Subendothelial Matrix Stiffening by Lysyl Oxidase Enhances RAGE-Mediated Retinal Endothelial Activation in Diabetes

Abstract

Endothelial cell (EC) activation is a crucial determinant of retinal vascular inflammation associated with diabetic retinopathy (DR), a major microvascular complication of diabetes. We previously showed that, similar to abnormal biochemical factors, aberrant mechanical cues in the form of lysyl oxidase (LOX)-dependent subendothelial matrix stiffening also contribute significantly to retinal EC activation in diabetes. Yet, how LOX is itself regulated and precisely how it mechanically controls retinal EC activation in diabetes is poorly understood. Here we show that high glucose-induced LOX upregulation in human retinal ECs (HRECs) is mediated by proinflammatory RAGE (receptor for advanced glycation end products/AGEs). HRECs treated with methylglyoxal (MGO), an active precursor to the AGE MG-H1, exhibited LOX upregulation that was blocked by a RAGE inhibitor, thus confirming the ability of RAGE to promote LOX expression. Crucially, as a downstream effector of RAGE, LOX was found to mediate both the proinflammatory and matrix remodeling effects of MGO/RAGE, primarily through its ability to crosslink/stiffen matrix. Finally, using decellularized HREC-derived matrices and a mouse model of diabetes, we demonstrate that LOX-dependent matrix stiffening feeds back to enhance RAGE, thereby achieving its autoregulation and proinflammatory effects. Collectively, these findings provide fresh mechanistic insights into the regulation and proinflammatory role of LOX-dependent mechanical cues in diabetes while simultaneously implicating LOX as an alternative (downstream) target to block AGE/RAGE signaling in DR.

Stiffness Measurement of Retinal Capillaries and Subendothelial Matrix using Atomic Force Microscopy

“…3B) . As we have previously reported, stiffness (Young’s modulus) of subendothelial matrices obtained from retinal EC cultures is also calculated in the aforementioned manner 5,6 .…”

“…This protocol, adapted from the Bruker NanoWizard® Series user manual and reported in our recent studies 4,5 , details the acquisition and analysis of AFM stiffness data from retinal capillaries and subendothelial matrix using Bruker’s NanoWizard® 4 XP Bioscience AFM. Although the steps outlined below are based on this specific model of AFM, the underlying principles are generally applicable to all AFM models.…”

“…This protocol, adapted from the Bruker NanoWizard® Series user manual and reported in our recent studies 4,5 , details the acquisition and analysis of AFM stiffness data from retinal capillaries and subendothelial matrix using Bruker's NanoWizard® 4 XP Bioscience AFM.…”

“…Specifically, the diabetes-induced increase in retinal capillary stiffness is caused by upregulation of collagen crosslinking enzyme lysyl oxidase (LOX) in retinal endothelial cells (ECs), which stiffens the subendothelial matrix (basement membrane) [4][5][6] . Matrix stiffening, in turn, stiffens the overlying retinal ECs (mechanical reciprocity), thus leading to the overall increase in retinal capillary stiffness 4 .…”

“…Vascular inflammation contributes significantly to retinal capillary degeneration in DR. Past studies have demonstrated an important role for aberrant molecular and biochemical cues in diabetes-induced retinal vascular inflammation 2,3 . However, our recent work has introduced a new paradigm for DR pathogenesis that identifies retinal capillary stiffening as a crucial yet previously unrecognized determinant of retinal vascular inflammation and degeneration [4][5][6] .…”