Cystathionine beta synthase (CBS) and angiogenesis

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如动脉粥样硬化、血栓形成、中风等的发病机制有关。CBS缺陷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，CBS缺陷导致这种病理结果的潜在分子机制几乎是未知的。我们假设CBS的促血管生成特性在维持血管张力和内皮细胞健康方面是至关重要的，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而直接或间接地发挥促血管生成作用。 CBS是参与含硫氨基酸如甲硫氨酸（Met）代谢的代谢酶。它是一种吡哆醛5-磷酸依赖性酶，是转硫途径中的第一个限速酶，催化L-丝氨酸和L-同型半胱氨酸（Hcy）缩合生成硫胱苷肽，这是半胱氨酸（Cys）合成的中间步骤。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CBS的药理学抑制或遗传缺失/突变损害转硫途径可能导致：a）高同型半胱氨酸血症（HHcy）; B）由于缺乏其前体Cys而导致细胞谷胱甘肽GSH水平降低;以及c）H2S产生减少。事实上，杂合和纯合缺失CBS敲除小鼠分别表现出轻度至重度HHcy。高同型半胱氨酸（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）这些酶的维生素辅因子如叶酸、吡啶多辛（维生素B6）、钴胺素（维生素B12）的营养缺乏;（iii）过量摄入富含甲硫氨酸的饮食。叶酸和维生素B12促进Hcy再甲基化为Met，而维生素B6支持转硫为胱硫醚。重要的是，多项大规模随机临床试验（RCT）表明，补充维生素虽然能降低血浆Hcy水平，但未能预防心血管或神经血管结局。这些发现强调了理解CBS功能机制的迫切需要，CBS功能的突变/缺失导致CVD和NVD独立于HHcy。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抑制体外HUVEC中的增殖、迁移和管形成。此外，补充GSH可诱导HUVEC增殖。B）CBS通过与VEGF/VEGFR 2轴的串扰维持促血管生成性质，因为沉默CBS抑制VEGF 165诱导的HUVEC增殖，在转录水平和可能在翻译水平下调VEGFR 2，并下调ERK- 1/2磷酸化。此外，通过siRNA沉默CBS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的目标，以确定CBS的促血管生成功能的机制。目的1：探讨CBS在体外调控血管生成的机制。目的2：研究CBS和VEGF通路之间的相互作用。目的3：确定CBS在体内调节血管生成中的作用。 因此，理解CBS功能的分子机制将不仅提供新的治疗途径来预防由于CBS缺陷引起的心血管和神经血管结果，而且还对抗血管生成依赖性疾病，例如黄斑变性、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是非常重要的，这项研究的影响远远超出了心血管和神经血管疾病。

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.