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Mediterranean G6PD variant mitigates expression of DNA methyltransferases and right heart pressure in experimental model of pulmonary hypertension

Open AccessPublished:November 10, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102691

      Abstract

      DNA methylation potentially contributes to the pathogenesis of pulmonary hypertension (PH). However, the role of DNA methyltransferases (DNMTs: 1, 3a, and 3b), the epigenetic writers, in modulating DNA methylation observed in PH remains elusive. Our objective was to determine DNMT activity and expression in the lungs of an experimental rat models of PH. Because the activity of DNMTs is metabolically driven, another objective was to determine the role of glucose-6-phosphate dehydrogenase (G6PD) in regulating DNMT expression and activity in the lungs of novel loss-of-function Mediterranean G6PD variant (G6PDS188F) rats. As outlined for modeling PH, rats injected with sugen5416 (SU) were placed in a hypoxia (Hx) chamber set at 10% oxygen for 3 weeks and then returned to normoxia (Nx) for 5 weeks (SU/Hx/Nx). Rats kept in atmospheric oxygen and treated with SU were used as controls. We assessed the activity and expression of DNMTs in the lungs of rats exposed to SU/Hx/Nx. Wild-type rats exposed to SU/Hx/Nx developed hypertension and exhibited increased DNMT activity and Dnmt1 and Dnmt3b expression. In G6PDS188F rats, which developed less of a SU/Hx/Nx-induced increase in right ventricle pressure and hypertrophy than wild-type rats, we observed a diminished increase in expression and activity of DNMTs, DNA hypomethylation, increased histone acetylation and methylation, and increased expression of genes encoding NOS3 and SOD2 – vascular-protective proteins. Collectively, increased DNMTs contribute to reduced expression of protective genes and to the pathogenesis of SU/Hx/Nx-induced experimental PH. Notably, G6PD regulates the expression of DNMTs and protective proteins in the lungs of hypertensive rats.

