Mechanisms of Transmigration of HIV Infected Cells Across the Blood Brain Barrier

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DESCRIPTION (provided by applicant): With the success of HAART, HIV infected individuals are living longer and HIV mediated damage to the CNS, including HIV Associated Dementia (HAD) and other forms of neurocognitive impairment, are increasingly presenting as a devastating consequence of HIV infection. Although a late clinical finding, compromise of the blood brain barrier (BBB) and infiltration into the CNS parenchyma by HIV infected monocytes have been shown to occur early in infection. In fact, cognitive impairment correlates more directly with neuroinflammation than with the presence of virus within the CNS. CCL2, the predominate monocyte chemoattractant, has been shown to be substantially elevated in patients with HAD and HIV Encephalitis. Thus, understanding the mechanisms of HIV and CCL2 mediated monocyte transmigration into the CNS and concomitant disruption of the BBB will enable the identification of potential targets of therapy, with the ultimate goal of preventing HIV related neurocognitive decline. We hypothesize that HIV infection of monocytes alters their expression of CCR2, cell adhesion molecules, and adherens and tight junction proteins, and that transmigration of these infected monocytes in response to CCL2 in the CNS results in aberrant monocyte-BBB endothelial cell interactions, promoting enhanced migration and BBB disruption. The goal of this research is to define the molecular interactions responsible for the enhanced diapedesis by HIV infected monocytes into the CNS in response to the CCL2 chemokine and the subsequent BBB breakdown. The specific goals of this research are: (1) to identify differentially expressed genes or changes to protein localization in monocytes as a result of HIV infection and CCL2 chemotactic stimulation that may cofitribute to their enhanced diapedesis using microarray, RT-PCR, Western blot, and confocal imaging; (2) to characterize the effects of CCL2 on brain microvascular endothelial cell junction proteins that may potentiate monocyte diapedesis and disrupt BBB integrity using microarray, RT-PCR, Western blot, confocal imaging, and coimmunoprecipitation (ColP) studies; (3) to characterize the dynamic interactions between junctional proteins, adaptor proteins, and the cytoskeleton in HIV infected monocytes and in human brain microvascular endothelial cells that contribute to the enhanced transmigration properties of monocytes and disruption of BBB integrity in response to CCL2 using radiolabeling, ColP, Western blot, autoradiography, confocal imaging, and tissue staining; and (4) to determine the phosphorylation changes in cell juction proteins during transmigration of HIV infected monocytes across the BBB in response to CCL2 using radiolabeling, immunoprecipitation, autoradiography, and kinase and phosphatase inhibitors.