Transglutaminase 2 S-Nitrosylation: Role in Age-Related Vascular Stiffness

描述（由申请方提供）：血管僵硬已明确确定为心血管疾病的风险因素，并且是心血管发病率和死亡率的独立预测因子。衰老与血管僵硬度增加和单纯收缩期高血压相关，后者是由血管壁所有成分（包括内皮、血管平滑肌和基质）的性质改变引起的。虽然这两个动态（内皮功能的改变和对血管平滑肌收缩性的影响），以及结构（例如，弹性蛋白的断裂）已经在衰老中有所描述，但对与年龄相关的血管硬度的分子机制仍然知之甚少。因此，靶向治疗仍然难以捉摸。在血管内皮细胞、平滑肌细胞和成纤维细胞中表达的组织转氨酶（TG 2，tTG）在细胞外基质蛋白之间酶促形成交联，并且可能促成这种病理生物学。TG 2的Ca 2+依赖性活化依赖于其外化至细胞表面。最近，已经证明S-亚硝基化（半胱氨酸残基的氧化还原敏感性翻译后修饰）导致TG 2酶抑制。在初步数据中，我们证明TG 2在细胞模型中是S-亚硝基化的，这导致细胞表面定位降低和交联活性降低。此外，使用TG 2-/-小鼠，我们表明，TG 2是主要的TGase介导的管道动脉的硬度，它是由内皮源性NO。我们进一步证明，在老龄大鼠和人类主动脉，TG 2活性增加，它的S-亚硝基化降低，尽管不变的TG 2丰度。最后，抑制老年大鼠中的TG可降低血管硬度。现在已经很好地确定，在衰老过程中，NO的生物利用度降低，活性氧（ROS）增加。考虑到亚硝基氧化还原平衡的这种变化，我们假设衰老与TG 2 S-亚硝基化减少有关，因此，增加了外部化，增加了基质交联活性和TG 2依赖的下游信号传导。总之，这些导致血管硬度增加，并最终损害血管功能，这是衰老的标志。在这项研究中，我们建议确定NO在调节TG 2的位置，活性和下游信号传导中的作用，并确定这种酶是否是与年龄相关的血管僵硬的关键目标。我们将使用分层方法，包括内皮细胞，血管组织从年轻和老年Fischer 344大鼠和TG 2-/-和NOS 3-/-小鼠，并在这些动物模型中的血管特性的侵入性和非侵入性措施。本研究的具体目的如下：1）探讨内皮依赖性NO在调节TG 2亚细胞分布和活性中的作用。2)确定TGF 2在下游TGF 2介导的血管信号传导中的作用。3)在动物模型中，使用复杂的体内血管特性测量方法，确定TG 2及其受NO调节在年龄相关血管僵硬度中的作用。4)确定老年人主动脉中的TG 2活性。

DESCRIPTION (provided by applicant): Vascular stiffness has been clearly established as a risk factor for cardiovascular disease, and is an independent predictor of cardiovascular morbidity and mortality. Aging is associated with increased vascular stiffness and isolated systolic hypertension, which results from alterations in the properties of all elements of the vascular wall including endothelium, vascular smooth muscle, and matrix. Although both dynamic (alterations in endothelial function and effects on vascular smooth contractility), as well as structural (eg. fracturing of elastin) have been described in aging, molecular mechanisms underlying age-related vascular stiffness remain poorly understood. Thus targeted therapy remains elusive. Tissue transglutaminase (TG2, tTG), expressed in vascular endothelial, smooth muscle cells, and fibroblasts, enzymatically form cross-links between extracellular matrix proteins, and may contribute to this pathobiology. Ca2+dependent activation of TG2 is dependent on its externalization to the cell surface. Recently, it has been demonstrated that S- nitrosylation, a redox-sensitive post-translational modification of cysteine residues, leads to TG2 enzyme inhibition. In Preliminary Data, we demonstrate that TG2 is S-nitrosylated in cellular models and that this leads to decreased cell-surface localization and decreased cross-linking activity. Furthermore, using TG2-/- mice, we show that TG2 is the primary TGase mediating stiffness of conduit arteries, and that it is regulated by endothelium-derived NO. We further demonstrate that in aging rat and human aorta, TG2 activity is increased, and it's S-nitrosylation is decreased despite unchanged TG2 abundance. Finally, inhibition of TG in old rats reduces vascular stiffness. It is now well established that NO bioavailability is diminished and reactive oxygen species (ROS) are increased in aging. Given this change in the nitroso-redox balance, we hypothesize that aging is associated with decreased TG2 S-nitrosylation and therefore, increased externalization and increased matrix cross-linking activity and TG2-dependent downstream signaling. Together, these result in increased vessel stiffness, and ultimately impaired vascular function, a hallmark of aging. In this grant, we propose to determine the role of NO in the regulation of TG2 location, activity and downstream signaling, and determine whether this enzyme is a critical target in age-related vascular stiffness. We will use a hierarchical approach including endothelial cells, vascular tissue from young and old Fischer 344 rats and TG2-/- and NOS3-/- mice, and invasive and non-invasive measures of vascular characteristics in these animal models. The following are the specific aims: 1) To determine the role of endothelium-dependent NO in the of regulation of TG2 subcellular distribution and activity. 2) To determine the role of TGF2 in downstream TG2 mediated vascular signaling. 3) To determine the role of TG2 and its regulation by NO in age-related vascular stiffness in animal models using sophisticated measures of vascular properties in vivo. 4) To determine TG2 activity in the aorta of aging humans.