      Key words

      Introduction

      DNA methylation is regulated by DNA methyltransferase (DNMT; 1, 3a, and 3b) and DNA demethylase or ten-eleven translocation (TET; 1, 2, and 3) methylcytosine dioxygenases (
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      • Cartron P.F.
      • Serandour A.A.
      • Hervouet E.
      From 1957 to Nowadays: A Brief History of Epigenetics.
      ). Recently, we and others reported that differential DNA methylation and a reduction in TET2 expression may be key to the pathogenesis of pulmonary hypertension (PH) in mice and humans (
      • Joshi S.R.
      • Kitagawa A.
      • Jacob C.
      • Hashimoto R.
      • Dhagia V.
      • Ramesh A.
      • Zheng C.
      • Zhang H.
      • Jordan A.
      • Waddell I.
      • Leopold J.
      • Hu C.J.
      • McMurtry I.F.
      • D'Alessandro A.
      • Stenmark K.R.
      • Gupte S.A.
      Hypoxic activation of glucose-6-phosphate dehydrogenase controls the expression of genes involved in the pathogenesis of pulmonary hypertension through the regulation of DNA methylation.
      ,
      • Potus F.
      • Pauciulo M.W.
      • Cook E.K.
      • Zhu N.
      • Hsieh A.
      • Welch C.L.
      • Shen Y.
      • Tian L.
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      • Mewburn J.
      • D'Arsigny C.L.
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      • Nichols W.C.
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      • Archer S.L.
      Novel Mutations and Decreased Expression of the Epigenetic Regulator TET2 in Pulmonary Arterial Hypertension.
      ). In those studies, our laboratory found that DNA methylation-dependent transcription of PH-related genes and Tet2 expression is suppressed in the lungs of hypoxic mice (
      • Joshi S.R.
      • Kitagawa A.
      • Jacob C.
      • Hashimoto R.
      • Dhagia V.
      • Ramesh A.
      • Zheng C.
      • Zhang H.
      • Jordan A.
      • Waddell I.
      • Leopold J.
      • Hu C.J.
      • McMurtry I.F.
      • D'Alessandro A.
      • Stenmark K.R.
      • Gupte S.A.
      Hypoxic activation of glucose-6-phosphate dehydrogenase controls the expression of genes involved in the pathogenesis of pulmonary hypertension through the regulation of DNA methylation.
      ). Moreover, we observed that inhibiting glucose-6-phosphate dehydrogenase (G6PD) activity increases pulmonary Tet2 expression in hypoxic mice and reduces hypoxia-induced PH (
      • Joshi S.R.
      • Kitagawa A.
      • Jacob C.
      • Hashimoto R.
      • Dhagia V.
      • Ramesh A.
      • Zheng C.
      • Zhang H.
      • Jordan A.
      • Waddell I.
      • Leopold J.
      • Hu C.J.
      • McMurtry I.F.
      • D'Alessandro A.
      • Stenmark K.R.
      • Gupte S.A.
      Hypoxic activation of glucose-6-phosphate dehydrogenase controls the expression of genes involved in the pathogenesis of pulmonary hypertension through the regulation of DNA methylation.
      ). However, two questions arise from those studies: 1) does the DNMT expression and activity increase or decrease in PH? and 2) is G6PD involved in regulating the expression and activity of DNA methylation writers in experimental models of PH?
      While loss-of-function mutations in TET2 are associated with diverse blood cell malignancies in humans (
      • Solary E.
      • Bernard O.A.
      • Tefferi A.
      • Fuks F.
      • Vainchenker W.
      The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases.
      ), loss of a single TET enzyme is not sufficient to efficiently promote malignancy (
      • An J.
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      • Chawla A.
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      • Li W.
      • Goodell M.A.
      • Chavez L.
      • Ko M.
      • Rao A.
      Acute loss of TET function results in aggressive myeloid cancer in mice.
      ). We, therefore, hypothesized that alteration of DNMT-catalyzed DNA methylation to modify gene expression must occur in PH. To test that idea, we assessed Dnmt (1, 3a, and 3b) expression in lungs of rat exposed to sugen5614/hypoxia/normoxia (SU/Hx/Nx), a model of experimental PH (
      • Abe K.
      • Toba M.
      • Alzoubi A.
      • Ito M.
      • Fagan K.A.
      • Cool C.D.
      • Voelkel N.F.
      • McMurtry I.F.
      • Oka M.
      Formation of plexiform lesions in experimental severe pulmonary arterial hypertension.
      ). In addition, to determine whether Dnmt expression is driven by G6PD, we performed experiments in a novel rat model harboring the loss-of-function Mediterranean G6pd variant (G6PDS188F), which we recently generated (
      • Kitagawa A.
      • Kizub I.
      • Jacob C.
      • Michael K.
      • D'Alessandro A.
      • Reisz J.A.
      • Grzybowski M.
      • Geurts A.M.
      • Rocic P.
      • Gupte R.
      • Miano J.M.
      • Gupte S.A.
      CRISPR-Mediated Single Nucleotide Polymorphism Modeling in Rats Reveals Insight Into Reduced Cardiovascular Risk Associated With Mediterranean G6PD Variant.
      ), and in their wild-type (WT) littermates. G6PD deficiency is a common enzymopathy in humans, and there are conflicting reports regarding the effects of G6PD deficiency on the progression and severity of PH. Some studies suggest the Mediterranean G6PDS188F variant makes no contribution to sickle cell anemia-associated PH, while others suggest the frequency of G6PD mutation is higher among idiopathic PH patients than the general population (
      • Chen L.Y.
      • Lai E.J.
      • Collins D.R.
      • Ostrow D.N.
      • Sreenivasan G.M.
      A young woman with episodic angioedema, papilledema, and eosinophilia.
      ,
      • Dahoui H.A.
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      • Muwakkit S.A.
      • Saab R.H.
      • Bissar A.N.
      • Jumaa N.M.
      • Farhat F.S.
      • Dabbous I.A.
      • Bitar F.F.
      • Abboud M.R.
      Pulmonary hypertension in children and young adults with sickle cell disease: evidence for familial clustering.
      ,
      • Kurdyukov S.
      • Eccles C.A.
      • Desai A.A.
      • Gonzalez-Garay M.
      • Yuan J.X.
      • Garcia J.G.N.
      • Rafikova O.
      • Rafikov R.
      New cases of Glucose-6-Phosphate Dehydrogenase deficiency in Pulmonary Arterial Hypertension.
      ). We therefore used Mediterranean G6PDS188F variant rats to assess the role of G6PD in the progression and severity of SU/Hx/Nx-induced experimental PH.
      Our findings suggest that increased DNMT-catalyzed DNA methylation potentially contributes to the development and progression of PH pathology. What is more, they further suggest that G6PD contributes to the increase in DNMT expression and activity as well as modify expression of linker histone1H1 and increase histone acetylation/methylation resulting in the higher expression of genes encoding endothelial nitric oxide synthase 3 (NOS3), mitochondrial superoxide dismutase 2 (SOD2), and other vascular biology-related proteins in the lungs of rats with SU/Hx/Nx-induced experimental PH.

