Microvascular research · 2026
Identification of Fscn2 as a candidate gene regulating endothelial cytoskeletal organization and angiogenic competence through a genome-wide association study (GWAS) in mice.
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Marik Binata, Bielenberg Diane R, Birsner Amy E, Fu Haojie, D'Amato Robert J
Аннотация
PURPOSE: Polymorphisms in genes that regulate angiogenesis, notably vascular endothelial growth factor (VEGFA) and basic fibroblast growth factor (FGF2), play a major role in the susceptibility and progression of angiogenic diseases such as cancer and ocular neovascular disorders. Previous studies from our laboratory reported that genetic variation among different mouse strains alters their angiogenic responses to growth factors. In this study, we hypothesized that genetic diversity among inbred mouse strains could be compared using GWAS to identify candidate genes associated with differences in VEGFA-induced angiogenic phenotypes.
METHODS: VEGFA-induced corneal neovascularization was assessed across 37 inbred mouse strains, and efficient mixed-model association analysis was used to identify genomic loci linked to variation in blood vessel area. Candidate genes were prioritized by predicted functional consequence and endothelial expression. The top candidate, Fscn2, was silenced by siRNA in human umbilical vein endothelial cells (HUVEC) and human retinal microvascular endothelial cells (HRMEC). Knockdown efficiency was confirmed by immunoblotting and immunofluorescence. The effects of FSCN2 depletion were assessed using endothelial cell proliferation, migration, Matrigel-based tube formation, and F-actin staining. In vivo angiogenic function was evaluated using a Matrigel plug assay in immunodeficient Nu/J mice, with angiogenesis quantified by human CD31 and mouse hemoglobin measurements and confirmed by histology.
RESULTS: Mouse GWAS identified a significant locus on chromosome 11 containing Fscn2 as a candidate gene associated with variation in VEGFA-induced corneal angiogenesis. The Fscn2 encodes fascin-2, an actin-bundling protein involved in filopodial extension and cytoskeletal organization. In HUVEC, FSCN2 siRNA reduced protein expression by approximately 80% relative to controls. FSCN2 knockdown significantly impaired proliferation, migration, tube formation, and F-actin organization in vitro. Comparable reductions in proliferation and migration were observed in HRMEC, indicating a conserved endothelial function. In vivo, Matrigel plugs containing FSCN2-depleted HUVEC showed significantly reduced angiogenesis, as evidenced by decreased human CD31 and mouse hemoglobin levels and fewer lumen-containing vascular structures.
CONCLUSIONS: Our GWAS identifies Fscn2 as a novel candidate gene associated with strain-dependent variation in a VEGFA-induced angiogenic phenotype in mice. Functional experiments in human endothelial cells and in vivo validation show that FSCN2 supports endothelial proliferation, migration, tube formation, and cytoskeletal organization. These findings expand the biological role of FSCN2 beyond sensory tissues and suggest that fascin-dependent cytoskeletal regulation contributes to endothelial angiogenic competence. Direct effects of FSCN2 in VEGFA signaling remains to be established.
METHODS: VEGFA-induced corneal neovascularization was assessed across 37 inbred mouse strains, and efficient mixed-model association analysis was used to identify genomic loci linked to variation in blood vessel area. Candidate genes were prioritized by predicted functional consequence and endothelial expression. The top candidate, Fscn2, was silenced by siRNA in human umbilical vein endothelial cells (HUVEC) and human retinal microvascular endothelial cells (HRMEC). Knockdown efficiency was confirmed by immunoblotting and immunofluorescence. The effects of FSCN2 depletion were assessed using endothelial cell proliferation, migration, Matrigel-based tube formation, and F-actin staining. In vivo angiogenic function was evaluated using a Matrigel plug assay in immunodeficient Nu/J mice, with angiogenesis quantified by human CD31 and mouse hemoglobin measurements and confirmed by histology.
RESULTS: Mouse GWAS identified a significant locus on chromosome 11 containing Fscn2 as a candidate gene associated with variation in VEGFA-induced corneal angiogenesis. The Fscn2 encodes fascin-2, an actin-bundling protein involved in filopodial extension and cytoskeletal organization. In HUVEC, FSCN2 siRNA reduced protein expression by approximately 80% relative to controls. FSCN2 knockdown significantly impaired proliferation, migration, tube formation, and F-actin organization in vitro. Comparable reductions in proliferation and migration were observed in HRMEC, indicating a conserved endothelial function. In vivo, Matrigel plugs containing FSCN2-depleted HUVEC showed significantly reduced angiogenesis, as evidenced by decreased human CD31 and mouse hemoglobin levels and fewer lumen-containing vascular structures.
CONCLUSIONS: Our GWAS identifies Fscn2 as a novel candidate gene associated with strain-dependent variation in a VEGFA-induced angiogenic phenotype in mice. Functional experiments in human endothelial cells and in vivo validation show that FSCN2 supports endothelial proliferation, migration, tube formation, and cytoskeletal organization. These findings expand the biological role of FSCN2 beyond sensory tissues and suggest that fascin-dependent cytoskeletal regulation contributes to endothelial angiogenic competence. Direct effects of FSCN2 in VEGFA signaling remains to be established.