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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 AGEs on endothelial permeability.HUVECs were either (a,b) treated with100 μg/mL of AGEs for 2, 4, 6, or 8 h, or (c,d) stimulated with AGEs at 25, 50, 100, or 200 μg/mL for 8 h, and those incubated with culture medium were used as control. Permeability coefficient of the transflux of tracer FITC-dextran (Pd) and trans-endothelial electronic resistance (TER) were measured. n = 3, *P < 0.05 versus control.
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f1: Influences of AGEs on endothelial permeability.HUVECs were either (a,b) treated with100 μg/mL of AGEs for 2, 4, 6, or 8 h, or (c,d) stimulated with AGEs at 25, 50, 100, or 200 μg/mL for 8 h, and those incubated with culture medium were used as control. Permeability coefficient of the transflux of tracer FITC-dextran (Pd) and trans-endothelial electronic resistance (TER) were measured. n = 3, *P < 0.05 versus control.

Mentions: We have previously reported that AGEs exerted dose- and time-dependent effects on monolayer permeability of human dermal microvascular endothelial (HMVECs)7. Here, human umbilical vein endothelial cells (HUVECs) representing venous endothelial cells were used and the concentration- and time-dependent effects of AGE modified bovine serum albumin (AGE-BSA) on monolayer permeability were explored. Firstly, HUVECs were treated with100 μg/mL of 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). We found that Pd was gradually increased from 2 h to 8 h and the changes became significant at 6 h and 8 h (Fig. 1a). Accordingly, TER was gradually decreased with significant differences at 6 h and 8 h (Fig. 1b). These data indicated a time-dependent increase in endothelial permeability induced by AGE-BSA. When cells were incubated with different concentrations of AGE-BSA for 8 h, Pd was increased and TER was decreased gradually, with significant differences at 100 μg/mL and 200 μg/mL (Fig. 1c,d), indicating AGE-BSA induced endothelial hyperpermeability in a dose-dependent fashion. These data were important since the generation of AGEs was increased with aging and elevating of blood glucose.


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 AGEs on endothelial permeability.HUVECs were either (a,b) treated with100 μg/mL of AGEs for 2, 4, 6, or 8 h, or (c,d) stimulated with AGEs at 25, 50, 100, or 200 μg/mL for 8 h, and those incubated with culture medium were used as control. Permeability coefficient of the transflux of tracer FITC-dextran (Pd) and trans-endothelial electronic resistance (TER) were measured. n = 3, *P < 0.05 versus control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Influences of AGEs on endothelial permeability.HUVECs were either (a,b) treated with100 μg/mL of AGEs for 2, 4, 6, or 8 h, or (c,d) stimulated with AGEs at 25, 50, 100, or 200 μg/mL for 8 h, and those incubated with culture medium were used as control. Permeability coefficient of the transflux of tracer FITC-dextran (Pd) and trans-endothelial electronic resistance (TER) were measured. n = 3, *P < 0.05 versus control.
Mentions: We have previously reported that AGEs exerted dose- and time-dependent effects on monolayer permeability of human dermal microvascular endothelial (HMVECs)7. Here, human umbilical vein endothelial cells (HUVECs) representing venous endothelial cells were used and the concentration- and time-dependent effects of AGE modified bovine serum albumin (AGE-BSA) on monolayer permeability were explored. Firstly, HUVECs were treated with100 μg/mL of 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). We found that Pd was gradually increased from 2 h to 8 h and the changes became significant at 6 h and 8 h (Fig. 1a). Accordingly, TER was gradually decreased with significant differences at 6 h and 8 h (Fig. 1b). These data indicated a time-dependent increase in endothelial permeability induced by AGE-BSA. When cells were incubated with different concentrations of AGE-BSA for 8 h, Pd was increased and TER was decreased gradually, with significant differences at 100 μg/mL and 200 μg/mL (Fig. 1c,d), indicating AGE-BSA induced endothelial hyperpermeability in a dose-dependent fashion. These data were important since the generation of AGEs was increased with aging and elevating of blood glucose.

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