VEGF-induced neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia (original) (raw)

The major finding of this study is that the administration of VEGF to rats undergoing focal cerebral ischemia reduces infarct size and enhances neurogenesis and cerebral angiogenesis. Of particular note, these effects were observed with delayed administration of VEGF, which was given beginning at day 1 and continued until day 3 after ischemia. This regimen was adopted because a previous study showed that delayed, but not early, postischemic administration of VEGF improved neurological recovery after stroke, apparently because early VEGF treatment promoted brain edema, hemorrhagic transformation of the infarct, and spread of the ischemic lesion (13). Neuroprotection following such delayed treatment is unusual, although it has been reported (32, 33). One explanation may be that most experimental neuroprotective agents have been directed against early events in ischemia, such as acute excitotoxicity, whereas VEGF may act on later events to prevent delayed cell death. In addition, in the model we employed, ischemia may evolve more slowly than it does after more prolonged MCAO, thereby expanding the time window for cell rescue. VEGF also produced a strikingly delayed effect on neurogenesis, which was apparent only after 28 days. This suggests a mechanism of action that, while triggered within the first 3 days after ischemia, requires considerably longer to become manifest.

VEGF and neuroprotection in ischemic brain. At least four previous studies have examined the effect of VEGF on cerebral infarct size. Hayashi et al. (12) applied VEGF topically to the surface of the rat brain after 90 minutes of MCAO, followed by reperfusion, and found that infarct volume at 24 hours was reduced approximately 45%. Bao et al. (14) used a different approach, in which transient MCAO in the rat was followed by intraventricular infusion of an anti-VEGF Ab for 7 days; in this case, the Ab increased infarct volume approximately 35%, providing indirect evidence for a role of VEGF in limiting infarct size. In contrast, van Bruggen et al. (34) reported that intraperitoneal administration of mFLT(1–3)-IgG, a fusion protein that sequesters VEGF, led to a decrease in infarct size of approximately 30% at 8–12 weeks after MCAO in mice. Finally, Zhang et al. (13) found a biphasic effect, wherein intravenous administration of VEGF to rats increased infarct size approximately 25% at 9 days when given 1 hour after MCAO, but improved neurological function at 7–28 days when given 48 hours after MCAO. These different results are not surprising considering that the studies cited differ with respect to species, whether MCAO was followed by reperfusion, whether VEGF or a VEGF-binding protein was given, the route and timing of administration, duration of follow up, and whether histological or functional criteria were used to evaluate outcome. One major determinant of the effect of VEGF on the ischemic brain (and a likely source of variability in the studies mentioned above) is the prominence of VEGF-induced edema, which is related to VEGF’s ability to enhance vascular permeability (13, 34). Our finding that intraventricular VEGF reduces infarct volume and improves neurological outcome at 3–28 days after transient focal ischemia is most consistent with the studies by Hayashi et al. (12) and Bao et al. (14), and with the delayed administration protocol used by Zhang et al. (13), and suggests that under the proper conditions, VEGF can protect the brain against ischemia. Although the mechanisms underlying VEGF-induced reduction of infarct size are uncertain, several lines of evidence, cited above, point to a direct protective effect on neurons. In previous studies, we found that VEGF reduced neuronal death from hypoxia in vitro and that this involved activation of the VEGFR2/Flk1 receptor and PI3K and decreased activation of caspase-3 (19, 20).

VEGF and neurogenesis in ischemic brain. Palmer et al. (24) first proposed a role for VEGF in neurogenesis, observing that in the DG new neurons were often observed in the vicinity of blood vessels. Louissaint et al. (25) went on to show that in the adult songbird brain, testosterone-induced angiogenesis was associated with increased production of brain-derived neurotrophic factor by endothelial cells and that an inhibitor of the VEGFR2/Flk1 tyrosine kinase blocked both the angiogenesis- and neurogenesis-promoting effects of testosterone in the higher vocal center of the canary brain. We reported that VEGF stimulated neurogenesis both in mouse brain cultures in vitro and in neuroproliferative regions (SVZ and SGZ) of the nonischemic mouse brain in vivo (26). In the latter case, VEGF was infused by the intracerebroventricular route for 3 days, BrdU was given by the intraperitoneal route over the same period, and neurogenesis was measured 1 week later. The observed increase in BrdU labeling of cells that coexpressed neuronal markers suggested that VEGF acted at an early (proliferative) phase of neurogenesis.

