Systemic sclerosis (SSc) is characterized by the accumulation of excess collagen and other matrix proteins in the skin and other organs as well as widespread vascular pathology and immune cell activation. The investigators' studies have focused on understanding the basis for excessive matrix biosynthesis. They have used, as a working hypothesis, the concept that immune or other events, in addition to stimulating fibroblasts, lead to clonal selection of high collagen producing fibroblast subpopulations which then manifest a fibrotic phenotype. The investigators have shown that fibroblasts are heterogeneous in their proliferative capacity, response to cytokines, and synthesis of collagen, matrix metalloproteinases, and other products. This heterogeneity was present at both the protein and mRNA level and that it represented a stable clonal phenotype. The investigators showed that immune cell products, in vitro, can lead to long-term alterations in fibroblast phenotype and that the clonal makeup of fibroblasts is skewed towards high collagen producing cells in SSc. Finally, they cloned the human cDNA and genomic DNA for a collagen transcription factor, cKROX. The investigators wish to continue and extend these studies to gain further understanding of the pathophysiology of fibrotic disease. In the next project period, they propose to explore further mechanisms leading to fibroblast abnormalities in SSc. They will investigate the human collagen transcription factor, cKROX, that they have cloned, and examine its expression in SSc and in clonal populations, as well as its regulation. They will examine differences in gene expression between scleroderma-involved and -uninvolved skin fibroblasts using differential display. They hope to identify among the expressed genes detected by differential display possible regulatory genes underlying abnormal fibroblast metabolism. They will test the hypothesis that apoptosis plays a role in leading to selective overgrowth of fibroblast populations in SSc. They will determine whether SSc fibroblasts have abnormal responses to apoptotic signals and whether these are based on different expression of proteins important in apoptosis such as Fas, bcl-2, p53, and c-myc. They will look at the role that hypoxia might play in altering fibroblast metabolism in SSc. They anticipate that these studies will improve the understanding of the interactions of fibroblasts, their milieu in inflammatory states, in general, and in SSc, in particular. The first specific aim is to examine the possibility that differences in the synthesis of collagen between SSc and normal cells are due to differential expression of cKROX. Stable transfectants will be used to evaluate cKROX as a transcription factor. Known factors that regulate collagen synthesis will be evaluated for their influence on cKROX expression. Lesional skin, nonlesional skin, normal skin, and clonal populations of fibroblasts will be evaluated using gel shift analysis, Northern blots, and RT-PCR for cKROX expression. In the second specific aim, differential display libraries will be developed from cloned and normal fibroblasts and lesional and nonlesional fibroblasts. The genes identified will be cloned and characterized. The third specific aim is to evaluate selective influences for clonal proliferation of fibroblasts including apoptosis and hypoxia.
Showing the most recent 10 out of 33 publications
