129. Vector and Cell Mediated BMPR2 Therapy Ameliorates PAH

Abstract

Inherited pulmonary arterial hypertension is a fatal disease caused by mutations in, and reduced expression of, the bone morphogenetic type 2 receptor (BMPR2), leading to abnormal proliferation of pulmonary vascular cells and obliteration of small pulmonary vessels. BMPR2 expression is also reduced in secondary forms of PAH. We previously showed that upregulation of BMPR2 expression in the pulmonary vascular endothelium achieved by an endothelialtargeted adenoviral vector ameliorated PAH in rat models. In the current work, we evaluated a similar vector approach in a transgenic, BMPR2-mutation related PAH model, further characterised signaling mechanisms involved in the rat model, and extended the approach to the use of ex vivo transduced endothelial progenitor cells (EPCs) as a cell therapy approach to BMPR2 upregulation.Methods: SM22-tet / R899x mice were fed doxycycline-containing chow to induce expression of BMPR2 dominant negative protein. Mice were then injected via the tail vein with a pulmonary vascular targeted AdBMPR2-conjugate complex (or control vector), and right ventricular pressure measured (RVSP) by transthoracic puncture. For the EPC approach, rat bone marrow-derived EPCs were cultured in endothelial selective media then transduced ex-vivo with AdBMPR2 for 48 hours. The transduced cells (or untransduced controls) were then injected via tail vein into rats that had induced PAH via subcutaneous monocrotaline injection (60mg/kg) 10 days previously.Results: In the murine experiment, BMPR2 delivery led to a 24% reduction of RVSP compared to disease and vector control animals. Analysis of whole lung lysate using RT-PCR revealed an increase in eNOS expression in the AdBMPR2 treated group. This effect was confirmed in vitro by transduction of cultured endothelial cells, and further confirmed by demonstration of increased NO production. The Ad vector approach in the rat MCT model ameliorated PAH as previously described, an effect associated with an increase in Smad1/5/8 phosphorylation and decreased Smad3 phosphorylation. In the EPC approach, PAH was also ameliorated following EPC+AdBMPR2 treatment, demonstrated by significant reduction compared to MCT only and EPC only for Fulton Index (26% and 16% respectively), RVSP (34% and 36%) and mPAP (25% and 16%), while analysis of downstream signaling is ongoing.Conclusion: These findings indicate that gene delivery strategies via direct vector approaches or cell therapy to upregulate in BMPR2 in the pulmonary vasculature have therapeutic potential in PAH.