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Pericytes contribute to the disruption of the cerebral endothelial barrier via increasing VEGF expression: implications for stroke.

Bai Y, Zhu X, Chao J, Zhang Y, Qian C, Li P, Liu D, Han B, Zhao L, Zhang J, Buch S, Teng G, Hu G, Yao H - PLoS ONE (2015)

Bottom Line: Our findings demonstrated that treatment of human brain microvascular pericytes with sodium cyanide (NaCN) and glucose deprivation resulted in increased expression of vascular endothelial growth factor (VEGF) via the activation of tyrosine kinase Src, with downstream activation of mitogen activated protein kinase and PI3K/Akt pathways and subsequent translocation of NF-κB into the nucleus.Conditioned medium from NaCN-treated pericytes led to increased permeability of endothelial cells, and this effect was significantly inhibited by VEGF-neutralizing antibody.Understanding the regulation of VEGF expression could open new avenues for the development of potential therapeutic targets for stroke and other neurological disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Medical School of Southeast University, Nanjing, China.

ABSTRACT
Disruption of the blood-brain barrier (BBB) integrity occurring during the early onset of stroke is not only a consequence of, but also contributes to the further progression of stroke. Although it has been well documented that brain microvascular endothelial cells and astrocytes play a critical role in the maintenance of BBB integrity, pericytes, sandwiched between endothelial cells and astrocytes, remain poorly studied in the pathogenesis of stroke. Our findings demonstrated that treatment of human brain microvascular pericytes with sodium cyanide (NaCN) and glucose deprivation resulted in increased expression of vascular endothelial growth factor (VEGF) via the activation of tyrosine kinase Src, with downstream activation of mitogen activated protein kinase and PI3K/Akt pathways and subsequent translocation of NF-κB into the nucleus. Conditioned medium from NaCN-treated pericytes led to increased permeability of endothelial cells, and this effect was significantly inhibited by VEGF-neutralizing antibody. The in vivo relevance of these findings was further corroborated in the stroke model of mice wherein the mice, demonstrated disruption of the BBB integrity and concomitant increase in the expression of VEGF in the brain tissue as well as in the isolated microvessel. These findings thus suggest the role of pericyte-derived VEGF in modulating increased permeability of BBB during stroke. Understanding the regulation of VEGF expression could open new avenues for the development of potential therapeutic targets for stroke and other neurological disease.

No MeSH data available.


Related in: MedlinePlus

NaCN mediated up-regulation of VEGF in primary human pericytes.(A) NaCN mediated induction of VEGF expression in primary human pericytes. Cells were incubated with NaCN (2mM) for different time points (6, 12 and 24 hours), followed by collection of media for assay of VEGF expression by Luminex assay. (B) Pericytes were exposed to glucose-deprived culture media for 6, 12 and 24 hours followed by Luminex assay for detection of VEGF. (C) Dose curve of NaCN on the expression of VEGF in pericytes by Luminex assay. (D) Effect of NaCN on the expression of VEGF in primary human pericyte by western Blot. All the data are presented as mean±SD of three individual experiments (n = 3). *p<0.05, **p<0.01, ***p<0.001 vs control group.
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pone.0124362.g001: NaCN mediated up-regulation of VEGF in primary human pericytes.(A) NaCN mediated induction of VEGF expression in primary human pericytes. Cells were incubated with NaCN (2mM) for different time points (6, 12 and 24 hours), followed by collection of media for assay of VEGF expression by Luminex assay. (B) Pericytes were exposed to glucose-deprived culture media for 6, 12 and 24 hours followed by Luminex assay for detection of VEGF. (C) Dose curve of NaCN on the expression of VEGF in pericytes by Luminex assay. (D) Effect of NaCN on the expression of VEGF in primary human pericyte by western Blot. All the data are presented as mean±SD of three individual experiments (n = 3). *p<0.05, **p<0.01, ***p<0.001 vs control group.

Mentions: Previous study has demonstrated that NaCN (an inhibitor of mitochondrial respiration used to mimic hypoxia) plays a critical role in the pathogenesis of stroke [30,40]. However, whether NaCN induces activation of pericytes and the mechanisms involved in this process, remain largely unknown. In order to examine the effect of NaCN on the release of cytokines/chemokines from pericytes, supernatants from NaCN-treated pericytes was subjected to human cytokine antibody array analysis. Treatment of pericytes with NaCN resulted in dramatic induction of VEGF expression using Luminex assay as shown in Fig 1A as well as in Table 1. To validate the effect of NaCN on the expression of VEGF in pericytes, we cultured pericytes under conditions of glucose deprivation to mimic stroke and assessed the expression of VEGF. As shown in Fig 1B, similar to NaCN exposure, glucose deprivation also resulted in up-regulation of VEGF protein expression. We next examined the concentration curve of NaCN mediated up-regulation of VEGF in pericytes. Pericytes were treated with different concentrations of NaCN (0.5, 1 and 2mM) for 24 hours and monitored for expression of VEGF protein by Luminex analysis. As shown in Fig 1C, NaCN induced a significant increase in VEGF expression at a concentration of 1mM and 2mM. We next determined the optimal time of NaCN-mediated up-regulation of VEGF in pericytes. Primary human pericytes were exposed to NaCN (2mM) at varying time points and assessed for VEGF expression by western blotting. As shown in Fig 1D, there was a time-dependent increase of VEGF expression following NaCN treatment, with the maximal expression of VEGF observed at 24 hours after NaCN treatment. These data thus demonstrated that NaCN treatment resulted in increased expression of VEGF in primary human pericytes.


