Contribution of caveolin to diabetic vascular dysfunction

在过去的15年里，与心血管疾病相关的2型糖尿病的发病率 翻了一番还多。基础病理包括内皮细胞和血管平滑肌(VSM)。 我们在两种不同的糖尿病小鼠模型中观察到的功能障碍：瘦素受体缺陷 (db/db)和链脲佐菌素诱导。与非糖尿病小鼠相比，两种糖尿病小鼠模型的主动脉 减少对内皮NO介导的血管扩张剂的松弛，更多地对5-羟色胺和其他血管收缩剂的收缩， 部分是通过增加Rho/Rho激酶通路的激活来实现的。尽管研究表明 糖尿病患者的收缩功能增强，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功能障碍的细胞机制 糖尿病患者的VSM。血管收缩5-羟色胺能受体和RhoA已被发现与 VSM特殊质膜微区中的Caveolin-1。我们实验室的研究表明 小窝蛋白-1(Cav-1)基因缺陷小鼠主动脉对5-羟色胺的收缩明显增加 在Rho激酶抑制剂的作用下，这些细胞恢复正常。依赖于Rho的收缩在 糖尿病。我们的总体假设是5-羟色胺受体及其下游信号分子 处于非活动状态的定位于小窝内的。这种受体和RhoA在小窝中的隔离减少 在糖尿病中，有助于VSM更大的激活和收缩。在这份提案中，我们将评估 糖尿病对肺泡微区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VSM中5-羟色胺受体亚型是否发生变化(链脲佐菌素- 诱导或瘦素受体缺陷)。最后，我们将确定是否改变了具体的表达方式 小窝内受体亚型或其下游信号成分有助于增强 糖尿病患者的血管收缩。为了验证这一假设，我们将解决以下具体目标： 特异性目的1：确定Rho/Rho激酶通路在肺泡内是否发生受体介导的激活 血管平滑肌中的微域。我们的工作假设是激活Rho/Rho的受体 激酶途径被隔离在小窝中。我们将评估特定的5-羟色胺的表达和定位 野生型和小窝蛋白-1缺陷小鼠主动脉微区受体、RhoA和Rho激酶 在基础和刺激条件下。 特异性目的2：确定VSM空泡复合体在糖尿病中是否改变以增加受体 通过Rho/Rho激酶通路进行信号传递。我们的工作假设是5-羟色胺的定位 空泡微区中的受体和/或RhoA抑制它们的激活，这种激活在糖尿病中会减少。我们 将比较5-羟色胺受体、RhoA和Rho激酶与Cav-1在脂筏中的表达 在基础和刺激条件下，非糖尿病和糖尿病野生型或Cav-1缺陷的主动脉。 特异性目的3：确定糖尿病血管功能障碍是否与小窝蛋白-1的变化有关 5-羟色胺受体或RhoA的区隔。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激活在糖尿病动脉中增加。我们将比较 病毒转染法抑制(SiRNA)或过表达小窝蛋白-1对血管反应的影响 非糖尿病和糖尿病小鼠的特定5-羟色胺受体激动剂或直接激活RhoA。

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.