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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

Disrupted BBB and increased expression of VEGF in the brains of stroked mice.(A) Representative image of significant signal loss in the lesioned area of the brain on T2-weight images at 24 hours after injection of SPIO in the stroked mice upper panel) and the quantification of signal loss (lower panel). (B) Pretreatment of mice with anti-VEGF neutralizing antibody significantly attenuated the increased permeability of BBB in stroke mice. n = 6 per group. All the data are presented as mean±SD. **p<0.01 vs control group; #p<0.05 vs sham group. (C) Expression of VEGF in the striatum isolated from sham and stroked mice by western blotting. Representative immunoblots and densitometric analyses of VEGF/β-actin from 4 mice/group are presented. Contra:contralateral; Ipsi:ipsilateral. (D) Double immunofluorescence staining specific for VEGF and pericyte marker PDGF-βR in isolated microvessles from sham and stroke mice. VEGF: red; PDGF-βR: green. Scale bar = 100μm. n = 8 per group. All the data are presented as mean±SD. *p<0.05, **p<0.01, ***p<0.001 vs control group; #p<0.05 vs sham group.
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pone.0124362.g006: Disrupted BBB and increased expression of VEGF in the brains of stroked mice.(A) Representative image of significant signal loss in the lesioned area of the brain on T2-weight images at 24 hours after injection of SPIO in the stroked mice upper panel) and the quantification of signal loss (lower panel). (B) Pretreatment of mice with anti-VEGF neutralizing antibody significantly attenuated the increased permeability of BBB in stroke mice. n = 6 per group. All the data are presented as mean±SD. **p<0.01 vs control group; #p<0.05 vs sham group. (C) Expression of VEGF in the striatum isolated from sham and stroked mice by western blotting. Representative immunoblots and densitometric analyses of VEGF/β-actin from 4 mice/group are presented. Contra:contralateral; Ipsi:ipsilateral. (D) Double immunofluorescence staining specific for VEGF and pericyte marker PDGF-βR in isolated microvessles from sham and stroke mice. VEGF: red; PDGF-βR: green. Scale bar = 100μm. n = 8 per group. All the data are presented as mean±SD. *p<0.05, **p<0.01, ***p<0.001 vs control group; #p<0.05 vs sham group.

Mentions: For the in vivo relevance of our in vitro findings, we next examined barrier integrity in the focal stroke model. Briefly, twenty-four hours following surgery to initiate the focal stroke, BBB integrity was examined in the sham and stroke groups of mice using tail vein injection of SPIO particles. As demonstrated in Fig 6A, and as expected, there was a disruption of the BBB integrity in the lesioned area of the brain as shown by the fact that there was significant loss of signal evidenced by T2-weight images at 24 hours post SPIO injection. In order to examine whether anti-VEGF antibody exert potential protection of the BBB integrity, mice were injected with anti-VEGF neutralizing antibody or the IgG isotype control antibody for 2 days before stroke operation. As shown in Fig 6B, anti-VEGF neutralizing antibody significantly attenuated the damage of BBB integrity in stroke mice, but not the IgG isotype control antibody treated group. Further validation of the in vitro findings involved assessment of expression of VEGF in the brain homogenates of the contralateral and ipsilateral of brain in sham and stroke groups. As shown in Fig 6C, there was increased expression of VEGF in the brain region in the mice with stroke compared with the sham group. Interestingly, there was increased expression of VEGF in the both contralateral and ipsilateral side of stroke compared with sham group, though there was only a small increase of VEGF expression in the contralateral side as noted. For further validation that the pericytes were the source of increased VEGF, microvessels from stroke versus sham mice were stained for VEGF and platelet-derived growth factor-β receptor (PDGF-βR, a well known pericyte marker) by double immunofluorescent staining. As shown in Fig 6D, in the microvessels from stroke mice, there was increased expression of VEGF co-localized with PDGF-βR compared with sham mice, suggesting thereby that the primary source of increased expression of VEGF was, at least part, from the pericytes, in the stroke model.


