O-GlcNAcylation regulates vascular smooth muscle cells in diabetic vasculopathy

描述（由申请方提供）：糖尿病患者普遍存在血管钙化，这会增加其发病率和死亡率。高血压是糖尿病的标志，可导致不良血管并发症。新兴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仍不清楚。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，已报告诱导培养中的VSMC钙化。我们已经发现，氧化应激和高糖增加成骨转录因子Runx 2的表达，这是必要的，足以诱导VSMC钙化在体外和体内。在人和小鼠的糖尿病动脉中观察到O-连接的N-乙酰基-葡萄糖胺（O-GlcNAc化）引起的蛋白质修饰增加，这与Runx 2增加和血管钙化有关。O-GlcNAc酰化是一个动态的、受严格调控的过程，它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-GlcNAc酰化由两种酶催化，O-GlcNAc转移酶（OGT）和O-GlcNAc酶（OGA），其分别催化靶蛋白上O-GlcNAc的转移和去除。通过己糖胺生物合成途径代谢的葡萄糖增加了UDP-GlcNAc的产生，UDP-GlcNAc是O-GlcNAc酰化的活性糖供体，可提高O-GlcNAc酰化。增加的O-GlcNAc酰化与糖尿病的不良心血管作用相关。我们的初步结果表明，高糖和氧化应激增加VSMC O-GlcNAc酰化和钙化。通过OGA敲低提高O-GlcNAc化促进VSMC钙化。此外，OGT的缺失不仅降低高糖和氧化应激诱导的VSMC蛋白O-GlcNAc酰化，而且阻断VSMC钙化。因此，我们推测，通过OGT缺失抑制VSMC中的O-GlcNAc化可降低糖尿病患者的血管钙化。使用我们新产生的可诱导SMC特异性OGT敲除小鼠模型，我们将1）确定OGT介导的O-GlcNAc酰化在体内糖尿病血管钙化中的作用;和2）阐明调节血管钙化的O-GlcNAc酰化依赖性分子信号。这些研究将为开发新的策略或药物提供重要的分子见解，以预防或治疗糖尿病和其他血管疾病中的血管钙化，这些血管疾病的特点是O-GlcNAc酰化增加。

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.