Glutaminase in Arterial Injury and Disease

项目摘要 该提案的广泛的长期目标是将酶谷氨酰胺酶-1（GLS 1）确立为关键的酶。 动脉损伤和疾病中损伤形成的介质。GLS 1是一种线粒体酶， 谷氨酰胺（Gln）转化为谷氨酸（Glu）和氨（NH3）。GLS 1在神经传递、酸传递和神经元代谢中起着关键作用。 碱平衡，血管生成，肝纤维化和肿瘤生长，但很少有人知道的功能， GLS 1在血管平滑肌细胞（SMC）中的表达。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。也是 发现SMC仅表达GLS 1亚型，GLS 1的过表达刺激SMC GLS 1活性或表达的抑制阻断SMC DNA合成或迁移， 分别此外，Glu而不是NH3替代Gln促进SMC增殖和胶原形成 合成.另外的实验表明，生长因子和葡萄糖刺激血管中的GLS 1活性， SMC。最后的初步实验还表明，动脉损伤和糖尿病诱导表达 GLS 1在血管壁中的表达，并且抑制GLS 1活性或GLS 1的基因缺失可减弱新生内膜 在糖尿病动物中，动脉损伤以及动脉纤维化和硬化后的血管形成。基于这些 研究结果表明，GLS 1通过刺激SMC在异常动脉重塑中起着不可或缺的作用 增殖、迁移、胶原合成和通过将Gln代谢为Glu和NH3而存活。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，GLS 1在调节SMC功能中的作用将是 测定这些研究将研究GLS 1基因递送对SMC增殖、迁移， 胶原蛋白的合成和存活，并确定各种GLS 1产品在这些过程中的作用。 他们还将探索生长因子诱导GLS 1是否有助于其促进SMC的能力 增殖、迁移和胶原蛋白合成。在目的2中，GLS 1在调节动脉反应中的作用 伤害将成立。这些研究将检查受伤啮齿动物中GLS 1表达的时间过程 颈动脉，并确定药物抑制GLS 1活性，沉默GLS 1表达，或 SMC中GLS 1基因缺失减弱动脉损伤后的重塑反应。在目标3中， 将研究GLS 1在异常动脉重构和糖尿病高血压中的作用。这些研究 将检测葡萄糖对培养的血管平滑肌细胞和动脉中GLS 1表达的影响， 糖尿病小鼠此外，他们还将研究GLS 1是否有助于葡萄糖介导的SMC改变。 功能，动脉重塑和高血压。预计这些研究将 确立GLS 1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 还鉴定了GLS 1及其产物作为动脉损伤后损伤形成的新贡献者， 确立GLS 1作为治疗糖尿病异常重构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.