H2S promotes developmental brain angiogenesis via the NOS/NO pathway in zebrafish
Weiqing Jiang, Chen Liu, Mingzhu Deng, Fei Wang, Xiao Ren, Yilin Fan, Jiulin Du, Yonggang Wang
doi: 10.1136/svn-2020-000584
Stroke & Vascular Neurology（SVN）新近上线文章“H2S promotes developmental brain angiogenesis via the NOS/NO pathway in zebrafish”，来自上海交通大学医学院附属仁济医院王永刚教授团队。
硫化氢已被广泛认为是继一氧化氮和一氧化碳后的第三个气体递质家族成员。细胞实验和动物缺血模型已证明，硫化氢可通过促进内皮细胞的生长、迁移和管状结构形成等方面达到促血管生成的作用；在体实验也表明生理性硫化氢具有促进肌肉组织血管生成的作用。然而，内源性硫化氢在脑血管新生中的作用尚不明确。本研究利用斑马鱼为模式生物，旨在明确内源性硫化氢对脑血管发育的作用。
研究发现，利用反义吗啉环寡聚核苷酸（morpholino oligomer, MO）技术和CRISPR/Cas9技术分别下调和敲除斑马鱼硫化氢产生酶 —— 胱硫醚β合成酶（Cystathionine-β-synthase, cbs）和胱硫醚γ裂解酶（Cystathionine-γ-lyase, cth），均能抑制斑马鱼胚胎脑血管发育。Figure 1. Knockdown and knockout of cbs or cth impair brain vascular development of larval zebrafish. (A) In situ hybridisation of whole zebrafish larvae at 3 dpf showing the ubiquitous expression of cbs and cth in the brain (lateral view). (B and C) Effects of morpholino oligonucleotide-mediated cbs or cth knockdown on brain vascular development. (B) Representative midbrain vessel structures reconstructed from confocal images of cbs or cth morphants at 3–5 dpf of the same larva. (C) Summary of data. The experiments were repeated three times, and six embryos were examined for each group at each time. (D and E) Effects of cbs or cth knockout (F0) on brain vascular development. (D) Representative midbrain vessel structures reconstructed from confocal images of the same F0 mutant larva at 3–5 dpf. (E) Summary of data. Six embryos were examined for each group. (F and G) Effects of cbs or cth homomutants on brain vascular development. (F) Representative midbrain vessel structures reconstructed from confocal images of the same cbs or cth homomutant larva at 3 and 5 dpf. (G) Summary of data. Six embryos were examined for each group. Scale bar, 300 µm (A), 25 µm (B, D, F). Error bars, SEM. *P<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student’s t-test).硫化氢供体GYY4137可改善下调cbs和cth所导致的脑血管发育障碍。Figure 2. Rescue effect of GYY4137 on the defects of brain vascular development in cbs and cth morphants. (A) Representative projected confocal images showing that GYY4137 treatment ameliorated the impaired brain vascular development in cbs and cth morphants. Confocal images were taken at 3–5 dpf of the same larva. (B) Summary of the rescue effect of GYY4137. Six embryos were examined for each group. Scale bar, 50 µm (A). Error bars, SEM. *P<0.05, **p<0.01, ***p<0.001 (one-way analysis of variance). MO, morpholino oligonucleotide.
利用软件分析斑马鱼中脑血管网络特征，结果显示，硫化氢在脑血管发育过程中主要作用在促血管生成方面，而并非脑血管网络拓扑结构的形成。
Figure 3. Structure analysis of the midbrain vasculature in cbs and cth morphants. (A) Image of a 3-dpf larva showing the midbrain position delineated with dashed lines (upper left), and representative midbrain vasculature centerlines of 3-dpf larvae of Ctrl morpholino oligonucleotide (MO; upper right), cbs MO (lower left), and cth MO (lower right). C, caudal; L, lateral. (B–E) Summary of changes in the total vessel length (B), vessel segment number (C), weighted average segment Strahler order (D) and internal vessel loop number (E) of the midbrain vasculature in groups of Ctrl MO, cbs MO and cth MO. Six embryos were examined for each group. Error bars, SEM. ***P<0.001 (unpaired two-tailed Student’s t-test).
下调cbs和cth后，斑马鱼胚胎编码一氧化氮合酶（nitric oxide synthase, NOS）的基因nos2a mRNA表达和总NO产生明显减少，且通过共注射斑马鱼nos2a mRNA可以改善下调cbs和cth所导致的脑血管发育障碍。
Figure 4. The nitric oxide synthase (NOS)/nitric oxide (NO) pathway is involved in cbs and cth knockdown-induced defects of brain vascular development. (A and B) Effects of cbs and cth knockdown on the NOS/NO signalling pathway. Summary of nos1, nos2a and nos2b RNA expression (A) and total NO production (B) in 3-days post fertilisation (dpf) embryos. (C and D) Rescue effect of nos2a mRNA on the brain vascular developmental defects in cbs and cth morphants. Summary of increased total NO production (C) and midbrain vessel density (D) in 3-dpf embryos coinjected with cbs or cth MO and nos2a mRNA in comparison with those injected with cbs or cth MO. Six embryos were examined for each group. (E and F) No effect of cbs and cth knockdown on the protein expression of p-ERK1/2, ERK1/2 and VEGF. Representative blots (E) and summary (F) of Western blotting data. Error bars, SEM. *P<0.05, **p<0.01 (unpaired two-tailed Student’s t-test).以上研究结果表明，内源性硫化氢通过NOS/NO通路促进斑马鱼脑血管生成。
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