Abstract
The disruption of microvascular barrier in response to advanced glycation end products (AGEs) stimulation contributes to vasculopathy associated with diabetes mellitus. Here, to study the role of Src and its association with moesin, VE-cadherin and focal adhesion kinase (FAK) in AGE-induced vascular hyperpermeability, we verified that AGE induced phosphorylation of Src, causing increased permeability in HUVECs. Cells over-expressed Src displayed a higher permeability after AGE treatment, accompanied with more obvious F-actin rearrangement. Activation of Src with pcDNA3/flag-SrcY530F alone duplicated these effects. Inhibition of Src with siRNA, PP2 or pcDNA3/flag-SrcK298M abolished these effects. The pulmonary microvascular endothelial cells (PMVECs) isolated from receptor for AGEs (RAGE)-knockout mice decreased the phosphorylation of Src and attenuated the barrier dysfunction after AGE-treatment. In vivo study showed that the exudation of dextran from mesenteric venules was increased in AGE-treated mouse. This was attenuated in RAGE knockout or PP2-pretreated mice. Up-regulation of Src activity induced the phosphorylation of moesin, as well as activation and dissociation of VE-cadherin, while down-regulation of Src abolished these effects. FAK was also proved to interact with Src in HUVECs stimulated with AGEs. Our studies demonstrated that Src plays a critical role in AGE-induced microvascular hyperpermeability by phosphorylating moesin, VE-cadherin, and FAK respectively.
Highlights
Microvascular barrier dysfunction and endothelial hyperpermeability are the crucial events in the development of inflammatory diseases, such as trauma, ischemia-reperfusion injury, arteriosclerosis, and especially, diabetes mellitus (DM)
We have previously reported that advanced glycation end products (AGEs) exerted dose- and time-dependent effects on monolayer permeability of human dermal microvascular endothelial (HMVECs)[7]
human umbilical vein endothelial cells (HUVECs) were treated with[100] μ g/mL of AGE modified bovine serum albumin (AGE-BSA) for different duration, and endothelial monolayer permeability was evaluated by dextran trans-endothelial flux using permeability coefficient for dextran (Pd) and trans-endothelial electric resistance (TER)
Summary
Microvascular barrier dysfunction and endothelial hyperpermeability are the crucial events in the development of inflammatory diseases, such as trauma, ischemia-reperfusion injury, arteriosclerosis, and especially, diabetes mellitus (DM). The disrupted barrier in response to a variety of stimuli causes endothelial hyperpermeability, exudation of vascular contents and inflammatory factors, transmigration of inflammatory cells, resulting in tissue edema and organ dysfunction. Recent studies showed that SFK signaling is important in the regulation of microvascular barrier function and various endothelial responses to a diversity of inflammatory mediators[11,12]. Src is one of the most widely studied members in SFKs. Inhibition of Src has been reported to abolish the increases in albumin permeability caused by C5a-activated neutrophils, which indicates its significance in vascular hyper-permeability[15]. We demonstrated Src acted as a signaling node which transduced the signal of AGEs on the ligation of RAGE to moesin, VE-cadherin, and FAK, resulting in the disruption of endothelial barrier and the increase of vascular permeability
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