The Role of Microvascular Pericytes in the Generation of Pro-fibrotic Connective Tissue Cells Investigations in vitro and in Reactive Tissues in vivo

Abstract: Pericytes are cells of mesenchymal origin located on the abluminal side, juxtapositioned to the endothelial cells in capillaries, venules and small arterioles. They are important for maintaining vessel integrity in resting tissues as well as the formation and stabilization of new vessels. They have been suggested to function as mesenchymal stem cells thereby contributing to the connective tissue cell population in reactive tissues. In this thesis the role of pericytes as progenitors for fibroblasts was further defined both in vitro and in vivo. In the first study connective tissue cells of mesenchymal origin were investigated based on their marker expression and relation to the microvasculature. The expression of alpha smooth muscle actin (?-SMA), a marker for myofibroblasts, was compared to the expression of certain integrins in three reactive conditions in human tissues. There was a co-localization of ?-SMA and ?1?1 integrins, indicating that ?1 integrin was important for acquiring the ?-SMA myofibroblast phenotype. To further investigate this, two animal models for carcinoma growth and wound healing using ?1 deficient mice were employed. Reduction/lack of ?-SMA expressing myofibroblasts substantiated or findings in human tissues, strengthening the hypothesis that the ?1 integrin is important for the differentiation of ?-SMA expressing myofibroblasts. In study two the effects of the HDAC inhibitor valproic acid (VPA) on pericyte function in vitro was investigated. This revealed that VPA had an inhibitory effect on pericyte proliferation, migration and differentiation into collagen type I producing fibroblasts. In addition qPCR array studies on angiogenesis related gene expression identified an up-regulation of genes involved in vessel stabilization in VPA treated pericytes. This suggests that VPA promotes a pericyte phenotype favoring vessel stability. In study three the differentiation from early mesenchymal stem cell like pericyte to fully differentiated fibroblast was further defined by flow cytometry marker analysis. By isolating pericytes from human placenta with a phenotype resembling the in vivo phenotype the differentiation pathway could be defined in five consecutive steps. The five steps were defined by their marker expression and their ability to give rise to the other cell populations in the differentiation lineage, as well as their slow cycling characteristics. A better understanding of how connective tissue cells are derived in fibrotic conditions may be beneficial in trying to modulate the outcome of the healing process towards optimal tissue regeneration with minimal fibrosis.