Contribution of caveolin to diabetic vascular dysfunction

描述（由申请人提供）： 在过去的15年中，与心血管疾病相关的2型糖尿病的发病率增加了一倍多。潜在的病理学包括内皮和血管平滑肌（VSM）功能障碍，我们已经在两种不同的糖尿病小鼠模型中观察到：瘦素受体缺陷（db/db）和链脲佐菌素诱导的。与非糖尿病小鼠相比，两种糖尿病小鼠模型的主动脉对内皮NO介导的血管扩张剂的舒张较少，对5-羟色胺和其他血管收缩剂的收缩更多，部分是由rho/rho激酶途径的活化增加介导的。虽然研究表明糖尿病患者的收缩功能增强，但这种异常的细胞机制尚不清楚。我研究的长期目标是了解导致糖尿病VSM功能障碍的细胞机制。在VSM的特化质膜微区中发现血管收缩剂β-肾上腺素能受体和rhoA与小窝蛋白-1相关。本实验室的研究表明，从小窝蛋白-1（cav-1）基因缺陷小鼠分离的主动脉收缩显著增加，5-羟色胺可通过rho激酶抑制剂恢复正常。Rho依赖性收缩在糖尿病中也同样增加。我们的总体假设是，5-羟色胺受体和它们的下游信号分子定位于处于非活性状态的小窝内。在糖尿病中，这种受体和rhoA在小窝中的隔离减少，从而导致VSM的更大激活和收缩。在这项提案中，我们将评估糖尿病对小窝微区中rhoA、rho激酶和5-羟色胺受体表达的影响。我们还将确定糖尿病患者（链脲佐菌素诱导或瘦素受体缺陷）中VSM中5-羟色胺受体亚型是否发生变化。最后，我们将确定是否在小窝内的特定受体亚型或其下游信号成分的表达的变化有助于增强糖尿病血管收缩。为了验证这一假设，我们将解决以下具体目标：具体目标1：建立受体介导的rho/rho激酶通路的激活是否发生在血管平滑肌的小窝微区。我们的工作假设是激活rho/rho激酶通路的受体被隔离在小窝中。我们将评估特定的5-羟色胺受体，rhoA和rho激酶的表达和定位在小窝微结构域在主动脉野生型和小窝蛋白-1缺陷小鼠在基础和刺激条件下。特定目的2：确定糖尿病中VSM小窝复合物是否改变以增加通过rho/rho激酶途径的受体信号传导。我们的工作假设是，5-羟色胺受体和/或rhoA在小窝微区的定位抑制了它们的激活，这在糖尿病中减少。我们将比较基础和刺激条件下非糖尿病和糖尿病野生型或cav-1缺陷主动脉中与cav-1相关的5-羟色胺受体、rhoA和rho激酶的表达。具体目标3：确定糖尿病患者的血管功能障碍是否与小窝蛋白-1对5-羟色胺受体或rhoA的区室化改变有关。我们的工作假设是小窝蛋白-1的缺失将增强受体介导的rhoA激活，并且这在糖尿病动脉中增加。我们将比较使用病毒转染抑制（siRNA）或过度表达小窝蛋白-1对非糖尿病和糖尿病小鼠中特异性5-羟色胺受体激动剂或直接激活rhoA的血管反应的影响。

DESCRIPTION (provided by applicant): In the last 15 years, the incidence of Type 2 diabetes associated with cardiovascular disease has more than doubled. The underlying pathology includes both endothelial and vascular smooth muscle (VSM) dysfunction which we have observed in two different mouse models of diabetes: leptin receptor deficient (db/db) and streptozotocin-induced. Compared to non-diabetic mice, aorta from both diabetic mouse models relax less to endothelial NO-mediated vasodilators and contract more to serotonin and other vasoconstrictors, in part, mediated by increased activation of the rho/rho kinase pathway. Although studies have demonstrated increased contractile function in diabetes, the cellular mechanisms underlying this abnormality are unknown. The long-term goal of my research is to understand the cellular mechanisms that contribute to the dysfunction of VSM in diabetes. Vasoconstrictor serotonergic receptors and rhoA have been found associated with caveolin-1 in specialized plasma membrane microdomains of VSM. Studies in our laboratory demonstrate that contractions of aorta isolated from caveolin-1 (cav-1) gene deficient mice are markedly increased to serotonin which are restored to normal by inhibitors of rho kinase. Rho-dependent contractions are similarly increased in diabetes. Our overall hypothesis is that serotonin receptors and their downstream signaling molecules are localized within caveolae in an inactive state. This sequestering of receptors and rhoA in caveolae is reduced in diabetes contributing to greater activation and contraction of VSM. In this proposal we will assess the impact of diabetes on the expression of rhoA, rho kinase and serotonin receptors in caveolar microdomains. We will also determine if the subtype of serotonin receptors changes in VSM in diabetics (streptozotocin- induced or leptin receptor deficient). Finally, we will determine whether a change in the expression of specific receptor subtypes or their downstream signaling components within caveolae contributes to the enhanced vasoconstriction in diabetes. To test this hypothesis, we will address the following specific aims: SPECIFIC AIM 1: To establish if receptor-mediated activation of the rho/rho kinase pathway occurs in caveolar microdomains in vascular smooth muscle. Our working hypothesis is that receptors that activate the rho/rho kinase pathway are sequestered in caveolae. We will assess expression and localization of specific serotonin receptors, rhoA and rho kinase in caveolar microdomains in aorta from wild type and caveolin-1 deficient mice under basal and stimulated conditions. SPECIFIC AIM 2: To determine whether VSM caveolar complexes change in diabetes to increase receptor signaling through the rho/rho kinase pathway. Our working hypothesis is that localization of serotonin receptors and/or rhoA in caveolar microdomains suppresses their activation which is reduced in diabetes. We will compare expression of serotonin receptors, rhoA and rho kinase in association with cav-1 in lipid rafts in non-diabetic and diabetic wild type or cav-1 deficient aorta under basal and stimulated conditions. SPECIFIC AIM 3: To determine whether the vascular dysfunction in diabetes is related to a change in caveolin-1 compartmentalization of serotonin receptors or rhoA. Our working hypothesis is that deletion of caveolin-1 will augment receptor mediated rhoA activation and this is increased in diabetic arteries. We will compare the effects of inhibition (siRNA) or overexpression of caveolin-1 using viral transfection on vascular responses to specific serotonin receptor agonists or direct activation of rhoA in non-diabetic and diabetic mice.