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，即由三种不同且独立的刺激物（17-雌二醇（E2）、葡糖胺（GlcN）和选择性O-GlcNAc酶抑制剂O-（乙酰氨基-2-脱氧-D-吡喃葡萄糖基亚基）氨基-N-苯基氨基甲酸酯（PUGNAc））诱导的蛋白质的O-连接的<$-N-乙酰葡糖胺（O-GlcNAc）修饰在球囊损伤的大鼠颈动脉中具有抗炎作用。这些新的观察结果提供了刺激性证据，即蛋白质的增强的O-GlcNAc修饰在急性腔内血管损伤的情况下具有抗炎和血管保护作用。我们有强有力的证据表明，增加蛋白O-GlcNAc修饰响应GlcN治疗与TNF-α诱导的表达的炎症介质在分离的大鼠主动脉平滑肌细胞（RASMC）的衰减在E2以前报道的方式。我们已经利用TNF-α处理的RASMC作为急性血管损伤反应的体外模型，并且已经开始定义刺激蛋白O-GlcNAc修饰的干预的机制，即，E2和GlcN抑制对TNF-α的炎症反应。我们专注于NF？B信号通路，已知其被TNF-α和急性血管损伤激活。初步实验表明，GlcN抑制TNF-α诱导的NF？RASMC中的B活化。随后的研究表明，预处理与GlcN抑制TNF-α诱导的磷酸化和降解的I？Ba在RASMCs，而E2预处理与初始减少，随后加速再现的I？Ba在TNF-α处理的细胞，可能反映了新的蛋白质合成介导的激活NF？B。目前的研究将直接测试的假设，O-GlcNAc修饰的蛋白质，包括I？Ba在体内调节对腔内动脉损伤的炎症反应和体外调节对分离的RASMC中TNF-α刺激的炎症反应中起机制作用。具体目标是：具体目标1：为了验证增加蛋白O-GlcNAc修饰通过抑制NF保护动脉免受体内急性腔内损伤相关炎症应激的假设？B信令。具体目标二：为了验证增加蛋白O-GlcNAc修饰通过抑制NF：B信号转导抑制体外RASMC中TNF-α诱导的炎症反应的假设，并确定NF：B信号转导中的精确位点。B信号级联反应是这种效应的原因。具体目标3：鉴定RASMC中O-GlcNAc修饰的特异性蛋白靶点，其在GlcN和E2的抗炎作用中发挥功能性作用。在成功完成这些目标后，将阐明负责O-GlcNAc修饰的抗炎和血管保护作用的细胞/分子机制，并将其与损伤反应的程度（即，炎症和新生内膜形成）。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：该提案将检验一种新的机制，即蛋白质的O-连接乙酰葡糖胺（O-GlcNAc）修饰，在受损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，并将鉴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.