Vascular endothelial growth factor (VEGF) is a secreted growth factor that plays an important regulatory role in vascular development. VEGF is a significant factor in angiogenesis in the periphery and in tumors and it has already been used clinically for peripheral vascular deficiencies. The proposed experiments will advance our understanding of VEGF and its receptors with respect to brain development and plasticity and may determine if the growth factor plays multiple roles in the CNS. Presently, there exists a paucity of work regarding its direct cellular actions on CNS tissues. The broad long-term goals of this research program are to understand the cellular and molecular interactions of VEGF with CNS tissue with a dual in vitro approach. Experiments use a serum-free organotypic explant system to determine VEGF effects on neuropil at different perinatal developmental stages as well as primary culture assays of neurons, astroglia and isolated CNS vessels. Preliminary data indicate that applied VEGF causes significant angiogenesis in fetal and adult CNS. Unexpectedly, VEGF application also produces significant astroglial proliferation mediated by its flt-1 receptor and enhanced neuronal expression of developmentally regulated proteins and as well as significant neurite outgrowth mediated by its flk-1 receptor. S.A. 1 will examine the angiogenic capacity of applied VEGF to CNS explants as well as the regulation of VEGF receptors and the integrity of the blood-brain barrier. S.A. 2 will test the hypothesis that VEGF acts as a mitogen for astroglia and is a growth factor for CNS neurons in organotypic explants. VEGF receptor and neuronal and astroglial markers will be examined with confocal microscopy, in situ hybridization and quantified by immunoblotting and immunoaffinity chromatography. An innovative technology utilizes microculture chambers to permit real-time microdialysis to determine if VEGF induces production of other cytokines/growth factors. Application of non-specific proteins, anti-sense oligodeoxynucleotides and receptor binding proteins will determine VEGF/receptor specificity in the system. We will also initiate experiments for analysis of receptor signal transduction pathways. S.A. 3 will test comparable parameters in primary cultures in order to verify VEGF's direct effects on CNS elements, absent potential indirect actions between cell types. As an angiogenic factor VEGF could have potential use in cerebrovascular disorders but may also have trophic effects upon astroglia and neurons. The results can lay important cell biological groundwork for understanding how brain tissue might respond to VEGF-based therapy.
