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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且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。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 该研究项目的目的是通过敲低或抑制运输单核细胞中的 MMP-9 来开发 HIV 疗法。猕猴感染人类免疫缺陷病毒 (HIV) 的同源物——猿猴免疫缺陷病毒 (SIV)，是用于研究 HIV 神经发病机制的既定模型。 与 HIV 一样，SIV 感染与脑炎 (SIVE) 相关，其特征是受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根据 MMP-9 的表达谱准确检查浸润 CNS 的细胞的免疫表型，作为这些细胞的侵袭性和血脑屏障 (BBB) 破坏潜力的指标。 将检查外周血单核细胞 (PBMC) 中 MMP-9+ 细胞的身份，作为 HIVE/SIVE 的诊断和预测标记。 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大脑，导致 BBB 完整性破裂并引发炎症反应。外周血中这些高度侵袭性细胞的检测可能可以预测艾滋病相关的中枢神经系统疾病。 HAD 与 CD14high CD16+ 单核细胞的优先扩增和 sCD14 水平升高相关。 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 MMP-9，其在浸润细胞和 HAD 脑脊液中的表达也上调。通过体外 BBB 模型中的基因沉默或在 HIV nef 相关 BBB 功能障碍的大鼠模型中使用特定 MMP-9 抑制剂来阻断 MMP-9 活性的研究表明，BBB 完整性得到改善。 外周免疫细胞和活化的小胶质细胞中 MMP-9 活性的上调可能对于 HIV 感染者中受感染的单核细胞通过促进小血管基底膜的分解而渗入 CNS 至关重要。在本提案中，将以 SIV 作为 HIV 动物模型，对 MMP-9 上调与 AIDS 神经病理学的相关性进行全面研究。 本研究的最终目标是表征 SIV 中表达 MMP-9 的炎症细胞的表型，该表型可用于诊断感染早期和 HAD 发展之前的 AIDS 神经病理并发症。还将讨论 MMP-9 抑制在通过减弱或消除 HIVE/SIVE 来预防和/或治疗 HAND 中的治疗意义。 我们假设单核细胞 MMP-9 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 HIVE/SIVE 发展的预测标记，并且干扰 MMP-9 活性将具有针对 HIVE/SIVE 的保护作用。我们正在通过流式细胞术纵向检查感染 SIV 的恒河猴 PBMC 中 MMP-9 的表达，并将检查与晚期疾病时大脑神经元损伤的相关性。用于确定 MMP-9 相对浓度和活性的蛋白质印迹和酶谱分析将测试 SIV 感染动物的血浆和脑脊液。将通过来自 PBMC 过夜培养物的条件组织培养基 (cTCM) 的酶谱检查 SIV 感染动物的 PBMC 的 MMP-9 表达和释放，并评估其与脑炎一致的脑损伤的相关性。 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品的Transwell系统的插入物中培养细胞来进行Matrigel侵袭测定。通过用 MMP-9 抑制剂阻断 MMP-9 表达细胞的迁移来证实其侵袭性的特异性。高侵袭性 PBMC 中免疫细胞的主要亚型将通过一组标记物的流式细胞术分析来定义。将使用 Transwell 系统插入物中的单层脑微血管内皮细胞 (BMVEC) 在体外模型中检查 SIV 感染动物血浆或 CSF 中的 MMP-9 对 BBB 造成的损害。将在感染 SIV 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，并将这些细胞跨 BBB 体外模型（Matrigel 或 Transwell 系统中的单层内皮细胞​​）进行迁移。检查了。 MMP-9 的过表达、siRNA 基因敲低、MMP-9 特异性抑制剂和治疗剂量的米诺环素将用于确认 MMP-9 参与迁移测定的特异性。在不存在或存在 MMP-9 抑制剂的情况下，将检查 SIV 感染和/或 MMP-9 过表达的单核细胞对健康和 SIV 感染的恒河猴大脑的浸润。单核细胞将被近红外染料标记，并且通过在红外成像仪上扫描脑组织切片和切片或通过对 Percoll 梯度制备的细胞悬浮液进行流式细胞术来检查大脑中这些细胞的存在。结果将通过 siRNA 基因敲低、特异性抑制剂或米诺环素来阻断 MMP-9 活性来证实。