RAAV1-mediated Gene Overexpression Of VEGF-165 And Angiopoietin-1 In Rat Cerebral Ischemia Model: An Investigation On Its Efficacy And Possible Mechanisms

IntroductionVascular endothelial growth factor (VEGF), a potent endothelial cell mitogen associated with angiogenesis, is strongly induced after acute focal cerebral ischemia in rats. And it has been suggested as a promising pharmacological agent for stroke therapy due to its strong angiogenic and neuroprotective capacities. In addition, early intervention of therapeutic factors within the time window of therapy is considered as a main treatment principal for stroke to rescue the ischemic penumbra. However, a few of previous studies clearly indicated that acute VEGF therapy via intraluminal way during reperfusion significantly aggravated ischemic injury by increased leakage of blood-brain barrier (BBB) and intracerebral hematoma formation in experimental stroke. In contrast, several recent investigations have demonstrated that VEGF application via intraventricular approach at the early stage of stroke could exhibit neuroprotective effects on brain ischemia without increasing BBB permeability or inducing further cerebral edema. And several new reports also indicated that the VEGF-induced permeability effect in the acute stage of stroke could be counteracted by angiopioetin-1, which shows a synergic effect on inducing ischemic angiogenesis with VEGF. Fortunately, these results open the possibility that the intraventricular route may be a favourable way for VEGF to exert early protective effect on cerebral ischemic injury, since VEGF infusion by this way was believed to avoid acting directly on vascular endothelial cells to increase microvessel leakage. Or combination of Ang-1 and VEGF may provide a more adapted therapeutic strategy than the use of VEGF alone in the early therapy for stroke.However, it was also reported that large-molecule therapeutic agents with intracerebroventricular delivery only distributed to the ependymal surface of the ipsilateral ventricle and failed to significantly penetrate into cerebral parenchyma with a very small treatment volume less than one percent of the brain. Likewise, with a molecular weight as large as 45,000 Da and 72,000 Da, infused VEGF or Ang-1 protein in the cerebral ventricle may be unable to substantially transport across the BBB and reach its maximal efficiency in the treatment of cerebral ischemia either. In contrast, preischemic vector-mediated VEGF gene transfer via intraventricular way may be a preferable alternative for VEGF early treatment for ischemic stroke due to its increased ability to pass through BBB. In a previous study, it was reported that intraventricular infusion of rAAV1 vector carrying enhanced green fluorescence protein gene resulted in a multiple transduction in both choroids plexus and paraventricular regions of adult rat brain18. In the present study,we injected rAAVl-VEGF165 into the lateral ventricle of adult rats eight weeks before transient middle cerebral artery occlusion (tMCAO) for gene therapy and examine its possible effects on ischemic insults at the early stage of stroke.Materials and methodsPartâ… Ninety-three adult male Wistar rats (body weight 180-200 g) were divided randomly into there equal groups to be injected with rAAV-VEGF (2.0×10¹⁰vg),rAAV-null(2.0×10¹⁰vg),or physiological saline (NS) respectively. And tMCAO models were induced after 8 weeks. During the following 48 hours, intracranial pressure (ICP), brain water content, cerebral edema volume in MRI,cerebral infarct volume, modified neurological severity scores (NSS), and VEGF level were determined respectively and compared statistically with that of the control groups.Partâ…¡ Ninety-three adult male Wistar rats (body weight 180-200 g) were divided randomly into two equal groups to be injected with rAAV-VEGF(1.0×10¹⁰vg)/rAAV-Angl(1.0×10¹⁰vg) or rAAV-VEGF(1.0×10¹⁰vg)/rAAV-null(1.0×10¹⁰vg),respectively. And tMCAO models were induced after 8 weeks. During the following 48 hours, VEGF and Ang-1 level, BBB permeablilty,microvessle density,the cerebral blood flow in peri-infarct zone, AQP4 expression, c-fos expression, and occludin expression were determined respectively and compared statistically with that of the control group.ResultsPartâ… Intraventricular application of rAAV-VEGF eight weeks before tMCAO resulted in VEGF overexpression and microvessle density elevation in multiple brain areas. At 24 hours following tMCAO, the rAAV-VEGF group, with VEGF overexpression in the rats brain, showed a significantly increase in ICP, brain water content, and cerebral edema volume compared with two control groups (P<0.05). And the ICP significantly correlated with the brain water content in the infarct hemisphere in all three groups during 24 hours following tMCAO (r=0. 93, P<0. 05). At 48 hours followingtMCAO,a 1.4-fold larger infarct volume and 1. 3-fold higher NSS were observed in the rAAV-VEGF group than both control groups (P<0.05). Partâ…¡Intraventricular application of rAAV-VEGF/rAAV-Angl eight weeks before tMCAO resulted in VEGF and Ang-1 overexpression, and significantly decreased EB permeability and AQP4 expression following ischemia (P< 0.05).In addition, the cerebral blood flow was also signicantly elevated with increased microvessle density in peri-infarct zone in rAAV-VEGF/ rAAV-Angl group compared to rAAV-VEGF/ rAAV-null group(P<0.05). Moreover, we also observed that there were more positive cells expressing c-fos or occludin in the brain of rat coming from rAAV-VEGF/rAAV-Angl group than that from rAAV-VEGF/rAAV-null group(P<0.05).ConclusionsPartâ… Our results indicate that intraventricular rAAV-VEGF pretreatment can result in deleterious intracranial hypertension and augment secondary ischemic insults at the early stage of tMCAO, and preischemic VEGF gene transfer via intraventricular approach may not be a favorable therapeutic strategy for tMCAO which should be adopted with caution or avoided in experimental stroke.Partâ…¡In cerebral ischemia, the combination of Ang-1 and VEGF could be used early to promote the formation of mature neovessles and protect the injured cells without inducing side effects on BBB permeability. And the early intraventricualr injection of mixed rAAV-VEGF and rAAV-Angl may be a rational therapeutic strategy in the gene therapy for experimental stroke.