Excessive proliferation and impaired apoptosis of pulmonary artery smooth muscle cells (PASMC) contributes to vascular obstruction in patients and fawn-hooded rats (FHR) with pulmonary arterial hypertension (PAH). Expression and activity of mitochondrial superoxide dismutase-2 (SOD2), the major generator of H2O2, is known to be reduced in PAH; however, the mechanism and therapeutic relevance of this is unknown.
Methods and Results
SOD2 expression in PASMC is decreased in PAH patients and FHR with PAH. FHR PASMC have higher proliferation and lower apoptosis rates than Sprague-Dawley PASMC. Moreover, FHR PASMC have hyperpolarized mitochondria, low H2O2 production and a reduced cytoplasmic and mitochondrial redox state. Administration of SOD2 siRNA to normal PASMC recapitulates the FHR-PAH phenotype, hyperpolarizing mitochondria, decreasing H2O2 and inhibiting caspase activity. Conversely, SOD2 over-expression in FHR PASMC, or therapy with the SOD-mimetic MnTBAP, reverses the hyperproliferative PAH phenotype. Importantly, SOD-mimetic therapy regresses PAH in vivo. Investigation of the SOD2 gene revealed no mutation, suggesting a possible epigenetic dysregulation. Genomic bisulfite sequencing demonstrates selective hypermethylation of a CpG island in an enhancer region of intron 2 and another in the promoter. Differential methylation occurs selectively in PA versus aortic SMC and is reversed by the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine, restoring both SOD2 expression and the proliferation/apoptosis ratio. The expression of the enzymes that mediate gene methylation, DNA methyltransferases 1 and 3B, is upregulated in FHR lungs.
Tissue-specific, epigenetic SOD2 deficiency initiates and sustains a heritable form of PAH by impairing redox signaling and creating a proliferative, apoptosis-resistant PASMC. SOD augmentation regresses experimental PAH. The discovery of an epigenetic component to PAH may offer new therapeutic targets.