L-arginine metabolism in diabetes-induced coronary dysfunction

描述（由申请人提供）：我们的总体目标是确定精氨酸酶活性在糖尿病诱导的心血管疾病中的具体作用。这些研究将为开发预防导致糖尿病性心脏病的血管损伤的新策略奠定基础。内皮细胞（EC）功能障碍是糖尿病血管并发症的一个显著特征。我们的初步数据显示，糖尿病大鼠的冠状动脉血管舒张受损和血管周围纤维化/肥大，这与活性氧（ROS）的形成和精氨酸酶活性的增加有关，这将l -精氨酸从eNOS转移到鸟氨酸途径。我们的数据还表明，高糖和ROS增加精氨酸酶I的表达，他汀类HMG辅酶a还原酶抑制剂和补充l -瓜氨酸（L-cit）阻断了糖尿病增加ROS、诱导精氨酸酶表达和活性、导致冠状动脉纤维化和血管舒张受损的作用。研究表明糖尿病和高糖诱导的ROS激活小G蛋白RhoA，从而激活Rho激酶（ROCK），从而解释了这些影响。ROCK反过来激活精氨酸酶并抑制eNOS，而NO的产生抑制精氨酸酶/鸟氨酸途径，他汀类药物增强eNOS活性，NO的形成并抑制RhoA/ROCK的激活。此外，我们的研究表明，l - citit可以提高L-arg水平并抑制精氨酸酶活性，比L-arg本身更能维持NOS的L-arg利用率。基于这些数据，我们假设糖尿病和高糖通过RhoA/ rock介导的精氨酸酶/鸟氨酸途径的激活导致血管功能障碍。l -精氨酸对eNOS的可用性降低，使酶解偶联形成超氧化物而不是一氧化氮，从而降低血管舒张。精氨酸酶/鸟氨酸途径的加速活性诱导血管周围纤维化、肥大，导致血管硬化。该模型将使用分子、药理学和生化方法的结合来检验以下假设，这些假设将用于糖尿病动物、糖尿病患者的血管和血液以及培养细胞的实验。1)明确糖尿病激活精氨酸酶的分子机制。2)检测糖尿病是否通过激活精氨酸酶导致ec依赖性血管松弛受损。3)通过激活精氨酸酶检测糖尿病是否引起血管纤维化、肥大和硬化。4)通过激活RhoA/ROCK检测糖尿病是否激活精氨酸酶/鸟氨酸通路。公共卫生相关性：冠状动脉血管性心脏病可导致心脏病发作，是一个主要的健康问题，糖尿病患者的情况更糟。了解糖尿病中减少心脏血流量的因素和事件顺序对预防和治疗这种疾病至关重要。一种酶可以阻止心脏获得足够的血液，导致心脏血管增厚和僵硬，限制这种酶的作用为减少这种疾病的发病率提供了很大的希望。

DESCRIPTION (provided by applicant): Our overall aim is to define the specific role of arginase activity in diabetes-induced cardiovascular disease. These studies will set the stage for developing new strategies to prevent the vascular injury that leads to diabetic heart disease. Endothelial cell (EC) dysfunction is a prominent feature in the vascular complications of diabetes. Our preliminary data show impaired coronary vasorelaxation and perivascular fibrosis/hypertrophy in diabetic rats, which are associated with reactive oxygen species (ROS) formation and increased arginase activity, which diverts L-arg from eNOS to the ornithine pathway. Our data also show that high glucose and ROS increase arginase I expression and that statin HMG CoA reductase inhibitors and supplemental L-citrulline (L-cit) block the actions of diabetes in increasing ROS, inducing arginase expression and activity and causing coronary fibrosis and impaired vasorelaxation. These effects are explained by studies showing that diabetes and high glucose-induced ROS activate the small G protein RhoA, which activates Rho kinase (ROCK). ROCK in turn activates arginase and inhibits eNOS, whereas NO production inhibits the arginase/ornithine pathway and statins enhance eNOS activity, NO formation and inhibit activation of RhoA/ROCK. Additionally, our studies indicate that L-cit, which increases L-arg levels and inhibits arginase activity, maintains L-arg availability for NOS better than L-arg itself. Based on these data, we hypothesize that diabetes and high glucose cause vascular dysfunction by RhoA/ROCK-mediated activation of the arginase/ornithine pathway. Decreased L-arginine availability to eNOS uncouples the enzyme to form superoxide rather than NO which reduces vasodilation. Accelerated activity of the arginase/ornithine pathway induces perivascular fibrosis, hypertrophy, leading to vascular stiffening. This model will be examined using a combination of molecular, pharmacological and biochemical approaches to test the following hypotheses for experiments in diabetic animals, vessels and blood from diabetic patients and cultured cells. 1) Define the molecular mechanisms by which diabetes activates arginase. 2) Test whether diabetes causes impaired EC-dependent vasorelaxation by activating arginase. 3) Test whether diabetes causes vascular fibrosis, hypertrophy and stiffening by activating arginase. 4) Test whether diabetes activates the arginase/ornithine pathway by activation of RhoA/ROCK. PUBLIC HEALTH RELEVANCE: Coronary vascular heart disease which can cause heart attacks is a major health problem, which is much worse in people who have diabetes. Understanding the factors and sequence of events in diabetes which reduce blood flow to heart is critical to preventing and treating this disease. Limiting actions of an enzyme which can prevent the heart from getting enough blood and cause thickening and stiffness of heart blood vessels offers much promise for reducing the incidence of this disease.