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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 NF-κB activation.(A) Treatment of primary human pericytes with NaCN (2mM) resulted in time-dependent increase in translocation of the p65 subunit of NF-κB into the nuclear fraction (right panel) with concomitant decrease in the cytosolic fraction (middle panel). NaCN failed to exert significant effect on the expression of NF-kB in the total cell lysis from pericytes. (B) Exposure of primary human pericytes to glucose deprivation resulted in time-dependent increase in translocation of the p65 subunit of NF-κB into the nuclear fraction (right panel) with concomitant decrease in the cytosolic fraction (middle panel). NaCN failed to exert significant effect on the expression of NF-kB in the total cell lysis from pericytes. (C) Pretreatment of primary human pericytes with MEK1/2 (U0126, 10μM), p38 inhibitor (SB203580, 10μM), JNK inhibitor (SP600125, 10μM) and PI3K inhibitor (LY294002, 5μM) resulted in inhibition of NaCN-mediated NF-kB translocation of the p65 subunit of NF-κB into the nucleus. (D) Pretreatment with the Ikk2 inhibitor-SC514 (5μM) resulted in inhibition of NaCN-mediated induction of VEGF. All the data are 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, ###p<0.001 vs NaCN-treated group.
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pone.0124362.g004: NaCN-mediated induction of VEGF expression involves NF-κB activation.(A) Treatment of primary human pericytes with NaCN (2mM) resulted in time-dependent increase in translocation of the p65 subunit of NF-κB into the nuclear fraction (right panel) with concomitant decrease in the cytosolic fraction (middle panel). NaCN failed to exert significant effect on the expression of NF-kB in the total cell lysis from pericytes. (B) Exposure of primary human pericytes to glucose deprivation resulted in time-dependent increase in translocation of the p65 subunit of NF-κB into the nuclear fraction (right panel) with concomitant decrease in the cytosolic fraction (middle panel). NaCN failed to exert significant effect on the expression of NF-kB in the total cell lysis from pericytes. (C) Pretreatment of primary human pericytes with MEK1/2 (U0126, 10μM), p38 inhibitor (SB203580, 10μM), JNK inhibitor (SP600125, 10μM) and PI3K inhibitor (LY294002, 5μM) resulted in inhibition of NaCN-mediated NF-kB translocation of the p65 subunit of NF-κB into the nucleus. (D) Pretreatment with the Ikk2 inhibitor-SC514 (5μM) resulted in inhibition of NaCN-mediated induction of VEGF. All the data are 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, ###p<0.001 vs NaCN-treated group.

Mentions: Based on a previous report demonstrating activation of NF-kB pathway in the induction of VEGF [45], we next determined whether NF-kB pathway was also involved in NaCN-mediated induction of VEGF expression in primary human pericytes. As shown in Fig 4A, NaCN treatment of pericytes resulted in a rapid translocation of NF-kB p65 into the nucleus with concomitant reduction in the cytoplasmic level of NF-kB. However, NaCN treatment failed to exert significant effect on the expression of NF-kB in the total cell lysis from pericytes (Fig 4A). These findings were further confirmed in pericytes cultured in glucose deprivation condition as shown in Fig 4B. We next wanted to examine whether there existed a link between activation of ERK, p38, JNK MAPKs, and PI3K/Akt with NF-kB. Primary human pericytes were pretreated with ERK, p38, JNK or PI3K inhibitor(s) followed by treatment with NaCN and assessed for translocation of NF-kB. As shown in Fig 4C, ERK, p38, JNK and PI3K inhibitors significantly inhibited NaCN-mediated activation of NF-kB. Further validation of the role of NF-κB p65 in NaCN-mediated expression of VEGF was carried out by pretreating the cells with the Ikk-2 inhibitor-SC514 and assessing for induction of VEGF. As shown in Fig 4D, pretreatment of cells with SC514 (5μM) resulted in amelioration of NaCN-mediated induction of VEGF, thereby underscoring the involvement of NF-κB p65 in this process.


