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Role of Src in Vascular Hyperpermeability Induced by Advanced Glycation End Products.

Zhang W, Xu Q, Wu J, Zhou X, Weng J, Xu J, Wang W, Huang Q, Guo X - Sci Rep (2015)

Bottom Line: Activation of Src with pcDNA3/flag-Src(Y530F) alone duplicated these effects.Inhibition of Src with siRNA, PP2 or pcDNA3/flag-Src(K298M) abolished these effects.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology, Key Laboratory for Shock and Microcirculation Research of Guangdong Province, Southern Medical University, Guangzhou 510515, China.

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-Src(Y530F) alone duplicated these effects. Inhibition of Src with siRNA, PP2 or pcDNA3/flag-Src(K298M) 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.

No MeSH data available.


Related in: MedlinePlus

AGE-induced moesin activation requires Src.(a) Pretreatment of PP2 prevented AGE-induced moesin phosphorylation. ECs were pretreated with PP2 (15 μmol/L) for 90 min before exposed to 100 μg/mL AGEs for 1 h. (b) Pretreatment of Src siRNA prevented AGE-induced moesin phosphorylation. ECs were pretreated with Src siRNA or control siRNA for 48 h before exposed to 100 μg/mL AGEs for 1 h. (c) Effects of K298M and Y530F on AGE-induced moesin phosphorylation. ECs were transfected with K298M and Y530F for 48 h before exposed to 100 μg/mL AGEs for 1 h. Moesin and phosphorylated moesin were determined by western blotting. n = 3, *P < 0.05 versus control, #P < 0.05 versus AGEs or Mock + AGEs.
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f6: AGE-induced moesin activation requires Src.(a) Pretreatment of PP2 prevented AGE-induced moesin phosphorylation. ECs were pretreated with PP2 (15 μmol/L) for 90 min before exposed to 100 μg/mL AGEs for 1 h. (b) Pretreatment of Src siRNA prevented AGE-induced moesin phosphorylation. ECs were pretreated with Src siRNA or control siRNA for 48 h before exposed to 100 μg/mL AGEs for 1 h. (c) Effects of K298M and Y530F on AGE-induced moesin phosphorylation. ECs were transfected with K298M and Y530F for 48 h before exposed to 100 μg/mL AGEs for 1 h. Moesin and phosphorylated moesin were determined by western blotting. n = 3, *P < 0.05 versus control, #P < 0.05 versus AGEs or Mock + AGEs.

Mentions: Since moesin regulates endothelial permeability via modulating equilibrium between contractile forces generated by the endothelial cytoskeleton and adhesive forces produced at inter-endothelial junctions and cell-matrix attachment, here we tried to find out whether Src signaling influenced moesin phosphorylation. Consistent with our previous findings7, AGEs induced a significant increase in moesin phosphorylation. However, this increase was abolished by Src inhibitor PP2 or its siRNA (Fig. 6a,b). We also observed that AGE-induced moesin phosphorylation was decreased by K298M, while further increased by Y530F (Fig. 6c,d). These data suggested that Src signaling mediated AGE-induced moesin phosphorylation.


Role of Src in Vascular Hyperpermeability Induced by Advanced Glycation End Products.

Zhang W, Xu Q, Wu J, Zhou X, Weng J, Xu J, Wang W, Huang Q, Guo X - Sci Rep (2015)

AGE-induced moesin activation requires Src.(a) Pretreatment of PP2 prevented AGE-induced moesin phosphorylation. ECs were pretreated with PP2 (15 μmol/L) for 90 min before exposed to 100 μg/mL AGEs for 1 h. (b) Pretreatment of Src siRNA prevented AGE-induced moesin phosphorylation. ECs were pretreated with Src siRNA or control siRNA for 48 h before exposed to 100 μg/mL AGEs for 1 h. (c) Effects of K298M and Y530F on AGE-induced moesin phosphorylation. ECs were transfected with K298M and Y530F for 48 h before exposed to 100 μg/mL AGEs for 1 h. Moesin and phosphorylated moesin were determined by western blotting. n = 3, *P < 0.05 versus control, #P < 0.05 versus AGEs or Mock + AGEs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585381&req=5

f6: AGE-induced moesin activation requires Src.(a) Pretreatment of PP2 prevented AGE-induced moesin phosphorylation. ECs were pretreated with PP2 (15 μmol/L) for 90 min before exposed to 100 μg/mL AGEs for 1 h. (b) Pretreatment of Src siRNA prevented AGE-induced moesin phosphorylation. ECs were pretreated with Src siRNA or control siRNA for 48 h before exposed to 100 μg/mL AGEs for 1 h. (c) Effects of K298M and Y530F on AGE-induced moesin phosphorylation. ECs were transfected with K298M and Y530F for 48 h before exposed to 100 μg/mL AGEs for 1 h. Moesin and phosphorylated moesin were determined by western blotting. n = 3, *P < 0.05 versus control, #P < 0.05 versus AGEs or Mock + AGEs.
Mentions: Since moesin regulates endothelial permeability via modulating equilibrium between contractile forces generated by the endothelial cytoskeleton and adhesive forces produced at inter-endothelial junctions and cell-matrix attachment, here we tried to find out whether Src signaling influenced moesin phosphorylation. Consistent with our previous findings7, AGEs induced a significant increase in moesin phosphorylation. However, this increase was abolished by Src inhibitor PP2 or its siRNA (Fig. 6a,b). We also observed that AGE-induced moesin phosphorylation was decreased by K298M, while further increased by Y530F (Fig. 6c,d). These data suggested that Src signaling mediated AGE-induced moesin phosphorylation.

Bottom Line: Activation of Src with pcDNA3/flag-Src(Y530F) alone duplicated these effects.Inhibition of Src with siRNA, PP2 or pcDNA3/flag-Src(K298M) abolished these effects.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology, Key Laboratory for Shock and Microcirculation Research of Guangdong Province, Southern Medical University, Guangzhou 510515, China.

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-Src(Y530F) alone duplicated these effects. Inhibition of Src with siRNA, PP2 or pcDNA3/flag-Src(K298M) 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.

No MeSH data available.


Related in: MedlinePlus