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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

Influences of Src signaling on endothelial permeability.(a) PP2 prevented AGE-induced hyperpermeability. HUVECs were treated with PP2 90 min before AGEs (100 μg/mL, 8 h) application. Culture medium was used as control. n = 3 *P < 0.05 versus control, #P < 0.05 versus AGEs. (b) Src siRNA caused reduction in Src protein level compared with control siRNA. HUVECs were transfected with Src siRNA and control siRNA for 48 h, after which cells were lysed and detected for Src protein level by WB. (c) Src siRNA prevented AGE-induced hyper-permeability. HUVECs were transfected with siRNA for 48 h before exposure to AGEs (100 μg/mL) for 8 h. n = 3, *P < 0.05 versus control, #P < 0.05 versus control siRNA with AGEs. (d,f) The effects of pcDNA3/flag-Src (WT-Src), K298M and Y530F on expression of Src were detected by western blotting. (e,g,h) The effects of WT-Src, K298M and Y530F on EC permeability. Cells were transfected with WT-Src, K298M and Y530F with or without stimulation of 100 μg/mL AGEs for 8 h. TER and Pd were detected. n = 3, *P < 0.05 versus control or mock, #P < 0.05 versus AGEs or mock with AGEs. (i) The effects of Src on the distribution of F-actin induced by AGEs. ECs were treated with or without 100 μg/mL AGEs for 8 h, followed by rhodamine-phalloidin staining, and F-actin was revealed by a laser confocal microscopy.
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f3: Influences of Src signaling on endothelial permeability.(a) PP2 prevented AGE-induced hyperpermeability. HUVECs were treated with PP2 90 min before AGEs (100 μg/mL, 8 h) application. Culture medium was used as control. n = 3 *P < 0.05 versus control, #P < 0.05 versus AGEs. (b) Src siRNA caused reduction in Src protein level compared with control siRNA. HUVECs were transfected with Src siRNA and control siRNA for 48 h, after which cells were lysed and detected for Src protein level by WB. (c) Src siRNA prevented AGE-induced hyper-permeability. HUVECs were transfected with siRNA for 48 h before exposure to AGEs (100 μg/mL) for 8 h. n = 3, *P < 0.05 versus control, #P < 0.05 versus control siRNA with AGEs. (d,f) The effects of pcDNA3/flag-Src (WT-Src), K298M and Y530F on expression of Src were detected by western blotting. (e,g,h) The effects of WT-Src, K298M and Y530F on EC permeability. Cells were transfected with WT-Src, K298M and Y530F with or without stimulation of 100 μg/mL AGEs for 8 h. TER and Pd were detected. n = 3, *P < 0.05 versus control or mock, #P < 0.05 versus AGEs or mock with AGEs. (i) The effects of Src on the distribution of F-actin induced by AGEs. ECs were treated with or without 100 μg/mL AGEs for 8 h, followed by rhodamine-phalloidin staining, and F-actin was revealed by a laser confocal microscopy.

Mentions: To further clarify the role of Src in AGE-induced endothelial barrier disruption, HUVECs were pretreated with a pan inhibitor of Src, PP2, for 90 min before stimulation with AGE-BSA. We revealed that PP2 significantly attenuated AGE-induced endothelial hyperpermeability (Fig. 3a), indicating that Src was involved in endothelial hyperpermeability induced by AGEs. To confirm this role of Src, cells were transfected with Src siRNA or its overexpression vector (WT-Src) before AGE-BSA stimulation. Knockdown of Src significantly decreased AGE-induced endothelial hyperpermeability, while Src overexpression obviously increased it (Fig. 3b,c). Moreover, Src overexpression alone increased endothelial permeability in cells without AGEs stimulation (Fig. 3d,e). To clarify whether the tyrosine kinase activity of Src was linked with AGE-induced endothelial hyperpermeability, we constructed a kinase-deficient mutant at Lys298 (K298M) and a constitutive active mutant at Tyr530 (Y530F) (Fig. 3f). We found that K298M did not change endothelial permeability in the absence of AGE-BSA, while K298M completely abolished endothelial hyperpermeability induced by AGEs (Fig. 3g). On the contrary, Y530F increased endothelial permeability in the absence of AGE-BSA, and further increased it in the presence of AGEs (Fig. 3h). These data suggest that Src activation mediated AGE-induced endothelial hyperpermeability and it was the tyrosine kinase activity of Src that triggered the downstream signaling events.


