Cystathionine beta synthase (CBS) and angiogenesis

胱硫醚β合酶 (CBS) 和血管生成

• 批准号: 8877629
• 负责人: Resham Bhattacharya
• 金额: $ 41.71万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-15 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8877629
• 关键词: Address; Amino Acids; Anabolism; Animal Model; Atherosclerosis; Binding; Biochemical; Biochemical Genetics; Biological Assay; Blood Vessels; Blood capillaries; Blood flow; Cardiovascular system; Carotid Arteries; Cell physiology; Cells; Chemicals; Cystathionine; Cystathionine beta-Synthase; Cysteine; Cysteine Desulfhydrase; Data; Defect; Deletion Mutation; Development; Diabetic Retinopathy; Diet; Disease; EMSA; Endothelial Cells; Enzymes; Equilibrium; Exhibits; FarGo; Folic Acid; Generations; Genetic; Genetic Transcription; Glutathione; Goals; Health; Hindlimb; Homocysteine; Homocystine; Human; Hydrogen Sulfide; Hyperhomocysteinemia; Immigration; Impairment; In Vitro; Individual; Inherited; Injury; Intake; Ischemia; Knock-out; Knockout Mice; Lead; Luciferases; Macular degeneration; Maleimides; Malnutrition; Mesoderm; Messenger RNA; Metabolic; Metabolic Diseases; Methionine; Mitochondria; Modeling; Molecular; Mus; Mutation; Outcome; Oxidation-Reduction; Pathogenesis; Pathology; Pathway interactions; Pattern; Phenotype; Phosphorylation; Physical condensation; Plasma; Production; Property; Protein Isoforms; Proteins; Pyridoxal Phosphate; Randomized Clinical Trials; Regulation; Reverse Transcriptase Polymerase Chain Reaction; Role; Serine; Signal Transduction; Small Interfering RNA; Stroke; Sulfur Metabolism Pathway; Supplementation; System; Thrombosis; Transgenic Organisms; Tube; Umbilical vein; VEGF165; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factors; Vitamin B 12; Vitamin B6; Vitamins; Wound Healing; Zebrafish; angiogenesis; base; capillary; cardiovascular risk factor; chromatin immunoprecipitation; combat; density; endothelial dysfunction; genetic approach; in vivo; inhibitor/antagonist; liquid chromatography mass spectrometry; loss of function; mRNA Expression; migration; mutant; notochord development; novel therapeutics; overexpression; prevent; promoter; protein expression; research study; sulfhydration; transcription factor

DESCRIPTION (provided by applicant): Cystathionine beta synthase (CBS) deficiency has been implicated in the pathogenesis of many cardiovascular and neurovascular diseases such atherosclerosis, thrombosis, stroke and many more. Animal models of CBS deficiency exhibit endothelial dysfunction, impaired angiogenesis, atherosclerosis, thrombosis, dyslipidosis, etc. However, underlying molecular mechanisms of CBS deficiency causing such pathological outcome is virtually unknown. We hypothesize that the pro-angiogenic property of CBS is critical in maintaining vascular tone and endothelial cell heath and it exerts the pro-angiogenic effect either directly or indirectly by maintaining an intricate balance among cellular homocysteine (Hcy), glutathione (GSH) and hydrogen sulfide (H2S) levels. CBS is a metabolic enzyme involved in the metabolism of sulfur containing amino acid such as methionine (Met). It is a pyridoxal 5-phosphate dependent and the first rate-limiting enzyme in the transsulfuration pathway that catalyzes the condensation of L-serine and L-homocysteine (Hcy) to produce cystathionine, an intermediate step in the synthesis of cysteine (Cys). Recent studies have shown that CBS can efficiently produce H2S via condensation of Cys and Hcy to form cystathionine and H2S. Therefore, impairment of the transsulfuration pathway via pharmacological inhibition or genetic deletion/mutation of CBS may lead to, a) hyperhomocysteninemia (HHcy); b) reduction in cellular glutathione GSH level due to lack of availability of its precursor Cys; and c) reduction in H2S production. In fact, heterozygous and homozygous deleted CBS knockout mice exhibit mild to severe HHcy, respectively. HHcy, an elevation of plasma Hcy level, is an independent risk factor for cardiovascular (CVD) and neurovascular diseases (NVDs). Several factors that cause elevation of plasma Hcy level are; (i) genetic deficiencies/mutations in the enzymes responsible for remethylation/transsulfuration pathway of Hcy; (ii) nutritional deficiencies of the vitamin co-factors of these enzymes such as folic acid, pyridoxin (vitamin B6), cobalamine (vitamin B12); (iii) excessive intake of methionine rich diet. Folic acid and vitamin B12 facilitates remethylation of Hcy to Met, whereas vitamin B6 supports transsulfuration to cystathionine. Importantly, several large-scale randomized clinical trials (RCTs) demonstrated that although the vitamin supplementation lowered the plasma Hcy levels, but it failed to prevent the cardiovascular or neurovascular outcomes. These findings highlight the critical need to understand the mechanism of CBS function, mutations/deletions of which lead to CVDs and NVDs independent of HHcy. Our hypothesis is supported by our preliminary data that demonstrate, a) Silencing CBS with siRNA or chemical inhibitors inhibits proliferation, migration and tube formation in HUVECs in vitro. In addition, supplementation with GSH induced proliferation of HUVECs. b) CBS maintains pro-angiogenic property via cross talk with VEGF/VEGFR2 axis as silencing CBS inhibits VEGF165 induced proliferation of HUVECs, downregulates VEGFR2 at the transcriptional and possibly at the translational level and downregulates ERK- 1/2 phosphorylation. Furthermore, silencing of CBS by siRNA leads to increased ROS production in HUVECs; c) Knockdown of CBS in zebra fish exhibits defects in notochord development and vascular defects. We proposed three specific aims to determine the mechanism of pro-angiogenic function of CBS. Aim 1: Determine the mechanism by which CBS regulates angiogenesis in vitro. Aim 2: Investigating the cross-talk between the CBS and the VEGF pathway. Aim3: Determine a role for CBS in regulating angiogenesis in vivo. Thus, understanding the molecular mechanism of CBS function will not only provide new therapeutic avenues to prevent cardiovascular and neurovascular outcome due to CBS deficiency but combat angiogenesis dependent disorders such as macular degeneration, diabetic retinopathy, etc. as well. Therefore, the idea that CBS exerts the pro-angiogenic effect by maintaining an intricate balance among cellular homocysteine (Hcy), glutathione (GSH) and hydrogen sulfide (H2S) levels is highly significant and the impact of this study goes far beyond cardiovascular and neurovascular diseases.

