One of the major findings of this study is the identification of SOD2 methylation as a potential new, epigenetic mechanism for PAH in an animal model of spontaneous and heritable PAH. This may be relevant for clinical PAH, since SOD2 is also downregulated in human PAH (). We identified hypermethylation of a CpG island in an enhancer region within intron 2 and the promoter of SOD2 as the basis for SOD2 downregulation in FHR. This appears to reflect significantly higher expression of DNA methyltransferase1 and 3B in lungs and of methyltranferase 3B in isolated PASMCs of FHR (). This epigenetic down-regulation of SOD2 impairs H2
-mediated redox signaling, activates HIF-1α and creates a proliferative, apoptosis-resistant state (Figures ,,). Both the mechanism of SOD2 downregulation and its consequences parallel those recently discovered in human cancer16, 17
, but to our knowledge, this is the first report of an epigenetic cause of a pulmonary vascular disease. The epigenetic down-regulation of SOD2 was reversible by treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine. Furthermore, our study demonstrates that augmentation of SOD2 (by 3 complementary strategies) restores mitochondrial function, inhibits PASMC proliferation and increases cell apoptosis in vitro
. In vivo
, MnTBAP causes partial regression of established PAH and decreases the muscularization of pulmonary precapillary resistance vessels ().
The FHR’s SOD2 deficiency is the consequence of covalent cytosine methylation that occurs in dinucleotide CpGs (). Methylation at cytosine’s C5 atom inhibits gene expression by preventing the binding of transcription factors18
. Methylation is reversible, heritable and can be tissue-specific19
. Indeed, SOD2 methylation occurred in the FHR PA but not in the FHR aorta (), thus pointing to tissue specific epigenetic mechanisms. This finding may contribute to understanding the localization of the pathology in human and FHR PAH to the pulmonary circulation. The SOD2 promoter and several of its introns have CpG islands that offer potential methylation sites. Using genomic bisulfite sequencing we directly confirmed that FHR have 2 discrete sites of differential hypermethylation (one in intron 2, the other in the promoter) ( and Supplemental Figure 3
). Although many regions in the SOD2 gene are heavily methylated, these two were the only CpG islands that were both differentially methylated and demethylated by 5-aza-2′-deoxycytidine. The intron 2 site is in the same region of the SOD2 gene that has been identified in transformed human lung fibroblast cell lines20
. 5-aza-2′-deoxycytidine covalently binds and irreversibly inhibits DNA methyltransferases21
, resulting in reactivation of transcription of previously methylated genes after a requisite cell division. It is unclear why 5-aza-2′-deoxycytidine had no effect on other areas of methylation, but other studies have described similar observations17
CpG methylation is established and maintained by 3 DNA methyltransferases22
. DNA methyltransferase 1 is expressed in proliferating tissues and its activity is coupled to DNA replication, copying methylation patterns from the parental to the daughter strand. Therefore, it is considered primarily a maintenance methyltransferase. In contrast, DNA methyltransferases 3A and 3B are considered “de novo
” methyltransferases that can establish new methylation patterns. We identified an upregulation of DNA MT3B in FHR PASMC (), which would be an elegant explanation for the observed hypermethylation of the SOD2 gene. Interestingly, DNA MT3B depletion is sufficient to reactivate methylation-silenced genes and decrease proliferation in both human breast adenocarcinoma and A549 cells23
It is interesting that changes in the activity or expression of DNA methyltransferases can have a relatively specific outcome in terms of site-specific methylation and regulation of specific genes in certain tissues. Current models suggest that the specificity of DNA methyltransferase activity can depend on their expression levels or their interaction with other epigenetic regulators. In the current study, for example, we found that FHR animals with PAH had higher expression levels of DNA MT3B. This DNA methyltransferase has been shown to have distinct CpG methylation activity patterns, which depend on the tissue environment and on the expression of differentially spliced isoforms24
. Another level of DNA methylation specificity is achieved by the interaction of DNA methyltransferases with other epigenetic regulators such as histone deacetylases. For example, DNA MT3B contains an ATRX homology domain that interacts with histone deacetylase 125
. Interestingly, such an association between increased CpG methylation and decreased histone acetylation has been observed for the SOD2 gene16
Gene methylation has an established role in promoting pathological cell proliferation in cancer. SOD2, a candidate tumor-suppressor gene8, 9
, is silenced in several malignancies16, 17, 26
. In multiple myeloma and pancreatic carcinoma the epigenetic silencing of SOD2 is caused by hypermethylation of CpG islands within SOD2’s promoter17, 26, 27
. Demethylation of SOD2 in cancer restores SOD2, increases H2
and decreases cell proliferation and tumor growth16, 17, 26
, consistent with our observations ( and ). The effects of directly overexpressing SOD2 (by 3 complementary means) are concordant with the effects of SOD2 demethylation in cancer28
(). Our study demonstrates that reversing gene methylation is beneficial in FHR PASMC in vitro
. Treatment with 5-aza-2′-deoxycytidine decreases SOD2 methylation and causes a dose-dependent increase in SOD2 and Kv1.5 expression in FHR PASMC ().
