Activation of mechanosensitive TRPV4 channel inhibits retinal vascular inflammation in diabetes

Abstract

Purpose : Endothelial cell (EC) activation is a major determinant of retinal vascular inflammation, a hallmark of early diabetic retinopathy (DR). We recently discovered retinal capillary ‘stiffening’ in diabetic mice that contributes greatly to EC activation. However, the mechanisms underlying this mechanical control of retinal EC activation remains unknown. Since mechanosensitive transient potential vanilloid type 4 (TRPV4) channel is known to activate anti-inflammatory endothelial nitric oxide synthase (eNOS) in retinal ECs, we here examined its role in the mechanical control of retinal EC activation in early DR.

Methods : Mildly-fixed or unfixed retinal vessels were isolated from mice (6-10/group) viz. nondiabetic (ND), diabetic (D), and D+GSK1016790A (TRVP4 agonist, 10 μg/kg, every 3d for 10 wks) for stiffness measurements using an atomic force microscope (AFM) or RT-qPCR analysis, respectively. Leukostasis was determined following FITC-Con A injection. To assess TRPV4 levels, human or mouse retinal ECs (HREC or mREC) treated with normal (NG, 5.5 mM) or high (HG 30 mM, 10d) glucose were used for RT-qPCR analysis, Ca²⁺ microfluorimetry and patch-clamp assay. EC activation was assessed by measuring ICAM-1 and eNOS mRNA, intracellular NO, and monocyte-EC adhesion in the presence or absence of TRPV4 agonist GSK (300 nM) or antagonist HC-06 (20 μM).

Results : Our findings reveal that ‘D’ mice exhibit significantly stiffer retinal capillaries (by ~3-fold) that correlates with EC activation markers viz., ICAM1 upregulation, leukostasis, and eNOS inactivation. Ca²⁺ microfluorimetry and patch-clamp assay reveal that mouse RECs express functional TRPV4, whose levels are significantly reduced in both ‘D’ mice and HG-treated retinal EC cultures. Importantly, administration of TRPV4 agonist GSK in ‘D’ mice or HG-treated retinal ECs mitigated the loss of retinal vascular TRPV4 that, in turn, inhibited retinal vascular inflammation. Finally, our in vivo and in vitro studies confirmed that downregulation of mechanosensitive TRPV4 in retinal ECs is caused by retinal capillary stiffening.

Conclusions : These findings implicate mechanosensitive TRPV4 channel as a crucial mechanical regulator of retinal vascular inflammation in diabetes and provide the basis to examine this pathway as a novel anti-inflammatory strategy for effective DR management.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.