Effects of Nitric Oxide on Smooth Muscle Cell Proliferations

描述（由申请人提供）：动脉粥样硬化组织形成的一个关键事件是血管平滑肌细胞（SMC）的过度增殖。据报道，一氧化氮（NO）供体提供的一氧化氮（NO）通过减少多胺的产生、激活NO- cgmp信号通路和中和氧化应激，有利于抑制SMC增殖。临床批准的NO供体是硝酸盐化合物，已知其可诱导硝酸盐耐受。实验性和商业化的no -供体的半衰期相对较短。我们最近报道的n -亚硝基一氧化氮供体以可调速率持续和可控的方式释放一氧化氮。这些化合物的表观半衰期为39 ~ 88小时。我们准备了3个新的NO捐赠者家庭。通过改变附着在n -亚硝基上的部分的性质，可以改变每个NO供体的NO释放谱。从人类主动脉平滑肌细胞（HASMC）的细胞培养研究中获得的数据显示，使用n -亚硝基NO供体之一（80 pM）可显著（40%）降低SMC的增殖。更重要的是，与传统的no供体相比，这种抑制作用是在非常低的no供体浓度下实现的。建议的研究将处理下列问题。首先，将合成n -亚硝基NO给体（水溶性、不水溶性、低分子量、枝状、聚乙二醇化、杂化、聚合物），并确定其NO释放谱，建立构效关系。其次，这些新的NO供体释放的NO对HASMC的生理影响将通过各种细胞和生物化学试验进行评估，包括细胞活力测定、[3H]胸苷结合试验、精氨酸酶、一氧化氮合酶（NOS）和鸟氨酸脱羧酶（ODC）酶活性测定。测定细胞培养中cGMP和多胺水平，评价NO对培养的HASMC的影响。第三，联合使用精氨酸酶抑制剂、ABH和n -亚硝基NO供体，在培养的HASMC中达到“正确”的生理NO水平。第四，这些联合剂量将与非对称二甲基精氨酸（ADMA）水平升高一起使用，以确定与单独使用ABH和no供体相比，该联合方案的有益效果。因此，我们提出的研究将进一步了解n -亚硝基NO供体以缓慢、持续和速率可调的方式释放NO对HASMC以及其他SMC、癌症和神经元细胞的影响。

DESCRIPTION (provided by applicant): A crucial event in the formation of atherosclerotic tissues is excessive proliferation of vascular smooth muscle cells (SMC). Nitrogen monoxide (NO) provided by NO donors has been reported to be beneficial for inhibition of SMC proliferation by reducing polyamine production, activating the NO-cGMP signal pathway and neutralizing oxidative stress. Clinically approved NO donors are nitrate compounds, which are known to induce nitrate tolerance. Experimental and commercially available NO-donors exhibit relatively short half- lives. N-nitroso NO donors recently reported by us release NO in a sustained and controlled fashion with tunable rates. The apparent half-lives of these compounds ranged from 39h to 88h. We have prepared 3 new families of NO donors. The NO release profile of each of the NO-donors can be varied by changing the nature of the moieties attached to the N-nitroso group. Data obtained from cell culture studies with human aortic smooth muscle cells (HASMC), using one of the N-nitroso NO donors (80 pM) exhibited a significant (40%) decrease of SMC proliferation. More importantly, this inhibition was achieved at a very low NO-donor concentration compared to the conventional NO-donors. The proposed studies will address the following issues. First, N-nitroso NO donors (water soluble, water insoluble, low molecular weight, dendritic, pegylated, hybrid, polymeric) will be synthesized and their NO-release profiles will be determined to establish structure- activity relationships. Second, the physiological effect on HASMC of NO released by these novel NO donors will be evaluated by a variety of cell and biochemistry assays including cell viability assay, [3H] thymidine incorporation assay, and determination of arginase, nitric oxide synthase (NOS) and ornithine decarboxylase (ODC) enzyme activities. cGMP and polyamine levels in the cell culture will be determined to evaluate the effect of NO in cultured HASMC. Third, a combination arginase inhibitor, ABH, and N-nitroso NO donors will be used to achieve the "right" physiological NO level in cultured HASMC. Fourth, these combination doses will be used along with elevated level of asymmetric dimethylarginine (ADMA) to determine the beneficial effects of this combination protocol in comparison to those observed with ABH and NO-donor alone. Thus our proposed studies should provide further understanding of the effects of NO released in a slow, sustained and rate-tunable manner from the N-nitroso NO donors on HASMC as well as other SMC, cancer and neuronal cells. PUBLIC HEALTH RELEVANCE: This proposal will involve syntheses of a variety of N-nitroso NO donors and investigate their inhibition of SMC proliferation as well as the influence of structure on NO release profiles. Results of this study will benefit the use of NO donors for the prevention, diagnosis and treatment of human diseases strongly associated with impaired NO production, including cardiovascular disease, neurodegenerative diseases, chronic inflammatory diseases, and cancer.