VEGF Receptor-2-Linked PI3K/Calpain/SIRT1 Activation Mediates Retinal Arteriolar Dilations to VEGF and Shear Stress.
OBJECTIVE
Vasomotor responses of retinal arterioles to luminal flow/shear stress and VEGF have a critical role in governing retinal blood flow possibly via nitric oxide synthase (NOS) activation. However, the cellular mechanism for flow-sensitive vasomotor activity in relation to VEGF signaling in retinal arterioles has not been characterized. We used an isolated vessel approach to specifically address this issue.


METHODS
Porcine retinal arterioles were isolated, cannulated, and pressurized to 55 cm H2O luminal pressure by two independent reservoir systems. Luminal flow was increased stepwise by creating hydrostatic pressure gradients across two reservoirs. Diameter changes and associated signaling mechanisms corresponding to increased flow and VEGF receptor 2 (VEGFR2) activation were assessed using videomicroscopic, pharmacological, and molecular tools.


RESULTS
Retinal arterioles developed basal tone under zero-flow condition and dilated concentration-dependently to VEGF165. Stepwise increases in flow produced graded vasodilation. Vasodilations to VEGF165 and increased flow were abolished by endothelial removal, and inhibited by pharmacological blockade of VEGFR2, NOS, phosphoinositide 3-kinase (PI3K), calpains, or sirtuin-1 (SIRT1) deacetylase. A VEGF165 antibody blocked vasodilation to VEGF165 but not flow. Immunostaining indicated that VEGFR2 was expressed in the endothelial and smooth muscle layers of retinal arterioles.


CONCLUSIONS
Ligand-dependent and ligand-independent activation of VEGFR2 in the endothelium mediates NO-dependent dilations of porcine retinal arterioles in response to VEGF165 and luminal flow/shear stress, respectively. It appears that NOS stimulation via PI3K, calpain proteases, and SIRT1-dependent deacetylation downstream from VEGFR2 activation contributes to these vasodilator responses.
Department of Surgery Scott & White Eye Institute, College of Medicine, Texas A&M Health Science Center, Temple, Texas, United States 2Department of Ophthalmology, Scott & White Eye Institute, College of Medicine, Texas A&M Health Science Center, Temple.
Travis W
TW
Hein
Department of Surgery Scott & White Eye Institute, College of Medicine, Texas A&M Health Science Center, Temple, Texas, United States 2Department of Ophthalmology, Scott & White Eye Institute, College of Medicine, Texas A&M Health Science Center, Temple.
Robert H
RH
Rosa
Department of Surgery Scott & White Eye Institute, College of Medicine, Texas A&M Health Science Center, Temple, Texas, United States.
Yi
Y
Ren
Department of Surgery Scott & White Eye Institute, College of Medicine, Texas A&M Health Science Center, Temple, Texas, United States.
Wenjuan
W
Xu
Department of Surgery Scott & White Eye Institute, College of Medicine, Texas A&M Health Science Center, Temple, Texas, United States 2Department of Ophthalmology, Scott & White Eye Institute, College of Medicine, Texas A&M Health Science Center, Temple.
Lih
L
Kuo
D000818:Animals; D001160:Arterioles / Q000503:physiopathology; D015153:Blotting, Western; D002154:Calpain / Q000096:biosynthesis / Q000235:genetics*; D004195:Disease Models, Animal; D005260:Female; D005786:Gene Expression Regulation; D007150:Immunohistochemistry; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000503:physiopathology; D019869:Phosphatidylinositol 3-Kinases / Q000096:biosynthesis / Q000235:genetics*; D012313:RNA / Q000235:genetics; D012164:Retinal Diseases / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D012171:Retinal Vessels / Q000378:metabolism / Q000503:physiopathology; D056564:Sirtuin 1 / Q000235:genetics*; D013314:Stress, Mechanical; D013552:Swine; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000096:biosynthesis / Q000235:genetics; D014664:Vasodilation / Q000502:physiology*
D012313:RNA; D019869:Phosphatidylinositol 3-Kinases; D040301:Vascular Endothelial Growth Factor Receptor-2; D002154:Calpain; C447939:SIRT1 protein, human; D056564:Sirtuin 1