Mechanobiology of Retinal Vascular Inflammation and Degeneration in Diabetes - PROJECT ABSTRACT Diabetic retinopathy (DR) is a major microvascular complication of diabetes and the leading cause of vison loss in the working-age population. Current treatments, which target only the advanced stages of DR, pose significant limitations and remain ineffective in many patients. Thus, there is a growing recognition that more effective DR management can be achieved by tackling the disease at the early stage. An important clinically-recognized hallmark of early DR is inflammation-mediated retinal vascular degeneration. Retinal endothelial activation (ICAM-1 upregulation) plays a crucial role in the development of these vascular abnormalities of early DR, but how retinal endothelial cells (ECs) become activated in diabetes is not fully understood. Our recent work has identified diabetes-induced retinal capillary stiffening as a crucial ‘mechanical’ determinant of endothelial ICAM-1 upregulation, vascular inflammation, and capillary degeneration. This retinal capillary stiffening is caused by excessive lysyl oxidase (LOX)-dependent crosslinking and stiffening of basement membrane collagen IV. This retinal capillary stiffening activates NF-κB via activation of mechanosensitive and proinflammatory small GTPase RhoA and its downstream effector ROCK. Our new preliminary data reveal that stiffness- dependent Rho/ROCK activation is associated with inhibition of upstream mechanosensitive Ca2+ channel transient receptor potential vanilloid 4 (TRPV4) that is known to localize within EC-matrix focal adhesions. Importantly, inhibition of retinal vascular (subendothelial matrix) stiffness rescues endothelial TRPV4 loss in diabetes, thus confirming the ability of TRPV4 to sense changes in matrix stiffness. Crucially, pharmacological activation of TRPV4 alone blocks retinal EC activation in diabetic mice. Based on these preliminary findings, we hypothesize that diabetes-induced retinal capillary stiffening inhibits TRPV4, leading to RhoA/ROCK/NF-kB activation, EC activation, and inflammation-mediated development of retinal vascular lesions of early DR. The objective of this proposal is to more deeply understand the role of mechanosensitive TRPV4 in retinal vascular inflammation and degeneration associated with early DR. To accomplish this goal, we will (i) characterize the effect of endothelial TRPV4 inhibition on retinal capillaries (Aim 1), (ii) identify the mechanotransduction pathway that mediates diabetes-induced inhibition of retinal endothelial TRPV4 (Aim 2), and (iii) determine the extent to which TRPV4 upregulation using pharmacological or gene therapy approaches rescues the retinal vascular abnormalities of early DR (Aim 3). Successful completion of this research will not only uncover the previously unknown mechanistic link between diabetes, retinal endothelial mechanobiology, vascular inflammation, and development of vascular lesions of early DR but, crucially, identify potentially new anti- inflammatory targets (e.g. TRPV4) for more effective DR therapies in the future.
