mechanosensitive properties in endothelium and their roles in regulation of endothelial function...
TRANSCRIPT
![Page 1: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/1.jpg)
Mechanosensitive properties in endothelium and their roles in
regulation of endothelial function
Zhi-Ren Zhang M.D., Ph.D.
Harbin Medical University
![Page 2: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/2.jpg)
Sensors for shear stress on vascular Sensors for shear stress on vascular endothelial cellsendothelial cells
Kimiko Yamamoto. Circulation Journal. 2009
![Page 3: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/3.jpg)
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?
![Page 4: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/4.jpg)
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?
![Page 5: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/5.jpg)
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?
![Page 6: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/6.jpg)
Is TRPP2 localized in the cilia of vascular endothelial cells?
Human
Mouse Wissam A. et al. Cellular biology.2009
![Page 7: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/7.jpg)
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
![Page 8: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/8.jpg)
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
![Page 9: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/9.jpg)
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?
![Page 10: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/10.jpg)
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?
![Page 11: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/11.jpg)
Are cilia involved in [Ca2+]i increase in response
to alteration of shear stress in HUVECs?
30s0
10 dyne/cm2
![Page 12: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/12.jpg)
Is cilium involved in [NO]i increase in response
to alteration of shear stress?
0
10 dyne/cm2
~75s
![Page 13: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/13.jpg)
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). ).
![Page 14: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/14.jpg)
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
![Page 15: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/15.jpg)
C
Does TRPP2 or TRPV4 express in cilia of HUVECs?
![Page 16: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/16.jpg)
TRPP2
TRPV4
Merged
cilium
Are TRPP2 and TRPV4 co-localized on cilium?
![Page 17: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/17.jpg)
Are TRPP2 and TRPV4 co-localized on human mesenteric vascular endothelial cells?
![Page 18: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/18.jpg)
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?
![Page 19: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/19.jpg)
qtRT-PCR demonstrating gene silencing efficiency
![Page 20: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/20.jpg)
TRPP2
β-actin
TRPV4
β-actin
Ctrl Scr P2-shRNA Ctrl Scr P2/V4-shRNA
Ctrl Scr V4-shRNA Ctrl Scr P2/V4-shRNA
![Page 21: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/21.jpg)
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
![Page 22: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/22.jpg)
Does knock down TRPP2 or TRPV4 affect increase in [NO]i in response to alteration of shear stress in HUVECs?
0
10 dyne/cm2
![Page 23: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/23.jpg)
p-AMPK
AMPK
p-eNOS
e-NOS
Ctrl Scr ScrCtrl
Flow 10 min
![Page 24: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/24.jpg)
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
![Page 25: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/25.jpg)
![Page 26: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/26.jpg)
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
![Page 27: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/27.jpg)
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 (?).
![Page 28: Mechanosensitive properties in endothelium and their roles in regulation of endothelial function Zhi-Ren Zhang M.D., Ph.D. Harbin Medical University](https://reader035.vdocument.in/reader035/viewer/2022062322/56649eb55503460f94bbe522/html5/thumbnails/28.jpg)
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