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)

Disrupted BBB and increased expression of VEGF in the brains of stroked mice.(A) Representative image of significant signal loss in the lesioned area of the brain on T2-weight images at 24 hours after injection of SPIO in the stroked mice upper panel) and the quantification of signal loss (lower panel). (B) Pretreatment of mice with anti-VEGF neutralizing antibody significantly attenuated the increased permeability of BBB in stroke mice. n = 6 per group. All the data are presented as mean±SD. **p<0.01 vs control group; #p<0.05 vs sham group. (C) Expression of VEGF in the striatum isolated from sham and stroked mice by western blotting. Representative immunoblots and densitometric analyses of VEGF/β-actin from 4 mice/group are presented. Contra:contralateral; Ipsi:ipsilateral. (D) Double immunofluorescence staining specific for VEGF and pericyte marker PDGF-βR in isolated microvessles from sham and stroke mice. VEGF: red; PDGF-βR: green. Scale bar = 100μm. n = 8 per group. All the data are presented as mean±SD. *p<0.05, **p<0.01, ***p<0.001 vs control group; #p<0.05 vs sham group.
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pone.0124362.g006: Disrupted BBB and increased expression of VEGF in the brains of stroked mice.(A) Representative image of significant signal loss in the lesioned area of the brain on T2-weight images at 24 hours after injection of SPIO in the stroked mice upper panel) and the quantification of signal loss (lower panel). (B) Pretreatment of mice with anti-VEGF neutralizing antibody significantly attenuated the increased permeability of BBB in stroke mice. n = 6 per group. All the data are presented as mean±SD. **p<0.01 vs control group; #p<0.05 vs sham group. (C) Expression of VEGF in the striatum isolated from sham and stroked mice by western blotting. Representative immunoblots and densitometric analyses of VEGF/β-actin from 4 mice/group are presented. Contra:contralateral; Ipsi:ipsilateral. (D) Double immunofluorescence staining specific for VEGF and pericyte marker PDGF-βR in isolated microvessles from sham and stroke mice. VEGF: red; PDGF-βR: green. Scale bar = 100μm. n = 8 per group. All the data are presented as mean±SD. *p<0.05, **p<0.01, ***p<0.001 vs control group; #p<0.05 vs sham group.
Mentions: For the in vivo relevance of our in vitro findings, we next examined barrier integrity in the focal stroke model. Briefly, twenty-four hours following surgery to initiate the focal stroke, BBB integrity was examined in the sham and stroke groups of mice using tail vein injection of SPIO particles. As demonstrated in Fig 6A, and as expected, there was a disruption of the BBB integrity in the lesioned area of the brain as shown by the fact that there was significant loss of signal evidenced by T2-weight images at 24 hours post SPIO injection. In order to examine whether anti-VEGF antibody exert potential protection of the BBB integrity, mice were injected with anti-VEGF neutralizing antibody or the IgG isotype control antibody for 2 days before stroke operation. As shown in Fig 6B, anti-VEGF neutralizing antibody significantly attenuated the damage of BBB integrity in stroke mice, but not the IgG isotype control antibody treated group. Further validation of the in vitro findings involved assessment of expression of VEGF in the brain homogenates of the contralateral and ipsilateral of brain in sham and stroke groups. As shown in Fig 6C, there was increased expression of VEGF in the brain region in the mice with stroke compared with the sham group. Interestingly, there was increased expression of VEGF in the both contralateral and ipsilateral side of stroke compared with sham group, though there was only a small increase of VEGF expression in the contralateral side as noted. For further validation that the pericytes were the source of increased VEGF, microvessels from stroke versus sham mice were stained for VEGF and platelet-derived growth factor-β receptor (PDGF-βR, a well known pericyte marker) by double immunofluorescent staining. As shown in Fig 6D, in the microvessels from stroke mice, there was increased expression of VEGF co-localized with PDGF-βR compared with sham mice, suggesting thereby that the primary source of increased expression of VEGF was, at least part, from the pericytes, in the stroke model.

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