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 NF-κB activation.(A) Treatment of primary human pericytes with NaCN (2mM) resulted in time-dependent increase in translocation of the p65 subunit of NF-κB into the nuclear fraction (right panel) with concomitant decrease in the cytosolic fraction (middle panel). NaCN failed to exert significant effect on the expression of NF-kB in the total cell lysis from pericytes. (B) Exposure of primary human pericytes to glucose deprivation resulted in time-dependent increase in translocation of the p65 subunit of NF-κB into the nuclear fraction (right panel) with concomitant decrease in the cytosolic fraction (middle panel). NaCN failed to exert significant effect on the expression of NF-kB in the total cell lysis from pericytes. (C) Pretreatment of primary human pericytes with MEK1/2 (U0126, 10μM), p38 inhibitor (SB203580, 10μM), JNK inhibitor (SP600125, 10μM) and PI3K inhibitor (LY294002, 5μM) resulted in inhibition of NaCN-mediated NF-kB translocation of the p65 subunit of NF-κB into the nucleus. (D) Pretreatment with the Ikk2 inhibitor-SC514 (5μM) resulted in inhibition of NaCN-mediated induction of VEGF. All the data are 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, ###p<0.001 vs NaCN-treated group.
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Related In: Results  -  Collection

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pone.0124362.g004: NaCN-mediated induction of VEGF expression involves NF-κB activation.(A) Treatment of primary human pericytes with NaCN (2mM) resulted in time-dependent increase in translocation of the p65 subunit of NF-κB into the nuclear fraction (right panel) with concomitant decrease in the cytosolic fraction (middle panel). NaCN failed to exert significant effect on the expression of NF-kB in the total cell lysis from pericytes. (B) Exposure of primary human pericytes to glucose deprivation resulted in time-dependent increase in translocation of the p65 subunit of NF-κB into the nuclear fraction (right panel) with concomitant decrease in the cytosolic fraction (middle panel). NaCN failed to exert significant effect on the expression of NF-kB in the total cell lysis from pericytes. (C) Pretreatment of primary human pericytes with MEK1/2 (U0126, 10μM), p38 inhibitor (SB203580, 10μM), JNK inhibitor (SP600125, 10μM) and PI3K inhibitor (LY294002, 5μM) resulted in inhibition of NaCN-mediated NF-kB translocation of the p65 subunit of NF-κB into the nucleus. (D) Pretreatment with the Ikk2 inhibitor-SC514 (5μM) resulted in inhibition of NaCN-mediated induction of VEGF. All the data are 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, ###p<0.001 vs NaCN-treated group.
Mentions: Based on a previous report demonstrating activation of NF-kB pathway in the induction of VEGF [45], we next determined whether NF-kB pathway was also involved in NaCN-mediated induction of VEGF expression in primary human pericytes. As shown in Fig 4A, NaCN treatment of pericytes resulted in a rapid translocation of NF-kB p65 into the nucleus with concomitant reduction in the cytoplasmic level of NF-kB. However, NaCN treatment failed to exert significant effect on the expression of NF-kB in the total cell lysis from pericytes (Fig 4A). These findings were further confirmed in pericytes cultured in glucose deprivation condition as shown in Fig 4B. We next wanted to examine whether there existed a link between activation of ERK, p38, JNK MAPKs, and PI3K/Akt with NF-kB. Primary human pericytes were pretreated with ERK, p38, JNK or PI3K inhibitor(s) followed by treatment with NaCN and assessed for translocation of NF-kB. As shown in Fig 4C, ERK, p38, JNK and PI3K inhibitors significantly inhibited NaCN-mediated activation of NF-kB. Further validation of the role of NF-κB p65 in NaCN-mediated expression of VEGF was carried out by pretreating the cells with the Ikk-2 inhibitor-SC514 and assessing for induction of VEGF. As shown in Fig 4D, pretreatment of cells with SC514 (5μM) resulted in amelioration of NaCN-mediated induction of VEGF, thereby underscoring the involvement of NF-κB p65 in this process.

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