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Purinergic Signaling in Varicella Zoster Virus Vasculopathy

水痘带状疱疹病毒血管病中的嘌呤能信号传导

基本信息

批准号：
9128742
负责人：
Maria Acena Nagel
金额：
$ 34.02万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-01 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9128742
关键词：
ADP Receptors Adenosine Aneurysm Antiviral Agents Arteries Attenuated Binding Blood Vessels Brain hemorrhage Cell surface Cells Central Nervous System Diseases Cerebrovascular system Chemosensitization DNA DNA Virus Infections Data Elderly Event Extracellular Matrix Fibroblasts Ganglia Germ Cells Health Herpes zoster disease Herpesviridae Herpesvirus 1 Herpesvirus Type 3 Human In Vitro Incidence Infection Integrins Ischemia Lead Measurement Measures Medial Mediating Membrane Microscopy Modeling Myofibroblast Pathway interactions Population Purinoceptor RNA Virus Infections Receptor Activation Risk Role Signal Pathway Signal Transduction Simplexvirus Smooth Muscle Smooth Muscle Myocytes Staining method Stains Stroke Structure Testing Time Tunica Adventitia Vaccines Vascular remodeling Virus Virus Diseases Visual cell motility cerebral artery cerebrovascular clopidogrel extracellular in vivo inhibitor/antagonist matrigel migration novel prevent receptor research study stroke treatment tripolyphosphate varicella zoster virus vasculopathy viral DNA

项目摘要

DESCRIPTION (provided by applicant): Varicella zoster virus (VZV) is a ubiquitous, human alphaherpesvirus that can reactivate from ganglia, infect cerebral arteries, and cause stroke (VZV vasculopathy). Specifically, VZV-infected arteries have a thickened intima composed of myofibroblasts, likely originating from smooth muscle cells or adventitial fibroblasts, which can contribute to luminal occlusion and ischemia, and have loss of medial smooth muscle cells which can contribute to aneurysm formation and hemorrhagic stroke. Binding of extracellular adenosine 5' triphosphate (ATP) and its metabolites to specific purinergic receptors on the cell surface (purinergic signaling) has emerged as important in virus infection and pathological vascular remodeling. Our preliminary data show that: (1) compared to mock-infected cells, extracellular ATP is rapidly increased upon VZV infection of human cerebrovascular adventitial fibroblasts, which are the initial cells infected in arteries and which are the key regulators of vascular tone and structure, and (2) blockade of cellular ATP/ADP receptors, particularly with P2Y12 receptor inhibitor clopidogrel, attenuates virus infection. Thus, we hypothesize that specific purinergic receptor subtypes in cerebral vasculature mediate VZV infection of adventitial fibroblasts and also promote VZV-induced pathological cerebrovascular remodeling and stroke. Herein, we will identify the mechanism(s) by which activation of P2Y12 receptors promotes VZV infection in cerebrovascular adventitial fibroblasts (Aim 1). Since P2Y12 receptor activation has been shown to activate specific integrins downstream, including a3b2 and _b1, which can be used for virus entry, we will treat VZV-infected human cerebrovascular adventitial fibroblasts with select integrin inhibitors involved in the P2Y12 pathway, as well as in herpesvirus entry into cells. One and 3 days later, virus will be titered and viral DNA quantitated by PCR to identify integrin chains/heterodimers involved in VZV infection. After identification of these specific integrins, we will determine if specific integrin activation is downstream of P2Y12 receptor activation by examining possible potentiation effects of combined integrin/P2Y12 inhibitors on viral DNA and titers. We will also identify the specific purinergic receptor(s) that mediates VZV-induced cerebrovascular remodeling by measuring the migration of VZV or mock-infected human cerebrovascular adventitial fibroblasts and smooth muscle cells in vitro with and without specific purinergic receptor inhibitors using matrigel invasion chambers (Aim 2a). Finally, additional preliminary data showed that VZV-infection of cadaveric human cerebral arteries ex vivo mimics vessel wall changes seen in vivo in VZV vasculopathy (thickening of the intimal layer) which is absent in mock-infection - providing a model to identify specific purinergic receptors involved in VZV-induced remodeling within a whole artery (Aim 2b). Thus, we will VZV or mock-infect cadaveric human cerebral artery explants with and without specific purinergic receptor inhibitors and examine effects on vascular remodeling 2 and 4 weeks later by measuring intimal thickening.

描述（由申请方提供）：水痘带状疱疹病毒（VZV）是一种普遍存在的人α疱疹病毒，可从神经节重新激活，感染脑动脉，并导致卒中（VZV血管病变）。具体而言，VZV感染的动脉具有由肌成纤维细胞组成的增厚的内膜，可能来源于平滑肌细胞或外膜成纤维细胞，这可能导致管腔闭塞和缺血，并且具有中膜平滑肌细胞的损失，这可能导致动脉瘤形成和出血性中风。细胞外腺苷5'三磷酸（ATP）及其代谢产物与细胞表面特异性嘌呤能受体的结合（嘌呤能信号传导）在病毒感染和病理性血管重塑中已成为重要的。我们的初步数据显示：（1）与模拟感染的细胞相比，VZV感染人脑血管外膜成纤维细胞后，细胞外ATP迅速增加，成纤维细胞是动脉中感染的初始细胞，是血管张力和结构的关键调节因子，（2）阻断细胞ATP/ADP受体，特别是用P2 Y12受体抑制剂氯吡格雷，可减弱病毒感染。因此，我们假设脑血管中特定的嘌呤能受体亚型介导VZV感染外膜成纤维细胞，并促进VZV诱导的病理性脑血管重塑和中风。在此，我们将确定P2 Y12受体激活促进脑血管外膜成纤维细胞中VZV感染的机制（目的1）。由于P2 Y12受体激活已显示激活下游的特异性整合素，包括可用于病毒进入的a3 b2和_b1，我们将用参与P2 Y12途径以及疱疹病毒进入VZV的选择的整合素抑制剂治疗VZV感染的人脑血管外膜成纤维细胞。细胞1天和3天后，对病毒进行滴定，并通过PCR定量病毒DNA，以鉴定参与VZV感染的整联蛋白链/异源二聚体。在鉴定这些特异性整合素后，我们将通过检查整合素/P2 Y12抑制剂组合对病毒DNA和滴度的可能增强作用来确定特异性整合素活化是否在P2 Y12受体活化的下游。我们还将通过使用基质胶侵入室测量VZV或模拟感染的人脑血管外膜成纤维细胞和平滑肌细胞在体外有和没有特异性嘌呤能受体抑制剂的情况下的迁移来鉴定介导VZV诱导的脑血管重塑的特异性嘌呤能受体（Aim 2a）。最后，额外的初步数据显示，离体尸体人脑动脉的VZV感染模拟了体内VZV血管病变（内膜层增厚）中观察到的血管壁变化，这在模拟感染中是不存在的-提供了一个模型，以识别参与VZV诱导的整个动脉重塑的特异性嘌呤能受体（目的2b）。因此，我们将VZV或模拟感染尸体人脑动脉外植体与特异性嘌呤能受体抑制剂和检查血管重塑2和4周后，通过测量内膜增厚的影响。