The Guanylate Cyclase Soluble Subunit Alpha-1 Deficiency Impairs Angiogenesis in Zebrafishes and Mice: In Vivo and In Vitro Studies.

Abstract

Moyamoya disease (MMD) is caused by abnormal vascular development. Guanylate cyclase soluble subunit alpha-1 (GUCY1A3) gene variation is verified as a crucial susceptible gene in MMD. In this study, we investigated the impact of GUCY1A3 on angiogenesis. GUCY1A3-knockout (KO) models were established using CRISPR/Cas9 technology in zebrafishes and mice. Blood vessel distribution in GUCY1A3-KO zebrafishes and retinal angiogenesis in postnatal GUCY1A3-KO mice were analyzed. Anti-angiogenic behaviors, including cell proliferation, migration, and apoptosis, and changes in hypoxia-inducible factor-1α (HIF-1α) distribution were examined in GUCY1A3-knockdown (KD) mice brain microvascular endothelial cells (BMECs). GUCY1A3-KO significantly decreased intracranial central artery development in zebrafishes, delayed retinal vascularization in mice, reduced retinal vascular endothelial growth factor A (VEGFA) expression in mice, and abolished expression of the GUCY1A3-encoded protein, α1 subunit of soluble guanylate cyclase. GUCY1A3-KD significantly decreased cell proliferation (flow cytometry analysis) and migration (wound-healing and Transwell assays), but increased apoptosis (hypoxia-induced apoptosis assay) in the BMECs. Immunofluorescence of HIF-1α revealed that nuclear translocation and protein expression were significantly reduced in the GUCY1A3-KD BMECs. These findings indicated that decreased expression of GUCY1A3 resulted in anti-angiogenic activity through inhibiting VEGFA and HIF-1α expression and nuclear translocation, inhibiting endothelial cell proliferation and migration, and promoting endothelial cell apoptosis.