Abstract 15271: In vivo RNA Profiling of Pulmonary Endothelial Cells Links BRCA1 to the Pathogenesis of Pulmonary Arterial Hypertension
Rationale: Endothelial cell (EC) dysfunction plays a key role in the pathogenesis of pulmonary arterial hypertension (PAH). To avoid EC culture or whole lung samples, we have, for the first time, used antibody (ab) coated magnetic beads and in conjunction with genome scale RNA expression microarrays to profile ECs at any stage of PAH. Our goal was to identify novel mediators of PAH and genes linked to bone morphogenetic protein receptor 2 (BMPR2) signaling. We hypothesized that targeting early stages of the disease would identify novel mediators of PAH and genes linked to bone morphogenetic protein receptor 2 (BMPR2) signaling.
Methods: CD31 ab-coated magnetic beads were used to pull-down ECs from monocrotaline-treated rats (60mg/kg). RNA profiling, performed directly from bead-bound ECs, revealed DNA-repair associated tumor suppressor, breast cancer 1 (BRCA1) as a significant gene.
Results were confirmed by qPCR. Human pulmonary microvascular ECs (PMVECs) and siRNA strategies were used to study the role of BRCA1 in PMVEC and its role in BMPR2 dysfunction. Results: In-vivo microarray profiling of ECs at the early stages of PAH revealed significant upregulation of BRCA1 during the critical phase of disease induction. qPCR showed 4-fold increase at days 5 and 10 (P<0.05, N=3) post-monocrotaline initiation whereas expression levels are normalized by day 15. We hypothesized that induced and sustained BRCA1 levels are important for EC homeostasis. Compared to control BRCA1-deficient PMVECs show 3,5-fold increase in apoptosis (P<0.001, N=3) under stress. Furthermore, BRCA1-deficient ECs lose their proliferation capacity under serum stimulation. Interestingly, BMPR2 deficient PMVECs showed significant decrease in BRCA1 expression (control 1.760 ± 0.06658 vs. BMPR2-deficient 1.14 ± 0.078, N=3. P<0.01). BRCA1 deficient PMVECs showed substantial increase in BMPR2 levels (control 0,77 ± 0.0033 vs. BRCA1-deficient 1.10 ± 0.029, N=3, P<0.01), suggesting a regulatory feedback loop between these genes.
Conclusions: BMPR2 dysfunction leads to down-regulation of BRCA1, which has severe consequences for EC fate due to decreased survival and regeneration capacity, and could sensitize ECs to DNA-damage and somatic mutations during the course of PAH.
- © 2012 by American Heart Association, Inc.