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

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.

Fig 1. 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.

Fig 2. NaCN-mediated induction of VEGF involves Src kinase activation.

(A) NaCN (2mM) induced Src phosphorylation in a time-dependent manner in primary human pericytes. Representative immunoblots from four separate experiments are presented. (B) Exposure of pericytes to glucose deprivation induced Src phosphorylation in a time-dependent manner in primary human pericytes. Representative immunoblots from four separate experiments are presented. (C) Inhibition of Src activity by Src inhibitor-PP2 (10μM) resulted in amelioration of NaCN-mediated induction of VEGF. All the data are presented as mean±SD of four individual experiments (n = 4). *p<0.05, **p<0.01 vs control group; #p<0.05 vs NaCN-treated group.

Fig 3. 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.

Fig 4. 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.

Fig 5. Disruption of endothelial barrier integrity by NaCN-treated pericytes involves VEGF.

Conditioned media from pericytes treated with NaCN (2mM) increased endothelial barrier permeability, which was ameliorated in HBMECs pretreated with the VEGF-neutralizing antibody. VEGF (100ng/ml)-treated group was used a positive control. All the data are presented as mean±SD of four individual experiments (n = 4). **p<0.01 vs control group; #p<0.05 vs NaCN-treated group.

Fig 6. 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.

Fig 7. Schematic of the signaling pathways involved in NaCN-mediated induction of VEGF in primary human pericytes.

Exposure of pericytes to NaCN resulted in activation of Src, MAPKs and PI3K/Akt signaling pathways, with the downstream activation of the NF-kB transcription factor leading to enhanced expression of VEGF and subsequent increased permeability of endothelial cells.