Limits...
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 VE-cadherin activation requires Src.(a) Pretreatment of PP2 prevented AGE-induced VE-cadherin 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 VE-cadherin phosphorylation. ECs were transfected with Src siRNA or control siRNA for 48 h before exposed to 100 μg/mL AGEs for 1 h. (c,d) Effects of K298M and Y530F on AGE-induced VE-cadherin phosphorylation. ECs were transfected with K298M or Y530F for 48 h before exposed to 100 μg/mL AGEs for 1 h. VE-cadherin and its phosphorylation form were determined by western blotting. The ratio of immunointensity between the phosphorylation of VE-cadherin (p-VE658) and β-actin were calculated. n = 3, *P < 0.05 versus control, #P < 0.05 versus AGEs or Mock + AGEs. (e) Down-regulation of Src prevented AGE-induced adherence junction dissociation. HUVECs were pretreated with PP2 (15 μmol/L) for 90 min or transfected with Src siRNA for 48 h, followed by incubation with or without 100 μg/mL AGEs for 8 h. Immunostaining for VE-cadherin (green) and DAPI (blue) were shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4585381&req=5

f7: AGE-induced VE-cadherin activation requires Src.(a) Pretreatment of PP2 prevented AGE-induced VE-cadherin 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 VE-cadherin phosphorylation. ECs were transfected with Src siRNA or control siRNA for 48 h before exposed to 100 μg/mL AGEs for 1 h. (c,d) Effects of K298M and Y530F on AGE-induced VE-cadherin phosphorylation. ECs were transfected with K298M or Y530F for 48 h before exposed to 100 μg/mL AGEs for 1 h. VE-cadherin and its phosphorylation form were determined by western blotting. The ratio of immunointensity between the phosphorylation of VE-cadherin (p-VE658) and β-actin were calculated. n = 3, *P < 0.05 versus control, #P < 0.05 versus AGEs or Mock + AGEs. (e) Down-regulation of Src prevented AGE-induced adherence junction dissociation. HUVECs were pretreated with PP2 (15 μmol/L) for 90 min or transfected with Src siRNA for 48 h, followed by incubation with or without 100 μg/mL AGEs for 8 h. Immunostaining for VE-cadherin (green) and DAPI (blue) were shown.

Mentions: Given that Src acted as an upstream of VE-cadherin and caused the disruption of endothelial barrier in response to permeability-increasing agents such as bradykinin and histamine17, we investigated whether Src influenced the distribution of VE-cadherin under AGE treatment. We revealed that AGEs induced the dissociation of VE-cadherin, which was prevented by inhibition of Src signaling with PP2 or Src siRNA (Fig. 7e). We also assessed the effects of Src on the phosphorylation of VE-cadherin. Our data showed that AGEs promoted VE-cadherin phosphorylation, which was blocked by PP2, Src siRNA (Fig. 7a,b), or K298M, but significantly increased by Y530F (Fig. 7c,d). Moreover, cells transfected with Y530F displayed a higher level of VE-cadherin phosphorylation compared with mock-transfected cells (Fig. 7d). These data suggest Src played a role in AGE-induced VE-cadherin phosphorylation and dissociation.


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 VE-cadherin activation requires Src.(a) Pretreatment of PP2 prevented AGE-induced VE-cadherin 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 VE-cadherin phosphorylation. ECs were transfected with Src siRNA or control siRNA for 48 h before exposed to 100 μg/mL AGEs for 1 h. (c,d) Effects of K298M and Y530F on AGE-induced VE-cadherin phosphorylation. ECs were transfected with K298M or Y530F for 48 h before exposed to 100 μg/mL AGEs for 1 h. VE-cadherin and its phosphorylation form were determined by western blotting. The ratio of immunointensity between the phosphorylation of VE-cadherin (p-VE658) and β-actin were calculated. n = 3, *P < 0.05 versus control, #P < 0.05 versus AGEs or Mock + AGEs. (e) Down-regulation of Src prevented AGE-induced adherence junction dissociation. HUVECs were pretreated with PP2 (15 μmol/L) for 90 min or transfected with Src siRNA for 48 h, followed by incubation with or without 100 μg/mL AGEs for 8 h. Immunostaining for VE-cadherin (green) and DAPI (blue) were shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: AGE-induced VE-cadherin activation requires Src.(a) Pretreatment of PP2 prevented AGE-induced VE-cadherin 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 VE-cadherin phosphorylation. ECs were transfected with Src siRNA or control siRNA for 48 h before exposed to 100 μg/mL AGEs for 1 h. (c,d) Effects of K298M and Y530F on AGE-induced VE-cadherin phosphorylation. ECs were transfected with K298M or Y530F for 48 h before exposed to 100 μg/mL AGEs for 1 h. VE-cadherin and its phosphorylation form were determined by western blotting. The ratio of immunointensity between the phosphorylation of VE-cadherin (p-VE658) and β-actin were calculated. n = 3, *P < 0.05 versus control, #P < 0.05 versus AGEs or Mock + AGEs. (e) Down-regulation of Src prevented AGE-induced adherence junction dissociation. HUVECs were pretreated with PP2 (15 μmol/L) for 90 min or transfected with Src siRNA for 48 h, followed by incubation with or without 100 μg/mL AGEs for 8 h. Immunostaining for VE-cadherin (green) and DAPI (blue) were shown.
Mentions: Given that Src acted as an upstream of VE-cadherin and caused the disruption of endothelial barrier in response to permeability-increasing agents such as bradykinin and histamine17, we investigated whether Src influenced the distribution of VE-cadherin under AGE treatment. We revealed that AGEs induced the dissociation of VE-cadherin, which was prevented by inhibition of Src signaling with PP2 or Src siRNA (Fig. 7e). We also assessed the effects of Src on the phosphorylation of VE-cadherin. Our data showed that AGEs promoted VE-cadherin phosphorylation, which was blocked by PP2, Src siRNA (Fig. 7a,b), or K298M, but significantly increased by Y530F (Fig. 7c,d). Moreover, cells transfected with Y530F displayed a higher level of VE-cadherin phosphorylation compared with mock-transfected cells (Fig. 7d). These data suggest Src played a role in AGE-induced VE-cadherin phosphorylation and dissociation.

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