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. 2009 Apr;40(4):1467-73.
doi: 10.1161/STROKEAHA.108.534644. Epub 2009 Feb 19.

VEGF Stimulates the ERK 1/2 signaling pathway and apoptosis in cerebral endothelial cells after ischemic conditions

Purnima Narasimhan  1 , Jing Liu, Yun Seon Song, Justin L Massengale, Pak H Chan
Affiliations

VEGF Stimulates the ERK 1/2 signaling pathway and apoptosis in cerebral endothelial cells after ischemic conditions

Purnima Narasimhan et al. Stroke. 2009 Apr.

Abstract

Background and purpose: Cerebral endothelial cells that line microvessels play an important role in maintaining blood flow homeostasis within the brain-forming part of the blood-brain barrier. These cells are injured by hypoxia-induced reperfusion, leading to blood-brain barrier breakdown and exacerbation of ischemic injury. We investigated the roles of vascular endothelial growth factor (VEGF) and the downstream extracellular signal-regulated kinase (ERK) protein after oxygen-glucose deprivation (OGD) in primary endothelial cells.

Methods: Primary mouse endothelial cells were isolated and subjected to OGD. Western analysis of VEGF and ERK 1/2 protein levels was performed. Cells were transfected with VEGF small interference RNA. A terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) assay and DNA fragmentation assay were used on mouse endothelial cells that overexpress copper/zinc-superoxide dismutase (SOD1).

Results: VEGF protein expression was induced and its receptor, Flk-1, was stimulated by OGD. Phosphorylation of ERK 1/2 protein levels was upregulated. Inhibition of phosphorylated ERK (pERK) expression by U0126 reduced endothelial cell death by OGD. Transfection of small interfering RNA for VEGF also inhibited an increase in pERK, suggesting that VEGF acts via ERK. The TUNEL and DNA fragmentation assays showed a significant decrease in TUNEL-positivity in the SOD1-overexpressing endothelial cells compared with wild-type cells after OGD.

Conclusions: Our data suggest that OGD induces VEGF signaling via its receptor, Flk-1, and activates ERK via oxidative-stress-dependent mechanisms. Our study shows that in cerebral endothelial cells the ERK 1/2 signaling pathway plays a significant role in cell injury after OGD.

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Figures

Figure 1
Figure 1
OGD-induced death of CECs. Cell death was measured by percent of LDH release (A) and enzyme-linked immunosorbent assay detection of DNA fragments (B). *P<0.05. Viability was assessed using calcein AM (green) and ethidium homodimer-1 (red) (C). Living cells were stained green and dead cells were stained red. Con, indicates control. Scale bars, 100 μm.
Figure 2
Figure 2
A, Upregulation of hypoxia-inducible factor (HIF) -1α after OGD in endothelial cell protein levels shown by Western analysis. An increase in HIF-1α was seen as early as 5 minutes. TFIID was used as an internal control for HIF-1α. Densitometric analysis of HIF-1α showed a 2-fold increase. B, Upregulation of VEGF and its receptor, Flk-1, after OGD in endothelial cell protein levels shown by Western analysis. β-actin was used as an internal control for VEGF and Flk-1 to adjust for protein loading. An increase in VEGF and Flk-1 by 2-fold after OGD and 2 hours of reoxygenation was observed. C indicates control; A.U., arbitrary units. *P<0.05.
Figure 3
Figure 3
A, Representative Western blot of pERK 1/2 after OGD in WT and SOD1 Tg endothelial cells. Within 2 hours of reoxygenation, pERK was upregulated in the WT cells and stayed high after 24 hours of reoxygenation. This upregulation was not observed in cells from the SOD1 Tg mice. Quantitative analysis of pERK Western blot showing an upregulation of pERK 1/2 by 1.5-fold in WT cells. *P<0.05. B, Immunostaining of pERK in brain sections of the WT and SOD1 Tg mice subjected to transient FCI and 4 hours of reperfusion (I/R). Sections were stained with fluorescein isothiocyanate-labeled-lectin (an endothelial marker) and Texas Red-labeled pERK. In the WT mice after I/R, pERK staining was visible in the endothelial cells, whereas it was not present in the WT mice under normal conditions or in the SOD1 Tg mice after I/R. C indicates control; O.D., optical density. Scale bar, 50 μm.
Figure 4
Figure 4
ROS play a role in endothelial cell death after OGD. Apoptosis is reduced in SOD1 Tg CECs compared with WT cells after OGD. TUNEL immunostaining showed fewer TUNEL-positive SOD1 Tg cells than WT cells after OGD. Scale bar, 100 μm.
Figure 5
Figure 5
VEGF acts via ERK 1/2 signaling. A, Endothelial cell treatment with 10 μg/ml of the neutralizing VEGF antibody demonstrated a simultaneous decrease in pERK 1/2. B, Quantitative analysis demonstrates a significant reduction in death of cells treated with the VEGF antibody. C, Blocking VEGF by siRNA showed a simultaneous decrease in pERK but not p-p38 or p-JNK. C indicates control; O, OGD; V, VEGF antibody; Scr, scrambled RNA; SiV, siRNA against VEGF; A.U., arbitrary units. *P<0.05.
Figure 6
Figure 6
A, Representative Western blot showing decrease in pERK levels after U0126 treatment. C indicates control; O, OGD; U, U0126; O.D., optical density. β-actin was used as an internal control. B, Inhibition of ERK 1/2 by 10 μM of U0126 (MAPK/ERK inhibitor) ameliorated endothelial cell death after OGD as measured by DNA fragmentation. *P<0.05.
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