TRANSCRIPTIONAL BASIS OF CARDIAC ALLOGRAFT REMODELING

描述(改编自申请人的摘要)：主要目标 研究计划是调查转录之间的关系 血管平滑肌(VSM)α-肌动蛋白基因的调控 移植物重塑和慢性排斥反应。不恰当地表达 这种重要的平滑肌肌动蛋白亚型已知会损害收缩能力和 对心血管功能有不利影响。VSMα-肌动蛋白表达缺失是 移植血管硬化症的一个特征似乎是 由结合和抑制VSM的转录调节蛋白介导 新生内膜平滑肌细胞中的α-肌动蛋白基因启动子。审查 小鼠异位心脏移植显示额外的组织重塑 慢性排斥反应过程中与VSM升高相关的过程 移植物基质(心脏成纤维细胞)中α-肌动蛋白基因转录 实质(心肌细胞)细胞床。拟议的研究重点放在 小鼠VSMα-肌动蛋白基因的调控及其特性 其同源转录调节蛋白(VAC)的性质 分离的VSM细胞、心肌细胞和心脏成纤维细胞及其 移植后产生的基因重新编程的对应物。慢性 通过将分离的细胞暴露于 促炎细胞因子决定免疫细胞产品如何影响血管紧张素转换酶 蛋白质活性和肌动蛋白基因重编程。来自VSM的监管要素 α-肌动蛋白启动子将被鉴定、突变，并与一个报告基因连锁 编码组织学可检测的产物，并用于制备转基因 为原位转录检测提供供体心脏和主动脉的小鼠 在移植后的移植物基质、实质和动脉床中。 同种异体心脏移植重塑的空间方面将通过使用 VSMα-肌动蛋白和VAC蛋白系列偶联的组织学探针 显微切割法、共聚焦显微镜和原位杂交技术 同种异体移植物的细胞表型分布。这些研究是预期的 提供移植和许可后基因重新编程的全球视角 心脏移植物解剖部位和细胞床的早期识别 那些最有可能进行结构重塑的公司。拟议的研究将 扩大我们对基因转录控制机制的理解 调节心脏移植的早期细胞适应不良。诊断性 基于VSMα-肌动蛋白的检测和治疗方案 转录可能代表着识别和管理早期阶段的新工具 慢性排斥反应早在移植肾功能下降之前就开始了 临床上是可以检测到的。

DESCRIPTION (adapted from applicant's abstract): The major objective of the research plan is to investigate relationships between transcriptional modulation of the vascular smooth muscle (VSM) alpha-actin gene, cardiac and aortic allograft remodeling, and chronic rejection. Inappropriate expression of this essential smooth muscle actin isoform is known to impair contractility and adversely affect cardiovascular function. Loss of VSM alpha-actin expression is a characteristic feature of transplant vascular sclerosis that appears to be mediated by transcriptional regulatory proteins that bind and repress the VSM alpha-actin gene promoter in neointimal smooth muscle cells. Examination of mouse heterotopic cardiac allografts has revealed additional tissue remodeling processes during chronic rejection that are associated with elevated VSM alpha-actin gene transcription in graft stromal (cardiac fibroblast) and parenchymal (cardiomyocyte) cell beds. The proposed research focuses on the control of the mouse VSM alpha-actin gene with an emphasis on characterizing the properties of its cognate transcriptional regulatory (VAC) proteins in isolated VSM cells, cardiomyocytes, and cardiac fibroblasts as well as their genetically reprogrammed counterparts that arise following transplant. Chronic rejection conditions will be simulated by exposing isolated cells to pro-inflammatory cytokines to determine how immune cell products influence VAC protein activity and actin gene reprogramming. Regulatory elements from the VSM alpha-actin promoter will be identified, mutated, linked to a reporter gene encoding a histologically detectable product, and used to prepare transgenic mice for supplying donor hearts and aortae for in situ assay of transcription in the graft stroma, parenchyma, and arterial beds following transplant. Spatial aspects of cardiac allograft remodeling will be revealed by using histologic probes for VSM alpha-actin and VAC proteins coupled with serial microtomy, confocal microscopy, and in situ hybridization to map the distribution of cellular phenotypes in allografts. These studies are expected to provide a global view of gene reprogramming following transplant and permit early identification of anatomical regions and cellular beds in heart grafts that are most at risk for structural remodeling. The proposed research will extend our understanding of gene transcriptional control mechanisms that mediate very early cellular maladaptations to heart transplantation. Diagnostic assays and therapeutic protocols based on aspects of VSM alpha-actin transcription may represent new tools for recognizing and managing early-stage chronic rejection well before a functional decline in graft performance becomes clinically detectable.