Epigenetic mechanisms of pulmonary hypertension

Abstract

Epigenetics refers to changes in phenotype and gene expression that occur without alterations in DNA sequence. Epigenetic modifications of the genome can be acquired de novo and are potentially heritable. This review focuses on the emerging recognition of a role for epigenetics in the development of pulmonary arterial hypertension (PAH). Lessons learned from the epigenetics in cancer and neurodevelopmental diseases, such as Prader-Willi syndrome, can be applied to PAH. These syndromes suggest that there is substantial genetic and epigenetic cross-talk such that a single phenotype can result from a genetic cause, an epigenetic cause, or a combined abnormality. There are three major mechanisms of epigenetic regulation, including methylation of CpG islands, mediated by DNA methyltransferases, modification of histone proteins, and microRNAs. There is substantial interaction between these epigenetic mechanisms. Recently, it was discovered that there may be an epigenetic component to PAH. In PAH there is downregulation of superoxide dismutase 2 (SOD2) and normoxic activation of hypoxia inducible factor (HIF-1α). This decrease in SOD2 results from methylation of CpG islands in SOD2 by lung DNA methyltransferases. The partial silencing of SOD2 alters redox signaling, activates HIF-1α) and leads to excessive cell proliferation. The same hyperproliferative epigenetic abnormality occurs in cancer. These epigenetic abnormalities can be therapeutically reversed. Epigenetic mechanisms may mediate gene-environment interactions in PAH and explain the great variability in susceptibility to stimuli such as anorexigens, virus, and shunts. Epigenetics may be relevant to the female predisposition to PAH and the incomplete penetrance of BMPR2 mutations in familial PAH.

Keywords: CpG islands; DNA methyl transferases; histone acetylation; small inhibitor RNA; superoxide dismutase 2.

Figure 1

Trends in biomedical epigenetic research publications. PubMed citations containing the words epigenetic or epigenetics in the title or abstract were counted (www.ncbi.nlm.nih.gov/sites/entrez?db=PubMed). (a) Research articles, excluding review articles and editorials, were counted for each year up to 2010. (b) All publications covering epigenetic(s) are categorized to editorials, reviews and research articles. (c) Distribution of research articles published in 2010 for cancer, stem cells and their differentiation, neuronal diseases and cardiopulmonary diseases including asthma.

Figure 2

Schematic of the mechanisms of epigenetic regulation. DNA methylation, histone modifications, and RNA-mediated gene silencing constitute three distinct mechanisms of epigenetic regulation. DNA methylation is a covalent modification of the cytosine (C) that is located 5’ to a guanine (G) in a CpG dinucleotide. Histone (chromatin) modifications refer to covalent post-translational modifications of N-terminal tails of four core histones (H3, H4, H2A, and H2B). The most recent mechanism of epigenetic inheritance involves RNAs. Reproduced with permission from Z. Herzeg.[16]

Figure 3

Methylation of SOD2 in FHR PASMC is reversible by 5-AZA. (a) Schematic of the CG dinucleotide percentage and CpG islands within the SOD2 promoter and first 2 kb after the transcriptional start site. Seven 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 below the amplicon map. No methylated CpG pairs were identified in amplicons 3 to 6. *Differentially methylated CpG dinucleotides in FHRs within amplicon 7 vs BN1 tissue. (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), FHRs (n=3), and FHRs treated with 5-AZA (FHR-Tx; 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 polymerase chain reaction product is amplified). As indicated by the arrow, the cytidine in FHR PASMCs was methylated (and therefore remains a cytidine; top left); this is reversed by 5-AZA. The site is not methylated in FHR aortic SMCs 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 PASMCs have lower SOD2 mRNA levels vs consomic PASMCs. 5-AZA causes a dose-dependent increase in SOD2 expression. Reproduced with permission from Archer et al.[1]

Figure 4

DNA methyltransferase expression is increased in FHR lung and PASMCs. (a) DNA MT1 and 3B mRNA are increased in FHR vs SDR lungs (n=12 each). *P<0.05, **P<0.01. (b) In low-passage (3 to 4) PASMCs (n=8 in each group), FHRs had higher DNA MT3B expression and a trend toward increased DNA MT1. Reproduced with permission from Archer et al.[1]Black bars = FHR; White bars = SDR

Figure 5

Administration of the SOD analog, MnTBAP, regresses PAH in FHRs. (a) MnTBAP reduces mean PA pressure measured by Doppler (lengthens PA acceleration time [PAAT]) and decreases right ventricular (RV) thickness in FHRs treated for 4 weeks (n=5 per group). *P<0.05. (b) MnTBAP therapy reduces mean pulmonary artery pressure (PAP) and total pulmonary resistance (TPR). (c) FHRs treated with MnTBAP exercise longer on a graded treadmill (n=15 per group). (d) Lung sections were stained for von Willebrand factor (vWF; red), a-smooth muscle cell (SMC) actin (green), and DAPI (blue). Note the fully muscularized (white arrows), partially muscularized (yellow arrows), and nonmuscularized blood vessels (red arrows). Bottom, A representative fully muscularized PA in a vehicle-treated FHR (left) vs MnTBAP (right). The percent medial thickness of precapillary resistance PAs was reduced and the number of nonmuscularized resistance PAs was increased by MnTBAP. **P<0.01 vs control. Reproduced with permission from Archer et al.[1]

Figure 6

Schematic of pathophysiologic mechanisms leading to the development of PAH.