描述（由申请人提供）：胱硫醚β合酶（CBS）缺乏与许多心血管和神经血管疾病（例如动脉粥样硬化、血栓形成、中风等）的发病机制有关。 CBS 缺乏的动物模型表现出内皮功能障碍、血管生成受损、动脉粥样硬化、血栓形成、血脂异常等。然而，导致此类病理结果的 CBS 缺乏的潜在分子机制实际上尚不清楚。我们假设 CBS 的促血管生成特性对于维持血管张力和内皮细胞健康至关重要，并且它通过维持细胞同型半胱氨酸 (Hcy)、谷胱甘肽 (GSH) 和硫化氢 (H2S) 水平之间的复杂平衡来直接或间接发挥促血管生成作用。 CBS是一种参与含硫氨基酸（例如蛋氨酸（Met））代谢的代谢酶。它是吡哆醛 5-磷酸依赖性酶，也是转硫途径中的第一个限速酶，可催化 L-丝氨酸和 L-同型半胱氨酸 (Hcy) 缩合产生胱硫醚，这是合成半胱氨酸 (Cys) 的中间步骤。最近的研究表明，CBS可以通过Cys和Hcy缩合形成胱硫醚和H2S来有效地产生H2S。因此，通过药理抑制或 CBS 基因缺失/突变来损害转硫途径可能会导致：a) 高同型半胱氨酸血症 (HHcy)； b) 由于缺乏其前体半胱氨酸而导致细胞谷胱甘肽谷胱甘肽水平降低； c) 减少 H2S 的产生。事实上，杂合子和纯合子缺失的 CBS 敲除小鼠分别表现出轻度至重度 HHcy。 HHcy 是血浆 Hcy 水平升高，是心血管 (CVD) 和神经血管疾病 (NVD) 的独立危险因素。导致血浆 Hcy 水平升高的几个因素是： (i) 负责 Hcy 的再甲基化/转硫途径的酶的遗传缺陷/突变； (ii) 这些酶的维生素辅助因子的营养缺乏，例如叶酸、吡哆醇（维生素 B6）、钴胺（维生素 B12）； (iii)过量摄入富含蛋氨酸的饮食。叶酸和维生素 B12 促进 Hcy 重新甲基化为 Met，而维生素 B6 支持转硫为胱硫醚。重要的是，多项大型随机临床试验（RCT）表明，虽然补充维生素可降低血浆Hcy水平，但未能预防心血管或神经血管结局。这些发现强调了了解 CBS 功能机制的迫切需要，其突变/缺失会导致独立于 HHcy 的 CVD 和 NVD。我们的假设得到了初步数据的支持，这些数据表明：a) 用 siRNA 或化学抑制剂沉默 CBS 可抑制体外 HUVEC 的增殖、迁移和管形成。此外，补充 GSH 还可诱导 HUVEC 增殖。 b) CBS 通过与 VEGF/VEGFR2 轴的串扰维持促血管生成特性，因为沉默 CBS 抑制 VEGF165 诱导的 HUVEC 增殖，在转录水平和可能在翻译水平下调 VEGFR2，并下调 ERK-1/2 磷酸化。此外，通过 siRNA 沉默 CBS 会导致 HUVEC 中 ROS 产生增加； c) 斑马鱼中 CBS 的敲低表现出脊索发育缺陷和血管缺陷。我们提出了三个具体目标来确定 CBS 促血管生成功能的机制。目标 1：确定 CBS 体外调节血管生成的机制。目标 2：研究 CBS 和 VEGF 通路之间的串扰。目标 3：确定 CBS 在调节体内血管生成中的作用。 因此，了解 CBS 功能的分子机制不仅可以提供新的治疗途径来预防 CBS 缺乏导致的心血管和神经血管结局，还可以对抗血管生成依赖性疾病，如黄斑变性、糖尿病视网膜病变等。因此，CBS 通过维持细胞同型半胱氨酸 (Hcy)、谷胱甘肽 (GSH) 和硫化氢 (H2S) 水平之间的复杂平衡来发挥促血管生成作用的想法非常重要，并且这项研究的影响远远超出了心血管和神经血管疾病的范围。