O-GlyNAcylation: Novel Mechanism of Estrogen-Induced Vasoprotection

项目摘要/摘要 我们对O-连接N-乙酰氨基葡萄糖(O-GlcNAc)修饰进行了开创性的观察 三种不同和独立的刺激，17-雌二醇(E_2)，氨基葡萄糖(GlcN)和 选择性O-GlcNAcase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)，对球囊损伤大鼠颈动脉具有抗炎作用。这些新奇的观察 提供具有挑衅性的证据，证明增强的蛋白质O-GlcNAc修饰具有抗炎和 急性腔内血管损伤时的血管保护作用。我们有强有力的证据证明 GlcN处理后蛋白O-GlcNAc修饰增加与肿瘤坏死因子-2的降低有关 诱导炎性介质在体外培养的大鼠主动脉平滑肌细胞中的表达 之前为E2报告的方式。我们利用肿瘤坏死因子处理的RASMC作为体外模型。 急性血管损伤反应，并已开始确定干预措施刺激的机制 蛋白O-GlcNAc修饰，即E2和GlcN，可抑制对肿瘤坏死因子-β的炎症反应。我们已经专注于 在核因子B信号通路上，已知可由肿瘤坏死因子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-β诱导的RASMCs中核因子B的激活。后续 研究表明，GlcN预处理可抑制肿瘤坏死因子诱导的磷酸化和I？B的降解 在RASMC中，而E2预处理与最初的减少有关，随后是加速的 在经肿瘤坏死因子处理的细胞中重现I？B？，可能反映了激活的 目前的研究将直接检验O-GlcNAc修饰蛋白质的假设，包括 在体内对血管内膜损伤的炎症反应起着调节的机制作用。 对体外培养的RASMCs产生肿瘤坏死因子-β刺激。具体目标是：具体目标1：测试 增加蛋白O-GlcNAc修饰保护动脉免受炎性应激相关的假说 体内急性腔内损伤通过抑制核因子B信号转导。具体目标2：检验假设 增加蛋白O-GlcNAc修饰抑制肿瘤坏死因子-β诱导的RASMCs炎症反应 通过抑制核因子B信号，并确定在核因子B信号级联中负责的确切位置 为了达到这个效果。特定目标3：确定O-GlcNAc修饰的RASMCs中的特定蛋白质靶点 在谷氨酰胺和雌二醇的抗炎作用中起一定作用。在成功完成这些任务后 O-抗炎和血管保护作用的目的、细胞/分子机制 GlcNAc的修饰将被阐明，并将与损伤反应的程度(即炎症)相关 和新生内膜的形成)。我们推测O-GlcNAc修饰代表了一种新的机制 血管保护，可能有助于制定新的预防和治疗策略 心血管疾病。

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.