Abstract
Proliferative diabetic retinopathy (PDR) involves persistent, uncontrolled formation of premature blood vessels with reduced number of pericytes. Our previous work showed that advanced glycation endproducts (AGEs) induced angiogenesis in human umbilical vein endothelial cells, mouse retina, and aortic ring, which was associated with moesin phosphorylation. Here we investigated whether moesin phosphorylation may contribute to pericyte detachment and the development of PDR. Primary retinal microvascular pericytes (RMPs) were isolated, purified from weanling rats, and identified by cellular markers α-SMA, PDGFR-β, NG2, and desmin using immunofluorescence microscopy. Effects of AGE-BSA on proliferation and migration of RMPs were examined using CCK-8, wound healing, and transwell assays. Effects on moesin phosphorylation were examined using western blotting. The RMP response to AGE-BSA was also examined when cells expressed the non-phosphorylatable Thr558Ala mutant or phospho-mimicking Thr558Asp mutant of moesin or were treated with ROCK inhibitor Y27632. Colocalization and interaction between CD44, phospho-moesin, and F-actin were observed. Experiments with cultured primary RMPs showed that AGE-BSA inhibited the proliferation, enhanced the migration, and increased moesin phosphorylation in a dose- and time-dependent manner. AGE-BSA also triggered the rearrangement of F-actin and promoted the interaction of CD44 with phospho-moesin in RMPs. These effects were abrogated in cells expressing the non-phosphorylatable moesin mutant and the application of ROCK inhibitor Y27632 attenuated AGE-induced alteration in cultured RMPs by abolishing the phosphorylation of moesin. However, those AGE-induced pathological process occurred in RMPs expressed the phospho-mimicking moesin without AGE-BSA treatment. It is concluded that AGEs could activate ROCK to mediate moesin phosphorylation at Thr558, and resulting phospho-moesin interacts with CD44 to form CD44 cluster, which might stimulate the migration of RMPs and subsequent RMP detachment in microvessel. This pathway may provide new drug targets against immature neovessel formation in PDR.
Highlights
Diabetic retinopathy is one of the most common complications of diabetes, affecting roughly one third of adults with diabetes and causing a large proportion of cases of adult blindness [1,2,3]
ROCK inhibition significantly attenuated advanced glycation endproducts (AGEs)-induced moesin phosphorylation (Figure 4A) and migration (Figures 4B, C), without affecting cell viability in the presence or absence of AGE-bovine serum albumin (BSA) (Figure 4D). These results suggest that activation of RhoA/ROCK might play a critical role in AGE-induced moesin phosphorylation and retinal microvascular pericytes (RMPs) migration
Expression of Thr558Ala mutant of moesin prevented these AGE-induced reorganization of Factin, and similar results were observed when RMPs were treated with ROCK inhibitor Y27632. These results suggest that activation of RhoA/ROCK pathway and moesin phosphorylation at Thr558 are involved in AGE-induced pericyte mobility and subsequent migration
Summary
Diabetic retinopathy is one of the most common complications of diabetes, affecting roughly one third of adults with diabetes and causing a large proportion of cases of adult blindness [1,2,3]. The nonproliferative form of diabetic retinopathy involves microaneurysm formation and intraretinal hemorrhage but not abnormal retinal neovascularization. This form can progress to proliferative diabetic retinopathy (PDR), in which proliferation of endothelial cells leads to uncontrolled neovascularization and sprouts in the retina. This can lead to blood leakage from immature vessels into the vitreous, greatly increasing the probability of vision loss [4, 5]. Two sprouts join and initiate blood flow in the newly formed loop, and subsequent interactions between endothelial cells and pericytes trigger the construction of new basement membrane, leading to vessel maturation and stabilization [8, 9]. The resulting neovessels are malformed and show a markedly increased permeability and propensity to rupture [13], which triggers an even greater extent of aberrant angiogenesis in a vicious cycle
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