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.
Despite the availability of a zoster vaccine, zoster incidence continues to rise in the growing elderly population with an attendant 30% increased risk of stroke. The results of these studies are significant since the specific purinergic receptor(s) involved in VZV infection and cell migration can serve as targets for novel, antiviral agents to treat stroke caused by VZV and other herpesvirus central nervous system diseases. Results will also lead to further characterization of the downstream events that mediate infection and vascular remodeling.
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