Production of H2
is a critical link between SOD2 expression and regulation of proliferation (Supplemental Figure 5
provides a schematic representation of the cascade of the proposed transcriptional, metabolic and redox consequences of SOD2 downregulation). Simply knocking down SOD2 expression in a normal PASMC diminishes endogenous H2
production and leads to an associated activation of HIF-1α (). Moreover, an anti-SOD2 siRNA caused many of the other abnormalities seen in human and FHR PAH, including a decrease in expression of Kv1.5 channels and a rise in cytosolic calcium (Supplemental Figure 1B
). Conversely, augmenting SOD2 elevates production of H2
in FHR PAs () and reduces cell proliferation (). Similarly, in prostate cancer, over-expressing SOD2 increases H2
and reduces cell proliferation29
. Thus, in both PAH and cancer there is an inverse correlation between SOD2 activity and H2
-mediated cell proliferation29
. Catalase, which reduces H2
levels, prevents SOD2’s ability to inhibit cancer cell proliferation29
. These observations suggest that the effect of SOD2 augmentation in PAH and cancer is mediated by increasing H2
production (from a deficient starting level). We support this contention by showing that 5-aza-2′-deoxycytidine only increases H2
production in FHR PASMC, having little effect on normal PASMC ().
Our study also identifies the downstream mechanism by which this mitochondrial abnormality promotes cell proliferation-namely, normoxic activation of HIF-1α. HIF-1α activation has previously been identified in human PAH2, 6
and FHR PAH2
. The current study supports a key role for H2
as the link between SOD2 and cell proliferation, as schematized in Supplemental Figure 5
. Although ROS are toxic at high levels, there is a physiologic level of H2
production by mitochondria during normal oxidative metabolism12, 30
. Physiologic levels of H2
serve as redox signaling molecules, involved in oxygen-sensing12
. The evidence linking SOD2 levels to ROS in PAH is clear. Simply lowering SOD2 message levels (using siRNA) decreases mitochondrial H2
production (); conversely, demethylating the SOD2 gene enhancer in FHR PAs restores SOD2 expression, leading to increased ROS and mitochondrial H2
production (). Compartment-specific, redox-sensitive, green fluorescent proteins demonstrated that the net change in redox state in FHR (vs Sprague-Dawley) PASMC is reduction (both in the cytosol and mitochondria), consistent with the observed decreased ROS levels in FHR ().
Evidence for the relevance of SOD2 deficiency in PAH comes from the concordant benefit of 3 different strategies that restore SOD2 expression or SOD activity in our study. In FHR PASMC, SOD2 gene therapy, application of the SOD2 mimetic MnTBAP, or 5-aza-2′-deoxycytidine each led to HIF-1α inactivation and restoration of Kv1.5 expression (). Although these surrogate endpoints are important, the key therapeutic finding of our study is that MnTBAP treatment regresses PAH in vivo (). This hemodynamic benefit is associated with a reduction in right ventricular hypertrophy, improved functional capacity and lung histology. The concordant findings in response to complementary strategies to modulate SOD2 expression reduces the possibility of artifacts related to flaws in any single strategy such as the stress of intraperitoneal MnTBAP injections, the inflammation from the SOD2 adenovirus, and potential confounding effects of demethylation of non-target genes by 5-aza-2′- deoxycytidine.
