VIRAL CHEMOKINE RECEPTORS: TRANSPLANT VASCULAR SCLEROSIS

该项目的长期目标是确定与人巨细胞病毒(HCMV)加速移植血管硬化有关的机制。所有带血管器官移植中移植物丢失的主要原因是与慢性排斥反应相关的血管损伤。这种形式的血管病变被称为TVS，其特征是向心性新生内膜平滑肌细胞增殖，导致血管闭塞，最终导致移植物失败。到目前为止，治疗严重电视病毒的唯一方法是再次移植。对移植受者的临床研究已经证明，人巨细胞病毒与电视加速之间存在直接联系。基于这些发现，我们建立了一种心脏和小肠移植的大鼠模型，在该模型中，CMV感染会加速TVS的时间和严重程度。虽然CMV介导这一过程的确切机制尚不清楚，但本课题组最近的工作表明，CMV可能通过表达病毒编码的趋化因子受体(US28)诱导平滑肌细胞(SMC)向趋化因子产生部位迁移，从而促进TVS的发生。我们还观察到，大鼠和小鼠CMV也通过各自的病毒趋化因子受体R33和M33诱导SMC迁移，R33和M33是HCMV US28的功能同源物。我们假设CMV加速TVS的机制涉及病毒编码的趋化因子受体的表达导致内膜SMC迁移，从而导致TVS特有的血管病变。因此，我们将利用我们的体外和体内模型来扩大我们的观察范围，并确定病毒编码的趋化因子受体在三个特定目标的电视节目发展中的作用。首先，利用慢性排斥反应的大鼠心脏移植模型，我们将确定病毒对TVS疾病进展动力学的影响，病毒在组织中的表达程度以及参与这一过程的细胞类型。我们还将确定宿主因素，如趋化因子和细胞因子，以及R33在RCMV诱导的TVS发展的不同阶段所起的作用。其次，我们将描述参与诱导SMC迁移的信号转导的R33结构域以及作为激动剂或拮抗剂诱导细胞运动的配体。在最后一个特例中，我们将产生重组RCMV，它包含信号相关区域的突变，以了解它们对大鼠心脏移植模型TVS的贡献。最后，R33诱导的SMC体外迁移的拮抗剂将在体内大鼠模型中测试其阻断TVS的能力。这些研究将为理解巨细胞病毒在电视加速中的作用提供有价值的动物模型。此外，这些研究的完成将为新的和合理的治疗策略的设计奠定基础，以提高感染HCMV的移植受者的长期移植物存活率。

The long term goal of this project is to identify mechanisms involved with human cytomegalovirus (HCMV) acceleration of transplant vascular sclerosis. The primary cause of graft loss of all vascularized organ transplants is due to a vascular lesion associated with chronic rejection. This form of vasculopathy referred to as TVS is characterized by concentric neointimal smooth muscle cell proliferation that results in vessel occlusion and ultimately graft failure. To date the only therapy available to treat severe TVS is retransplantation. Clinical studies in transplant recipients have demonstrated a direct link between HCMV and the acceleration of TVS. Based on these findings, we have developed a rat model of heart and small bowel transplantation in which CMV infection accelerates the time and severity of TVS. While the exact mechanism through which CMV mediates this process is unknown, recent work from our group suggests that CMV may contribute to TVS through induction of smooth muscle cell (SMC) migration towards sites of chemokine production through expression of a virally encoded chemokine receptor (US28). We have also observed that Rat and Murine CMV also induce SMC migration through their respective viral chemokine receptors R33 and M33, which are functional homologues of HCMV US28. We hypothesize that the mechanism of CMV-accelerated TVS involves the expression of virally encoded chemokine receptors leading to intimal SMC migration, which results in the characteristic vascular lesions of TVS. Therefore, we will utilize our in vitro and in vivo models to extend our observations and determine the role of virally encoded chemokine receptors in the development of TVS in three specific aims. First, using a rat cardiac transplant model of chronic rejection, we will determine the effects of virus on the kinetics of disease progression of TVS and the extent of viral expression in tissues as well as the cell types involved in the process. We will also determine the host factors such as chemokines and cytokines, and the contribution of R33 involved at various stages of the development of RCMV-induced TVS. Secondly, we will characterize the R33 domains involved in signaling which induce SMC migration and the ligands, which serve as agonists or antagonists to induced cellular movement. In the last specific, we will generate recombinant RCMV which contain mutations in domains involved in signaling to understand their contribution to TVS in the rat cardiac transplant model. Lastly, antagonists of R33 induced SMC migration in vitro will be tested for their ability to block TVS in the in vivo rat model. These studies will provide a valuable animal model to understand the role of CMV in the acceleration of TVS. In addition completion of these studies will form the basis for novel and rational design of therapeutic strategies to enhance long-term graft survival in HCMV infected transplant recipients.