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Kaustabh Ghosh, Xiao Yang, Arun Bhaskaran, Arup Das, Timothy S Kern; Molecular mechanisms underlying the mechanical control of retinal endothelial activation associated with diabetes. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3202.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal endothelial activation is a hallmark of inflammation associated with diabetic retinopathy (DR). We have recently shown that high glucose (HG) increases lysyl oxidase (LOX)-dependent stiffness of retinal capillary basement membrane that, in turn, promotes retinal endothelial cell (EC) activation. Here we aim to uncover the underlying mechanotransduction pathway mediating this BM stiffening-dependent mechanical control of diabetic retinal endothelial activation, with a particular emphasis on the role of Rho/ROCK and mechanosensitive ion channel Transient Receptor Potential Vanilloid 4 (TRPV4), key mediators of cellular mechanotransduction.
Retinas and retinal capillaries were isolated from control and streptozotocin-induced diabetic mice for detection of ROCK and TRPV4 expression by Western Blotting (WB). Human retinal EC cultures were treated with normal glucose (5.5 mM) or high glucose (HG; 30 mM) for 10d and Rho and TRPV4 activities were assessed by G-LISA assay and Ca2+ microfluorimetry, respectively. TRPV4 expression was measured by WB. EC activation was assessed by measuring intracellular NO production, ICAM-1 expression, and monocyte-EC adhesion. TRPV4 and ROCK-2 activities were modulated using selective pharmacological antagonist and agonist.
Our findings reveal that diabetic retinal inflammation in vivo correlates with significant increase in retinal capillary stiffness, increased LOX expression, and significantly impaired TRPV4 expression. In vitro, HG treatment enhances LOX-dependent stiffening of retinal subendothelial matrix and ECs, which correlate strongly with increased Rho/ROCK expression and activity. Notably, these effects correlated with significant decrease in TRPV4 expression and activity. Importantly, pharmacological enhancement of TRPV4 or suppression of Rho/ROCK alone suppressed the inflammatory effects of HG-induced matrix and EC stiffening in vitro.
These findings reveal a critical role of Rho/ROCK/TRPV4-dependent endothelial mechanotransduction in the mechanical control of retinal inflammation in DR pathogenesis and provide the basis to examine this pathway as a novel anti-inflammatory strategy for effective DR management.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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