Hypoxia-induced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia

Abstract

We investigated the hypothesis that hypoxia induces angiogenesis and thereby may counteract the detrimental neurological effects associated with stroke. Forty-eight to seventy-two hours after permanent middle cerebral artery occlusion we found a strong increase in the number of newly formed vessels at the border of the infarction. Using the hypoxia marker nitroimidazole EF5, we detected hypoxic cells in the ischemic border of the neocortex. Expression of vascular endothelial growth factor (VEGF), which is the main regulator of angiogenesis and is inducible by hypoxia, was strongly up-regulated in the ischemic border, at times between 6 and 24 hours after occlusion. In addition, both VEGF receptors (VEGFRs) were up-regulated at the border after 48 hours and later in the ischemic core. Finally, the two transcription factors, hypoxia-inducible factor-1 (HIF-1) and HIF-2, known to be involved in the regulation of VEGF and VEGFR gene expression, were increased in the ischemic border after 72 hours, suggesting a regulatory function for these factors. These results strongly suggest that the VEGF/VEGFR system, induced by hypoxia, leads to the growth of new vessels after cerebral ischemia. Exogenous support of this natural protective mechanism might lead to enhanced survival after stroke.

Figure 1.

Immunohistochemistry for PECAM-1 (a, c, e, g) and Ki67 (b, d, f, h) in frontal brain sections of mice at various times after MCAO. a and b: control brain; c and d: brain 72 hours after occlusion; e–h: brain 48 hours after occlusion. Increased PECAM-1 (arrows in c) and Ki67 staining (d) are visible at the border of the infarction (marked by asterisks). PECAM-1 (e and g) and Ki67 (f and h) staining were performed on adjacent sections showing a pial vessel (e and f) and a neocortical vessel (g and h) invading the infarcted area. Arrows point to proliferating endothelial cells (f and h). Original magnifications: a–d, ×6.25; e–h, ×100.

Figure 2.

Time course of endothelial cell proliferation after MCAO in the ischemic penumbra. PECAM-1- and Ki67-positive cells were counted under the microscope in randomly chosen high-magnification fields (×100) in the control animals (C) and at various time points (12, 24, 48, 72, 168 hours) after MCAO. Each column and bar represents, respectively, the mean and SD of four to eight examined fields.

Figure 3.

Immunohistochemistry for the in vivo hypoxia marker EF5. The nitroimidazole compound EF5 was injected intravenously into mice 20 hours after MCAO (a and b) or into control animals (c). Four hours later, brains were removed and analyzed by immunofluorescence. a: Infarcted hemisphere; b: contralateral brain hemisphere of the same animal; c: brain from control animal. n.c., normal cortex; border and core region of the infarction are marked. Original magnification, ×25.

Figure 4.

RT-PCR analysis of VEGF (VEGF₁₂₀, 512 bp; VEGF₁₆₄, 644 bp), HIF-1α (187 bp), and β-actin (514 bp) transcripts. cDNA was synthesized from 1 μg of total RNA from contralateral (c) and ipsilateral (i) brain hemispheres at different times after occlusion. The RT-PCR presented is representative of results obtained from three individual experiments. −, no RT; m, molecular weight marker.

Figure 5.

Detection of VEGF mRNA in mouse brain at various times after MCAO. Frontal sections were hybridized in situ with ³⁵S-labeled RNA antisense (a, b, d–g) or sense (c) probes. Shown are (a) a bright-field image of a control animal and dark-field images of (b) a control animal and of animals (d) 12, (e) 24, (f) 72, and (g) 144 hours after MCAO, respectively. Note the strong expression of VEGF in choroid plexus in normal brain (arrowhead in b) and up-regulation of VEGF in the pia (arrowheads in d and e) and in the penumbra (arrows in d and e) during cerebral ischemia. Original magnification, ×6.25.

Figure 6.

Induction of VEGF protein during cerebral infarction. Brain lysates were prepared from contralateral (C) and ipsilateral cerebral hemispheres at various times after MCAO (0, 3, 12, 24, 48, 72, 168 hours). VEGF production was determined by a commercial immunoassay. Data show values in ipsilateral hemispheres (in percentage of the corresponding contralateral side) and are given as the mean of two independent experiments.

Figure 7.

Expression of VEGFR-1 mRNA during cerebral infarction: in situ hybridization with a VEGFR-1-specific probe. Shown are dark-field images of (a) a control animal and of animals (b) 24 and (c) 48 hours after MCAO and bright-field images of a control animal, showing VEGFR-1 expression in endothelial cells (arrow in d) and glial cells (arrow in e) but not in hippocampal neurons (arrowhead in e). Note the up-regulation of VEGFR-1 during infarction in pial vessels (arrowhead) and in vessels invading the ischemic core (arrow in c). Original magnifications: a–c, ×6.25; d and e, ×250.

Figure 8.

Expression of VEGFR-2 mRNA during cerebral infarction: in situ hybridization with a VEGFR-2-specific probe. Shown are dark-field images of (a) a control animal and of animals (b) 24, (c) 48, (d) 72, and (e) 168 hours after MCAO. Note the up-regulation of VEGFR-2 in pial vessels (arrowheads) and in vessels invading the ischemic core (arrows) in d. f: Contralateral hemisphere 24 hours after MCAO. Note strong VEGFR-2 expression in the hippocampus (arrow). g: Ipsilateral hippocampus 72 hours after MCAO: higher magnification of the area depicted in d. h: Bright-field image of g. i: Hybridization with sense probe, same area as in g. k: Higher magnification of h, showing VEGFR-2-expressing cells (arrowheads). Original magnifications: a–f, ×6.25; g–i, ×25; k, ×250.

Figure 9.

Expression of VEGFR-2 protein during cerebral infarction. Immunohistochemistry with a monoclonal anti-VEGFR-2 antibody of ischemic brain hemisphere (a) and contralateral side (b) 72 hours after MCAO. Note the up-regulation of VEGFR-2 in pial vessels (arrowheads) and in vessels invading the ischemic core (arrows) in a. c: Immunohistochemistry staining with anti-VEGFR-2 antibody (same region as in Figure 8g ▶ ). The VEGFR-2-positive area is marked by arrows. d: Immunohistochemistry staining for PECAM-1 (same region as in c). Positive endothelial cells are marked by arrowheads. Original magnifications: a and b, ×25; c and d, ×50.

Figure 10.

Expression of HIF-1α and HIF-2α mRNA during cerebral infarction: in situ hybridization with specific probes for (a, c, e) HIF-1α and (b, d, f) HIF-2α in animals 72 hours after MCAO. a: Dark-field image showing HIF-1α expression; c: higher magnification of a; e: bright-field image of c; b: dark-field image showing HIF-2α expression; d: higher magnification of b; f: bright-field image of d. Note that HIF-1α-positive cells line the infarction (arrows in e), and HIF-2α-positive cells are situated in vessel-like structures (arrows in f). Original magnifications: a and b, ×6.25; c–f, ×50.