Epigenetic Attenuation of Mitochondrial Superoxide Dismutase 2 in Pulmonary Arterial Hypertension

Abstract

Background— Excessive proliferation and impaired apoptosis of pulmonary artery (PA) smooth muscle cells (PASMCs) contribute to vascular obstruction in patients and fawn-hooded rats (FHRs) with PA hypertension (PAH). Expression and activity of mitochondrial superoxide dismutase-2 (SOD2), the major generator of H 2 O 2 , is known to be reduced in PAH; however, the mechanism and therapeutic relevance of this are unknown. Methods and Results— SOD2 expression in PASMCs is decreased in PAH patients and FHRs with PAH. FHR PASMCs have higher proliferation and lower apoptosis rates than Sprague-Dawley rat PASMCs. Moreover, FHR PASMCs have hyperpolarized mitochondria, low H 2 O 2 production, and reduced cytoplasmic and mitochondrial redox state. Administration of SOD2 small interfering RNA to normal PASMCs recapitulates the FHR PAH phenotype, hyperpolarizing mitochondria, decreasing H 2 O 2 , and inhibiting caspase activity. Conversely, SOD2 overexpression in FHR PASMCs or therapy with the SOD-mimetic metalloporphyrin Mn(III)tetrakis (4-benzoic acid) porphyrin (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 PAs versus aortic SMCs and is reversed by the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine, restoring both SOD2 expression and the ratio of proliferation to apoptosis. Expression of the enzymes that mediate gene methylation, DNA methyltransferases 1 and 3B, is upregulated in FHR lungs. Conclusions— 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.