In the present study, we found that VEGF had a delayed effect on the number of BrdU-labeled cells of neuronal lineage in the ischemic brain, consistent with increased survival of newborn neurons rather than increased proliferation. This discrepancy can be reconciled by considering that focal cerebral ischemia itself induces neuroproliferation in SVZ and SGZ (27). Thus, the neuroproliferative effect of VEGF that we observed in the nonischemic brain (27) may be masked by the effect of ischemia itself in the ischemic brain. Comparison of control and VEGF-treated ischemic brain, however, reveals another effect of VEGF: the delayed appearance, between 3 and 28 days, of a difference between the number of BrdU-labeled cells of neuronal lineage in both SVZ and SGZ, and on both sides of the brain, with more such cells surviving in VEGF-treated mice in each case. This difference implies that VEGF not only stimulates the proliferation of neuronal precursors in the nonischemic brain, but also promotes the survival of proliferating cells of neuronal lineage in the ischemic brain, consistent with the neurotrophic effects of VEGF demonstrated previously in other systems (15–18). Because VEGF was administered for only 3 days following ischemia, even the delayed effects of VEGF must have been triggered during this period. Previous studies on the role of trophic factors in neurogenesis provide clues as to how this might occur (35, 36). Thus, the VEGF-induced increase in survival of neuronal precursor cells could be due to either long latency, downstream effects of VEGF signaling in the same cells that are stimulated to proliferate by ischemia (including priming these cells for the subsequent effects of other growth factors), or by enhanced proliferation of a separate, VEGF-responsive subpopulation characterized by more robust survival capacity.

VEGF and angiogenesis in ischemic brain. The timing and distribution of VEGF expression correlate with angiogenesis in the normal and hypoxic developing adult rat brain (4) and the ischemic adult (9) rat brain. For example, VEGF protein expression in the ischemic penumbra increased progressively between 2 and 14 days after MCAO, and evidence of new vessel formation was present during days 7–28 (9). Intracerebral infusion of VEGF stimulates angiogenesis detectable by laminin immunostaining in rat cerebral cortex 3–7 days later (10, 11). In the ischemic rat brain, intravenously administered VEGF also triggers angiogenesis, as manifested by an increase in the number and volume of FITC-dextran–perfused cerebral cortical microvessels 7 days later (13). We found an increase in the density of vWF-immunoreactive blood vessels 7–28 days after focal ischemia plus VEGF treatment in the striatal ischemic penumbra. The vWF immunostaining increased roughly fourfold over control levels and roughly twofold over levels in ischemic penumbra of rats not given VEGF. Neither ischemia nor ischemia plus VEGF significantly affected vessel density in the DG, however. This may be because (a) the DG does not undergo ischemic injury in this model, as shown by cresyl violet and Klenow staining (27), and (b) an ischemia-induced increase in the expression of VEGF receptors may be required to maximize the angiogenic effect of VEGF (5, 7, 37). In the present context, the failure of ischemia or VEGF to stimulate angiogenesis in DG argues that angiogenesis is not required for the neuroproliferative response to ischemia or VEGF in this region, although angiogenesis might still promote neurogenesis from resident precursor cells in the ischemic penumbra.

Several studies have shown that cerebral ischemia increases VEGF expression (5, 6, 38–40), so it may be puzzling that administration of exogenous VEGF should be able to modify infarct size, neurogenesis, or angiogenesis in the ischemic brain. This apparent paradox may be explained by differences in concentration, distribution, or timing of exposure to endogenous and exogenous VEGF. For example, intraventricular delivery of exogenous VEGF, as in this study, may result in higher levels of VEGF in the vicinity of the brain’s neuroproliferative zones because of the proximity of both SVZ and DG to the lateral ventricles.

In conclusion, our findings suggest that in the ischemic rat brain, administration of exogenous VEGF (a) provides an early neuroprotective effect that reduces infarct size and improves neurological outcome, which is maximal by 3 days; (b) promotes the survival of nascent neurons arising in DG and SVZ, an effect that becomes evident between 3 and 28 days; and (c) stimulates angiogenesis in the ischemic penumbra, but not in neuroproliferative zones remote from the site of ischemia. Thus, direct neuroprotection may reduce ischemic injury in the acute phase, whereas neurogenesis, angiogenesis, or both may contribute to longer-term repair of the injured brain. For example, neurogenesis might have the capacity to enhance brain repair after ischemia by replacement of dead cells, and both neurogenesis and angiogenesis could lead to the release of growth factors that promote recovery indirectly. Moreover, the effects of VEGF on neurogenesis and angiogenesis could be interrelated: although we did not observe ischemia-induced angiogenesis in DG, its occurrence in the ischemic penumbra might promote neurogenesis from resident neuronal precursor cells. These findings may have implications for the possible role of VEGF as a therapeutic agent for stroke.