High Glucose Increases Lysyl Oxidase Expression and Activity in Retinal Endothelial Cells: Mechanism for Compromised Extracellular Matrix Barrier Function

Abstract

OBJECTIVEIn diabetes, retinal vascular basement membrane (BM) undergoes significant thickening and compromises vessel function including increased vascular permeability, a prominent lesion of early diabetic retinopathy. In this study we determined whether altered expression and activity of lysyl oxidase (LOX), a cross-linking enzyme, may compromise vascular basement membrane functional integrity under high-glucose (HG) conditions.RESEARCH DESIGN AND METHODSRat retinal endothelial cells (RRECs) grown in normal (5 mmol/l) or HG (30 mmol/l glucose) medium for 7 days were assessed for expression of LOX and proLOX by Western blot analysis and LOX enzyme activity. To determine whether HG alters cellular distribution patterns of LOX and proLOX, immunostaining with respective antibodies was performed. Similarly, cells grown in normal or HG medium were subjected to both LOX inhibition with β-aminopropionitrile (BAPN) and by small interfering RNA knockdown, and respectively examined for cell monolayer permeability. Additionally, retinas of streptozotocin (STZ)-induced diabetic rats were analyzed to determine if diabetes altered LOX expression.RESULTSWestern blot analysis revealed significantly increased LOX and proLOX expression in cells grown in HG medium compared with those grown in normal medium. The increased LOX level was strikingly similar to LOX upegulation in the diabetic retinas. In cells grown in HG medium, LOX activity and cell monolayer permeability was significantly increased, as were LOX and proLOX immunostaining. Small interfering RNA- or BAPN–induced-specific blockage of LOX expression or activity, respectively, reduced cell monolayer permeability.CONCLUSIONSHG-induced increased LOX expression and activity compromises barrier functional integrity, a prominent lesion of diabetic retinopathy.

Diabetes-related alterations in the enteric nervous system and its microenvironment

“…Most recent findings [187][188][189] provide a sufficient basis for the speculation that the different degrees of susceptibility of enteric neurons and microvessels to a pathological stimulus such as hyperglycemia might be related to the prevalence of bacteria in the different parts of the GI tract. Accordingly, the differences in prevalence of bacteria in different gut segments [188,190] are influenced by the oxygen supply of the small and large intestine [191] . Knowledge of species and functional composition of the gut microbiome is rapidly increasing thanks to technological advances in culture independent methods [189,[192][193][194][195] .…”

Animal Models of Diabetic Retinopathy

“…Chemically induced DM lacks specificity; has deleterious effects on the kidney, lung and liver; may cause tumors or necrosis; 90,91 and may lead to erroneous conclusions. Exposure to high concentration of glucose is widely used in cell-culture models of DM, [92][93][94] but there are no data showing that this method can be used to induce hyperglycemia in rodents. Most importantly, the immersion-induced diabetic zebrafish model mimics the fluctuations in blood glucose levels of human diabetic patients, who respond to dietary changes and insulin injections.…”

Blood Brain Barrier Injury in Diabetes: Unrecognized Effects on Brain and Cognition

“…Transcription and expression of the upstream transcription factor, hypoxia inducible factor 1 (HIF-1), is elevated by HG in mouse BMVECs and associated with down regulation of occludin and ZO-1 (Chronopoulos et al, 2010; Tien et al, 2014). A blockade of VEGF in these cells ameliorated the downstream impact on TJs, suggesting that VEGF is produced by BMVECs in response to HIF-1 and acts in an autocrine/paracrine fashion to increase BBB permeability (Figure 2).…”

“…Therefore, Cx43 down-regulation may contribute to the dysfunction of the blood-retina barrier through vascular permeability in diabetic retinopathy. Though this effect is best characterized in retinal tissue, several studies have demonstrated a significant role for Cx43 in regulation of TJ proteins, such as occludin, at the BBB (Chronopoulos et al, 2010), and ZO-1 in kidney (Xie et al, 2013). …”