cells? Human MouseWissam A. et al. Cellular biology.2009

7. Normal expression level and localization of TRPP2 in cilia is required for the endothelial cilia to sense fluid shear stress. Wissam A. et al. Cellular biology.2009

8. Fluid flow (shear stress) Cilia Polycystin-1 Polycystin-2 Ca 2+ PKC eNOS CaM Akt/PKB Ca 2+ /CaM Nitric oxide Vasodilator Mechanosensory cilia as microcompartments. Surya M, et al. International Journal of Vascular Medicine 2011

9. Are there other sensors for shear stress on vascular endothelial cells beyond what have found so far?

10. Control 5 mM Chloral hydrate (3 days) Nuclei α-tubulin Merged XZ view Do human umbilical vein endothelial cells (HUVECs) have primary cilia?

11. Are cilia involved in [Ca 2+ ] i increase in response to alteration of shear stress in HUVECs? 30s 0 10 dyne/cm 2

12. Is cilium involved in [NO] i increase in response to alteration of shear stress? 0 10 dyne/cm 2 ~75s

13. The data presented thus far indicate that there is another “secondary” sensor beyond primary cilium. What is it? We have previously indentified a functional channel complex formed by TRPP2 and TRPV4 in CCD isolated from mouse and this channel complex exerts dual functions (submitted to Cell).

14. TRPV4MergeNucleiTRPP2 WB: IP:Lysate TRPV4 IgGTRPP2 WB:TRPP2 IP:LysateIgGTRPV4 A B Do TRPP2 and TRPV4 physically interact in HUVECs? IgG

15. C Does TRPP2 or TRPV4 express in cilia of HUVECs?

16. TRPP2 TRPV4 Merged cilium Are TRPP2 and TRPV4 co-localized on cilium?

17. Are TRPP2 and TRPV4 co-localized on human mesenteric vascular endothelial cells?

18. The data presented thus far strongly suggest that TRPP2/TRPV4 physically interact in HUVECs, and that this complex expresses both in cilia and plasma membrane of HUVECs Question Does this complex play a role in response to macheno-stimuli in HUVECs?

19. qtRT-PCR demonstrating gene silencing efficiency

20. TRPP2 β-actin TRPV4 β-actin CtrlScr P2-shRNA CtrlScr P2/V4-shRNA CtrlScr V4-shRNA CtrlScr P2/V4-shRNA

21. Does knock down TRPP2 and/or TRPV4 affect [Ca 2+ ] i transient in response to alteration of shear stress in HUVECs ？ 0 10 dyne/cm 2 30s

22. Does knock down TRPP2 or TRPV4 affect increase in [NO] i in response to alteration of shear stress in HUVECs ？ 0 10 dyne/cm 2

23. p-AMPK AMPK p-eNOS e-NOS CtrlScr Ctrl Flow 10 min

24. p-AMPKα AMPKα CtrlScrCtrlScrCtrlScr p-eNOS e-NOS V4- shRNA P2- shRNA P2/V4- shRNA Flow 10 min p-eNOS e-NOS PI3K inhibitor (+) (-) (+) (-) (+) (-) CtrlScrCtrlScrCtrlScr P2- shRNA V4- shRNA P2/V4- shRNA

26. A Do TRPP2 and TRPV4 form a functional channel complex in HUVECs? baseline 100 mV TRPP2/TRPV4 forms a non-selective calcium permeable cation channel

27. There are several different machenosensors in vascular endothelial cells, including primary cilium. TRPP2 and TRPV4 assemble to form a functional channel complex in HUVECs. TRPP2/TRPV4 channel complex exerts dual functions, i. e. acting as a mechanosensor and regulating [Ca 2+ ] i and NO production in response to alternation of shear stress in HUVECs, via AMPK-p- eNOS (?).

28. Jia-Ning Zhang, Ph.D. candidate Dan Zhao, Ph.D. Associate Prof. Ying Zhao, Bing-Kun Zhang John Y.-J. Shyy, Ph.D., Prof. Univ. of California, Riverside Funding: CNSF, NIH