Mechanosensitive properties in endothelium and their roles in

regulation of endothelial function

Zhi-Ren Zhang M.D., Ph.D.

Harbin Medical University

Sensors for shear stress on vascular Sensors for shear stress on vascular endothelial cellsendothelial cells

Kimiko Yamamoto. Circulation Journal. 2009

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?

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?

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?

Is TRPP2 localized in the cilia of vascular endothelial cells?

Human

Mouse Wissam A. et al. Cellular biology.2009

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

Fluid flow(shear stress)

Cilia

Polycystin-1

Polycystin-2

Ca2+

PKC

eNOS

CaM

Akt/PKBCa2+

/CaM

Nitric oxideVasodilator

Mechanosensory cilia as microcompartments.

Surya M, et al. International Journal of Vascular Medicine 2011

Are there other sensors for shear Are there other sensors for shear stress on vascular endothelial cells stress on vascular endothelial cells

beyond what have found so far?beyond what have found so far?

Control

5 mM Chloral hydrate(3 days)

Nuclei α-tubulin Merged

XZ view

XZ view

Do human umbilical vein endothelial cells (HUVECs) have primary cilia?

Are cilia involved in [Ca2+]i increase in response

to alteration of shear stress in HUVECs?

30s0

10 dyne/cm2

Is cilium involved in [NO]i increase in response

to alteration of shear stress?

0

10 dyne/cm2

~75s

The data presented thus far indicate The data presented thus far indicate that there is another “secondary” that there is another “secondary”

sensor beyond primary ciliumsensor beyond primary cilium..

What is it?What is it?

We have previously indentified a functional We have previously indentified a functional channel complex formed by TRPP2 and TRPV4 in channel complex formed by TRPP2 and TRPV4 in

CCD isolated from mouse and this channel CCD isolated from mouse and this channel complex exerts dual functions (submitted to complex exerts dual functions (submitted to

CellCell). ).

TRPV4 MergeNuclei TRPP2

WB:

IP: Lysate

TRPV4

IgG TRPP2

WB: TRPP2

IP: Lysate IgG TRPV4

A

B

Do TRPP2 and TRPV4 physically interact in HUVECs?

IgG

C

Does TRPP2 or TRPV4 express in cilia of HUVECs?

TRPP2

TRPV4

Merged

cilium

Are TRPP2 and TRPV4 co-localized on cilium?

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

The data presented thus far strongly The data presented thus far strongly suggest that TRPP2/TRPV4 physically suggest that TRPP2/TRPV4 physically

interact in HUVECs, and that this interact in HUVECs, and that this complex expresses both in cilia and complex expresses both in cilia and

plasma membrane of HUVECsplasma membrane of HUVECs

QuestionQuestion

Does this complex play a role in response to Does this complex play a role in response to macheno-stimuli in HUVECs?macheno-stimuli in HUVECs?

qtRT-PCR demonstrating gene silencing efficiency

TRPP2

β-actin

TRPV4

β-actin

Ctrl Scr P2-shRNA Ctrl Scr P2/V4-shRNA

Ctrl Scr V4-shRNA Ctrl Scr P2/V4-shRNA

Does knock down TRPP2 and/or TRPV4 affect [Ca2+]i transient in response to

alteration of shear stress in HUVECs？

0

10 dyne/cm2

30s

Does knock down TRPP2 or TRPV4 affect increase in [NO]i in response to alteration of shear stress in HUVECs？

0

10 dyne/cm2

p-AMPK

AMPK

p-eNOS

e-NOS

Ctrl Scr ScrCtrl

Flow 10 min

p-AMPKα

AMPKα

Ctrl Scr Ctrl Scr Ctrl Scr

p-eNOS

e-NOS

V4-shRNA

P2-shRNA

P2/V4-shRNA

Flow 10 min

p-eNOS

e-NOS

PI3K inhibitor (+) (-) (+) (-) (+) (-)

Ctrl Scr Ctrl Scr Ctrl ScrP2-

shRNAV4-

shRNAP2/V4-shRNA

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

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 [Ca2+]i and NO production in response to

alternation of shear stress in HUVECs, via AMPK-p-

eNOS (?).

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