Introduction
Vascular endothelial growth factor (VEGF) is a major regulator of new blood vessel growth (angiogenesis) and is required for the ability of the mature vasculature to adapt to tissue hypoxia. In addition, recent reports suggest that VEGF also has neurotrophic and neuroprotective functions. Therapeutic angiogenesis with VEGF is a clinically promising strategy in ischemic disease. The pathophysiological consequences of enhanced vessel formation, however, are poorly understood. In addition, the use of VEGF as a therapeutic agent is hampered by the fact that VEGF is also a major inducer of vascular permeability leading to brain edema. In an attempt to dissect the molecular pathways in vivo between angiogenesis, neuroprotection, and vascular permeability inducing properties of VEGF in the brain we generated transgenic mice overexpressing VEGF specifically in the brain. The aim of the present study was to define the pathophysiological consequences of brain-selective VEGF overexpression in ischemic stroke.
Methods
Mice expressing human VEGF165 under a neuron-specific promoter were established, which exhibited an increased density of brain vessels under physiological conditions 1 . These mice were submitted to focal cerebral ischemia, as induced by 90 or 30 minutes of intraluminal middle cerebral artery (MCA) occlusion, followed by 24 (90 min) or 72 (30 min) hours of reperfusion 2 . Cerebral blood flow (CBF) was measured using the 14C-iodoantipyrine technique.
Results
Transgenic mice overexpressing VEGF165 under control of the rat neuron-specific enolase (NSE) promoter are viable and fertile. Transgenic VEGF expression was predominantly found in neuronal cells of the hippocampus, dentate gyrus and cortex while the endogenous mouse VEGF gene is mainly expressed in astrocytes. Analysis of new vessel growth revealed a significant increase in the capillary density in all brain areas analyzed. After transient focal ischemia, transgenic VEGF significantly alleviated neurological deficits and infarct volume and reduced disseminated neuronal injury and caspase-3 activity. Furthermore, Akt activity was increased, confirming earlier in vitro observations that VEGF has neuroprotective properties. Brain swelling was not influenced by VEGF expression, while sodium fluorescein extravasation was moderately increased; suggesting that the higher VEGF levels in transgenic animal induced a mild blood brain barrier leakage. To elucidate whether enhanced angiogenesis improves regional cerebral blood flow in the ischemic brain, 14C-iodoantipyrine autoradiography was performed. Autoradiographies revealed that VEGF induces hemodynamic steal phenomena with reduced blood flow in ischemic areas and increased flow values only outside the MCA territory.
Conclusions
Our data demonstrate that VEGF protects neurons from ischemic cell death by a direct phosphatidylinositol-3 kinase/ Akt-dependent neurotrophic action rather than by promoting angiogenesis. Our results suggest that strategies aiming at increasing vascular density in the whole brain, e.g. by VEGF overexpression, may worsen rather than improve cerebral hemodynamics after stroke.