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

Retinal capillary degeneration is a clinical hallmark of the early stages of diabetic retinopathy (DR). Our recent studies have revealed that diabetes-induced increase in retinal capillary stiffness plays a crucial and previously unrecognized causal role in inflammation-mediated degeneration of retinal capillaries. Retinal capillary stiffening results from overexpression of lysyl oxidase, an enzyme that crosslinks and stiffens the subendothelial matrix. Since tackling DR at the early stage is expected to prevent or slow down DR progression and associated vision loss, subendothelial matrix and capillary stiffness represent relevant and novel therapeutic targets for early DR management. Further, direct measurement of retinal capillary stiffness can serve as a crucial preclinical validation step for the development of new imaging techniques for non-invasive assessment of retinal capillary stiffness in animal and human subjects. With this view in mind, we here provide a detailed protocol for the isolation and stiffness measurement of mouse retinal capillaries and retinal subendothelial matrix using atomic force microscopy.

Mechanical Regulation of Retinal Vascular Inflammation and Degeneration in Diabetes.

Vascular inflammation is known to cause degeneration of retinal capillaries in early diabetic retinopathy (DR), a major microvascular complication of diabetes. Past studies investigating these diabetes-induced retinal vascular abnormalities have focused primarily on the role of molecular or biochemical cues. Here we show that retinal vascular inflammation and degeneration in diabetes are also mechanically regulated by the increase in retinal vascular stiffness caused by overexpression of the collagen-cross-linking enzyme lysyl oxidase (LOX). Treatment of diabetic mice with LOX inhibitor ß-aminopropionitrile (BAPN) prevented the increase in retinal capillary stiffness, vascular intracellular adhesion molecule-1 overexpression, and leukostasis. Consistent with these anti-inflammatory effects, BAPN treatment of diabetic mice blocked the upregulation of proapoptotic caspase-3 in retinal vessels, which concomitantly reduced retinal capillary degeneration, pericyte ghost formation, and the diabetes-induced loss of contrast sensitivity in these mice. Finally, our in vitro studies indicate that retinal capillary stiffening is sufficient to increase the adhesiveness and neutrophil elastase-induced death of retinal endothelial cells. By uncovering a link between LOX-dependent capillary stiffening and the development of retinal vascular and functional defects in diabetes, these findings offer a new insight into DR pathogenesis that has important translational potential.

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

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 receptor for advanced glycation end products (RAGE). HRECs treated with methylglyoxal (MGO), an active precursor to the advanced glycation end product (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 AGE/RAGE, primarily through its ability to crosslink or 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. ARTICLE HIGHLIGHTS: We investigated the regulation and proinflammatory role of retinal endothelial lysyl oxidase (LOX) in diabetes. Findings reveal that LOX is upregulated by advanced glycation end products (AGE) and receptor for AGE (RAGE) and mediates AGE/RAGE-induced retinal endothelial cell activation and subendothelial matrix remodeling. We also show that LOX-dependent subendothelial matrix stiffening feeds back to enhance retinal endothelial RAGE. These findings implicate LOX as a key proinflammatory factor and an alternative (downstream) target to block AGE/RAGE signaling in diabetic retinopathy.

Inflammatory markers in relation to nonalcoholic fatty liver disease in urban South Indians.

OBJECTIVE: This study assessed the association of inflammatory markers, high-sensitivity C-reactive protein (hsCRP), and total leukocyte count with nonalcoholic fatty liver disease (NAFLD) in urban South Indians. SUBJECTS AND METHODS: We randomly selected subjects with and without NAFLD (n=100 each) from the Chennai Urban Rural Epidemiology Study conducted in Chennai in south India. NAFLD was diagnosed by ultrasonography. hsCRP was measured by nephelometry, and leukocyte count was measured by flow cytometry. Insulin resistance was analyzed by homeostasis assessment model using the following expression: fasting insulin (µIU/mL)×fasting glucose (mmol/L)/22.5. RESULTS: Mean hsCRP values were significantly higher in subjects with NAFLD compared with those without (4.2±1.2 mg/L vs. 2.2±0.4 mg/L; P<0.001). Leukocyte count was also higher in subjects with NAFLD compared with those without (7.8±1.4×10(3)/µL vs 6.9±0.9×10(3)/µL, P<0.001). Both hsCRP (P<0.001) and leukocyte count (P<0.001) increased with increasing severity of NAFLD. Multiple logistic regression analysis was done using NAFLD as the dependent variable and hsCRP and leukocyte count as independent variables. Both hsCRP (odds ratio 1.293, 95% confidence interval 1.13-1.470, P<0.001) and leukocyte count (odds ratio 1.293, 95% confidence interval 1.069-1.564, P=0.008) had a significant association with NAFLD even after adjusting for waist circumference, insulin resistance, serum triglycerides, and presence of type 2 diabetes. CONCLUSIONS: hsCRP and leukocyte count are associated with NAFLD after adjusting for conventional cardiometabolic risk factors.