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O-GlyNAcylation: Novel Mechanism of Estrogen-Induced Vasoprotection

O-甘氨酰化：雌激素诱导血管保护的新机制

基本信息

批准号：
8204767
负责人：
Suzanne Oparil
金额：
$ 35.89万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-01-01 至 2013-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8204767
关键词：
Acetylglucosamine Acute Animals Anti-Inflammatory Agents Anti-inflammatory Arterial Injury Arteries Attenuated Binding Blood Vessels Cardiovascular Diseases Carotid Arteries Cells DNA Development Estradiol Estrogens Glucosamine Immunoblotting In Vitro Infiltration Inflammation Inflammation Mediators Inflammatory Inflammatory Response Injury Intervention Label Leukocytes Link Mediating Modification Molecular Nuclear Translocation Pathogenesis Pathway interactions Phenylcarbamates Phosphorylation Play Post-Translational Protein Processing Prevention strategy Protein Biosynthesis Proteins Proteomics Rattus Reporting Role Seminal Signal Pathway Signal Transduction Site Smooth Muscle Myocytes Stable Isotope Labeling Stimulus Stress TNF gene Techniques Testing Time Transcription Regulatory Protein Vascular Diseases abstracting attenuation cytokine in vitro Model in vivo inhibitor/antagonist injured monocyte neointima formation neutrophil novel peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase protective effect research study response response to injury synthetic peptide

项目摘要

Project Summary/Abstract We have made the seminal observation that O-linked ¿-N-acetylglucosamine (O-GlcNAc) modification of proteins induced by three distinct and independent stimuli, 17-¿ estradiol (E2), glucosamine (GlcN) and the selective O-GlcNAcase inhibitor O-(acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc), has anti-inflammatory effects in balloon injured rat carotid arteries. These novel observations provide provocative evidence that enhanced O-GlcNAc modification of proteins has anti-inflammatory and vasoprotective effects in the setting of acute endoluminal vascular injury. We have strong evidence that increased protein O-GlcNAc modification in response to GlcN treatment is associated with attenuation of TNF- ¿-induced expression of inflammatory mediators in isolated rat aortic smooth muscle cells (RASMCs) in a manner previously reported for E2. We have utilized the TNF-¿-treated RASMC as an in vitro model of the acute vascular injury response and have begun to define the mechanisms by which interventions that stimulate protein O-GlcNAc modification, i.e., E2 and GlcN, inhibit inflammatory responses to TNF-¿. We have focused on the NF¿B signaling pathway, which is known to be activated by both TNF-¿ and acute vascular injury. Initial experiments demonstrated that GlcN inhibits TNF-¿-induced NF¿B activation in RASMCs. Subsequent studies showed that pretreatment with GlcN inhibits TNF-¿-induced phosphorylation and degradation of I¿B¿ in RASMCs, while E2 pretreatment is associated with an initial reduction, followed by an accelerated reappearance of I¿B¿ in TNF-¿ treated cells, likely reflecting new protein synthesis mediated by activated NF¿B. The current study will test directly the hypothesis that O-GlcNAc modification of proteins, including I¿B¿, plays a mechanistic role in regulating the inflammatory response to endoluminal arterial injury in vivo and to TNF-¿ stimulation in isolated RASMCs in vitro. The Specific Aims are: Specific Aim 1: To test the hypothesis that increasing protein O-GlcNAc modification protects arteries from inflammatory stress related to acute endoluminal injury in vivo via inhibition of NF¿B signaling. Specific Aim 2: To test the hypothesis that increasing protein O-GlcNAc modification inhibits TNF-¿-induced inflammatory responses in RASMCs in vitro via inhibition of NF¿B signaling and define the precise sites in the NF¿B signaling cascade that are responsible for this effect. Specific Aim 3: To identify specific protein targets of O-GlcNAc modification in RASMCs that play a functional role in the anti-inflammatory effects of GlcN and E2. Upon successful completion of these Aims, cellular/molecular mechanisms responsible for the anti-inflammatory and vasoprotective actions of O- GlcNAc modification will be elucidated and will be related to the extent of the injury response (i.e., inflammation and neointima formation). We postulate that O-GlcNAc modification represents a novel mechanism of vasoprotection that may lend itself to the development of new strategies for the prevention and treatment of cardiovascular disease.

项目总结/摘要我们已经进行了开创性的观察，O-连接的<$-N-乙酰葡萄糖胺（O-GlcNAc）的修饰，由三种不同的和独立的刺激，17-雌二醇（E2），葡萄糖胺（GlcN）和选择性O-GlcNAc酶抑制剂O-（乙酰氨基-2-脱氧-D-吡喃葡萄糖基）氨基-N-苯基氨基甲酸酯（PUGNAc）对大鼠颈动脉球囊损伤具有抗炎作用。这些新颖的观察提供了刺激性的证据，即蛋白质的增强的O-GlcNAc修饰具有抗炎和急性腔内血管损伤的血管保护作用。我们有有力的证据表明对GlcN处理反应的蛋白质O-GlcNAc修饰增加与TNF-α的减弱有关。体外培养的大鼠主动脉平滑肌细胞（RASMCs）中，此前报道的E2。我们利用TNF-α处理的RASMC作为体外模型，急性血管损伤反应，并已开始定义的干预机制，刺激蛋白O-GlcNAc修饰，即，E2和GlcN抑制对TNF-α的炎症反应。我们重点在NF B信号通路上，已知NF B信号通路被TNF-α和急性血管损伤激活。初步实验表明GlcN抑制RASMC中TNF-诱导的NF B活化。后续研究表明，GlcN预处理可抑制TNF-B的磷酸化和降解在RASMCs中，E2预处理与初始减少有关，随后是加速的在TNF-α处理的细胞中，I B的重新出现，可能反映了活化的B细胞介导的新蛋白质合成。 NF B。目前的研究将直接测试蛋白质的O-GlcNAc修饰，包括 I B在体内调节对腔内动脉损伤的炎症反应中起机械作用， TNF-α刺激的RASMCs中。具体目标：具体目标1：测试增加蛋白质O-GlcNAc修饰保护动脉免受炎症应激相关假说通过抑制NF B信号传导在体内急性腔内损伤。具体目标2：检验以下假设：增加蛋白O-GlcNAc修饰抑制体外RASMCs中TNF-<$诱导的炎症反应通过抑制NF ² B信号传导并定义NF ² B信号传导级联中负责的精确位点为了这个效果。具体目的3：鉴定RASMC中O-GlcNAc修饰的特异性蛋白质靶标，在GlcN和E2的抗炎作用中发挥功能性作用。在成功完成这些目的：研究O-12抗炎和血管保护作用的细胞/分子机制。将阐明GlcNAc修饰并将其与损伤反应的程度相关（即，炎症和新生内膜形成）。我们假设O-GlcNAc修饰代表了一种新的机制，血管保护，这可能有助于开发新的预防和治疗策略，心血管疾病