Pericytes contribute to the disruption of the cerebral endothelial barrier via increasing VEGF expression: implications for stroke.

Bai Y, Zhu X, Chao J, Zhang Y, Qian C, Li P, Liu D, Han B, Zhao L, Zhang J, Buch S, Teng G, Hu G, Yao H - PLoS ONE (2015)

NaCN mediated up-regulation of VEGF in primary human pericytes.(A) NaCN mediated induction of VEGF expression in primary human pericytes. Cells were incubated with NaCN (2mM) for different time points (6, 12 and 24 hours), followed by collection of media for assay of VEGF expression by Luminex assay. (B) Pericytes were exposed to glucose-deprived culture media for 6, 12 and 24 hours followed by Luminex assay for detection of VEGF. (C) Dose curve of NaCN on the expression of VEGF in pericytes by Luminex assay. (D) Effect of NaCN on the expression of VEGF in primary human pericyte by western Blot. All the data are presented as mean±SD of three individual experiments (n = 3). *p<0.05, **p<0.01, ***p<0.001 vs control group.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4401453&req=5

pone.0124362.g001: NaCN mediated up-regulation of VEGF in primary human pericytes.(A) NaCN mediated induction of VEGF expression in primary human pericytes. Cells were incubated with NaCN (2mM) for different time points (6, 12 and 24 hours), followed by collection of media for assay of VEGF expression by Luminex assay. (B) Pericytes were exposed to glucose-deprived culture media for 6, 12 and 24 hours followed by Luminex assay for detection of VEGF. (C) Dose curve of NaCN on the expression of VEGF in pericytes by Luminex assay. (D) Effect of NaCN on the expression of VEGF in primary human pericyte by western Blot. All the data are presented as mean±SD of three individual experiments (n = 3). *p<0.05, **p<0.01, ***p<0.001 vs control group.
Mentions: Previous study has demonstrated that NaCN (an inhibitor of mitochondrial respiration used to mimic hypoxia) plays a critical role in the pathogenesis of stroke [30,40]. However, whether NaCN induces activation of pericytes and the mechanisms involved in this process, remain largely unknown. In order to examine the effect of NaCN on the release of cytokines/chemokines from pericytes, supernatants from NaCN-treated pericytes was subjected to human cytokine antibody array analysis. Treatment of pericytes with NaCN resulted in dramatic induction of VEGF expression using Luminex assay as shown in Fig 1A as well as in Table 1. To validate the effect of NaCN on the expression of VEGF in pericytes, we cultured pericytes under conditions of glucose deprivation to mimic stroke and assessed the expression of VEGF. As shown in Fig 1B, similar to NaCN exposure, glucose deprivation also resulted in up-regulation of VEGF protein expression. We next examined the concentration curve of NaCN mediated up-regulation of VEGF in pericytes. Pericytes were treated with different concentrations of NaCN (0.5, 1 and 2mM) for 24 hours and monitored for expression of VEGF protein by Luminex analysis. As shown in Fig 1C, NaCN induced a significant increase in VEGF expression at a concentration of 1mM and 2mM. We next determined the optimal time of NaCN-mediated up-regulation of VEGF in pericytes. Primary human pericytes were exposed to NaCN (2mM) at varying time points and assessed for VEGF expression by western blotting. As shown in Fig 1D, there was a time-dependent increase of VEGF expression following NaCN treatment, with the maximal expression of VEGF observed at 24 hours after NaCN treatment. These data thus demonstrated that NaCN treatment resulted in increased expression of VEGF in primary human pericytes.

Bottom Line: Our findings demonstrated that treatment of human brain microvascular pericytes with sodium cyanide (NaCN) and glucose deprivation resulted in increased expression of vascular endothelial growth factor (VEGF) via the activation of tyrosine kinase Src, with downstream activation of mitogen activated protein kinase and PI3K/Akt pathways and subsequent translocation of NF-κB into the nucleus.Conditioned medium from NaCN-treated pericytes led to increased permeability of endothelial cells, and this effect was significantly inhibited by VEGF-neutralizing antibody.Understanding the regulation of VEGF expression could open new avenues for the development of potential therapeutic targets for stroke and other neurological disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Medical School of Southeast University, Nanjing, China.

ABSTRACT
Disruption of the blood-brain barrier (BBB) integrity occurring during the early onset of stroke is not only a consequence of, but also contributes to the further progression of stroke. Although it has been well documented that brain microvascular endothelial cells and astrocytes play a critical role in the maintenance of BBB integrity, pericytes, sandwiched between endothelial cells and astrocytes, remain poorly studied in the pathogenesis of stroke. Our findings demonstrated that treatment of human brain microvascular pericytes with sodium cyanide (NaCN) and glucose deprivation resulted in increased expression of vascular endothelial growth factor (VEGF) via the activation of tyrosine kinase Src, with downstream activation of mitogen activated protein kinase and PI3K/Akt pathways and subsequent translocation of NF-κB into the nucleus. Conditioned medium from NaCN-treated pericytes led to increased permeability of endothelial cells, and this effect was significantly inhibited by VEGF-neutralizing antibody. The in vivo relevance of these findings was further corroborated in the stroke model of mice wherein the mice, demonstrated disruption of the BBB integrity and concomitant increase in the expression of VEGF in the brain tissue as well as in the isolated microvessel. These findings thus suggest the role of pericyte-derived VEGF in modulating increased permeability of BBB during stroke. Understanding the regulation of VEGF expression could open new avenues for the development of potential therapeutic targets for stroke and other neurological disease.

No MeSH data available.


Related in: MedlinePlus