Shear stress, SIRT1, and Arterial Stiffening

描述(申请人提供)：动脉硬化，表现为导管动脉的增厚和弹性降低，是中风和心脏病的独立危险因素。尽管动脉硬化的病因与衰老有很好的相关性，但其潜在的细胞和分子基础仍不清楚。在体内，血流动力动态地作用于血管壁，极大地影响动脉的功能和力学特性。动脉硬化可能涉及血管内皮细胞对这些血流动力产生的剪切力的异常反应。我们最近发现，抗衰老调节因子SIRT1受血管内皮细胞(ECs)内切应力的调节。此外，钙调蛋白依赖的蛋白激酶(CaMKK)似乎是一种剪应力敏感的激酶，调节SIRT1。因此，我们假设切应力上调导管动脉内皮细胞中的SIRT1，从而改善导致动脉硬化的病理生理重塑。在上游，CaMKKb被激活，以响应生理上相关的剪应力。作为激活CaMKKb-SIRT1通路的结果，内皮型一氧化氮合酶(ENOS)衍生的NO生物利用度和PGC-1a调节的ROS清除剂的表达增强。因此，剪切力增强的SIRT1的有利作用包括减少氧化应激和细胞外基质的重塑。为了验证我们的假设，我们提出了三个具体目标。具体目标1将检测在培养的内皮细胞中诱导SIRT1的关键血流动力学因素。然后将进行分子信号实验，以研究CaMKKb调节内皮细胞SIRT1响应定义的剪应力的机制。具体目标2将阐明剪切力诱导的SIRT1在EC/血管平滑肌细胞共培养系统中发挥抗僵化作用的细胞和分子机制。我们将破译SIRT1和AMP激活的蛋白激酶(AMPK)在eNOS来源的NO生物利用度和PGC-1a调节的活性氧(ROS)清除剂中的协同作用。具体目标3将研究剪切力激活的SIRT1在小鼠模型动脉硬化中的作用。具体地说，我们将比较EC-SIRT1-/-基因敲除、TG-EC-SIRT1和CaMKKb-/-小鼠动脉硬化的时空变化。血液动力学因素、主动脉壁重构和基因表达谱中与动脉硬化相关的变化将被测量和关联。这些拟议研究的结果，如果如预期的那样，将有助于理解动脉硬化的机制和分子基础。 公共卫生相关性：动脉硬化与衰老过程相关，是许多心血管疾病的独立危险因素。SIRT1是参与许多抗应激反应的主要调节因子，我们最近发现血流动力学可以激活血管细胞中的SIRT1。这项研究将探讨SIRT1诱导血流的机制和分子基础，即抗动脉硬化。这一提议将显著增加我们对动脉硬化中血流与衰老过程相互作用的机制的理解。拟议的实验结果可能有助于预防和/或治疗该病的新疗法。

DESCRIPTION (provided by applicant): Arterial stiffening, manifested by thickening and reduced elasticity of conduit arteries, is an independent risk factor for stroke and heart disease. Although the etiology of arterial stiffening correlates well with aging, the underlying cellular and molecular basis remains unclear. In vivo, hemodynamic forces dynamically act on the vascular wall, which greatly affect arterial function and mechanical properties. Arterial stiffening may involves aberrant endothelial response to shear stress resulted from these hemodynamic forces. We recently found that SIRT1, an anti-aging regulator is modulated by shear stress in vascular endothelial cells (ECs). Moreover, calmodulin-dependent protein kinase (CaMKK) appears to be a shear stress-sensitive kinase that regulates SIRT1. We thus hypothesize that shear stress upregulates SIRT1 in the endothelium of conduit arteries, which ameliorates pathophysiological remodeling leading to arterial stiffening. At the upstream, CaMKKb is activated in response to the physiologically relevant shear stress. As a consequence of the activated CaMKKb-SIRT1 pathway, the endothelial nitric oxide synthase (eNOS)-derived NO bioavailability and PGC-1a-regulated ROS scavenger expression are augmented. Thus, the advantageous effects of shear stress-augmented SIRT1 include decreased oxidative stress and remodeling of extracellular matrices. To test our hypothesis, three Specific Aims are proposed. Specific Aim 1 will examine the hemodynamic factors critical for the induction of SIRT1 in cultured ECs. Molecular signaling experiments will then be conducted to study the mechanism by which CaMKKb regulates SIRT1 in ECs responding to the defined shear stress. Specific Aim 2 will elucidate the cellular and molecular mechanisms by which shear stress-induced SIRT1 exerts anti- stiffening effects in an EC/vascular smooth muscle cell co-culture system. We will decipher the synergistic effect of SIRT1 and AMP-activated protein kinase (AMPK) in eNOS-derived NO bioavailability and PGC-1a-regulated reactive oxygen species (ROS) scavengers. Specific Aim 3 will investigate the role of shear stress-activated SIRT1 in arterial stiffening in mouse models. Specifically, we will compare the spatiotemporal changes of arterial stiffening in EC-sirt1-/- knockout, Tg-EC-sirt1, and CaMKKb-/- mice. The arterial stiffening-associated changes in hemodynamic factors, aortic wall remodeling, and gene expression profiles will be measured and correlated. Results from these proposed studies, if as anticipated, will help to understand the mechano and molecular basis of arterial stiffening. PUBLIC HEALTH RELEVANCE: Arterial stiffening, associated with the aging process, is an independent risk factor for many cardiovascular diseases. SIRT1 is a master regulator involved in many anti-stress responses and we recently found that hemodynamic force can activate SIRT1 in vascular cells. This proposed study will investigate the mechano and molecular basis of flow induction of SIRT1, which would be anti-arterial stiffening. The proposal will significantly increase our understanding of mechanisms by which blood flow interplays with aging process in arterial stiffening. Results from the proposed experiments are likely to contribute to novel therapies for its prevention and/or treatment.