鳥糞嘌呤核苷酸交換蛋白BIG 1和BIG2 (Brefeldin A-inhibited guanine nucleotide-exchange protein)是腺嘌呤核苷二磷酸核糖化因子 (ARF) 的活化蛋白，而ARF是高基氏體 (Golgi) 與細胞膜之間囊泡形成以及囊泡運輸的關鍵調節因子。BIG1和BIG2會藉由其Sec7 domain將ARF上結合的GDP置換為GTP來活化ARF。血管新生是生長發育過程中正常而重要的過程;然而，它也是腫瘤從良性狀態轉變成惡性狀的基本步驟。血管新生中的化學刺激是由各種血管生成因子所進行，其中的血管內皮生長因子（vascular endothelial growth factor , VEGF）已被證明是血管生成中的主要生長因子。有文獻報導，VEGF-165在內質網與高基氏體之間的早期運輸需要ARF1進行調控，且使用布雷非德菌素A (brefeldin A)干擾ARF1之活化會抑制VEGF-165分泌。然而，是哪些鳥糞嘌呤核苷酸交換因子（guanine nucleotide exchange factors, GEFs）主要活化ARF1進而調節VEGF的分泌與血管發育目前尚未釐清。我們的研究目的是為了瞭解哪些GEFs是主要調控ARF1活性以介導VEGF分泌，進而影響血管發育。我們透過GST pull-down assay證實BIG1/2會影響ARF1的活性，也發現在下調BIG1/2表現的膠質母細胞瘤細胞(U251 cells)和人類臍靜脈內皮細胞(Human Umbilical Vein Endothelial Cells, HUVEC)中VEGF mRNA和蛋白質以及細胞外VEGF的表達顯著降低。為了確認BIGs的功能，我們利用管形成檢驗(tube formation assay)作為體外試驗模型並選擇斑馬魚作為體內試驗模型。下調BIGs 表現之HUVECs的管形成程度降低。利用morpholino下調arfgef2表現時，斑馬魚中血管母細胞(angioblast) 的遷移和體節間血管（intersegmental vessels, ISVs）的出芽生長受到影響。 同時，使用CRISPR / Cas9下調arfgef2基因的斑馬魚胚胎在外觀和血管發育方面皆有發現缺陷。綜合以上，我們的實驗結果顯示BIG1和BIG2可能參與在VEGF分泌中並調節血管生成。

Brefeldin A-inhibited guanine nucleotide-exchange protein BIG1 and BIG2 are well known as activator of ADP-ribosylation factor (ARF), the crucial regulator of vesicle formation and traffic between Golgi and plasma membrane. BIG1 and BIG2 replace ARF-bound GDP with GTP by their Sec7 domain to activate ARF. Angiogenesis is a normal and vital process in growth and development; however, it is also a fundamental step in the transition of tumors from benign state to malignant one. The chemical stimulation of angiogenesis is performed by various angiogenic growth factors, especially vascular endothelial growth factor (VEGF). Recent research showed that early trafficking of VEGF-165 between the endoplasmic reticulum and the Golgi requires ARF1 and treatment cells with brefeldin A to interfere with ARF1 activation inhibited VEGF-165 secretion. However, it remains unknown which guanine nucleotide exchange factors (GEFs) participate in regulation of ARF1 activation to mediate VEGF secretion and then affect vascular development. In the present study, we first confirmed that knockdown of BIG1 and/or BIG2 by shRNA led to a reduction of ARF1 activation by GST pull-down assay. The amounts of VEGF secretion to extracellular medium, and the expression of VEGF mRNA and protein were significantly decreased in glioblastoma U251 cells and HUVECs that depletion of BIG1 and/or BIG2. To explore the possible role(s) of BIGs in angiogenesis, we used HUVEC tube formation assay as in vitro model and chose zebrafish as in vivo model. Capillary tubule formation in HUVECs were markedly inhibited when depletion of BIGs proteins. Angioblast migration and intersegmental vessels (ISVs) sprouting in zebrafish were impaired when arfgef2 was knockdowned by morpholino. Depletion arfgef2 by using CRISPR/Cas9, also caused zebrafish embryos have defects on phenotype and vascular development. Taken together, these data reveal that BIG1 and BIG2 may through regulation of VEGF expression and secretion to modulate angiogenesis.

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