      Results

      SU/Hx/Nx augments folate pathway protein expression in lungs of wild-type but not G6PDS188F rats

      As illustrated (Figure 1A), G6PD-NADPH-dependent one-carbon metabolism and polyamine pathway metabolites (s-adenosylmethionine and spermine, respectively) are methyl-group donors and regulators of DNA methylation (
      • Amelio I.
      • Cutruzzola F.
      • Antonov A.
      • Agostini M.
      • Melino G.
      Serine and glycine metabolism in cancer.
      ,
      • Puleston D.J.
      • Villa M.
      • Pearce E.L.
      Ancillary Activity: Beyond Core Metabolism in Immune Cells.
      ) and are elevated in lungs/fibroblasts/SMCs of PH patients (
      • Joshi S.R.
      • Kitagawa A.
      • Jacob C.
      • Hashimoto R.
      • Dhagia V.
      • Ramesh A.
      • Zheng C.
      • Zhang H.
      • Jordan A.
      • Waddell I.
      • Leopold J.
      • Hu C.J.
      • McMurtry I.F.
      • D'Alessandro A.
      • Stenmark K.R.
      • Gupte S.A.
      Hypoxic activation of glucose-6-phosphate dehydrogenase controls the expression of genes involved in the pathogenesis of pulmonary hypertension through the regulation of DNA methylation.
      ,
      • D'Alessandro A.
      • El Kasmi K.C.
      • Plecita-Hlavata L.
      • Jezek P.
      • Li M.
      • Zhang H.
      • Gupte S.A.
      • Stenmark K.R.
      Hallmarks of Pulmonary Hypertension: Mesenchymal and Inflammatory Cell Metabolic Reprogramming.
      ), we sought to determine the role of G6PD in modulating expression of enzymes of folate pathway and activity of DNMTs in lungs of SU/Hx/Nx rats. To accomplish that goal, we used a recently developed loss-of-function Mediterranean G6PD variant (G6PDS188F) rat (
      • Kitagawa A.
      • Kizub I.
      • Jacob C.
      • Michael K.
      • D'Alessandro A.
      • Reisz J.A.
      • Grzybowski M.
      • Geurts A.M.
      • Rocic P.
      • Gupte R.
      • Miano J.M.
      • Gupte S.A.
      CRISPR-Mediated Single Nucleotide Polymorphism Modeling in Rats Reveals Insight Into Reduced Cardiovascular Risk Associated With Mediterranean G6PD Variant.
      ). As expected, G6PD activity was 70-80% lower in the heart and lungs of G6PDS188F than wild-type (WT) rats (Figure 1B). Next, because G6PD deficiency causes hemolytic anemia, we compared hematocrit levels between G6PDS188F and WT rats and found no difference in the hematocrit between both genotypes (Figure 1C). Further, we did not observe hemolysis in G6PDS188F rats and the hematocrit between SU/Hx/Nx G6PDS188F (49±1%) and WT (49±1%) rats was not different. Interestingly, quantitative MS proteomics results revealed enzymes that convert s-adenosyl-l-homocysteine to homocysteine (SAHH) increased and methionine to s-adenosylmethionine (MAT2A) and s-adenosyl-l-homocysteine to homocysteine (SAHH2) trend to increase lungs of SU/Hx/Nx WT rats as compared to SU/Nx WT rats (Figure 1D). In contrast, MAT2A and SAHH decreased in SU/Hx/Nx G6PDS188F rats as compared to WT rats. While we did not find differences in the expression levels of enzymes in the one-carbon and folate pathway by Western blotting (Figure 2A).
      Figure thumbnail gr1
      Figure 1Elevated pulmonary folate pathway proteins in SU/Hx/Nx wild type but not G6PDS188F rats. (A) A schematic illustration connecting G6PD-derived NADPH-dependent one-carbon metabolism and polyamine pathway to DNA methylation. (B) G6PD activity in the heart and lungs of Mediterranean G6PD variant (G6PDS188F) rats is considerably lower (<70-80%) than in wild-type (WT) rats. (C) Hematocrit is not different between G6PDS188F and WT rats. Rats receiving sugen5416 (SU) were kept in normoxia (21% O2) or exposed to hypoxia (10% O2) for 3 weeks followed by normoxia (21% O2) for 5 weeks. Expression levels of one carbon metabolism/folate pathway proteins methionine adenosyl transferase 2A (MAT2A), S-Adenosyl-L-Homocysteine Hydrolase (SAHH), and S-Adenosyl-L-Homocysteine Hydrolase-2 (SAHH2) in lungs were determined by Western blotting and by quantitative proteomics. (D) Quantitative proteomics revealed MAT2A, SAHH, and SAHH2 are increased or showed increasing trend in SU/Hx/Nx than SU/Nx in WT but not in G6PDS188F rats. N = 5 in each group. Comparisons among all four groups were made using two-way ANOVA and between two groups using Student’s t test. **P<0.005 and ****P<0.0001.
      Figure thumbnail gr2
      Figure 2Elevated pulmonary DNMT expression and activity in SU/Hx/Nx wild type but not G6PDS188F rats. Rats receiving sugen5416 (SU) were kept in normoxia (21% O2) or exposed to hypoxia (10% O2) for 3 weeks followed by normoxia (21% O2) for 5 weeks. (A) Western blot of three separate experiments show no differences in expression of MAT2A or SAHH but higher expression of DNMT1 and DNMT3B between WT and G6PDS188F rats exposed to SU/Hx/Nx and SU/Nx. (B) Summary results showing higher expression of DNMT1 and DNMT3B in lungs of rats exposed to SU/Hx/Nx and SU/Nx. DNMT3A was not detected. (C) Pulmonary DNMT activity was higher in lungs of wild type (WT), but not G6PDS188F, rats that received SU (20 mg/kg; I.P.) kept in hypoxia + normoxia (Hx; blue bar) than in normoxia (Nx; red bar). N = 5 in each group. (D-F) RT-PCR analysis showing that pulmonary expression of Dnmt1, Dnmt3a, and Dnmt3b in wild-type and G6PDS188F rats with SU exposed to hypoxia changes as compared to their respective normoxia control. Pulmonary expression of Dnmt1 and Dnmt3b, but not Dnmt3a, was higher in wild-type but not in G6PDS188F rats with SU kept in hypoxia + normoxia (Hx; blue bar) than in normoxia (Nx; red bar). Comparisons among all four groups were made using two-way ANOVA. *P<0.05; **P<0.005; ***P<0.0005; and ****P<0.0001

