Cystathionine beta synthase (CBS) and angiogenesis

胱硫醚β合酶 (CBS) 和血管生成

基本信息

批准号：
9276099
负责人：
Resham Bhattacharya
金额：
$ 42.13万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-15 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9276099
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 Hindlimb Homocysteine Human Hydrogen Sulfide Hyperhomocysteinemia Impairment In Vitro Individual Inherited Injury Intake Ischemia KDR gene Knock-out Knockout Mice Lead Luciferases MAPK3 gene Macular degeneration Maleimides Malnutrition Mesoderm Messenger RNA Metabolic Metabolic Diseases Methionine Mitochondria Modeling Molecular Mus Mutation Outcome Oxidation-Reduction Pathogenesis Pathologic Pathology Pathway interactions Pattern Pharmacology 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 Transcriptional Activation Transgenic Organisms Tube Umbilical vein VEGF165 Vitamin B 12 Vitamin B6 Vitamin Deficiency Vitamins Wound Healing Zebrafish angiogenesis base capillary cardiovascular risk factor chromatin immunoprecipitation combat density endothelial dysfunction experimental study genetic approach in vivo inhibitor/antagonist knock-down liquid chromatography mass spectrometry loss of function mRNA Expression migration mouse model mutant neurovascular notochord development novel therapeutics overexpression prevent promoter protein expression public health relevance sulfhydration transcription factor virtual

项目摘要

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）：胱淀粉β合酶（CBS）缺乏与许多心血管和神经血管疾病的发病机理有关，例如动脉粥样硬化，血栓形成，中风等。 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，几乎未知，CBS缺乏的基本分子机制实际上是未知的。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荒地至关重要，并且通过在细胞同心半胱氨酸（HCY），谷胱甘肽（GSH）（GSH）（GSH）和硫化物（H2S）中保持复杂的平衡来直接或间接发挥促血管生成作用。 CBS是一种代谢酶，参与含有氨基酸的硫代谢，例如蛋氨酸（Met）。这是一种5-磷酸吡啶毒素依赖性，并且是转硫途径中的第一次限制酶，它催化L-丝氨酸和L-联合系统（HCY）的凝结，以产生胱硫硫氨酸，从而在cysteine（Cys）合成的中间步骤（Cys）。最近的研究表明，CBS可以通过CYS和HCY的凝结有效地产生H2s，形成胱淀粉和H2S。因此，通过药理学抑制或CBS的遗传缺失/突变可能导致跨硫化途径的损害可能导致a））高粒细胞肾上腺素（HHCY）； b）由于缺乏其前体Cys的可用性，细胞谷胱甘肽GSH水平的降低； c）减少H2S生产。实际上，杂合和纯合删除的CBS基因敲除小鼠分别表现出轻度至重度HCY。 HHCY是血浆HCY水平的升高，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二甲基化/转移途径的酶中的遗传缺陷/突变；（ii）这些酶的维生素辅助因子的营养缺乏，例如叶酸，吡ido毒素（维生素B6），钴拉明（维生素B12）；（iii）过度摄入富含蛋氨酸的饮食。叶酸和维生素B12促进了HCY的将HCY的再甲基化，而维生素B6则支持硫氨酸的转化。重要的是，一些大规模的随机临床试验（RCT）表明，尽管补充维生素降低了血浆HCY水平，但未能阻止心血管或神经血管结局。这些发现突出了了解CBS功能机制的迫切需要，其突变/缺失导致CVD和NVD独立于HHCY。我们的假设得到了我们的初步数据的支持，该数据证明，a）用siRNA或化学抑制剂沉默的CBS抑制了HUVEC在体外的HUVEC中的增殖，迁移和管形成。另外，补充GSH诱导的HUVEC增殖。 b）CBS通过与VEGF/VEGFR2轴的交叉交谈来维持促血管生成特性，因为沉默CBS抑制VEGF165诱导HUVEC的增殖，在转录上下调VEGFR2在转录水平，可能在翻译水平上，并下调ERK-1/2磷酸化。此外，siRNA对CBS的沉默导致HUVEC的ROS产生增加。 c）斑马鱼中CBS的敲低表现出脊索发育和血管缺陷中的缺陷。我们提出了三个特定的目的，以确定CBS的促血管生成功能的机理。目标1：确定CBS在体外调节血管生成的机制。 AIM 2：研究CBS和VEGF途径之间的串扰。 AIM3：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将提供新的治疗途径，以防止CBS缺乏症引起的心血管和神经血管结局，而且还可以打击依赖血管生成的疾病，例如黄斑变性，糖尿病性视网膜病变等。因此，CBS通过在细胞同型半胱氨酸（HCY），谷胱甘肽（GSH）和硫化氢（H2S）水平之间保持复杂的平衡来发挥促血管生成作用的想法非常重要，这项研究的影响远远超出了心血管疾病和神经血管疾病。