Abstract 600: Activation of p53 in Pulmonary Arterial Endothelial Cells with Dysfunctional BMPR2 Rescues DNA Damage, Apoptosis and Persistent Pulmonary Hypertension

Abstract

Pulmonary arterial (PA) hypertension (PAH) is a life-threatening disease characterized by loss of microvessels, the result of endothelial cell (EC) apoptosis associated with dysfunction or a mutation in BMPR2. We identified p53 as critical in the formation of a DNA damage-inducible transcriptionally active complex with PPARγ, an important downstream factor of BMPR2 signaling. The p53-PPARγ complex regulates genes promoting PAEC homeostasis. Upon reoxygenation after hypoxia, PAEC with defective BMPR2 fail to stabilize p53 leading to mitochondrial dysfunction, DNA damage, endothelial apoptosis and persistent pulmonary hypertension (PH) in mice. We therefore hypothesized that in PAEC, pharmacological activation of p53 by the small molecule compound Nutlin-3 would stabilize the PPARγ-p53 complex downstream of BMPR2 to reverse the PH phenotype. Indeed, we showed that in PAEC transfected with BMPR2 siRNA, disruption of the PPARγ-p53 complex upon reoxygenation was prevented by Nutlin-3 treatment. This was associated with a reduction in DNA damage as assessed by Histone 2AX phosphorylation. In PAEC isolated from PAH patients with a BMPR2 mutation, Nutlin-3 treatment induced repair of prevalent DNA damage as determined by the Comet assay, and improved PAEC survival as measured by Caspase 3/7 activity. In PAEC with a BMPR2 mutation, Nutlin treatment stabilized p53, and induced PPARγ-p53 complex formation and expression of target genes, i.e., apelin (a pro-angiogenic factor), GADD45A (a DNA repair enzyme) and p21 (a cell cycle inhibitor) as detected by quantitative PCR and western immunoblots. In EC-specific BMPR2 knockout mice, we have observed persistent chronic hypoxia induced PH even after a month of reoxygenation in room air. We now show that this can be reversed by daily i.p. treatment with Nutlin-3a (12 mg/kg) during reoxygenation. In conclusion, pharmacological stabilization of p53 by Nutlin-3 in PAEC with dysfunctional BMPR2 induces repair of DNA damage, improves PAEC survival and rescues persistent pulmonary hypertension. These results auger well for pursuing the use of Nutlin-3 in pulmonary hypertension and other vascular diseases associated with DNA damage such as atherosclerosis.