O-GlcNAcylation regulates vascular smooth muscle cells in diabetic vasculopathy

描述（申请人提供）：血管钙化在糖尿病患者中普遍存在，增加了糖尿病患者的发病率和死亡率。高血糖症是糖尿病的标志，可导致不良的血管并发症。新出现的临床和基础研究支持高血糖和血管钙化之间的强相关性。然而，高血糖在调节血管钙化中的确切作用及其潜在的分子机制尚不清楚。血管平滑肌细胞（VSMC）的成骨分化和钙化指导糖尿病血管中膜和内膜的血管钙化。与糖尿病相关的条件，包括高葡萄糖和氧化应激，已经报道在培养中诱导VSMC钙化。我们发现氧化应激和高糖增加成骨转录因子Runx2的表达，这是体外和体内诱导VSMC钙化的必要和充分条件。在人类和小鼠的糖尿病动脉中观察到O-linked ss- n -乙酰氨基葡萄糖（o - glcnac酰化）的蛋白修饰增加，这与Runx2增加和血管钙化有关。O- glcn酰化是一个动态的、受到严格调控的过程，与磷酸化一样普遍存在，在多种生物过程的调控中起着关键作用。o - glcn酰化由O-GlcNAc转移酶（OGT）和O-GlcNAcase （OGA）两种酶催化，它们分别催化O-GlcNAc在靶蛋白上的转移和去除。葡萄糖通过己糖胺生物合成途径代谢增加了UDP-GlcNAc的产生，而UDP-GlcNAc是o - glcn酰化的活性糖供体，从而提高了o - glcn酰化。o - glcn酰化升高与糖尿病的不良心血管效应有关。我们的初步结果表明，高葡萄糖和氧化应激增加了VSMC的o - glcn酰化和钙化。o - glcn酰化通过OGA敲低促进VSMC钙化。此外，OGT的缺失不仅降低了VSMC中高糖和氧化应激诱导的蛋白o - glcn酰化，而且阻断了VSMC的钙化。因此，我们假设通过OGT缺失抑制VSMC中的o - glcn酰化可降低糖尿病的血管钙化。利用我们新建立的smc特异性OGT敲除小鼠模型，我们将1)确定OGT介导的o - glcn酰化在糖尿病血管钙化中的作用；2)阐明调节血管钙化的o - glcn酰化依赖的分子信号。这些研究将为开发新的策略或药物来预防或治疗糖尿病和其他血管疾病的血管钙化提供重要的分子见解。

DESCRIPTION (provided by applicant): Vascular calcification is prevalent in patients with diabetes mellitus, which increases their morbidity and mortality. Hyperglycemia is a hallmark of diabetes that leads to adverse vascular complications. Emerging clinical and basic investigations support a strong correlation between hyperglycemia and vascular calcification. However, the precise role of hyperglycemia in regulating vascular calcification and the underlying molecular mechanisms remain unknown. Osteogenic differentiation and calcification of vascular smooth muscle cells (VSMC) directs vascular calcification in the media and intima of diabetic vasculature. Conditions associated with diabetes, including high glucose and oxidative stress, have been reported to induce VSMC calcification in culture. We have found that oxidative stress and high glucose increase the expression of the osteogenic transcription factor Runx2, which is essential and sufficient to induce VSMC calcification in vitro and in vivo. Increased protein modification by O-linked ss-N-acetyl-glucosamine (O-GlcNAcylation) was observed in diabetic arteries from human and mice, which was associated with increased Runx2 and vascular calcification. O- GlcNAcylation is a dynamic and tightly regulated process, which is as common and ubiquitous as phosphorylation and plays a key role in the regulation of diverse biological processes. O-GlcNAcylation is catalyzed by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which catalyze the transfer and removal of O-GlcNAc on target proteins, respectively. Glucose metabolized via the hexosamine biosynthesis pathway increases the production of UDP-GlcNAc, an active sugar donor for O-GlcNAcylation, which elevates O-GlcNAcylation. Increased O-GlcNAcylation has been associated with the adverse cardiovascular effects of diabetes. Our preliminary results demonstrated that high glucose and oxidative stress increased VSMC O-GlcNAcylation and calcification. Elevation of O-GlcNAcylation via OGA knockdown promoted VSMC calcification. Moreover, deletion of OGT not only decreased high glucose and oxidative stress-induced protein O-GlcNAcylation in VSMC, but also blocked VSMC calcification. Therefore, we hypothesize that inhibition of O-GlcNAcylation by OGT deletion in VSMC decreases vascular calcification in diabetes. Using our newly generated inducible SMC-specific OGT knockout mouse model, we will 1) determine the role of OGT-mediated O-GlcNAcylation in diabetic vascular calcification in vivo; and 2) elucidate O-GlcNAcylation-dependent molecular signals that regulate vascular calcification. These studies will provide important molecular insights into developing new strategies or drugs to prevent or treat vascular calcification in diabetes and other vascular diseases featuring increased O-GlcNAcylation.