Hepatoma derived growth factor and vascular smooth muscle cell proliferation

描述(申请人提供)：血管平滑肌细胞(VSMC)增殖是血管损伤病理生理反应的关键组成部分。这一过程的核心是生长因子的产生和激活。其中一种VSMC生长因子--肝癌衍生生长因子(HDGF)在球囊损伤后高表达于新生内膜细胞的胞核，并在人动脉粥样硬化斑块中与增殖的VSMC共表达。然而，HDGF刺激VSMC增殖的机制尚不清楚。这项建议的总体目标是阐明HDGF在体外刺激VSMC增殖和对损伤做出反应的核机制。在我们的初步研究中，我们发现HDGF被蛋白激酶C磷酸化，蛋白激酶C是血管损伤的关键信号中间体。我们现在也有证据表明，HDGF是一种核结合蛋白，起着转录抑制因子的作用。在这些研究的基础上，我们建议确定PKC在体外和体内对HDGF有丝分裂功能的翻译后调节机制以及作为DMA结合、转录抑制因子的机制。首先，我们将通过腺病毒表达候选PKC丝氨酸(S)到丙氨酸(A)或天冬氨酸(D)突变的单个或组合突变、PKCA缺陷敲除小鼠和磷酸化特异的HDGF抗体来验证PKC磷酸化对高密度脂蛋白生长因子有丝分裂功能至关重要的假说。其次，我们将通过基于核磁共振的结构/功能分析来验证以下假设：HDGF是一个DNA结合蛋白，作为转录抑制因子通过有争议的PWWP结构域与HDGF靶基因的启动子结合，转录抑制是否通过HDAC途径介导，这些活性是否受PKC磷酸化的调节。这项研究的意义在于，通过将HDGF的激活与PKC联系起来，揭示了HDGF在血管损伤中作用的关键步骤，并为血管疾病的新治疗策略提供了基础。与公共健康相关：心血管疾病是美国头号死亡原因，尽管有降胆固醇药物的干预和血管内支架的开发。这是因为患者受损动脉中的血管细胞持续有害生长，最终导致脑血管或心脏血管功能不全。我们已经确定了一种由病变动脉中的血管细胞产生的蛋白质(HDGF)，它可以促进血管细胞的生长。我们建议确定该蛋白活性的调节机制，为将来设计治疗心血管疾病的新药提供依据。

DESCRIPTION (provided by applicant): Smooth muscle cell (VSMC) proliferation is a key component of the pathophysiologic response to vascular injury. Central to this process is the production and activation of growth factors. One such VSMC growth factor, hepatoma derived growth factor (HDGF), becomes highly expressed in the nucleus of neointimal cells after balloon injury and is co-expressed with PCNA in proliferating VSMC in human atherosclerotic plaques. However, the mechanisms by which HDGF stimulates VSMC proliferation are unknown. The overall goal of this proposal is to elucidate the nuclear mechanisms by which HDGF stimulates VSMC proliferation in vitro and in response to injury. In our pilot studies, we found that HDGF is phosphorylated by protein kinase C, a key signaling intermediate in vascular injury. We also now have evidence that HDGF is a nuclear binding protein, functioning as a transcriptional repressor. Based upon these studies, we propose to determine the mechanisms of PKC mediated post-translational regulation of HDGF mitogenic function in vitro and in vivo and as a DMA binding, transcriptional repressor. First, we will test the hypothesis that PKC phosphorylation s critical to HDGF mitogenic function in isolated VSMC and in the wire injured mouse carotid using adenoviral expression of single or combination mutations of candidate PKC serines (S) to alanine (A, loss of phosphorylation) or aspartic acid (D, phosphorylation mimic) mutations, PKCa deficient knockout mice, and phospho-specific HDGF antibodies. Second, we will test the hypotheses that HDGF is a DNA binding protein using NMR based structure/function analyses, functions as a transcriptional repressor binding to the promoters of HDGF target genes via the controversial PWWP domain, whether transcriptional repression is mediated by the HDAC pathway and whether these activities are regulated by PKC phosphorylation. The significance of the proposed studies is that by linking HDGF activation with PKC, a critical step in the function of HDGF in vascular injury is revealed and provides the basis for new therapeutic strategies in vascular disease. Relevance to public health: Cardiovascular disease is the number one cause of death in the United States despite interventions with cholesterol lowering agents and the development of endovascular stents. This is because of the continued detrimental growth of vascular cells in the damaged arteries of patients; eventually, leading to vascular insufficiency of the brain or heart. We have identified a protein (HDGF) produced by vascular cells in diseased arteries that increases the growth of vascular cells. We propose here to determine the mechanism regulating the activity of this protein to provide the basis for the future design of new drugs to treat cardiovascular disease.