This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The purpose of this research project is to develop a therapy for HIVE through knockdown or inhibition of MMP-9 in trafficking monocytes. Macaque infection with the homologue of human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), is an established model used to study HIV neuropathogenesis. Like HIV, SIV infection is associated with encephalitis (SIVE), characterized by infiltration of central nervous system (CNS) by infected monocytes. The main goal in this study is to accurately examine the immune phenotype of cells infiltrating the CNS based on the expression profile of MMP-9 as an indicator of the invasiveness of these cells and potential for disruption of blood brain barrier (BBB). The identity of MMP-9+ cells in peripheral blood mononuclear cells (PBMC) will be examined as a diagnostic and predictive marker of HIVE/SIVE. MMP-9 will also be evaluated as a therapeutic target for adjunct therapy for HIV Both viral loads and peripheral immune activation are linked to HIV neuropathogenesis. MMP-9 expressing cells transmigrate from peripheral blood to the brain, causing a breach in the BBB integrity and starting an inflammatory response. Detection of these highly invasive cells in the peripheral blood may be predictive of AIDS-related CNS disease. HAD is associated with preferential expansion of CD14high CD16+ monocytes and increased sCD14 levels. CD16+ monocytes produce CCL2 and IL-6, which have also been associated with increased risk of HAD. These cells produce MMP-9, which is also upregulated in infiltrating cells and in the CSF with HAD. Studies that have blocked MMP-9 activity through gene silencing in an in vitro BBB model or with the use of specific MMP-9 inhibitors in a rat model of HIV nef-related BBB dysfunction have demonstrated improved BBB integrity. Upregulated MMP-9 activity in peripheral immune cells and in activated microglia is likely essential for the infiltration of infected monocytes into the CNS in HIV-infected individuals by promoting break down of the basement membrane of small blood vessels. In the present proposal a comprehensive investigation on the correlation of MMP-9 upregulation with the neuropathology of AIDS will be conducted on SIV as an animal model of HIV. The ultimate goal in this study is to characterize the phenotype of MMP-9 expressing inflammatory cells in SIV that may be used in the diagnosis of neuropathologic complications of AIDS early in the infection and prior to the development of HAD. Therapeutic implications of MMP-9 inhibition in prevention and/or treatment of HAND by attenuation or elimination of HIVE/SIVE will also be addressed. We hypothesize that differential monocyte MMP-9 expression and activity will constitute a positive risk factor and serve as a predictive marker for the development of HIVE/SIVE early in the infection and that interference with MMP-9 activity will be protective against HIVE/SIVE. We are in the process of examining MMP-9 expression in PBMCs longitudinally in SIV-infected rhesus by flow cytometry and will examine correlations with the neuronal injuries in the brain at terminal disease. Western blotting and zymography to determine the relative concentration and activity of MMP-9 will test plasma and CSF of SIV infected animals. MMP-9 expression and release from the PBMC of SIV infected animals will be examined by zymography of the conditioned tissue culture media (cTCM) from overnight cultures of PBMC and its correlation with the brain injuries consistent with encephalitis will be evaluated. PBMC of SIV infected animals will be subjected to Matrigel invasion assay by culturing the cells in the inserts of Transwell system covered with Matrigel as an in vitro substitute for the basement membrane. The specificity of the invasiveness for MMP-9 expressing cells will be confirmed by blocking their transmigration by MMP-9 inhibitors. The predominant subtypes of the immune cells among the highly invasive PBMC will be defined by flow cytometric analysis of a panel of markers. Damages to the BBB caused by the MMP-9 from plasma or CSF of SIV infected animals will be examined in an in vitro model using a monolayer of brain microvascular endothelial cells (BMVEC) in the insert of a Transwell system. Expression of a reporter gene (EGFP) driven by MMP-9 promoter will be monitored in peripheral monocytes infected with SIV and transmigration of these cells across an in vitro model of BBB (Matrigel or a monolayer of endothelial cells in a transwell system) will be examined. Overexpression of MMP-9, siRNA gene knockdown, MMP-9 specific inhibitors, and therapeutic dose of minocycline will be used to confirm the specificity of MMP-9 involvement in migration assays. Infiltration of healthy and SIV infected rhesus brain by SIV infected and/or MMP-9 overexpressed monocytes will be examined in the absence or presence of MMP-9 inhibitors. The monocytes will be labeled by a near infrared dye and the presence of these cells in the brain will be examined by scanning the slices and sections of brain tissue on an infrared imager or by flow cytometry on cell suspensions prepared by Percoll gradients. Results will be confirmed by siRNA gene knockdown, specific inhibitors, or minocycline to block the MMP-9 activity.
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