PKC Epsilon in Vascular Dysfunction

描述(由申请人提供)： 动脉粥样硬化是发达国家的主要死亡原因。糖尿病显著增加动脉粥样硬化并发症的风险。新的证据表明，抵抗素是一种与胰岛素抵抗素相关的新型脂肪因子，在糖尿病人群中导致动脉粥样硬化性疾病和不良的干预结果。我们和其他人已经证明，抵抗素显著诱导血管平滑肌细胞(VSMC)功能障碍，这是内膜增生和再狭窄的关键步骤。然而，人们对其潜在机制知之甚少，治疗方案也在很大程度上缺乏。最近，我们发现抵抗素的细胞效应是由PKC-[介导的。我们的研究小组还表明，激活PKC-1对移植心肌的缺血/再灌注损伤具有保护作用，抑制PKC-1可以减轻大鼠移植心肌的内膜增生。尽管靶向PKC-1治疗动脉粥样硬化并发症在很大程度上是未知的。基于我们新颖的、看似有争议的观察，我们认为PKC-参与心血管疾病是一个动态的过程。急性激活PKC可保护缺血/再灌注所致的细胞损伤，而持续抑制PKC可最大限度地减少抵抗素诱导的内膜增生和再狭窄。我们的基本假设是，特定时间的PKC调节可减少抵抗素诱导的内膜增生和再狭窄。为了追寻这一假说，我们提议进行一项更全面的研究，以阐明血管损伤后抵抗素夸大的细胞应激中PKC-β调节的分子机制、细胞效应和体内影响。提出了三个具体目标(SA)。SA 1：确定PKC在抵抗素诱导的细胞效应中的作用。在这个SA中，我们将首先确认我们的初步发现，并确定VSMC中特定时间的PKC-调制。然后，我们将使用一种新的激活的巨噬细胞-VSMC共培养系统来探索PKC-β的调节作用。最后，我们将验证抵抗素和PKC-β在体外对人颈动脉斑块的调节作用。SA 2：研究依赖PKC的抵抗素诱导的细胞窘迫的分子机制。利用HCASMC模型，我们将研究PKC-β在抵抗素诱导的SA2a中ROS过度产生中的作用。我们还将通过检测已知的抵抗素诱导的SA2b信号通路中特定时间的PKC-？调制来扩大我们的初步观察。最后，我们将用无偏倚的蛋白质组学方法探索新的依赖PKC的下游信号通路(S)，并确定一个新的PKC介导的分子相互作用，即线粒体乙醛脱氢酶是否参与抵抗素诱导的细胞功能障碍(SA2C)。SA 3：在小鼠模型中评估PKC对抵抗素增强的损伤后内膜增生的影响。我们将在心房阻断前和血管干预后独立地调节PKC，以使用转基因小鼠模型来确定特定时间的PKC调节对抵抗素加重的内膜增生的体内影响。通过成功完成我们的目标，在特定时间点潜在应用新型PKC特异性多肽调节剂，代表了一种新的治疗选择。破译血管内膜增生的临床相关机制(S)，并最终将其转化为抑制疾病进展的新治疗策略，支持我们将心血管疾病并发症降至最低和改善心血管手术临床结果的长期目标。

DESCRIPTION (provided by applicant): Atherosclerosis is the leading cause of death in the developed countries. Diabetes mellitus markedly increases the risk of atherosclerotic complications. Emerging evidence suggests that resistin, a novel adipokine implicated in insulin resistin, contributes to atherosclerotic disease and the poor interventional outcomes among diabetic population. We and others have shown that resistin significantly induces vascular smooth muscle cell (VSMC) dysfunction, a key step in intimal hyperplasia and restenosis. However, little is known about the underlying mechanisms and the treatment option is largely lacking. Recently, we demonstrated that the cellular effect of resistin was mediated by PKC-[. Our research team also showed that activating PKC-¿ protected against ischemia/reperfusion injury of transplanted myocardium and inhibiting PKC-¿ mitigated intimal hyperplasia in rat. Although therapeutically targeting PKC-¿ in the treatment of atherosclerotic complications is largely unknown. Based on our novel, seemingly controversial observations, we believe that the involvement of PKC-¿ in cardiovascular disease is a dynamic process. Acute activation of PKC-¿ protects against ischemia/reperfusion-induced cellular injuries whereas sustained inhibition of PKC-¿ following procedures can minimize resistin-induced intimal hyperplasia and restenosis. It is our fundamental hypothesis that time-specific PKC-¿ modulation reduces resistin-induced intimal hyperplasia and restenosis. To pursue this hypothesis, we propose a more comprehensive investigation to elucidate the molecular mechanisms, cellular effects, and in vivo influences of PKC-¿ modulation in resistin-exaggerated cellular stress following vascular injury. Three specific aims (SA) are proposed. SA 1: Determine the role of PKC-¿ in resistin-induced cellular effects. In this SA, we will first confirm our preliminary findings and determine time-specific PKC-¿ modulation in VSMC. We will then explore the modulating effect of PKC-¿ using a novel activated macrophage-VSMC co-culture system. Lastly, we will verify the effects of resistin and PKC-¿ modulation in ex vivo human carotid plaques. SA 2: Characterize the molecular mechanisms of PKC-¿-dependent resistin-induced cellular distress. Using a HCASMC model, we will study the involvement of PKC-¿ in resistin-induced ROS over-production in the SA2a. We will also expand our preliminary observation by examining time-specific PKC-¿ modulation in known resistin-induced signaling pathways in SA2b. Lastly, we will explore novel PKC-¿-dependent downstream signaling pathway(s) using an unbiased proteomics approach and determine whether a novel PKC-¿-mediated molecular interaction, mitochondria aldehyde dehydrogenase (ALDH2), is involved in resistin- induced cellular dysfunction (SA2c). SA 3: Evaluate the effects PKC-¿ on resistin-augmented post-injury intimal hyperplasia in a murine model. We will independently modulate PKC-¿ before atrial clamping and after vascular interventions to determine the in vivo effects of time-specific PKC-¿ modulation on resistin-exacerbated intimal hyperplasia using a transgenic murine model. The potential application of novel PKC-¿ specific peptide modulators at specific time points, justified by successful completion of our aims, represents a novel therapeutic option. Deciphering clinically-relevant mechanism(s) of intimal hyperplasia and ultimately translating these into a novel therapeutic strategy to suppress disease progression supports our long-term goal of minimizing complications of cardiovascular diseases and improving the clinical outcome of cardiovascular procedures.