BACKGROUND: Different mutations of the angiopoietin-1 gene (ANGPT1) have been associated with the occurrence of hereditary angioedema (HAE). OBJECTIVE: The purpose of the study is to clarify whether the ANGPT1 A119S variant plays its role via haploinsufficiency or a dominant negative effect. METHODS: The ability of ANGPT1 A119S variant to affect the endothelial barrier function was assessed by immunocytochemistry. Inter-endothelial gap formation molecules primarily responsible for cell-cell adhesions of HUVECs, vascular endothelial (VE)-cadherin and β-catenin, and reorganization of the F-actin cytoskeletal were evaluated. RESULTS: In in vitro conditions mimicking the heterozygous state, the p.A119S variant significantly reduced the capability to bind its natural receptor (80.7% of normal), less than the homozygous condition (59.1%). After stimulation of VEGF or bradykinin, the addiction to equimolar amounts of wtANGPT1 and ANGPT1 p.A119S clearly reduced the expression of VE-cadherin on the endothelial cell surface (31% and 24% respectively). Likewise, cell surface expression of β-catenin was reduced and severe gap formation between adjacent HUVECs developed. In cultured cells, β-catenin expression was mostly observed along the cell surface. Treatment with equimolar amounts of wtANGPT1 and ANGPT1 p.A119S failed to restore the reorganization of the F-actin cytoskeletal elements. ANGPT1 p.A119S variant in homozygous condition further diminished VE-cadherin and β-catenin expression and failed to reduce stress fibre formation significantly affecting the endothelial barrier functionality. CONCLUSIONS AND CLINICAL RELEVANCE: Present data show that in a heterozygous state the p.A119S substitution results in a pathogenic loss of function of the protein due to a mechanism of haploinsufficiency. The ANGPT1 reduced ability to counteract the increment of endothelial permeability produced by inducers, such as VEGF and bradykinin, stimulate vascular leakage and reorganization of the F-actin cytoskeletal elements. As a result, a partial impairment of the ANGPT1 functionality, like when dominant mutations occur, represents a pathophysiological cause of HAE.
BACKGROUND: Different mutations of the angiopoietin-1 gene (ANGPT1) have been associated with the occurrence of hereditary angioedema (HAE). OBJECTIVE: The purpose of the study is to clarify whether the ANGPT1A119S variant plays its role via haploinsufficiency or a dominant negative effect. METHODS: The ability of ANGPT1A119S variant to affect the endothelial barrier function was assessed by immunocytochemistry. Inter-endothelial gap formation molecules primarily responsible for cell-cell adhesions of HUVECs, vascular endothelial (VE)-cadherin and β-catenin, and reorganization of the F-actin cytoskeletal were evaluated. RESULTS: In in vitro conditions mimicking the heterozygous state, the p.A119S variant significantly reduced the capability to bind its natural receptor (80.7% of normal), less than the homozygous condition (59.1%). After stimulation of VEGF or bradykinin, the addiction to equimolar amounts of wtANGPT1 and ANGPT1p.A119S clearly reduced the expression of VE-cadherin on the endothelial cell surface (31% and 24% respectively). Likewise, cell surface expression of β-catenin was reduced and severe gap formation between adjacent HUVECs developed. In cultured cells, β-catenin expression was mostly observed along the cell surface. Treatment with equimolar amounts of wtANGPT1 and ANGPT1p.A119S failed to restore the reorganization of the F-actin cytoskeletal elements. ANGPT1p.A119S variant in homozygous condition further diminished VE-cadherin and β-catenin expression and failed to reduce stress fibre formation significantly affecting the endothelial barrier functionality. CONCLUSIONS AND CLINICAL RELEVANCE: Present data show that in a heterozygous state the p.A119S substitution results in a pathogenic loss of function of the protein due to a mechanism of haploinsufficiency. The ANGPT1 reduced ability to counteract the increment of endothelial permeability produced by inducers, such as VEGF and bradykinin, stimulate vascular leakage and reorganization of the F-actin cytoskeletal elements. As a result, a partial impairment of the ANGPT1 functionality, like when dominant mutations occur, represents a pathophysiological cause of HAE.