Towards cell-specific therapy for restenosis: CCN5 in development and vascular injury

SMC hyperplasia is responsible for the failure due to restenosis of a substantial fraction (as high as 30%) of many vascular surgical procedures, including percutaneous transluminal coronary angioplasty (PTCA), coronary artery bypass grafts (CABG), arterio-venous shunts, endarterectomies, and heart transplants. Recent advancements in therapies to prevent restenosis have met with some success, however non-specific inhibition of vascular smooth muscle cell (VSMC) and endothelial cell (EC) proliferation prevents adequate re-endothelialization in these therapies, leading to increased rates of thrombosis. Vascular smooth muscle cell-specific therapies that inhibit VSMC proliferation but allow EC regeneration would prevent restenosis but allow the endothelium to regenerate.;The six proteins of the CCN family have important roles in development, angiogenesis, cell motility, proliferation, and other fundamental cell processes. CCN5 is a growth arrest-specific gene in vascular smooth muscle cells (VSMC) that strongly inhibits adult smooth muscle cell proliferation and motility in vitro. In vivo, CCN5 expression dramatically decreases in proliferating VSMCs in a rat carotid artery balloon vascular injury model.;I have hypothesized that CCN5 is an important down-regulator of SMC proliferation and motility in vivo. If this is true then: (1) CCN5 levels should be low in the developing vascular system but should increase as vessels approach adult morphology; (2) VSMC CCN5 levels should decrease following vascular injury as the cells begin to proliferate; (3) Over-expression of CCN5 in a vascular injury model should reduce the size of a vascular lesion.;To date, CCN5 distribution in developing rodents and humans has not been mapped comprehensively. In contrast to our initial prediction, I demonstrated that CCN5 was widely expressed throughout the developing embryo, including within the vasculature in both developing mouse and human tissues. CCN5 protein expression was initially present in most tissues, and tissue-specific expression differences were observed later in development. I demonstrated both protein and mRNA expression throughout all major organs of developing GD14.5 mice. Although CCN2 and CCN5 protein expression patterns in some adult pathologic conditions are inversely expressed, this expression pattern was not found in developing mouse and human tissues. My observations of CCN5 expression imply interesting new routes of investigation for CCN5 biology, including previously unsuspected roles in bronchiole morphogenesis and myotendinous junctions. I also demonstrated for the first time CCN5 protein localization within nuclei, suggesting that CCN5 may have previously unsuspected roles in regulating transcription.;I demonstrated that, following mouse carotid ligation vascular injury, vascular media CCN5 protein expression is reduced as early as 2 days following injury, and does not increase to pre-injured levels as long as 3 months following injury. While I initially predicted that CCN5 expression in vivo would be tightly correlated to proliferation as was observed in VSMC in vitro, I demonstrated that CCN5 protein expression is globally reduced in all cells of the vascular media following vascular injury, not simply in those that incorporate the proliferation marker BrdU and are thus acutely proliferating. Thus, CCN5 loss may indicate an "injured" or non-quiescent cell or tissue that may be primed to enter the cell cycle due to a recent change in the local environment, though may not necessarily mark cell cycle status. Finally, I demonstrated that overexpression of CCN5 in vascular injury is able to limit neointimal formation in a carotid ligation model. BrdU incorporation was minimal in vascular media treated with AdCCN5, but was present within neointima and adventitia, suggesting that AdCCN5 treatment is able to selectively limit vascular smooth muscle cell proliferation but not inhibit endothelial cell regeneration following vascular injury. Thus, I demonstrated that CCN5 may be a good candidate molecule for VSMC-specific therapies to prevent restenosis following surgical vascular procedures.;While it is clear that the biological functions as well as the physiologic roles for CCN5 are complex and likely to be tissue-specific, the data presented in this thesis suggest a role for CCN5 in the control of mammalian development and in the pathophysiology of restenosis following vascular procedures.