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.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 本研究的目的是通过下调或抑制单核细胞转运过程中的基质金属蛋白酶-9的表达，开发一种治疗疱疹的方法。猕猴感染与人类免疫缺陷病毒(HIV)、猴免疫缺陷病毒(SIV)同源，是一种用于研究HIV神经发病机制的成熟模型。与HIV一样，SIV感染与脑炎(SIVE)有关，其特征是受感染的单核细胞渗透到中枢神经系统(CNS)。本研究的主要目的是基于基质金属蛋白酶-9的表达谱，准确地检测渗透到中枢神经系统的细胞的免疫表型，以此作为这些细胞侵袭性和血脑屏障(BBB)破坏的潜在指标。外周血单个核细胞(PBMC)中MMP9+细胞的特性将被检测为HIVE/SIVE的诊断和预测标记物。基质金属蛋白酶-9也将被评估为艾滋病毒辅助治疗的治疗靶点 病毒载量和外周免疫激活都与HIV神经发病有关。表达基质金属蛋白酶-9的细胞从外周血转移到大脑，导致血脑屏障完整性的破坏，并启动炎症反应。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CD14高CD16+单核细胞优先扩增和sCD14水平升高有关。CD16+单核细胞产生CCL2和IL-6，这也与HAD风险增加有关。这些细胞产生基质金属蛋白酶-9，在浸润性细胞和HAD患者的脑脊液中也表达上调。在体外血脑屏障模型中通过基因沉默或在HIV nef相关血脑屏障功能障碍的大鼠模型中使用特定的基质金属蛋白酶-9抑制剂来阻断基质金属蛋白酶-9活性的研究已经证明改善了血脑屏障的完整性。HIV感染者外周免疫细胞和激活的小胶质细胞中的MMP-9活性上调，可能是通过促进小血管基底膜的破坏，使感染的单核细胞渗透到中枢神经系统中所必需的。本研究拟以SIV作为HIV的动物模型，对其表达上调与艾滋病神经病理的关系进行全面的研究。本研究的最终目的是确定SIV中表达炎性细胞的MMP9的表型特征，该表型可用于艾滋病感染早期和HAD发生前的神经病理并发症的诊断。还将讨论通过抑制HIVE/SIVE来预防和/或治疗手部的基质金属蛋白酶-9的治疗意义。 我们推测，单核细胞表达和活性的差异将构成一个积极的危险因素，并作为感染早期发生HIVE/SIVE的预测标志，干扰MMP9的活性将对HIVE/SIVE具有保护作用。我们正在用流式细胞术检测SIV感染恒河猴外周血单个核细胞中的基质金属蛋白酶-9的纵向表达，并将研究其与终末期疾病时脑神经损伤的相关性。免疫印迹和酶谱检测MMP9的相对浓度和活性将检测SIV感染动物的血浆和脑脊液。我们将用过夜培养的条件组织培养液来检测SIV感染动物的PBMC中基质金属蛋白酶-9的表达和释放，并将评估其与脑炎所致脑损伤的相关性。将SIV感染动物的PBMC接种于覆盖有Matrigel的Transwell系统中，作为基底膜的体外替代物，进行Matrigel侵袭实验。表达基质金属蛋白酶-9的细胞的侵袭性的特异性将通过用基质金属蛋白酶-9抑制剂阻断其迁移来证实。在高度侵袭性的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确定。SIV感染动物的血浆或脑脊液中的基质金属蛋白酶-9对血脑屏障的损伤将在体外模型中进行检测，该模型使用植入Transwell系统的单层脑微血管内皮细胞(BMVEC)。由基质金属蛋白酶-9启动子驱动的报告基因(EGFP)在感染SIV的外周血单核细胞中的表达将被监测，并将检测这些细胞在体外BBB(Transwell系统中的Matrigel或单层内皮细胞)模型中的迁移。过表达的基质金属蛋白酶-9，siRNA基因敲除，基质金属蛋白酶-9的特异性抑制物，以及治疗剂量的米诺环素，将被用来证实基质金属蛋白酶-9参与迁移试验的特异性。在没有或存在MMP9抑制剂的情况下，SIV感染和/或MMP9过度表达的单核细胞对健康和SIV感染的恒河猴脑的渗透将被检测。单核细胞将被近红外染料标记，通过在红外成像仪上扫描脑组织切片或通过Percoll梯度制备的细胞悬液上的流式细胞术来检查这些细胞在大脑中的存在。结果将通过siRNA基因敲除、特异性抑制剂或米诺环素来阻断基质金属蛋白酶-9的活性而得到证实。