P039 - ENDOTHELIAL CELL-SPECIFIC LOSS OF BREAST CANCER SUSCEPTIBILITY GENE 2 EXACERBATES ANGIOTENSIN II-INDUCED ENDOTHELIAL DYSFUNCTION

Background: Mutations in the breast cancer susceptibility gene 2 (BRCA2) increase the risk of breast cancer, with emerging evidence linking BRCA2 deficiency to cardiovascular complications, including hypertension. Evidence suggests that BRCA2 mutation promotes endothelial dysfunction, a key determinant of vascular health. Angiotensin II (Ang II) - a regulator of blood pressure, induces endothelial dysfunction through reactive oxygen species (ROS), inflammation, and impaired nitric oxide (NO) production, contributing to the development and progression of hypertension. Endothelial cell-specific loss of BRCA2 is also known to induce ROS, inflammation, apoptosis and reduce NO production; however, the specific role of BRCA2 in endothelial dysfunction in hypertension remains unknown. Accordingly, we hypothesize that loss of endothelial cell-specific BRCA2 exacerbates Ang II-induced endothelial dysfunction.

METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVECs) were transfected with scrambled control or siBRCA2 for 48 hours, followed by 24-hour treatment with AngII (2µM) or vehicle. Quantitative PCR (qPCR), immunoblotting (IB), immunofluorescence (IF), and functional assays were performed. IF assessed DNA damage (γH2AX), BRCA2-silencing, and cell proliferation. Transcript levels evaluated by qPCR, and protein levels were quantified by IB for markers of apoptosis (p53, cleaved caspase-3), inflammation (ICAM-1, VCAM-1) endothelial function (phospho-eNOS/eNOS), and BRCA2 expression. Functional assays included, tube formation, migration (scratch assay), proliferation (Brdu assay), and NO production (DAF-FM assay). Migration rate (µm/hr) was quantified by scratch closure over time. Data was analyzed using two-way ANOVA.

AngII treatment significantly reduced migration, tube formation, proliferation, and NO production compared to control, with the greatest reductions in AngII + siBRCA2 group (***p < 0.001). IB showed decreased eNOS expression in the siBRCA2 group compared to controls (*p < 0.05), and siBRCA2+ AngII group compared to Ang II treated controls (#p < 0.05). ICAM-1 and VCAM-1 were also upregulated in BRCA2-deficient AngII-treated cells, supporting increased inflammatory activation. IF showed increased γH2AX in nuclei upon AngII treatment, with the most in AngII + siBRCA2 group (***p < 0.001). Data on pro-apoptotic p53 showed elevated p53 expression in AngII-treated BRCA2-deficient endothelial cells, which coincided with increased apoptosis in this group compared to AngII-treated controls.

Conclusion: AngII treatment significantly impaired migration, tube formation, proliferation, and NO production in BRCA2-deficient endothelial cells. Loss of BRCA2 was also associated with increased AngII-induced DNA damage, inflammation and apoptosis. Our data support a protective role of BRCA2 in endothelial cells and suggest that targeting BRCA2-related pathways could improve endothelial function in individuals with BRCA2 mutations or at risk due to hypertension.