ANTIMITOGENIC MECHANISMS OF PKG IN VASCULAR SM CELLS

血管平滑肌细胞(PASMC)的过度增殖会导致患有肺动脉高压的新生儿和患有多种形式先天性心脏病的婴儿和儿童出现异常的肺动脉重塑和高血压。NO供体可抑制体外丝裂原刺激的SMC增殖。最近观察到吸入一氧化氮(NO)治疗可以减少血管损伤动物的肺动脉细胞增殖。尽管研究表明，NO信号通过激活cGMP依赖的蛋白激酶(PKG)来抑制SMC的增殖，但其机制尚不完全清楚。这一建议的广泛、长期目标是阐明PKG激活抑制PASMC增殖的分子机制。具体目的1研究PKG激活如何调节增殖中的PASMC的细胞周期进程，并确定特定的PKG敏感的细胞周期调节因子。利用流式细胞术、[~3H]胸腺嘧啶核苷掺入DNA的研究以及细胞周期调节蛋白的活性和/或表达的分析，将探讨血清刺激的PASMC中PKG激活的抗增殖机制。特定目的2测试PKG激活是否调节丝裂原活化蛋白激酶(MAPK)信号通路中的级联。通过PASMC表达PKG、增殖实验和MAPK信号通路的特异性抑制物，可以确定PKG信号对ERK、SAPK/JNK和p38-信号的影响。特异靶3确定特定的PKG磷酸化靶点，通过该靶点PKG可抑制PASMC的增殖。例如，如果PKG激活调节MAPK信号级联，将使用表达PKG的增殖的PASMC来评估这一途径的组成成分的丰度和酶活性。这些结果将对肺血管疾病的病因学中细胞异常增殖的基本机制提供重要的见解。该奖项将允许申请者利用理想的研究培训环境，获得细胞和分子生物学方面的新知识和技能。此外，还制定了精心设计的培训计划，使研究肺血管疾病的基本机制的独立研究事业得以成功发展。

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.