The benefits of MnTBAP are consistent with those of tempol (another membrane permeable SOD mimetic) which decreases hypoxic pulmonary hypertension in rats31
and recombinant SOD1, which ameliorates persistent pulmonary hypertension in newborn lambs32
. Bowers et al
. have also noted decreased SOD2 in the PAs in human PAH4
; however, their interpretation was that PAH is a condition of increased oxidative stress (in part because other oxidant markers were elevated). Our data indicate that in FHR, H2
is subphysiologic due to both a primary effect, methylation-induced reduction of the SOD2 gene expression, and a secondary effect, pyruvate dehydrogenase kinase-mediated inhibition of oxidative metabolism2
. Identification of the therapeutic potential for 5-aza-2′-deoxycytidine is particularly relevant because 5-aza-2′-deoxycytidine (Decitabine) is approved for human use in myeloproliferative disorders (where it demethylates p15).
There are limitations in using human tissue, notably we are unsure of their smoking status. We acknowledge smoking could change SOD2 levels33
, however this is unlikely to be an important confounder as the rodent data are consistent with the human data. We acknowledge that the net effect of loss of SOD2 is controversial (oxidation in SOD2 knockout mice34
versus reduction in our paper). Interestingly the SOD2 haploinsufficient mouse has a doubling of cancer risk34
, supporting our central finding that downregulation of this mitochondrial enzyme is a proliferative, anti-apoptotic signal. We suspect the differences in ROS relate to the duration and severity of SOD2 loss (life long and more profound) for the knockout mouse versus acquired and more modest in FHR.
Finally, pyruvate dehydrogenase kinase activation in PAH thwarts mitochondrial respiration, limiting input to the mitochondrial electron transport chain. While in FHR low ROS and a reduced state activate HIF-1α, others find that ROS stabilizes HIF-1α35-37
Interestingly, Oberley’s group noted in cancer that siSOD2 activates HIF-1α, consistent with our findings. However, they found that siSOD2 increased superoxide levels without changing H2
. This again may relate to the magnitude of the SOD2 decrease achieved and cell specific differences in metabolic activity, prolyl hydroxylase activity and the status of the many other antioxidant systems. Our findings are supported by earlier studies showing that short pre-exposure of cells to H2
selectively prevents hypoxia-induced accumulation of HIF-1α protein10
In summary, the recognition of a novel epigenetic mechanism of PAH (methylation-induced attenuation of SOD2 expression) may partially explain the excessive cell proliferation and decreased apoptosis in PAH and could offer new therapeutic targets.
Pulmonary arterial hypertension (PAH) is characterized by remodeling of small precapillary resistance arteries, leading to increased vascular resistance and right ventricular failure. PAH is increasingly seen as a disease in which vascular obstruction is caused not only by vasoconstriction and inflammation, but also by enhanced proliferation and impaired apoptosis of vascular cells. The discovery that most patients with familial PAH have mutations in the bone morphogenetic protein receptor type II gene highlighted a potential genetic basis for PAH. However, most sporadic PAH patients do not have these mutations. Here we report that both fawn hooded rats (a strain with spontaneous PAH) and humans with PAH have a fragmented mitochondrial network and decreased expression of mitochondrial superoxide dismutase 2 (SOD2) in their pulmonary artery smooth muscle cells (PASMC). SOD2 is an important generator of hydrogen peroxide, which at physiological levels is a signaling molecule. In cancer, SOD2 is considered a potential oncogene and its expression is depressed. Suppression of SOD2 in normal PASMC recapitulates the proliferative, PAH phenotype. Interestingly, SOD2 downregulation is not due to gene mutation, rather the SOD2 gene is epigenetically silenced by specific (and reversible) methylation of specific CpG islands in the promoter and intron 2. This epigenetic inhibition of gene transcription is associated with increased DNA methyltransferase expression. SOD supplementation or demethylation strategies reverse the hyperproliferative phenotype of the FHR PASMC and regress experimental PAH in vivo. This is the first demonstration of an epigenetic basis for a heritable vascular disease and has etiologic and therapeutic implications.