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)

Influences of Src signaling on endothelial permeability.(a) PP2 prevented AGE-induced hyperpermeability. HUVECs were treated with PP2 90 min before AGEs (100 μg/mL, 8 h) application. Culture medium was used as control. n = 3 *P < 0.05 versus control, #P < 0.05 versus AGEs. (b) Src siRNA caused reduction in Src protein level compared with control siRNA. HUVECs were transfected with Src siRNA and control siRNA for 48 h, after which cells were lysed and detected for Src protein level by WB. (c) Src siRNA prevented AGE-induced hyper-permeability. HUVECs were transfected with siRNA for 48 h before exposure to AGEs (100 μg/mL) for 8 h. n = 3, *P < 0.05 versus control, #P < 0.05 versus control siRNA with AGEs. (d,f) The effects of pcDNA3/flag-Src (WT-Src), K298M and Y530F on expression of Src were detected by western blotting. (e,g,h) The effects of WT-Src, K298M and Y530F on EC permeability. Cells were transfected with WT-Src, K298M and Y530F with or without stimulation of 100 μg/mL AGEs for 8 h. TER and Pd were detected. n = 3, *P < 0.05 versus control or mock, #P < 0.05 versus AGEs or mock with AGEs. (i) The effects of Src on the distribution of F-actin induced by AGEs. ECs were treated with or without 100 μg/mL AGEs for 8 h, followed by rhodamine-phalloidin staining, and F-actin was revealed by a laser confocal microscopy.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f3: Influences of Src signaling on endothelial permeability.(a) PP2 prevented AGE-induced hyperpermeability. HUVECs were treated with PP2 90 min before AGEs (100 μg/mL, 8 h) application. Culture medium was used as control. n = 3 *P < 0.05 versus control, #P < 0.05 versus AGEs. (b) Src siRNA caused reduction in Src protein level compared with control siRNA. HUVECs were transfected with Src siRNA and control siRNA for 48 h, after which cells were lysed and detected for Src protein level by WB. (c) Src siRNA prevented AGE-induced hyper-permeability. HUVECs were transfected with siRNA for 48 h before exposure to AGEs (100 μg/mL) for 8 h. n = 3, *P < 0.05 versus control, #P < 0.05 versus control siRNA with AGEs. (d,f) The effects of pcDNA3/flag-Src (WT-Src), K298M and Y530F on expression of Src were detected by western blotting. (e,g,h) The effects of WT-Src, K298M and Y530F on EC permeability. Cells were transfected with WT-Src, K298M and Y530F with or without stimulation of 100 μg/mL AGEs for 8 h. TER and Pd were detected. n = 3, *P < 0.05 versus control or mock, #P < 0.05 versus AGEs or mock with AGEs. (i) The effects of Src on the distribution of F-actin induced by AGEs. ECs were treated with or without 100 μg/mL AGEs for 8 h, followed by rhodamine-phalloidin staining, and F-actin was revealed by a laser confocal microscopy.
Mentions: To further clarify the role of Src in AGE-induced endothelial barrier disruption, HUVECs were pretreated with a pan inhibitor of Src, PP2, for 90 min before stimulation with AGE-BSA. We revealed that PP2 significantly attenuated AGE-induced endothelial hyperpermeability (Fig. 3a), indicating that Src was involved in endothelial hyperpermeability induced by AGEs. To confirm this role of Src, cells were transfected with Src siRNA or its overexpression vector (WT-Src) before AGE-BSA stimulation. Knockdown of Src significantly decreased AGE-induced endothelial hyperpermeability, while Src overexpression obviously increased it (Fig. 3b,c). Moreover, Src overexpression alone increased endothelial permeability in cells without AGEs stimulation (Fig. 3d,e). To clarify whether the tyrosine kinase activity of Src was linked with AGE-induced endothelial hyperpermeability, we constructed a kinase-deficient mutant at Lys298 (K298M) and a constitutive active mutant at Tyr530 (Y530F) (Fig. 3f). We found that K298M did not change endothelial permeability in the absence of AGE-BSA, while K298M completely abolished endothelial hyperpermeability induced by AGEs (Fig. 3g). On the contrary, Y530F increased endothelial permeability in the absence of AGE-BSA, and further increased it in the presence of AGEs (Fig. 3h). These data suggest that Src activation mediated AGE-induced endothelial hyperpermeability and it was the tyrosine kinase activity of Src that triggered the downstream signaling events.

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