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

转谷氨酰胺酶 2 S-亚硝基化：在年龄相关血管僵硬中的作用

• 批准号: 8307888
• 负责人: DAN E BERKOWITZ
• 金额: $ 39.41万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-20 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8307888
• 关键词: Age; Aging; Animal Model; Aorta; Arteries; Autopsy; Biological Availability; Blood Pressure; Blood Vessels; Buffers; Cardiovascular Diseases; Cardiovascular system; Cell model; Cell surface; Characteristics; Collagen; Cystamine; Cysteine; Data; Elastin; Elderly; Elements; Endothelial Cells; Endothelium; Enzyme Inhibition; Enzymes; Equilibrium; Exercise; Extracellular Matrix; Extracellular Matrix Proteins; Fibroblasts; Fracture; Grant; Heart; Human; Hypertension; Inbred F344 Rats; Isolated systolic hypertension; Lead; Location; Measurement; Measures; Mediating; Molecular; Morbidity - disease rate; Mus; Myocardial Infarction; NG-Nitroarginine Methyl Ester; NOS3 gene; Oxidation-Reduction; Pathway interactions; Perfusion; Physiologic pulse; Physiological; Post-Translational Protein Processing; Property; Proteins; Rattus; Reactive Oxygen Species; Regulation; Risk; Risk Factors; Role; SKIL gene; Signal Transduction; Silastic; Smooth Muscle Myocytes; Stroke; System; Tissues; Transglutaminases; Up-Regulation; Vascular Endothelial Cell; Vascular Endothelium; Vascular Smooth Muscle; age effect; age related; blood pump; crosslink; in vivo; inhibitor/antagonist; mortality; pressure; protein protein interaction; public health relevance; research study; scaffold; shear stress; transcription factor; transglutaminase 2

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. PUBLIC HEALTH RELEVANCE: As we age, our blood vessels become stiffer because of the changes that occur in all wall of the large blood vessels. When the aorta stiffens it is less able to buffer the pulse created by pumping of blood into it from the heart leading high blood pressure in the elderly which is very difficult to treat. This vascular stiffening and high blood pressure is associated with an increase risk of heart attack and stroke as we age. We have identified an enzyme that creates "bridges" or "scaffolds" between other proteins in the vessel wall contributing to its stiffening, and plan to study its mechanism of regulation, thereby helping us to determine whether blocking the enzyme might be important in treating age-related high blood pressure.

描述(由申请人提供)：血管僵硬已被明确确定为心血管疾病的危险因素，并是心血管发病率和死亡率的独立预测因素。衰老与血管僵硬增加和单纯的收缩期高血压有关，这是由于血管壁的所有元素包括内皮、血管平滑肌和基质的属性改变所致。虽然无论是动态的(内皮功能的改变和对血管平滑收缩的影响)，以及结构性的(例如，弹性蛋白断裂)在衰老中已有描述，但与年龄相关的血管僵硬的分子机制仍知之甚少。因此，靶向治疗仍然难以捉摸。组织谷氨酰胺转氨酶(TG2，tTG)在血管内皮细胞、平滑肌细胞和成纤维细胞中表达，在细胞外基质蛋白之间形成交联链，可能参与了这种病理生理过程。TG2的激活依赖于它对细胞表面的外化。最近的研究表明，半胱氨酸残基的氧化还原敏感的翻译后修饰--S亚硝化会导致TG2酶的抑制。在初步数据中，我们证明了TG2在细胞模型中是S亚硝化的，这导致细胞表面定位减少和交联剂活性降低。此外，在TG2-/-小鼠身上，我们证明TG2是介导管道动脉僵硬的主要TGase，并且它受内皮源性NO的调节。我们进一步证明，在衰老的大鼠和人的主动脉中，Tg2的活性增加，其S亚硝化反应减少，而Tg2的丰度没有变化。最后，抑制老年大鼠的甘油三酯可降低血管僵硬。现已证实，随着年龄的增长，生物利用度不会降低，而活性氧物种(ROS)会增加。鉴于亚硝基-氧化还原平衡的这种变化，我们假设衰老与Tg2-S亚硝化减少有关，因此，增加了外部化，增加了基质交联活性和Tg2依赖的下游信号转导。总而言之，这些都会导致血管僵硬增加，并最终损害血管功能，这是衰老的标志。在这项授权中，我们建议确定NO在调节TG2的位置、活性和下游信号中的作用，并确定该酶是否是与年龄相关的血管僵硬的关键靶点。我们将使用分层方法，包括来自年轻和老年Fischer 344大鼠以及TG2-/-和NOS3-/-小鼠的内皮细胞、血管组织，以及在这些动物模型中对血管特征的侵入性和非侵入性测量。具体目的如下：1)确定内皮依赖性NO在调节TG2亚细胞分布和活性中的作用。2)研究TGF2在TG2下游血管信号转导中的作用。3)通过体内复杂的血管特性测量，确定TG2在增龄性血管僵硬动物模型中的作用以及NO对其调节作用。4)测定增龄人主动脉中TG2活性。 与公共健康相关：随着我们年龄的增长，我们的血管会变得更加僵硬，因为所有大血管的管壁都会发生变化。当主动脉变硬时，从心脏向其泵入血液所产生的脉搏的缓冲能力就会减弱，从而导致老年人高血压，这是很难治疗的。随着年龄的增长，这种血管僵硬和高血压与心脏病发作和中风的风险增加有关。我们已经确定了一种酶，它可以在血管壁中其他有助于其硬化的蛋白质之间建立“桥梁”或“支架”，并计划研究其调节机制，从而帮助我们确定阻断这种酶是否在治疗年龄相关性高血压方面可能是重要的。