O-GlyNAcylation: Novel Mechanism of Estrogen-Induced Vasoprotection

描述(由申请人提供)：我们已经进行了开创性的观察，即O-连接-N-乙酰氨基葡萄糖(O-GlcNAc)对三种不同和独立的刺激，17-雌二醇(E2)，氨基葡萄糖(GlcN)和选择性O-GlcNAcase抑制剂O-(乙酰氨基-2-脱氧-D-氨基葡萄糖)氨基-N-苯基氨基甲酸酯(PUGNAc)，在球囊损伤的大鼠颈动脉具有抗炎作用。这些新的观察结果提供了具有挑衅性的证据，表明增强的O-GlcNAc蛋白质修饰在急性腔内血管损伤的背景下具有抗炎和血管保护作用。我们有强有力的证据表明，GlcN治疗后蛋白O-GlcNAc修饰的增加与肿瘤坏死因子-a诱导的炎性介质在分离的大鼠主动脉平滑肌细胞(RASMCs)中的表达减弱有关，就像以前报道的E2的方式一样。我们利用经肿瘤坏死因子-α处理的RASMC作为急性血管损伤反应的体外模型，并已开始确定刺激蛋白O-GlcNAc修饰的干预措施，即E2和GlcN，抑制对肿瘤坏死因子-α的炎症反应的机制。我们将重点放在核因子？B信号通路上，已知该通路可被肿瘤坏死因子-a和急性血管损伤激活。初步实验表明，GlcN可抑制肿瘤坏死因子-a诱导的RASMCs中核因子？B的激活。随后的研究表明，GlcN可抑制TNF-a诱导的RASMCs内I？BA的磷酸化和降解，而E_2的作用与细胞内I？BA的初始减少有关，随后细胞内I？BA的表达加速，这可能反映了活化的核因子？B介导的新的蛋白质合成。本研究将直接验证O-GlcNAc修饰蛋白(包括I？BA)在调节血管内膜损伤和体外培养的RASMCs对炎症反应的机制。其具体目的是：特定目的1：验证增加蛋白O-GlcNAc修饰在体内通过抑制核因子？B信号保护动脉免受与急性腔内损伤相关的炎症应激的假说。具体目的2：验证增加蛋白O-GlcNAc修饰通过抑制NF：B信号通路抑制肿瘤坏死因子-a诱导的RASMCs炎症反应的假说，并确定在NF？B信号级联反应中与这一效应有关的确切位置。具体目标3：确定O-GlcNAc修饰的RASMCs中在GlcN和E2的抗炎作用中起功能作用的特定蛋白质靶点。在成功完成这些目标后，O-GlcNAc修饰的抗炎和血管保护作用的细胞/分子机制将被阐明，并将与损伤反应的程度(即炎症和新生内膜形成)相关。我们推测O-GlcNAc修饰代表了一种新的血管保护机制，可能有助于开发预防和治疗心血管疾病的新策略。 公共卫生相关性：这项建议将检验一种新的机制，即O-连接的乙酰氨基葡萄糖(O-GlcNAc)修饰蛋白质，在受损的动脉和细胞因子刺激的平滑肌细胞(SMC)中具有抗炎和血管保护作用的假设，并将确定负责这些保护作用的特定蛋白质。

DESCRIPTION (provided by applicant): 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-a-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-a-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-a. We have focused on the NF?B signaling pathway, which is known to be activated by both TNF-a and acute vascular injury. Initial experiments demonstrated that GlcN inhibits TNF-a-induced NF?B activation in RASMCs. Subsequent studies showed that pretreatment with GlcN inhibits TNF-a-induced phosphorylation and degradation of I?Ba in RASMCs, while E2 pretreatment is associated with an initial reduction, followed by an accelerated reappearance of I?Ba in TNF-a 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?Ba, plays a mechanistic role in regulating the inflammatory response to endoluminal arterial injury in vivo and to TNF-a 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-a-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. PUBLIC HEALTH RELEVANCE: This proposal will test the hypothesis that a novel mechanism, O-linked acetylglucosamine (O-GlcNAc) modification of proteins, has anti-inflammatory and vasoprotective effects in injured arteries and cytokine- stimulated smooth muscle cells (SMCs) and will identify the specific proteins that are responsible for these protective effects.