An IFN-y-Integrin-Growth Factor Axis in GA Biomarker Development

移植物动脉硬化(GA)的标志是血管重塑，包括内膜和中膜 通过基质沉积和中层平滑肌细胞(VSMC)增殖并迁移到 新生内膜。干扰素-γ在免疫介导的重塑中起重要作用 在GA中观察到的典型反应。我们的基因芯片和免疫组织化学分析 动脉病变，移植的人冠状动脉识别血管内皮生长因子 血管内皮生长因子受体-2(VEGFR2)和神经粘蛋白-2与血管内皮细胞和血管内皮细胞 细胞源性神经粘蛋白样分子(ESDN)，作为潜在的GA特异性标志物。因为已知的 为了研究VEGFR2-α-v-β3的相互作用以及α-v-β3在VSMC迁移/增殖中的作用，我们利用人动脉移植/SCID小鼠GA嵌合模型来研究α-v-β3在GA过程中是否受到调控。我们发现α-v-beta3、ESDN和VEGFR2都作为GA的早期事件被高度上调和/或激活，这与细胞增殖动力学一致。血管内皮生长因子可诱导Th1细胞极化并激活α-v-β3 整合素。Alpha-v-beta3参与可以反过来增强VEGFR2介导的信号转导。我们假设血管内皮生长因子， 由组织缺氧和/或同种异体反应诱导，有助于T细胞分化，干扰素-γ的分泌和 随之而来的是类似DTH的反应。同时，血管内皮细胞生长因子通过其经典受体和神经粘蛋白样蛋白 分子，调节VSMC整合素激活和α-v-β3介导的细胞增殖/迁移，这是一个标志 GA的特性。现在的具体建议包括：(1)确定血管内皮生长因子是否有助于 利用血管内皮生长因子/血管内皮生长因子受体拮抗剂在嵌合SCID中诱导产生干扰素的T细胞和GA的研究 小鼠/人动脉移植模型，以及同种异体T细胞-内皮细胞培养；(2)评估病因 SCID/人动脉移植中α-v-β3整合素激活/功能在外周血单核细胞和干扰素-γ诱导的GA中的作用及其与干扰素-γ和VEGFR信号转导的关系 模型和α-v-beta3抗体介导的阻断，以及激活的α-v-beta3靶向试剂和microSPECT技术在体内的GA成像；(3)确定GA中ESDN的诱导是由干扰素介导的还是 在嵌合小鼠模型中利用同种异体人PBMC或干扰素-γ刺激血管内皮生长因子依赖，以及 ESDN表达是否增强或终止最大GA反应；以及(4)解决干扰素-γ- 心脏移植患者VEGFR/ESDN-α-v-β3轴与血管内超声记录的GA的相关性 微血管定位的干扰素、干扰素调节的趋化因子、血管内皮生长因子受体、α-v-β3和ESDN的表达 心内膜心肌活检标本中，以及分泌分子的血清样本中。分子机制 在AIMS 1-3中确定的体内途径中，将在体外进行研究，并将与人类GA相关 拟议的研究将建立干扰素-γ-α-v-β3-VEGFR/ESDN轴在 遗传算法的发展，从而定义了早期遗传算法的敏感和实用的标记。

The hallmark of graft arteriosclerosis (GA) is vascular remodeling, which includes intimal and medial expansion by matrix deposition and medial smooth muscle cell (VSMC) proliferation with migration into the neointima. Interferon-gamma (IFN-y) plays a causative and key role in immune-mediated remodeling responses typical of those observed in GA. Our cDNA array and immunohistochemical analyses of arteriopathic, explanted human allograft coronary vessels identified vascular endothelial growth factor receptor-2 (VEGFR2) and Neuropilin-2, both VEGF-responsive receptors, and endothelial/smooth muscle cell-derived neuropilin-like molecule (ESDN), as potential specific GA markers. Because of the known VEGFR2-alpha-v--beta3 interactions and the role of alpha-v-beta3 in VSMC migration/proliferation, we utilized the chimeric human arterial transplant/SCID mouse model of GA to address whether alpha-v-beta3 is modulated during GA. We demonstrated that alpha-v-beta3, ESDN and VEGFR2 are all highly upregulated and/or activated as early events in GA, consistent with cell proliferation kinetics. VEGF can induce Th1 cell polarization and activate alpha-v-beta3 integrin. alpha-v-beta3 engagement can, in turn, enhance VEGFR2-mediated signaling. We hypothesize that VEGF, induced by tissue hypoxia and/or alloreactivity, contributes to T cell differentiation, IFN-y elaboration and consequent DTH-like responses. In parallel, VEGF, through its classical receptors and neuropilin-like molecules, modulates VSMC integrin activation and alpha-v-beta3-mediated cell proliferation/migration, a hallmark feature of GA. Specific proposals now include to: (1) determine whether VEGF contributes to the development of IFN-y- producing T cells and GA utilizing VEGF/VEGFR antagonists in the chimeric SCID mouse/human arterial graft model, and in allogeneic T cell-endothelial cell cultures; (2) assess the causative role of alpha-v-beta3 integrin activation/function in allogeneic peripheral blood mononuclear cell (PBMC)- and IFN-y-induced GA and its relationship to IFN-yR and VEGFR signaling, using the SCID/human arterial transplant model and alpha-v-beta3 antibody-mediated blockade, as well as in vivo GA imaging with an activated alpha-v-beta3-targeted agent and microSPECT technology; (3) determine whether ESDN induction in GA is IFN-y-mediated or VEGF-dependent, utilizing the allogeneic human PBMC or IFN-y stimuli in the chimeric mouse model, and whether ESDN expression enhances or terminates maximal GA responses; and (4) address the IFN-y- VEGFR/ESDN-alpha- v-beta3 axis in cardiac transplant patients, correlating intravascular ultrasound- documented GA with microvessel-localized IFN-y, IFN-y-regulated chemokines, VEGF/VEGFRs, alpha-v-beta3 and ESDN expression in endomyocardial biopsy specimens and, for secreted molecules, serum samples. Molecular mechanisms of the pathways identified in vivo in Aims 1-3 will be studied in vitro, and relevance to human GA will be addressed in Aim 4. The proposed studies will establish the validity of an IFN-y - alpha-v-beta3 - VEGFR/ESDN axis in the development of GA, thus defining sensitive and practical markers of early GA.