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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 induction of VEGF expression involves MAPKs and PI3K/Akt cell signaling pathways.(A) Western blot analysis of time-dependent activation ERK, p38, JNK MAPKs MAPKs and PI3K/Akt pathways by NaCN (2mM) in primary human pericytes. (B) Glucose deprivation resulted in activation ERK, p38, JNK MAPKs and PI3K/Akt pathways in primary human pericytes. (C) Inhibition of the ERK, p38, JNK MAPKs and Akt pathways by MEK1/2 (U0126, 10μM), p38 inhibitor (SB203580, 10μM), JNK inhibitor (SP600125, 10μM) and PI3K inhibitor (LY294002, 5μM) resulted in amelioration of NaCN-mediated induction of VEGF. (D) Pretreatment of pericytes with another MEK inhibitor-PD98059 (10μM) significantly inhibited NaCN-mediated induction of VEGF. (E) Pretreatment of primary human pericytes with Src inhibitor-PP2 resulted in inhibition of NaCN-mediated phosphorylation of ERK, p38, JNK and Akt pathways. Representative immunoblots and densitometric analyses of pERK/ERK, pp38/p38, pJNK/JNK and pAkt/Akt from 4 separate experiments are presented. All the data are indicated as mean±SD of four individual experiments (n = 4). *p<0.05; **p<0.01, ***p<0.001 vs control group; #p<0.05; ##p<0.01 vs NaCN-treated group.
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pone.0124362.g003: NaCN-mediated induction of VEGF expression involves MAPKs and PI3K/Akt cell signaling pathways.(A) Western blot analysis of time-dependent activation ERK, p38, JNK MAPKs MAPKs and PI3K/Akt pathways by NaCN (2mM) in primary human pericytes. (B) Glucose deprivation resulted in activation ERK, p38, JNK MAPKs and PI3K/Akt pathways in primary human pericytes. (C) Inhibition of the ERK, p38, JNK MAPKs and Akt pathways by MEK1/2 (U0126, 10μM), p38 inhibitor (SB203580, 10μM), JNK inhibitor (SP600125, 10μM) and PI3K inhibitor (LY294002, 5μM) resulted in amelioration of NaCN-mediated induction of VEGF. (D) Pretreatment of pericytes with another MEK inhibitor-PD98059 (10μM) significantly inhibited NaCN-mediated induction of VEGF. (E) Pretreatment of primary human pericytes with Src inhibitor-PP2 resulted in inhibition of NaCN-mediated phosphorylation of ERK, p38, JNK and Akt pathways. Representative immunoblots and densitometric analyses of pERK/ERK, pp38/p38, pJNK/JNK and pAkt/Akt from 4 separate experiments are presented. All the data are indicated as mean±SD of four individual experiments (n = 4). *p<0.05; **p<0.01, ***p<0.001 vs control group; #p<0.05; ##p<0.01 vs NaCN-treated group.

Mentions: Given that MAPK kinase and PI3K/Akt pathways play critical roles in expression of VEGF [43,44], we next sought to examine whether these pathways contributed to NaCN-mediated induction of VEGF. As shown in Fig 3A, treatment of primary human pericytes with NaCN resulted in increased time-dependent phosphorylation of ERK, p38, JNK and Akt. Further validation of this finding was performed by exposure of pericytes to glucose deprivation. As shown in Fig 3B, glucose deprivation resulted in phosphorylation of ERK, p38, JNK in pericytes. In order to examine the role of these pathways in NaCN-mediated up-regulation of VEGF, primary human pericytes were pretreated with inhibitors specific for the respective signaling pathways prior to stimulation with NaCN and assessed for expression of VEGF. As shown in Fig 3C, pretreatment of cells with MEK (U0126, 10μM), p38 (SB203580, 10μM), JNK (SP600125, 10μM) and PI3K (LY294002, 5μM) inhibitors significantly decreased NaCN-mediated induction of VEGF. The functional role of NaCN-induced ERK activation in mediating VEGF expression was also corroborated using another MEK inhibitor-PD98059 (Fig 3D). These findings thus underpinned the role of ERK, p38, JNK MAPKs and PI3K/Akt in NaCN-mediated induction 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 induction of VEGF expression involves MAPKs and PI3K/Akt cell signaling pathways.