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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

Involvement of RAGE in AGE-induced phosphorylation of Src and hyperpermeability.(a) Knockout of RAGE prevented AGE-induced PMVEC hyper-permeability. PMVECs of wild type mice and RAGE knockout mice were treated with or without 100 μg/mL AGEs for 90 min and TER was measured. (b) Knockout of RAGE prevented AGE-induced Src phosphorylation. PMVECs from wild type and RAGE knockout mice were treated with or without 100 μg/mL AGEs for 1 h. Phosphorylation of Src was detected by western blotting. The ratio of immunointensity between the phosphorylation of Src (p-Src 419) and β-actin were calculated. n = 3, *P < 0.05 versus wild type without AGEs, #P < 0.05 versus wild type with AGEs.
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f4: Involvement of RAGE in AGE-induced phosphorylation of Src and hyperpermeability.(a) Knockout of RAGE prevented AGE-induced PMVEC hyper-permeability. PMVECs of wild type mice and RAGE knockout mice were treated with or without 100 μg/mL AGEs for 90 min and TER was measured. (b) Knockout of RAGE prevented AGE-induced Src phosphorylation. PMVECs from wild type and RAGE knockout mice were treated with or without 100 μg/mL AGEs for 1 h. Phosphorylation of Src was detected by western blotting. The ratio of immunointensity between the phosphorylation of Src (p-Src 419) and β-actin were calculated. n = 3, *P < 0.05 versus wild type without AGEs, #P < 0.05 versus wild type with AGEs.

Mentions: To investigate whether RAGE is required for Src activation in AGE-induced endothelial dysfunction, pulmonary microvascular endothelial cells (PMVECs) isolated from wild type and RAGE knockout mice were used. Our data showed that the elevated endothelial permeability by AGEs was completely abolished by RAGE knockout (Fig. 4a). Moreover, AGE-induced Src phosphorylation was inhibited in RAGE-knockout cells (Fig. 4b). These results suggest that AGE-evoked Src activation and consequent endothelial hyperpermeability was through the ligation of RAGE.


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)

Involvement of RAGE in AGE-induced phosphorylation of Src and hyperpermeability.(a) Knockout of RAGE prevented AGE-induced PMVEC hyper-permeability. PMVECs of wild type mice and RAGE knockout mice were treated with or without 100 μg/mL AGEs for 90 min and TER was measured. (b) Knockout of RAGE prevented AGE-induced Src phosphorylation. PMVECs from wild type and RAGE knockout mice were treated with or without 100 μg/mL AGEs for 1 h. Phosphorylation of Src was detected by western blotting. The ratio of immunointensity between the phosphorylation of Src (p-Src 419) and β-actin were calculated. n = 3, *P < 0.05 versus wild type without AGEs, #P < 0.05 versus wild type with AGEs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Involvement of RAGE in AGE-induced phosphorylation of Src and hyperpermeability.(a) Knockout of RAGE prevented AGE-induced PMVEC hyper-permeability. PMVECs of wild type mice and RAGE knockout mice were treated with or without 100 μg/mL AGEs for 90 min and TER was measured. (b) Knockout of RAGE prevented AGE-induced Src phosphorylation. PMVECs from wild type and RAGE knockout mice were treated with or without 100 μg/mL AGEs for 1 h. Phosphorylation of Src was detected by western blotting. The ratio of immunointensity between the phosphorylation of Src (p-Src 419) and β-actin were calculated. n = 3, *P < 0.05 versus wild type without AGEs, #P < 0.05 versus wild type with AGEs.
Mentions: To investigate whether RAGE is required for Src activation in AGE-induced endothelial dysfunction, pulmonary microvascular endothelial cells (PMVECs) isolated from wild type and RAGE knockout mice were used. Our data showed that the elevated endothelial permeability by AGEs was completely abolished by RAGE knockout (Fig. 4a). Moreover, AGE-induced Src phosphorylation was inhibited in RAGE-knockout cells (Fig. 4b). These results suggest that AGE-evoked Src activation and consequent endothelial hyperpermeability was through the ligation of RAGE.

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