      SU/Hx/Nx increases total DNMT activity and Dnmt1 and Dnmt3b expression in lungs of wild-type rats

      Next, we measured DNMT expression and activity in the lungs of rats exposed to SU/Hx/Nx, SU/Nx, hypoxia (Hx; 10% O2), and normoxia (Nx: 21% O2). SU/Hx/Nx increased DNMT1 and DNMT3B expression (Figure 2A and B). DNMT3A was not detectable. Although pulmonary DNMT activity was similar in hypoxic and normoxic rats, it was significantly higher in SU/Hx/Nx than SU/Nx rats (Figure 2C). While Hx increased pulmonary expression of Dnmt3a (Nx: 0.20±0.04 and Hx: 0.54±0.20; P<0.05) and Dnmt3b (Nx: 0.009±0.002 and Hx: 0.026±0.007; P<0.05) but not Dnmt1 (Nx: 1.68±0.63 and Hx: 2.79±1.20; NS), SU/Hx/Nx increased expression of Dnmt1 and Dnmt3b (Figure 2D and 2F), as compared to respective SU/Nx control, in rats. SU/Hx/Nx did not significantly affect Dnmt3a expression in rats (Figure 2E).

      SU/Hx/Nx does not increase DNMT activity and Dnmt1 and Dnmt3b expression in lungs of G6PDS188F rats

      Since we found G6PDS188F variant prevented SU/Hx/Nx-elicited expression of protein involved in one carbon metabolism or folate pathway (Figure 1E) and correspondingly pulmonary DNMT activity did not increase in SU/Hx/Nx (22.7±0.63 OD/min/mg Protein) as compared with normoxic (20.2±0.88 OD/min/mg Protein) G6PDS188F rats (Figure 2A), we measured expression of genes encoding DNMTs in the lungs of SU/Hx/Nx and SU/Nx G6PDS188F rats. Interestingly, there was no difference in pulmonary expression of Dnmt1 (Figure 2B), Dnmt3a (Figure 2C), and Dnmt3b (Figure 2D) in SU/Hx/Nx as compared to SU/Nx G6PDS188F rats. Consistently, expression of DNMT1 and DNMT3B did not increase in SU/Hx/Nx G6PDS188F rats (Figure 2E and F).

