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

Effects of AGEs on Src phosphorylation.HUVECs were either (a,c) incubated with 100 μg/mL of AGEs for 10, 30, 60, 90, or 120 min, or (d,f) stimulated with 12.5, 25, 50, 100, or 200 μg/mL AGEs for 60 min. Specific antibody was used to detect P-Src 419 (a,d) and P-Src 530 (c,f) by western blotting. The effect of BSA alone on Src 419 phosphorylation was also detected as BSA control by incubating the cells with (b) 100 μg/mL of BSA for 10, 30, 60, 90, 120 min, or with (e) 12.5, 25, 50, 100, 200 μg/mL BSA for 60 min. Those incubated with culture medium were used as blank control. The ratio of immunointensity between the phosphorylation of Src (p-Src 419, p-Src 530) and total Src were calculated. n = 3, *P < 0.05 versus control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Effects of AGEs on Src phosphorylation.HUVECs were either (a,c) incubated with 100 μg/mL of AGEs for 10, 30, 60, 90, or 120 min, or (d,f) stimulated with 12.5, 25, 50, 100, or 200 μg/mL AGEs for 60 min. Specific antibody was used to detect P-Src 419 (a,d) and P-Src 530 (c,f) by western blotting. The effect of BSA alone on Src 419 phosphorylation was also detected as BSA control by incubating the cells with (b) 100 μg/mL of BSA for 10, 30, 60, 90, 120 min, or with (e) 12.5, 25, 50, 100, 200 μg/mL BSA for 60 min. Those incubated with culture medium were used as blank control. The ratio of immunointensity between the phosphorylation of Src (p-Src 419, p-Src 530) and total Src were calculated. n = 3, *P < 0.05 versus control.

Mentions: Once activated through phosphorylation at Tyr 419, the tyrosine kinase of Src can phosphorylate many proteins involved in the regulation of endothelial permeability. We asked whether Src activation was involved in the signal events evoked by AGEs in HUVECs. Cells were incubated with 100 μg/mL of AGE-BSA for different duration and phosphorylation of Src was determined by western blotting. The results showed that phosphorylation of Src 419 was rapidly enhanced at 10 min, reached a peak at 60 min, and then returned to baseline level at 120 min, while phosphorylation of Src 530 showed the contrary tendency during 120 min period. Incubation with BSA (Bovine Serum Albumin) alone had no effect on Src 419 phosphorylation (Fig. 2a–c). Src activation was then examined in HUVECs stimulated with different concentrations of AGE-BSA for 60 min. We found that Src 419 phosphorylation was significantly increased by AGE-BSA at the concentrations ranging from 25 μg/mL to 200 μg/mL, with the peak from 50 to 100 μg/mL. On the contrary, Src 530 phosphorylation was decreased by AGE-BSA at the same concentrations range, with the peak at 50 μg/mL. Different doses of BSA alone showed no significant effects on phosphorylation of both Src 419 and Src 530 (Fig. 2d–f). These data strongly suggest that Src activation was linked with AGE-induced disruption of endothelial barrier. We also noticed that, compared to 100 μg/mL, the endothelial monolayer permeability increases further at 200 μg/mL, but Src phosphorylation decreases at 200 μg/mL relative to 100 μg/mL. It is possible that some other signal pathways, not only Src, might participate in high dose AGE-induced hyperpermeability, which needs to be investigated in the future.


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)

Effects of AGEs on Src phosphorylation.HUVECs were either (a,c) incubated with 100 μg/mL of AGEs for 10, 30, 60, 90, or 120 min, or (d,f) stimulated with 12.5, 25, 50, 100, or 200 μg/mL AGEs for 60 min. Specific antibody was used to detect P-Src 419 (a,d) and P-Src 530 (c,f) by western blotting. The effect of BSA alone on Src 419 phosphorylation was also detected as BSA control by incubating the cells with (b) 100 μg/mL of BSA for 10, 30, 60, 90, 120 min, or with (e) 12.5, 25, 50, 100, 200 μg/mL BSA for 60 min. Those incubated with culture medium were used as blank control. The ratio of immunointensity between the phosphorylation of Src (p-Src 419, p-Src 530) and total Src were calculated. 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

f2: Effects of AGEs on Src phosphorylation.HUVECs were either (a,c) incubated with 100 μg/mL of AGEs for 10, 30, 60, 90, or 120 min, or (d,f) stimulated with 12.5, 25, 50, 100, or 200 μg/mL AGEs for 60 min. Specific antibody was used to detect P-Src 419 (a,d) and P-Src 530 (c,f) by western blotting. The effect of BSA alone on Src 419 phosphorylation was also detected as BSA control by incubating the cells with (b) 100 μg/mL of BSA for 10, 30, 60, 90, 120 min, or with (e) 12.5, 25, 50, 100, 200 μg/mL BSA for 60 min. Those incubated with culture medium were used as blank control. The ratio of immunointensity between the phosphorylation of Src (p-Src 419, p-Src 530) and total Src were calculated. n = 3, *P < 0.05 versus control.
Mentions: Once activated through phosphorylation at Tyr 419, the tyrosine kinase of Src can phosphorylate many proteins involved in the regulation of endothelial permeability. We asked whether Src activation was involved in the signal events evoked by AGEs in HUVECs. Cells were incubated with 100 μg/mL of AGE-BSA for different duration and phosphorylation of Src was determined by western blotting. The results showed that phosphorylation of Src 419 was rapidly enhanced at 10 min, reached a peak at 60 min, and then returned to baseline level at 120 min, while phosphorylation of Src 530 showed the contrary tendency during 120 min period. Incubation with BSA (Bovine Serum Albumin) alone had no effect on Src 419 phosphorylation (Fig. 2a–c). Src activation was then examined in HUVECs stimulated with different concentrations of AGE-BSA for 60 min. We found that Src 419 phosphorylation was significantly increased by AGE-BSA at the concentrations ranging from 25 μg/mL to 200 μg/mL, with the peak from 50 to 100 μg/mL. On the contrary, Src 530 phosphorylation was decreased by AGE-BSA at the same concentrations range, with the peak at 50 μg/mL. Different doses of BSA alone showed no significant effects on phosphorylation of both Src 419 and Src 530 (Fig. 2d–f). These data strongly suggest that Src activation was linked with AGE-induced disruption of endothelial barrier. We also noticed that, compared to 100 μg/mL, the endothelial monolayer permeability increases further at 200 μg/mL, but Src phosphorylation decreases at 200 μg/mL relative to 100 μg/mL. It is possible that some other signal pathways, not only Src, might participate in high dose AGE-induced hyperpermeability, which needs to be investigated in the future.

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