MATRIX DEPENDENT CONTROL OF PULMONARY VASCULAR SMOOTH MUSCLE CELL RESPONSE

新生儿持续性肺动脉高压患者表现为 肺血管平滑肌细胞增生，增强 血管张力和细胞外基质（ECM）积累增加 肺血管中的成分。 这项建议是根据 假设ECM可能有助于这种疾病的病因学， 调节PVSM细胞的收缩性和生长， 血管调节剂 这个概念是基于最近的发现， 分子控制细胞内化学信号的设定点 路径（例如，Na +/H +交换、磷酸肌醇周转、cGMP 水平）被可溶性血管收缩剂（例如，内皮素-1， PDGF）和血管松弛剂（例如，一氧化氮）改变PVSM细胞 收缩和生长。 PVSM细胞将在化学- 确定的培养基，用不同密度的 纯化的ECM分子（例如，纤连蛋白，层粘连蛋白，不同胶原 类型，合成的含RGD的肽）或ECM包被的微珠， 改变细胞-ECM接触形成和整合素结合， 方式 血管紧张素（细胞骨架）的ECM依赖性控制 僵硬）的存在或不存在不同的血管收缩剂， 血管舒张剂将在培养的PVSM细胞中直接测量， 磁扭转细胞仪 这些研究的结果将进行比较 并与对DNA合成和细胞内 信号通路（例如，Na +/H +交换，肌醇脂质合成 和分解，Ca 2+释放，cGMP水平，肌球蛋白轻链激酶 激活、肌球蛋白磷酸化、蛋白酪氨酸激酶激活）。 抗整联蛋白抗体和合成肽将用于绘制 跨膜信号传导的特定途径，并确定 特异性整合素受体在控制 血管收缩与生长。 免疫荧光显微镜将是 用于识别信号分子分布的变化， 它们被募集到病灶内的整合素结合位点 粘附复合物响应于变化的细胞-ECM接触形成， 添加血管活性剂。 最后，一种新开发的方法， 分离生物化学活性粘着斑复合物将用于 研究整合素和受体如何传递信号， 血管收缩剂在细胞内整合。 研究还将 开始探索整联蛋白拮抗剂作为抑制剂的效用 肺血管重塑的研究进展 总之，我们希望能更好地 了解肺动脉高压是如何发展的， 新生儿损伤，并开发新的治疗方法， 通过表征ECM调节信号的机制， 转导，并因此控制PVSM细胞的生长和收缩 应答

Patients with persistent pulmonary hypertension of newborn exhibit pulmonary vascular smooth muscle (PVSM) cell hyperplasia, enhanced vascular tone, and increased accumulation of extracellular matrix (ECM) components in their lung vessels. This proposal is based on the hypothesis that in ECM may contribute to the etiology of this disease by regulating PVSM cell contractility and growth in response to soluble vasoregulators. This concept is based on the recent finding that ECM molecules control the ~set point~ of intracellular chemical signaling pathways (e.g., Na +/H + exchange, phosphoinositide turnover, cGMP levels) which are used by soluble vasoconstrictors (e.g., endothelin-1, PDGF) and vasorelaxants (e.g., nitric oxide) to alter PVSM cell contractility and growth. PVSM cells will be cultured in chemically- defined medium on dishes that are coated with different densities of purified ECM molecules (e.g., fibronectin, laminin, different collagen types, synthetic RGD-containing peptides) or ECM-coated microbeads to vary cell-ECM contact formation and integrin binding in a controlled manner. ECM-dependent control of vasomotor tone (cytoskeletal stiffness) in the presence or absence of different vasoconstrictors and vasodilators will be measured directly in cultured PVSM cells using magnetic twisting cytometry. Results of these studies will be compared and contrasted with effects on DNA synthesis and on intracellular signaling pathways (e.g., Na +/H + exchange, inositol lipid synthesis and breakdown, Ca2+ release, cGMP levels, myosin light chain kinase activation, myosin phosphorylation, protein tyrosine kinase activation). Anti-integrin antibodies and synthetic peptides will be used to map out specific paths of transmembrane signaling and to determine the importance of specific integrin receptors during control of vasoconstriction versus growth. Immunofluorescence microscopy will be used to identify changes in the distribution of signaling molecules and their recruitment to the site of integrin binding within the focal adhesion complex in response to varying cell-ECM contact formation and addition of vasoactive agents. Finally, a newly developed method for isolating biochemically active focal adhesion complexes will be used to study how signals transmitted by integrins and receptors for vasoconstrictors integrate inside the cell. Studies also will be initiated to explore the utility of integrin antagonists as inhibitors of pulmonary vascular remodeling in vivo. In summary, we hope to better understand how pulmonary hypertension may develop in response to neonatal injury and to develop new approaches for treatment of this disease by characterizing the mechanism by which ECM regulates signal transduction and hence, controls PVSM cell growth and contractile responses.