Rho/ROCK-mediated Retinal Endothelial Stiffening Impairs TRPV4 Signaling and Promotes Diabetic Retinal Inflammation

Abstract

Purpose : Retinal inflammation is a hallmark of diabetic retinopathy (DR). We have recently shown that loss of mechanosensitive ion channel Transient Receptor Potential Vanilloid 4 (TRPV4) activity contributes to hyperglycemia-induced retinal endothelial cell (EC) activation, a critical early event in diabetic retinal inflammation. However, the mechanotransduction pathway involved in TRPV4-mediated retinal EC activation remains unknown. Since Rho is a key molecular player in mechanotransduction and increased Rho/ROCK promotes EC activation by suppressing endothelial nitric oxide (NO) production, we here examined the role of Rho/ROCK in TRPV4-mediated diabetic retinal EC activation.

Methods : Retinal capillaries were isolated from control and streptozotocin-induced diabetic mice for detection of ROCK expression by Western Blot (WB). Human retinal EC cultures were treated with normal glucose (5.5 mM) or high glucose (HG; 30 mM) for 10d and Rho activity was assessed by G-LISA assay. Stiffness of isolated mouse retinal capillaries and cultured human ECs, which is directly regulated by Rho/ROCK, was measured by atomic force microscopy. EC activation was assessed by measuring the levels of intracellular NO, ICAM-1 expression, and monocyte-EC adhesion. Expression and activity of TRPV4 was determined by WB and Ca²⁺ microfluorimetry, respectively. TRPV4 and ROCK activities were modulated using selective pharmacological antagonist and agonist.

Results : Diabetic mouse retinal capillaries exhibited significantly greater ROCK expression and stiffness, which correlated with Rho/ROCK activation and stiffening of HG-treated human retinal ECs. The HG-treated stiffer ECs underwent significant activation, which was consistent with a marked decrease in expression and activity of TRPV4. Importantly, inhibition of ROCK activity alone caused significant recovery of TRPV4 expression/activity, endothelial NO production and suppression of monocyte-EC adhesion. Conversely, pharmacological enhancement of TRPV4 suppressed Rho-mediated EC stiffening and the inflammatory effects in vitro.

Conclusions : These findings indicate that Rho/ROCK acts as both an effector and target of TRPV4 and implicate Rho/ROCK/TRPV4 axis as a crucial regulator of diabetic retinal inflammation associated with DR.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.