Mechanisms of CD40/CD40L in vascular injury and repair

描述(申请人提供)：该项目的长期目标是更好地了解血管损伤和修复的炎症机制，并开发新的治疗策略来治疗血管疾病患者。大量证据表明，CD40-CD40配体(CD40L)通路在免疫和炎症反应中起着关键作用。循环中CD40L水平的升高与高胆固醇血症、糖尿病、急性冠脉综合征以及再狭窄的可能性增加有关。到目前为止，还没有关于CD40L通路是否以及如何参与血管损伤和修复过程的直接实验证据。我们的初步研究结果表明，在动脉粥样硬化的背景下，CD40L阻断对动物的血管损伤反应具有多重有益作用，这与在非动脉粥样硬化的小鼠损伤模型中的有限证据相反。根据我们的初步数据，这一建议的总体假设是循环CD40L(包括可溶性CD40L和血小板相关CD40L)升高，并在动脉粥样硬化环境中血管损伤后促进促炎反应和新生内膜形成。为了验证我们的假设并实现我们的长期目标，我们将在以下三个具体目标中使用遗传学、药理学和生化方法在具有良好特征的细胞和动物模型中。目的1在非动脉粥样硬化环境中，观察CD40L在apoE-/-CD40L-/-小鼠和apoE-/-CD40L+/+小鼠动脉损伤(颈动脉丝和FeCl3损伤)后炎症和新生内膜形成中的作用。骨髓来源细胞和血管壁细胞对CD40L表达的相对贡献将通过骨髓移植创造嵌合动物来确定。目的2将确定在动脉粥样硬化环境中，可溶性CD40L和血小板相关CD40L在血管损伤的炎症和血栓反应中的作用。我们将检测apoE-/-CD40L-/-小鼠和apoE-/-CD40L+/+小鼠的血小板-白细胞的激活和相互作用以及组织因子(TF)的表达，然后确定重组sCD40L或含CD40L的血小板在小鼠静脉内重建对动脉损伤处血小板-白细胞聚集和组织因子(TF)表达的影响。目的探讨CD40(CD40L受体)在体外中性粒细胞活化和炎症功能中的作用，以及在体内血管损伤后的炎症和血栓反应中的作用。我们将利用CD40表达的中性粒细胞和CD40缺陷的中性粒细胞在体外确定CD40L对中性粒细胞的影响是否需要CD40，然后确定CD40在CD40缺陷(CD40-/-和apoE-/-CD40-/-)小鼠动脉损伤后白细胞募集和新生内膜形成中的作用。此外，我们还将确定体内是否需要CD40来诱导CD40L介导的转铁蛋白。这些研究的结果将从机制上深入了解CD40/CD40L信号在动脉粥样硬化背景下血管损伤反应中的作用，并可能为开发新的治疗策略来预防和治疗人类血管疾病提供有价值的信息。项目简介：临床再狭窄，定义为血管初始管腔大小丧失50%，仍然是冠状动脉成形术和支架置入术后患者的主要临床问题。该项目旨在利用已建立的血管损伤和修复的小鼠模型更好地了解再狭窄的机制。所获得的结果将为研究再狭窄的机制提供新的见解，并有望为开发预防和治疗人类血管疾病的新的治疗策略提供有价值的信息。

DESCRIPTION (provided by applicant): The long-term goal of this project is to better understand inflammatory mechanisms of vascular injury and repair and to develop new therapeutic strategies to treat patients with vascular diseases. Abundant evidence indicates that the CD40-CD40 ligand (CD40L) pathway plays a pivotal role in immune and inflammatory responses. Elevated levels of CD40L in the circulation have been associated with hypercholesterolemia, diabetes, acute coronary syndromes, and an increased probability of restenosis. To date there is no direct experimental evidence addressing if and how the CD40L pathway contributes to vascular injury and repair process. The results of our preliminary studies indicate multiple beneficial effects of CD40L blockade on the vascular injury response in animals on an atherogenic background, which is contrary to the limited evidence in a non-atherogenic mouse injury model. Based on our preliminary data, the overall hypothesis of this proposal is that circulating CD40L (both soluble CD40L and platelet-associated CD40L) is elevated and contributes to proinflammatory responses and neointimal formation after vascular injury in an atherogenic environment. To test our hypothesis and achieve our long-term goal, we will employ genetic, pharmacologic, and biochemical approaches in well-characterized cellular and animal models in the following three Specific Aims. Aim 1 will establish the role of CD40L in inflammation and neointima formation after arterial injury (carotid wire and FeCl3 injury) in apoE-/-CD40L-/- mice and apoE-/-CD40L+/+ mice vs in mice deficient in CD40L in a non-atherogenic environment. The relative contribution of CD40L expression by bone- marrow derived cells and vascular wall cells will be determined by creation of chimeric animals by bone marrow transplantation. Aim 2 will establish the contribution of soluble CD40L and platelet-associated CD40L in the inflammatory and thrombotic responses to vascular injury in an atherogenic milieu. We will examine platelet-leukocyte activation and interactions as well as tissue factor (TF) expression in apoE-/-CD40L-/- mice and apoE-/-CD40L+/+ mice, and then determine effects of intravenous reconstitution with recombinant sCD40L or CD40L-containing platelets in the mice on platelet-leukocyte leukocyte recruitment and TF expression at sites of arterial injury. Aim 3 will establish the role of CD40 (the receptor for CD40L) in neutrophil activation and inflammatory functions in vitro, and in the inflammatory and thrombotic responses to vascular injury in vivo. We will determine if CD40 is required for CD40L-mediated effects on neutrophils in vitro using CD40- expressing and CD40-deficient neutrophils, and then determine the role of CD40 in leukocyte recruitment and neointima formation after arterial injury in CD40-deficient (CD40-/- and apoE-/-CD40-/-) mice. In addition, we will determine if CD40 is required for CD40L-mediated TF induction in vivo. The results of these studies will provide mechanistic insight into the role of CD40/CD40L signaling in the vascular response to injury on an atherogenic background and may provide valuable information for the development of novel therapeutic strategies to prevent and treat vascular disease in humans. Project Narrative: Clinical restenosis, defined as e 50% loss of the initial lumen size of vessels, remains a major clinical problem in patients after coronary angioplasty and stent placement. This project is designed to better understand the mechanisms of restenosis using the well established mouse model of vascular injury and repair. The results obtained will provide novel insights into the mechanisms of restenosis and promise to provide valuable information for the development of novel therapeutic strategies to prevent and treat vascular disease in humans.