Purinergic Signaling in Varicella Zoster Virus Vasculopathy

 描述(申请人提供)：水痘带状疱疹病毒(VZV)是一种普遍存在的人类甲型疱疹病毒，可从神经节重新激活，感染脑动脉，并导致中风(VZV血管病)。具体地说，VZV感染的动脉内膜增厚，由肌成纤维细胞组成，可能起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，并有中膜平滑肌细胞的丧失，这可能导致动脉瘤形成和出血性中风。细胞外三磷酸腺苷(ATP)及其代谢产物与细胞表面特定的嘌呤能受体的结合(嘌呤能信号)在病毒感染和病理性血管重塑中起重要作用。我们的初步数据显示：(1)与模型感染细胞相比，VZV感染人脑血管外膜成纤维细胞后，细胞外ATP迅速增加，而脑血管外膜成纤维细胞是动脉中感染血管的初始细胞，是血管张力和结构的关键调节细胞；(2)阻断细胞内的ATP/ADP受体，特别是与P2Y12受体抑制剂氯吡格雷一起阻断细胞内的ATP/ADP受体，可减轻病毒感染。因此，我们假设脑血管中特定的嘌呤能受体亚型介导外膜成纤维细胞感染VZV，并促进VZV诱导的病理性脑血管重构和卒中。在这里，我们将确定激活P2Y12受体促进脑血管外膜成纤维细胞感染VZV的机制(S)(目标1)。由于p2y12受体的激活已被证明可以激活下游的特定整合素，包括可用于病毒进入的a3b2和_b1，我们将用参与p2y12途径的部分整合素抑制剂以及疱疹病毒进入的途径来治疗vzv感染的人脑血管外膜成纤维细胞。 细胞。1天和3天后，分别进行病毒滴度和病毒DNA定量，以确定与VZV感染有关的整合素链/异源二聚体。在鉴定了这些特定的整合素后，我们将通过检测整合素/P2Y12联合抑制剂对病毒DNA和滴度的可能增强作用来确定是否在P2Y12受体激活的下游有特定的整合素激活。我们还将通过使用基质侵袭小室(AIM 2a)在体外检测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有和没有特定嘌呤能受体抑制剂的情况下的迁移来确定介导VZV诱导的脑血管重塑的特定的嘌呤能受体(目标2a)。最后，更多的初步数据显示，VZV体外感染身体人类大脑动脉模拟了VZV血管病变(内膜层增厚)在体内看到的血管壁变化，这在模拟感染中是不存在的-提供了一个模型来识别参与VZV诱导的全动脉重塑的特定嘌呤能受体(目标2b)。因此，我们将VZV或模拟感染的身体人大脑动脉外植体加或不加特定的嘌呤能受体抑制剂，并在2周和4周后通过测量内膜增厚来检测对血管重塑的影响。

 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.