A defect in microvessel function appears to be responsible for the brain edema that occurs in children with lead poisoning and may also contribute to the neurotoxicity that results from exposure to low levels of lead. Our long-term goal is to determine the mechanisms by which inorganic lead damages the immature blood-brain barrier. We have found that low levels of lead activate protein kinase C (PKC) in neural microvessels. The focus of the current proposal is to investigate the functional significance of this activation and determine whether PKC mediates the inhibition by lead of neural microvessel formation that we find in cell culture. We will determine if lead induces PKC-dependent protein phosphorylation and immediate early gene expression in microvessels isolated from immature brain and further characterize the inhibitory effect of lead on in-vitro microvessel morphogenesis in co-cultures of endothelial cells and astrocytes. Experiments will be carried out using two in-vitro systems developed in this laboratory - microvessels isolated from immature rat brain and co-cultures of endothelial cells and astrocytes that model neural microvessel formation. Using synthetic peptide PKC substrates, we will measure lead induced PKC activity in both preparations. The phosphorylation of endogenous microvascular protein substrates after exposure to lead will be characterized by two dimensional gel electrophoresis. Inhibitors of PKC will be used to evaluate the specificity of this reaction. We will determine which immediate early genes known to be regulated by PKC are induced by exposure of the model microvascular systems to lead. Seven of these genes - c-myc, c-fox, c-jun, c-egr, beta-actin, fibronectin and the fibronectin receptor - will be studied using antibodies directed against their protein products. Finally, we will determine if the inhibitory action of lead upon in vitro angiogenesis is mediated by PKC and prevented by inhibitors of PKC. These experiments should provide new insights to an afferent limb of the metabolic responsive brain to lead exposure.
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