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.
