Glutaminase in Arterial Injury and Disease

项目总结 这项提议的广泛的长期目标是将谷氨酰胺酶-1(GLS1)确立为关键的 动脉损伤和疾病中病变形成的介体。GLS1是一种线粒体酶，负责代谢 谷氨酰胺(Gln)为谷氨酸(Glu)和氨(NH3)。GLS1在神经传递中起关键作用，酸性- 碱基平衡、血管生成、肝纤维化和肿瘤生长，但对其功能知之甚少 血管平滑肌细胞(SMC)表达GLS1。然而，初步研究表明， SMC的增殖、迁移和存活高度依赖于Gln的存在。它也是 发现SMC只表达GLS1亚型，过表达GLS1刺激SMC 迁移，抑制GLS1活性或表达阻止SMC DNA合成或迁移， 分别进行了分析。此外，在促进SMC增殖和胶原方面，Glu而不是NH3是Gln的替代品 综合。进一步的实验表明，生长因子和葡萄糖能刺激血管中的GLS1活性 SMCS。最后的初步实验还表明，动脉损伤和糖尿病会诱导血管内皮生长因子的表达 血管壁中的GLS1，抑制GLS1的活性或GLS1的基因缺失可以减弱新生内膜 糖尿病动物动脉损伤后的形成以及动脉纤维化和硬化。基于这些 结果表明，GLS1通过刺激SMC在异常动脉重塑中起着不可或缺的作用。 通过将谷氨酰胺代谢成谷氨酸和氨来促进细胞的增殖、迁移、胶原合成和存活。这一假设 将在三个相互关联的具体目标中进行测试。在目标1中，GLS1在调节SMC功能中的作用将是 下定决心。这些研究将探讨GLS1基因对SMC增殖、迁移、 胶原蛋白的合成和存活，并决定各种GLS1产物在这些过程中的作用。 他们还将探索生长因子诱导GLS1是否有助于他们促进SMC的能力 增殖、迁移和胶原合成。在目标2中，GLS1在调节动脉反应中的作用 将会被确立为伤害。这些研究将检测GLS1在受损啮齿动物中的表达时程 颈动脉，并确定是否药物抑制GLS1活性，沉默GLS1表达，或 SMC中GLS1基因缺失可减弱动脉损伤后的重塑反应。在目标3中， 我们将研究GLS1在糖尿病患者异常动脉重构和高血压中的作用。这些研究 将研究葡萄糖对培养的血管SMC和动脉中GLS1表达的影响。 糖尿病小鼠。此外，他们还将研究GLS1是否有助于SMC中葡萄糖介导的改变 糖尿病小鼠的功能、动脉重塑和高血压。预计这些研究将 建立GLS1作为SMC增殖、迁移、胶原合成和存活的关键调节因子。他们可能会 也确定GLS1及其产物是动脉损伤后病变形成的新的贡献者，以及 建立GLS1作为治疗糖尿病重构异常和高血压的新的翻译靶点。

PROJECT SUMMARY The broad long-term goal of this proposal is to establish the enzyme glutaminase-1 (GLS1) as a critical mediator of lesion formation in arterial injury and disease. GLS1 is a mitochondrial enzyme that metabolizes glutamine (Gln) to glutamate (Glu) and ammonia (NH3). GLS1 plays a critical role in neurotransmission, acid- base balance, angiogenesis, hepatic fibrosis, and tumor growth, but little is known regarding the function of GLS1 in vascular smooth muscle cells (SMCs). However, preliminary studies demonstrated that the proliferation, migration, and survival of SMCs is highly dependent on the presence of Gln. It was also discovered that SMCs exclusively express the GLS1 isoform, that overexpression of GLS1 stimulates SMC migration, and that inhibition of GLS1 activity or expression blocks SMC DNA synthesis or migration, respectively. Moreover, Glu, but not NH3, substitutes for Gln in promoting SMC proliferation and collagen synthesis. Additional experiments revealed that growth factors and glucose stimulate GLS1 activity in vascular SMCs. Final pilot experiments also demonstrated that arterial injury and diabetes induces the expression of GLS1 in the vessel wall, and that inhibition of GLS1 activity or genetic depletion of GLS1 attenuates neointima formation following arterial injury as well as arterial fibrosis and stiffening in diabetic animals. Based on these findings, it is proposed that GLS1 plays an integral role in aberrant arterial remodeling by stimulating SMC proliferation, migration, collagen synthesis, and survival by metabolizing Gln to Glu and NH3. This hypothesis will be tested in three interrelated specific aims. In aim 1, the role of GLS1 in regulating SMC function will be determined. These studies will investigate the effect of GLS1 gene delivery on SMC proliferation, migration, collagen synthesis and survival, and determine the role of the various GLS1 products on these processes. They will also explore if the induction of GLS1 by growth factors contributes to their ability to promote SMC proliferation, migration, and collagen synthesis. In aim 2, the role of GLS1 in regulating the arterial response to injury will be established. These studies will examine the time-course of GLS1 expression in injured rodent carotid arteries, and determine if pharmacological inhibition of GLS1 activity, silencing GLS1 expression, or genetic deletion of GLS1 in SMCs attenuates the remodeling response following arterial injury. In aim 3, the role of GLS1 in aberrant arterial remodeling and hypertension in diabetes will be investigated. These studies will examine the effect of glucose on GLS1 expression both in cultured vascular SMCs and in arteries from diabetic mice. In addition, they will examine if GLS1 contributes to glucose-mediated alterations in SMC function, arterial remodeling, and hypertension in diabetic mice. It is anticipated that these studies will establish GLS1 as a key regulator of SMC proliferation, migration, collagen synthesis, and survival. They may also also identify GLS1 and its products as novel contributors to lesion formation following arterial injury, and establish GLS1 as a new translational target in treating abnormal remodeling and hypertension in diabetes.