(A) Western blot analysis of time-dependent activation ERK, p38, JNK MAPKs MAPKs and PI3K/Akt pathways by NaCN (2mM) in primary human pericytes. (B) Glucose deprivation resulted in activation ERK, p38, JNK MAPKs and PI3K/Akt pathways in primary human pericytes. (C) Inhibition of the ERK, p38, JNK MAPKs and Akt pathways by MEK1/2 (U0126, 10μM), p38 inhibitor (SB203580, 10μM), JNK inhibitor (SP600125, 10μM) and PI3K inhibitor (LY294002, 5μM) resulted in amelioration of NaCN-mediated induction of VEGF. (D) Pretreatment of pericytes with another MEK inhibitor-PD98059 (10μM) significantly inhibited NaCN-mediated induction of VEGF. (E) Pretreatment of primary human pericytes with Src inhibitor-PP2 resulted in inhibition of NaCN-mediated phosphorylation of ERK, p38, JNK and Akt pathways. Representative immunoblots and densitometric analyses of pERK/ERK, pp38/p38, pJNK/JNK and pAkt/Akt from 4 separate experiments are presented. All the data are indicated as mean±SD of four individual experiments (n = 4). *p<0.05; **p<0.01, ***p<0.001 vs control group; #p<0.05; ##p<0.01 vs NaCN-treated group.
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Related In: Results  -  Collection

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pone.0124362.g003: NaCN-mediated induction of VEGF expression involves MAPKs and PI3K/Akt cell signaling pathways.(A) Western blot analysis of time-dependent activation ERK, p38, JNK MAPKs MAPKs and PI3K/Akt pathways by NaCN (2mM) in primary human pericytes. (B) Glucose deprivation resulted in activation ERK, p38, JNK MAPKs and PI3K/Akt pathways in primary human pericytes. (C) Inhibition of the ERK, p38, JNK MAPKs and Akt pathways by MEK1/2 (U0126, 10μM), p38 inhibitor (SB203580, 10μM), JNK inhibitor (SP600125, 10μM) and PI3K inhibitor (LY294002, 5μM) resulted in amelioration of NaCN-mediated induction of VEGF. (D) Pretreatment of pericytes with another MEK inhibitor-PD98059 (10μM) significantly inhibited NaCN-mediated induction of VEGF. (E) Pretreatment of primary human pericytes with Src inhibitor-PP2 resulted in inhibition of NaCN-mediated phosphorylation of ERK, p38, JNK and Akt pathways. Representative immunoblots and densitometric analyses of pERK/ERK, pp38/p38, pJNK/JNK and pAkt/Akt from 4 separate experiments are presented. All the data are indicated as mean±SD of four individual experiments (n = 4). *p<0.05; **p<0.01, ***p<0.001 vs control group; #p<0.05; ##p<0.01 vs NaCN-treated group.
Mentions: Given that MAPK kinase and PI3K/Akt pathways play critical roles in expression of VEGF [43,44], we next sought to examine whether these pathways contributed to NaCN-mediated induction of VEGF. As shown in Fig 3A, treatment of primary human pericytes with NaCN resulted in increased time-dependent phosphorylation of ERK, p38, JNK and Akt. Further validation of this finding was performed by exposure of pericytes to glucose deprivation. As shown in Fig 3B, glucose deprivation resulted in phosphorylation of ERK, p38, JNK in pericytes. In order to examine the role of these pathways in NaCN-mediated up-regulation of VEGF, primary human pericytes were pretreated with inhibitors specific for the respective signaling pathways prior to stimulation with NaCN and assessed for expression of VEGF. As shown in Fig 3C, pretreatment of cells with MEK (U0126, 10μM), p38 (SB203580, 10μM), JNK (SP600125, 10μM) and PI3K (LY294002, 5μM) inhibitors significantly decreased NaCN-mediated induction of VEGF. The functional role of NaCN-induced ERK activation in mediating VEGF expression was also corroborated using another MEK inhibitor-PD98059 (Fig 3D). These findings thus underpinned the role of ERK, p38, JNK MAPKs and PI3K/Akt in NaCN-mediated induction 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