Epigenetic attenuation of mitochondrial superoxide dismutase 2 in pulmonary arterial hypertension: a basis for excessive cell proliferation and a new therapeutic target

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.

Figure 1. Decreased SOD2 expression in FHR and patients with WHO Category-1 PAH

A) Confocal microscopy shows decreased PASMC SOD2 expression and increased mitochondrial fragmentation in FHR PASMC. Note the loss of SOD2 (green) in FHR as evidenced by the reduced intensity and peak height. B) Confocal immunofluorescence images of 3 control patients (who died from non lung-related conditions, upper 3 rows) and 3 patients who died of PAH (lower rows). Note the medial hypertrophy and plexiform lesions in the PAH patients. SOD2 expression (red) is decreased in the media and adventitia of small PAs and in the plexiform lesions. α-smooth muscle actin (green) is increased in PAH. C) mean±SEM of red fluorescence intensity showing decreased SOD2 expression in the media of small PAs. * p<0.05.

Figure 2. Inhibition of SOD2 expression in normal Sprague-Dawley PASMC recapitulates the PAH phenotype

A) siRNAs reduce SOD2 mRNA and H₂O₂ production. B) siSOD2 hyperpolarizes ΔΨm (increased red TMRM intensity). C) SOD2 siRNA activates HIF-1α (note translocation to the nucleus-red in middle panel) and decreases Kv1.5 expression (decreased green, left panel). D) Immunoblotting confirmed that siSOD2 decreases Kv1.5 expression (left panel). In line with loss of Kv channels, siSOD2 increased intracellular K⁺ concentration (middle panel). siSOD2 also reduced caspase activity (right panel).

Figure 3. Methylation of SOD2 in FHR PASMC is reversible by 5-aza-2′-deoxycytidine

A) Schematic of the CG dinucleotide percentage and CpG islands within the SOD2 promoter and first 2 KB after the transcriptional start site. 7 amplicons were surveyed within the SOD2 gene. Their approximate locations are represented by solid, horizontal lines. The positions of individual CpG dinucleotides are shown as vertical tick marks in the panel below the amplicon map. * denotes the differentially methylated CpG dinucleotides in FHR within amplicon 7 compared to BN1 tissue. No methylated CpG pairs were identified in amplicons 3-6. B) Corresponding methylation percentage of the differentially methylated CpG in intron 2. Results are expressed as a frequency of cytosine methylation in PAs from consomic control BN1 rats (n=2), FHR (n=3) and FHR treated with 5-aza-2′-deoxycytidine (n=3). C) Representative sequencing traces of genomic DNA from cultured PASMCs. Only methylated cytidines are protected against bisulfite-mediated deamination of cytidine into uridine (which is recognized as thymidine when the PCR product is amplified). As indicated by the arrow, the cytidine in FHR PASMCs was methylated (and therefore remains a cytidine, upper left) and this is reversed by 5-Azacytidine. The site is not methylated in FHR aortic SMC or in SDR PASMCs. The bar graph shows the mean data indicating the reversibility and tissue specificity of this SOD2 methylation in intron 2 in cultured PASMCs. D) FHR PASMC have lower SOD2 mRNA levels versus consomic PASMC. 5-aza-2′-deoxycytidine (5-AZA) causes a dose-dependent increase in SOD2 expression.

Figure 4. 5-aza-2′-deoxycytidine restores SOD2 expression, reduces proliferation and enhances apoptosis in FHR PASMC

A-B) Representative and mean data showing that FHR PASMC express less SOD2 than SDR PASMC and this is partially reversible with 5-AZA treatment, n=4 per group. *p<0.05. C) 5-aza-2′-deoxycytidine-induced, dose-dependent increase in Kv1.5 mRNA in FHR PASMC, *p<0.05. D-E) 5-aza-2′-deoxycytidine (black bars) increases apoptosis and decreases proliferation in FHR PASMC and lung cancer cells (A549), but not SDR PASMC, n=4-6/group. *p<0.05 between baseline and 5-aza-2′-deoxycytidine, † significant difference from SDR PASMC. F) 5-aza-2′-deoxycytidine increases O₂-consumption in FHR and SDR PASMC, but not A549 cells (n=3-8/group).

Figure 5. Lower ROS levels and reduced cellular environment in FHR PASMC

A) Decreased ROS levels in freshly isolated resistance PAs from FHR versus consomic rats, measured using L-012 chemiluminescence at 37°C. B) PASMCs were transfected with redox-sensitive GFP constructs targeted to the cytoplasm (upper panel) and mitochondria (lower panel). FHR PASMC had a reduced cytoplasm and mitochondria, relative to Sprague Dawley PASMC. Dithiothreitol and tert-butyl hydroperoxide were used to completely reduce and oxidize the cells, respectively. C) ROS mean data and representative trace (inset) showing the CMH triplet signal. Peak height (EPR amplitude units) is proportional to ROS levels and is normalized to wet weight. PAs from FHR treated with 5-aza-2′-deoxycytidine (black bars) have increased production of ROS (left) and H₂O₂ (right) versus untreated FHR. Control rats show no induction of ROS or H₂O₂, n=5/group.

Figure 6. DNA methyltransferase expression is increased in FHR lung and PASMCs

A)DNA MT1 and 3B mRNA is increased in FHR versus SDR lungs (n=12 each). *, **p<0.05 and 0.01, respectively. In low passage (3-4) PASMCs (n=8 in each group), FHR had higher DNA MT3B expression and a trend toward increased DNA MT1.

Figure 7. SOD2 supplementation inactivates HIF-1α and increases Kv1.5 expression in FHR PASMC

A) The SOD2 virus and a control GFP virus are effective, increasing expression of SOD2 and GFP mRNA, respectively, in FHR PASMC. B) Immunofluorescence using an Alexa647 secondary antibody shows that the SOD2 adenovirus increases human SOD2 expression (green, left lower panel). This restores Kv1.5 expression (green) and inactivates HIF-1α (red nuclei). Nuclei are counterstained blue (DAPI). C-D) 48-hours incubation with MnTBAP decreases nuclear localization of HIF-1α and restores Kv1.5 expression.

Figure 8. MnTBAP regresses PAH in FHR

A) MnTBAP reduces mean PA pressure measured by Doppler (lengthens PAAT) and decreases right ventricular thickness in FHR treated for 4 weeks, n=5/group. * p<0.05. B) MnTBAP therapy reduces mean PAP and total pulmonary resistance (TPR). C) FHR treated with MnTBAP exercise longer on a graded treadmill, n=15/group. D) Lung sections were stained for von Willebrand factor (red), alpha smooth muscle cell actin (green) and DAPI (blue). Note the fully muscularized (white arrows), partially muscularized (yellow arrows) and non-muscularized blood vessels (red arrows). Lower panel: A representative fully muscularized PA in a vehicle-treated FHR (left) versus MnTBAP (right). The % medial thickness of precapillary resistance PAs was reduced and the number of non-muscularized resistance PAs was increased by MnTBAP. ** p<0.01 versus control.