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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 FAK activation requires Src.(a) Pretreatment of PP2 prevented AGE-induced FAK 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 FAK phosphorylation. ECs were pretreated 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 FAK phosphorylation. ECs were pretreated with K298M and Y530F for 48 h before exposed to 100 μg/mL AGEs for 1 h. FAK and its phosphorylation form were detected by western blotting. (e) AGE induced Src/FAK association in HUVECs. HUVECs were treated with 100 μg/mL AGEs for 1 h and lysed, and co-immunoprecipitation (co-IP) for FAK was performed. Increased expression of Src was detected by western blotting (upper panel). HUVECs were treated with 100 μg/mL AGEs for 1 h and co-IP for Src was performed. Increased expression of FAK was detected by western blotting (lower panel). (f) PF573228 prevented AGE-induced hyper-permeability of endothelial cells. HUVECs were treated with PF573228 (20 μmol/L) for 60 min before exposed to 100 μg/mL AGEs for 8 h. Culture medium was used as control. Permeability coefficient of the transflux of trace FITC-dextran (Pd) and TER were measured. The ratio of immunointensity between the phosphorylation of FAK (p-FAK 925) and β-actin were calculated. n = 3, *P < 0.05 versus control, #P < 0.05 versus AGEs group or Mock + AGEs.
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f8: AGE-induced FAK activation requires Src.(a) Pretreatment of PP2 prevented AGE-induced FAK 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 FAK phosphorylation. ECs were pretreated 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 FAK phosphorylation. ECs were pretreated with K298M and Y530F for 48 h before exposed to 100 μg/mL AGEs for 1 h. FAK and its phosphorylation form were detected by western blotting. (e) AGE induced Src/FAK association in HUVECs. HUVECs were treated with 100 μg/mL AGEs for 1 h and lysed, and co-immunoprecipitation (co-IP) for FAK was performed. Increased expression of Src was detected by western blotting (upper panel). HUVECs were treated with 100 μg/mL AGEs for 1 h and co-IP for Src was performed. Increased expression of FAK was detected by western blotting (lower panel). (f) PF573228 prevented AGE-induced hyper-permeability of endothelial cells. HUVECs were treated with PF573228 (20 μmol/L) for 60 min before exposed to 100 μg/mL AGEs for 8 h. Culture medium was used as control. Permeability coefficient of the transflux of trace FITC-dextran (Pd) and TER were measured. The ratio of immunointensity between the phosphorylation of FAK (p-FAK 925) and β-actin were calculated. n = 3, *P < 0.05 versus control, #P < 0.05 versus AGEs group or Mock + AGEs.

Mentions: To explore whether FAK was the downstream target of Src in AGE-treated HUVECs, cells were transfected with Src siRNA or pretreated with PP2 before the application of AGE-BSA. We found that AGE-BSA treatment induced a significant increase in FAK phosphorylation and this increase was inhibited by PP2 or Src siRNA (Fig. 8a,b). These findings were confirmed by the usages of the kinase deficient vector K298M and the constitutive active vector Y530F. Accordingly, FAK phosphorylation was inhibited by K298M, while further increased by Y530F in AGE-treated cells (Fig. 8c,d). To figure out whether FAK was phosphorylated via the formation of the Src-FAK complex, co-immunoprecipitation assay was performed. As shown in Fig. 8e, Src was associated with FAK in AGE-treated cells, not in control cells. Taken together, these data suggested that AGEs induced FAK phosphorylation by the ligation of Src and the tyrosine kinase Src.


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 FAK activation requires Src.(a) Pretreatment of PP2 prevented AGE-induced FAK 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 FAK phosphorylation. ECs were pretreated 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 FAK phosphorylation. ECs were pretreated with K298M and Y530F for 48 h before exposed to 100 μg/mL AGEs for 1 h. FAK and its phosphorylation form were detected by western blotting. (e) AGE induced Src/FAK association in HUVECs. HUVECs were treated with 100 μg/mL AGEs for 1 h and lysed, and co-immunoprecipitation (co-IP) for FAK was performed. Increased expression of Src was detected by western blotting (upper panel). HUVECs were treated with 100 μg/mL AGEs for 1 h and co-IP for Src was performed. Increased expression of FAK was detected by western blotting (lower panel). (f) PF573228 prevented AGE-induced hyper-permeability of endothelial cells. HUVECs were treated with PF573228 (20 μmol/L) for 60 min before exposed to 100 μg/mL AGEs for 8 h. Culture medium was used as control. Permeability coefficient of the transflux of trace FITC-dextran (Pd) and TER were measured. The ratio of immunointensity between the phosphorylation of FAK (p-FAK 925) and β-actin were calculated. n = 3, *P < 0.05 versus control, #P < 0.05 versus AGEs group or Mock + AGEs.
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Related In: Results  -  Collection

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Show All Figures
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f8: AGE-induced FAK activation requires Src.(a) Pretreatment of PP2 prevented AGE-induced FAK 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 FAK phosphorylation. ECs were pretreated 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 FAK phosphorylation. ECs were pretreated with K298M and Y530F for 48 h before exposed to 100 μg/mL AGEs for 1 h. FAK and its phosphorylation form were detected by western blotting. (e) AGE induced Src/FAK association in HUVECs. HUVECs were treated with 100 μg/mL AGEs for 1 h and lysed, and co-immunoprecipitation (co-IP) for FAK was performed. Increased expression of Src was detected by western blotting (upper panel). HUVECs were treated with 100 μg/mL AGEs for 1 h and co-IP for Src was performed. Increased expression of FAK was detected by western blotting (lower panel). (f) PF573228 prevented AGE-induced hyper-permeability of endothelial cells. HUVECs were treated with PF573228 (20 μmol/L) for 60 min before exposed to 100 μg/mL AGEs for 8 h. Culture medium was used as control. Permeability coefficient of the transflux of trace FITC-dextran (Pd) and TER were measured. The ratio of immunointensity between the phosphorylation of FAK (p-FAK 925) and β-actin were calculated. n = 3, *P < 0.05 versus control, #P < 0.05 versus AGEs group or Mock + AGEs.
Mentions: To explore whether FAK was the downstream target of Src in AGE-treated HUVECs, cells were transfected with Src siRNA or pretreated with PP2 before the application of AGE-BSA. We found that AGE-BSA treatment induced a significant increase in FAK phosphorylation and this increase was inhibited by PP2 or Src siRNA (Fig. 8a,b). These findings were confirmed by the usages of the kinase deficient vector K298M and the constitutive active vector Y530F. Accordingly, FAK phosphorylation was inhibited by K298M, while further increased by Y530F in AGE-treated cells (Fig. 8c,d). To figure out whether FAK was phosphorylated via the formation of the Src-FAK complex, co-immunoprecipitation assay was performed. As shown in Fig. 8e, Src was associated with FAK in AGE-treated cells, not in control cells. Taken together, these data suggested that AGEs induced FAK phosphorylation by the ligation of Src and the tyrosine kinase Src.

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