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: 8146069
• 负责人: DAN E BERKOWITZ
• 金额: $ 38.85万
• 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/8146069
• 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。衰老与血管僵硬增加和孤立性收缩期高血压有关，这是由血管壁所有成分（包括内皮、血管平滑肌和基质）性质的改变引起的。虽然既有动态的（内皮功能的改变和对血管平滑收缩性的影响），也有结构性的(如：弹性蛋白断裂在衰老过程中已被描述，但与年龄相关的血管僵硬的分子机制仍然知之甚少。因此，靶向治疗仍然难以捉摸。组织转谷氨酰胺酶（TG2, tTG）在血管内皮细胞、平滑肌细胞和成纤维细胞中表达，通过酶作用在细胞外基质蛋白之间形成交联，并可能参与这种病理生物学。TG2的Ca2+依赖性激活依赖于其外化到细胞表面。最近有研究表明，半胱氨酸残基的翻译后氧化还原敏感修饰S-亚硝基化可导致TG2酶抑制。在初步数据中，我们证明了TG2在细胞模型中被s-亚硝基化，这导致细胞表面定位降低和交联活性降低。此外，通过使用TG2-/-小鼠，我们发现TG2是介导导管动脉僵硬的主要TGase，并且它受内皮源性NO的调节。我们进一步证明，在衰老大鼠和人主动脉中，TG2活性增加，s -亚硝基化降低，但TG2丰度不变。最后，抑制老年大鼠的TG可降低血管硬度。现在已经确定，随着年龄的增长，NO的生物利用度降低，活性氧（ROS）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-亚硝基化减少有关，因此，外化增加，基质交联活性增加，以及TG2依赖的下游信号传导增加。这些因素共同导致血管僵硬增加，并最终损害血管功能，这是衰老的标志。在本研究中，我们拟确定NO在调节TG2位置、活性和下游信号传导中的作用，并确定该酶是否是年龄相关血管僵硬的关键靶点。我们将采用分层方法，包括来自年轻和年老Fischer 344大鼠、TG2-/-和NOS3-/-小鼠的内皮细胞、血管组织，以及这些动物模型中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)确定TGF2在下游TG2介导的血管信号传导中的作用。3)通过对体内血管特性的复杂测量，确定TG2及其通过NO调控在动物模型中与年龄相关的血管硬度中的作用。4)测定老龄人主动脉中TG2活性。