Cystathionine beta synthase (CBS) and angiogenesis

描述(由申请人提供)：胱硫醚β合成酶(CBS)缺乏与许多心血管和神经血管疾病的发病机制有关，如动脉粥样硬化、血栓形成、中风等。CBS缺乏的动物模型表现为内皮功能障碍、血管生成障碍、动脉粥样硬化、血栓形成、血脂异常等。然而，CBS缺乏导致这些病理结果的潜在分子机制几乎是未知的。我们推测，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至关重要，它通过维持细胞内同型半胱氨酸(Hcy)、谷胱甘肽(GSH)和硫化氢(H2S)水平之间的复杂平衡，直接或间接地发挥促血管生成作用。CBS是一种代谢酶，参与蛋氨酸等含硫氨基酸的代谢。它是一种依赖于5-磷酸吡哆醛的酶，也是跨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)缩合生成半胱氨酸(Cystathionine)，这是半胱氨酸(Cys)合成的中间步骤。最近的研究表明，CBS可以通过半胱氨酸和同型半胱氨酸缩合生成胱硫醚和硫化氢来高效地产生硫化氢。因此，通过药物抑制或CBS的基因缺失/突变导致的跨硫途径的损害可能导致：a)高同型半胱氨酸血症；b)由于其前体Cys的缺乏而导致细胞内谷胱甘肽GSH水平的降低；以及c)H 2S的产生减少。事实上，杂合子和纯合子缺失的CBS基因敲除小鼠分别表现出轻度到重度的HHcy。同型半胱氨酸是血浆同型半胱氨酸水平的升高，是心血管疾病和神经血管疾病的独立危险因素。导致血浆同型半胱氨酸水平升高的几个因素是：(I)负责同型半胱氨酸再甲基化/硫化途径的酶的遗传缺陷/突变；(Ii)这些酶的维生素辅助因子，如叶酸、吡哆醇(维生素B6)、钴胺(维生素B12)的营养缺乏；(Iii)过量摄入蛋氨酸丰富的饮食。叶酸和维生素B12促进同型半胱氨酸重新甲基化为蛋氨酸，而维生素B6支持硫代转化为胱硫醚。重要的是，多项大规模随机临床试验(RCT)表明，补充维生素虽然降低了血浆Hcy水平，但不能预防心血管或神经血管结局。这些发现突显了了解CBS功能的机制的迫切需要，CBS功能的突变/缺失导致心血管疾病和非心脏疾病独立于HHcy。我们的假设得到了我们的初步数据的支持，这些数据表明，a)用siRNA或化学抑制剂沉默CBS可以抑制体外培养的HUVEC的增殖、迁移和管状形成。此外，补充GSH可诱导人脐静脉内皮细胞增殖。B)CBS通过与VEGFR2轴的相互作用维持促血管生成的特性，沉默CBS抑制VEGF165诱导的HUVECs增殖，在转录和翻译水平下调VEGFR2，并下调ERK-1/2的磷酸化。此外，siRNA沉默CBS导致HUVECs中ROS产生增加；c)斑马鱼中CBS的敲除表现出脊索发育和血管缺陷。我们提出了三个具体的目标来确定CBS促血管生成的机制。目的1：确定CBS在体外调节血管生成的机制。目的2：研究CBS与血管内皮细胞生长因子途径之间的相互作用。目的：确定CBS在体内调节血管生成中的作用。因此，了解CBS功能的分子机制不仅将为预防CBS缺乏症所致的心血管和神经血管疾病提供新的治疗途径，而且还将为防治黄斑变性、糖尿病视网膜病变等血管生成依赖型疾病提供新的治疗途径。因此，CBS通过维持细胞内同型半胱氨酸(Hcy)、谷胱甘肽(GSH)和硫化氢(H2S)水平的复杂平衡来发挥促血管生成作用的想法具有非常重要的意义，而且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.