MMP-9 ROLE IN SIVE

MMP-9 在 SIVE 中的作用

• 批准号: 8358006
• 负责人: Susan V. Westmoreland
• 金额: $ 6.84万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8358006
• 关键词: Acquired Immunodeficiency Syndrome; Address; Animal Model; Animals; Basement membrane; Blood - brain barrier anatomy; Blood Vessels; Brain; Brain Injuries; CCL2 gene; Cells; Central Nervous System Diseases; Culture Media; Cultured Cells; Detection; Development; Diagnosis; Diagnostic; Dose; Dyes; Encephalitis; Endothelial Cells; FCGR3B gene; Flow Cytometry; Functional disorder; Funding; Gelatinase B; Gene Silencing; Genes; Goals; Grant; HIV; Homologous Gene; Immigration; Immune; In Vitro; Individual; Infection; Infiltration; Inflammatory; Inflammatory Response; Interleukin-6; Investigation; Label; Link; Macaca; Macaca mulatta; Matrigel Invasion Assay; Microglia; Migration Assay; Minocycline; Modeling; Molecular Profiling; Monitor; National Center for Research Resources; Neuraxis; Neuronal Injury; Neuropathogenesis; New England; Percoll; Peripheral; Peripheral Blood Mononuclear Cell; Phenotype; Plasma; Prevention; Primates; Principal Investigator; Process; Rattus; Relative (related person); Reporter Genes; Research; Research Infrastructure; Research Project Grants; Resources; Risk; Risk Factors; SIV; Scanning; Slice; Small Interfering RNA; Source; Specificity; Suspension substance; Suspensions; System; Terminal Disease; Testing; Therapeutic; United States National Institutes of Health; Up-Regulation; Viral Load result; Virus Diseases; Western Blotting; attenuation; base; brain tissue; cost; immune activation; improved; in vitro Model; inhibitor/antagonist; matrigel; monocyte; monolayer; neuropathology; overexpression; peripheral blood; promoter; therapeutic target; tissue culture; trafficking

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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 子项目的主要研究者可能是由其他来源提供的， 包括其他NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 本研究的目的是通过敲低或抑制运输单核细胞中的MMP-9来开发治疗HIVE的方法。猕猴感染人类免疫缺陷病毒（HIV）的同源物猴免疫缺陷病毒（SIV）是研究HIV神经发病机制的一种已建立的模型。 与HIV一样，SIV感染与脑炎（SIVE）相关，其特征在于被感染的单核细胞浸润中枢神经系统（CNS）。本研究的主要目标是基于MMP-9的表达谱准确地检查浸润CNS的细胞的免疫表型，MMP-9作为这些细胞的侵袭性和破坏血脑屏障（BBB）的潜力的指标。 将检查外周血单核细胞（PBMC）中MMP-9+细胞的身份，作为HIVE/SIVE的诊断和预测标志物。 MMP-9也将作为HIV辅助治疗的治疗靶点进行评估 病毒载量和外周免疫激活都与HIV神经发病机制有关。表达MMP-9的细胞从外周血迁移到脑，导致BBB完整性的破坏并开始炎症反应。在外周血中检测到这些高侵袭性细胞可能预示着艾滋病相关的中枢神经系统疾病。HAD与CD 14高CD 16+单核细胞的优先扩增和sCD 14水平升高相关。CD 16+单核细胞产生CCL 2和IL-6，这也与HAD的风险增加有关。这些细胞产生MMP-9，其在浸润细胞和具有HAD的CSF中也上调。在体外BBB模型中通过基因沉默阻断MMP-9活性或在HIV nef相关BBB功能障碍的大鼠模型中使用特异性MMP-9抑制剂的研究已经证明改善了BBB完整性。 MMP-9在外周免疫细胞和活化的小胶质细胞中的活性上调可能是HIV感染个体中感染的单核细胞通过促进小血管基底膜的破坏而浸润到CNS中所必需的。在本提案中，将对作为HIV动物模型的SIV进行关于MMP-9上调与AIDS神经病理学的相关性的全面调查。 本研究的最终目标是表征SIV中表达MMP-9的炎性细胞的表型，其可用于在感染早期和HAD发展之前诊断AIDS的神经病理学并发症。MMP-9抑制在预防和/或治疗HAND通过衰减或消除HIVE/SIVE的治疗意义也将得到解决。 我们假设，差异单核细胞MMP-9的表达和活性将构成一个积极的危险因素，并作为一个预测指标的发展HIVE/SIVE感染的早期和MMP-9活性的干扰将保护对HIVE/SIVE。我们正在研究MMP-9在SIV感染恒河猴的PBMC中的表达，并将通过流式细胞术研究与晚期疾病脑神经元损伤的相关性。蛋白质印迹法和酶谱法测定MMP-9的相对浓度和活性将检测SIV感染动物的血浆和CSF。将通过来自PBMC过夜培养物的条件组织培养基（cTCM）的酶谱法检查MMP-9从SIV感染动物的PBMC的表达和释放，并将评价其与符合脑炎的脑损伤的相关性。SIV感染动物的PBMC将通过在覆盖有Matrigel作为基底膜的体外替代物的Transwell系统的插入物中培养细胞来进行Matrigel侵袭测定。通过MMP-9抑制剂阻断MMP-9表达细胞的迁移来证实MMP-9表达细胞的侵袭特异性。高度侵袭性PBMC中免疫细胞的主要亚型将通过一组标志物的流式细胞术分析来定义。将在体外模型中使用在Transwell系统的插入物中的单层脑微血管内皮细胞（BMVEC）检查由来自SIV感染动物的血浆或CSF的MMP-9引起的对BBB的损伤。将在用SIV感染的外周单核细胞中监测由MMP-9启动子驱动的报告基因（EGFP）的表达，并将检查这些细胞穿过BBB的体外模型（Matrigel或transwell系统中的单层内皮细胞）的迁移。MMP-9的过表达、siRNA基因敲低、MMP-9特异性抑制剂和米诺环素的治疗剂量将用于确认MMP-9参与迁移测定的特异性。在MMP-9抑制剂不存在或存在的情况下，检查SIV感染和/或MMP-9过表达的单核细胞对健康和SIV感染的恒河猴脑的浸润。将用近红外染料标记单核细胞，并通过在红外成像仪上扫描脑组织切片和切片或通过流式细胞术对Percoll梯度制备的细胞悬液进行检查，以检查脑中是否存在这些细胞。结果将通过siRNA基因敲除、特异性抑制剂或米诺环素阻断MMP-9活性来证实。