It is postulated that vascular disease involves a disturbance in the homeostatic balance of factors regulating vascular tone and structure. Recent developments in gene transfer techniques have emerged as an exciting therapeutic option to treat vascular disease. Several studies have established the feasibility of direct in vivo gene transfer into the vasculature by using reporter genes such as 8-galactosidase or luciferase. To date no study has documented therapeutic effects with in vivo gene transfer of a cDNA encoding a functional enzyme. This study tests the hypothesis that endothelium-derived nitric oxide is an endogenous inhibitor of vascular lesion formation. After denudation by balloon injury of the endothelium of rat carotid arteries, we restored endothelial cell nitric oxide synthase (ec-NOS) expression in the vessel wall by using the highly efficient Sendai virus/liposome in vivo gene transfer technique. ec-NOS gene transfection not only restored NO production to levels seen in normal untreated vessels but also increased vascular reactivity of the injured vessel. Neointima formation at day 14 after balloon injury was inhibited by 70%. These findings provide direct evidence that NO is an endogenous inhibitor ofvascular lesion formation in vivo (by inhibiting smooth muscle cell proliferation and migration) and suggest the possibility of ec-NOS transfection as a potential therapeutic approach to treat neointimal hyperplasia.The process of intimal hyperplasia is common to various forms of vascular diseases such as atherosclerosis, transplant vasculopathy, and restenosis following balloon angioplasty. The use of in vivo gene therapy for the treatment of vascular disorders has been speculated for several years. Recently, our laboratory and others have shown that neointima formation after balloon injury can be inhibited by antisense oligonucleotide transfection (1, 2). However, there has been no report of a "therapeutic" transfection of a functional gene whose product inhibits neointima formation. This study demonstrates the successful inhibition of neointimal hyperplasia by in vivo gene transfer by introducing the cDNA encoding the endothelial cell nitric oxide synthase (ec-NOS). Our study has two major implications: (i) that the prevention of neointimal hyperplasia by an in vivo gene transfer approach is feasible, and (ii) that NO is an effective in vivo inhibitor of vascular smooth muscle cell (VSMC) accumulation and that its enhanced expression by in vivo gene transfer may be a promising strategy for the treatment of vascular disease.Injury to the endothelium plays an essential role in the "response to injury" hypothesis (3, 4). Experimental studies have shown that vascular injury induces local expression of mitogens and chemotactic factors mediating neointima formation. The lesion is characterized in part by the abnormal migration and proliferation of VSMCs in the intima. In addition, it is postulated that endothelial denudation may result in the loss of constitutively expressed endotheliumderived...

iNOS expression occurs in failing human cardiac myocytes and may be involved in the pathophysiology of DCM, IHD, and VHD.

No abstract

Abstract-Cell cycle progression represents a key event in vascular proliferative diseases, one that depends on an increased rate of protein synthesis. An increase in phosphatidylinositol 3-kinase (PI 3-kinase) activity is associated with vascular smooth muscle cell proliferation, and rapamycin, which blocks the activity of the mammalian target of rapamycin, inhibits this proliferation in vitro and in vivo. We hypothesized that these 2 molecules converge on a critical pathway of translational regulation that is essential for successful upregulation of cell cycle-regulatory proteins in activated smooth muscle cells. p70 S6 kinase, a target of PI 3-kinase and the mammalian target of rapamycin, was rapidly activated on growth factor stimulation of quiescent coronary artery smooth muscle cells and after balloon injury of rat carotid arteries. The translational repressor protein 4E-binding protein 1 was similarly hyperphosphorylated under these conditions. These events were associated with increases in the protein levels of cyclin B1, cyclin D1, cyclin E, cyclin-dependent kinase 1, cyclin-dependent kinase 2, proliferating cell nuclear antigen, and p21Cip1 in vivo and in vitro, whereas inhibition of the PI 3-kinase signaling pathway with either rapamycin or wortmannin blocked the upregulation of these cell cycle proteins, but not mRNA, and arrested the cells in vitro before S phase. In contrast to findings in other cell types, growth factor-or balloon injury-induced downregulation of the cell cycle inhibitor p27

Objective-Although numerous diseases involving cellular proliferation are also associated with phenotypic changes, there has been little direct evidence that cell phenotype and the cell's response to external stimuli are modified during passage through different phases of the cell cycle. In this study, we demonstrate that an association exists between cell cycle progression and the expression of genes involved in cellular activation. VSMCs not only enter cell cycle and proliferate, thereby contributing to neointima formation, but also become "activated," a term referring to the expression of cytokines, adhesion molecules, chemoattractants, proteolytic enzymes, and other molecules not normally expressed in the quiescent, contractile VSMCs of the medial layer of the vessel wall. For example, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) have been documented on neointimal VSMCs of animal models of atherosclerosis, restenosis, and transplant vasculopathy, as well as in human plaques. 3-7 These molecules, in turn, likely mediate and facilitate a robust inflammatory response and the further proliferation and migration of VSMCs. 1,8 Because both activated VSMCs and proliferating VSMCs were detected within the same neointimal lesion, it was generally assumed that proliferating cells are activated cells. However, this supposition has never been proven. Furthermore, it is not known whether activation and proliferation of See page 804 cells during atherogenesis are parallel-occurring but independent phenomena, or whether the activation state of a cell is influenced by its entry into and its progression through the different phases of the cell cycle. In a series of recent studies, our laboratory and others have found that vascular cell cycle arrest not only inhibits cellular proliferation and, as a result, neointima formation, but also ameliorates changes in vascular cell phenotype and actually reduces the heightened susceptibility of certain vessels to atherosclerosis or vasculopathy. 9,10 The current study, therefore, tested the hypothesis that an association exists between cell cycle progression and the susceptibility of vascular cells to cytokine induced adhesion molecule expression on the cell surface. We were able to demonstrate that VCAM-1 and ICAM-1 expression were inhibited during the G1-phase and S-phase of cell cycle. Methods and Results-Early