ANTIMITOGENIC MECHANISMS OF PKG IN VASCULAR SM CELLS

过度的平滑肌细胞（PASMC）增殖导致患有肺动脉高压的新生儿以及患有多种形式的先天性心脏病的婴儿和儿童的异常肺动脉重构和高血压。 NO供体在体外降低丝裂原刺激的SMC增殖。 最近已观察到吸入一氧化氮（NO）治疗可降低血管损伤动物的肺动脉细胞增殖。 虽然研究表明NO信号通过激活cGMP依赖性蛋白激酶（PKG）来降低SMC增殖，但其机制尚不完全清楚。 本提案的广泛、长期目标是阐明PKG激活抑制PASMC增殖的分子机制。具体目标1检查PKG激活如何调节增殖PASMC的细胞周期进程，并确定特定的PKG敏感性细胞周期调节因子。 使用流式细胞术，[3 H]胸腺嘧啶掺入DNA的研究，和细胞周期调节蛋白的活性和/或表达的测定，PKG激活的抗增殖机制将在血清刺激的PASMC中进行研究。 具体目标2测试PKG活化是否调节丝裂原活化蛋白激酶（MAPK）信号通路中的级联。 使用表达PKG的PASMC、增殖测定和MAPK信号传导级联的特异性抑制剂，将确定PKG信号传导对ERK、SAPK/JNK和p38信号传导的影响。 特异性目的3鉴定了PKG通过其降低PASMC增殖的特异性PKG磷酸化靶点。 例如，如果PKG活化调节MAPK信号级联，则将使用表达PKG的增殖PASMC来评价该途径的组分的丰度和酶活性。 这些结果将为了解肺血管疾病的病理性细胞异常增殖的基本机制提供重要的信息。该奖项将允许申请人利用理想的研究培训环境，以获得细胞和分子生物学的新知识和技能。 此外，一个精心构建的培训计划已经制定，这将允许一个独立的研究职业生涯检查肺血管疾病的基本机制的成功发展。

Excessive smooth muscle cell (PASMC) proliferation causes abnormal pulmonary artery remodeling and hypertension in newborns with pulmonary hypertension and in infants and children with many forms of congenital heart disease. NO donors decrease mitogen-stimulated SMC proliferation in vitro. Recently inhaled nitric oxide (NO) treatment has been observed to decrease pulmonary artery cell proliferation in animals with vascular injury. Although studies suggest that NO-signaling decreases SMC proliferation via activation of cGMP-dependent protein kinase (PKG), the mechanism is incompletely understood. The BROAD, LONG-TERM OBJECTIVE of this proposal is to elucidate molecular mechanisms by which PKG activation inhibits PASMC proliferation. Specific aim 1 examines how PKG activation modulates the cell cycle progression of proliferating PASMC and identifies specific PKG-sensitive cell cycle regulators. Using flow cytometry, studies of [3H]thymidine-incorporation into DNA, and assays of the activity and / or expression of cell cycle regulatory proteins, the antiproliferative mechanisms of PKG activation will be investigated in serum-stimulated PASMC. Specific aim 2 tests whether PKG activation modulates cascades in the mitogen-activated protein kinase (MAPK) signaling pathway. Using PASMC expressing PKG, proliferation assays, and specific inhibitors of MAPK signaling cascades, the effect of PKG signaling on the ERK, SAPK/JNK and p38-signaling will be determined. Specific aim 3 identifies specific PKG- phosphorylation targets through which PKG decreases PASMC proliferation. For example, should PKG activation modulate the MAPK signaling cascade, the abundance and enzymatic activity of constituents of this pathway will be evaluated using PKG-expressing proliferating PASMC. These results will provide important insights into basic mechanisms of abnormal cell proliferation which is pathognomatic for pulmonary vascular disease. This award will permit the applicant to take advantage of an ideal research training environment for the acquisition of new knowledge and skills in cell and molecular biology. In addition, a carefully constructed training program has been developed that will permit successful development of an independent research career examining the basic mechanisms of pulmonary vascular diseases.