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.

R01HL120585

2013-08-15

2019-05-31