Abstract
Purpose:
Inflammation of retinal capillaries, which is characterized by leukostasis and hyperpermeability, is a critical early event in the pathogenesis of diabetic retinopathy (DR). However, the mechanism by which diabetic retinal endothelial inflammation is regulated is not fully understood. Since diabetes is associated with stiffening and inflammation of non-retinal vessels such as aorta, we hypothesize that retinal capillaries also undergo excessive high glucose-induced stiffening that, in turn, alters endothelial mechanobiology and promotes inflammation.
Methods:
RF/6A chorioretinal endothelial cells (ECs) were treated with normal glucose (NG; 5.5 mM) or high glucose (HG; 30 mM), followed by stiffness measurement of EC and EC-secreted basement membrane (BM) using atomic force microscope. Changes in BM stiffness were confirmed by measurement of the expression and activity of lysyl oxidase (LOX), a BM crosslinking (stiffening) enzyme. Activity of endothelial mechanosensitive ion channel (MIC) was evaluated by measurement of calcium influx. Endothelial inflammation was assessed by monocyte-EC adhesion, intracellular NO production, NF-κB activation and ICAM-1 expression. Endothelial LOX activity was pharmacologically inhibited by BAPN. To further confirm the role of BM stiffening in retinal endothelial inflammation, ECs were grown on synthetic matrices of low and high stiffness, and levels of NO and monocyte-EC adhesion analyzed.
Results:
Our in vitro findings indicate that HG treatment leads to retinal EC and LOX-mediated BM stiffening that correlates with impaired MIC activity and NO production, enhanced NF-kB activity and ICAM-1 expression, and significantly greater monocyte-EC adhesion. Remarkably, inhibition of LOX activity alone caused significant reduction of HG-induced EC and BM stiffening, recovery of endothelial MIC activity and NO production, and marked suppression of NF-κB activation and monocyte-EC adhesion. Our studies using synthetic BM further confirmed that BM stiffening alone is necessary and sufficient to induce retinal endothelial inflammation. Importantly, pharmacological modulation of MIC activity reversed the effects of BM stiffness on ECs.
Conclusions:
These findings reveal a hitherto unknown role of LOX-mediated BM stiffening in diabetic retinal endothelial inflammation and implicate LOX and endothelial MIC as new targets for effective DR therapies.