      SU/Hx/Nx increases expression DNMT1 and DNMT3B in occluded arteries and alveoli of wild-type rats

      Next, we performed immunohistochemistry to determine expression of DNMTs in lungs of WT and G6PDS188F rats. Immunohistochemical assay revealed DNMT1 expression (brown staining) increased in and around the arteries (Figure 3A-i and -ii) and in more alveoli (Figure 3B-i and -ii) of SU/Hx/Nx- rats as compared to SU/Nx-WT rats. While we do not see intense brown staining for DNMT3A (Figure 3C and D), which corroborated the Western blot results (Figure 2E and F), we found increased brown staining for DNMT3B around the arteries (Figure 3E-i and -ii) and in alveoli (Figure 3F-i and -ii) of SU/Hx/Nx-WT rats as compared to SU/Nx-WT rats. To this regard, we found less intense staining areas for all DNMTs around arteries (Figure 3A-, C-, and E-iii and -iv) and alveoli (Figure 3B-, D-, and F-iii and -iv) of SU/Hx/Nx-G6PDS188F rats as compared to SU/Nx-G6PDS188F rats.
      Figure thumbnail gr3
      Figure 3DNMTs increased in arteries and alveoli of SU/Hx/Nx wild type but not G6PDS188F rats. (A-F) A representative micrograph (4x and 40x magnification; 4x: scale bar is 100 μm and 40x: scale bar is 10 μm) of immunohistochemistry results from four different experiment show staining of different DNMTs in lungs of SU/Nx- and SU/Hx/Nx-WT (i and ii) and -G6PDS188F (iii and iv) rats. (A-i and ii) DNMT1 and (E-i and ii) DNMT3B expression (brown staining) increased in SU/Hx/Nx-WT as compared with SU/Nx-WT rat arteries and alveoli indicated by red arrows. (A-iii and iv) DNMT1 and (E-iii and iv) DNMT3B show less intense staining in SU/Hx/Nx- G6PDS188F and SU/Nx- G6PDS188F rat arteries and alveoli. (C- and D-iii and iv).

      SU/Hx/Nx increases methylation of the Sod2, Abra, and Serpine1 promoter in the lungs of wild-type but not G6PDS188F rats

      Because DNMT1 and DNMT3B expression and activity was higher in the lungs of SU/Hx/Nx than SU/Nx WT rats, we performed methylated DNA immunoprecipitation (MeDIP)-PCR to determine differential methylation status of the methylation-sensitive non-coding H19 gene imprinting center region 1 (H19ICR1) sequence in G6PDS188F versus WT rat genome. We found enriched methylated H19ICR gene region in lungs of SU/Hx/Nx (1.57±0.27) as compared with SU/Nx (1.08±0.15) WT rats but not G6PDS188F rats (SU/Hx/Nx: 1.13±0.11 and Su/Nx: 1.20±0.01). Next, we performed MS-PCR using primers designed (Table 1) to amplify methylated and unmethylated CpG islands in the middle and proximal regions of the Nos3, Sod2, Abra, and Serpine1 promoters in the lungs of SU/Hx/Nx vs. SU/Nx rats (Figure 4). We selected Nos3 and Sod2 promoters because products of these genes are implicated in the pathogenesis of pulmonary hypertension (
      • Archer S.L.
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      • Kim G.H.
      • Zhang H.J.
      • Toth P.T.
      • Svensson E.C.
      • Dyck J.R.
      • Gomberg-Maitland M.
      • Thebaud B.
      • Husain A.N.
      • Cipriani N.
      • Rehman J.
      Epigenetic attenuation of mitochondrial superoxide dismutase 2 in pulmonary arterial hypertension: a basis for excessive cell proliferation and a new therapeutic target.
      ,
      • Ke X.
      • Johnson H.
      • Jing X.
      • Michalkiewicz T.
      • Huang Y.W.
      • Lane R.H.
      • Konduri G.G.
      Persistent pulmonary hypertension alters the epigenetic characteristics of endothelial nitric oxide synthase gene in pulmonary artery endothelial cells in a fetal lamb model.
      ), and Abra and Serpine1 promoters because proteins encoded by these genes are involved in vascular smooth muscle biology and thrombosis (
      • Wallace M.A.
      • Lamon S.
      • Russell A.P.
      The regulation and function of the striated muscle activator of rho signaling (STARS) protein.
      ,
      • Law R.H.
      • Zhang Q.
      • McGowan S.
      • Buckle A.M.
      • Silverman G.A.
      • Wong W.
      • Rosado C.J.
      • Langendorf C.G.
      • Pike R.N.
      • Bird P.I.
      • Whisstock J.C.
      An overview of the serpin superfamily.
      ) and we recently observed that expression of Serpine1 and Abra was altered in aortas from rats fed a high-fat diet (
      • Dhagia V.
      • Kitagawa A.
      • Jacob C.
      • Zheng C.
      • D'Alessandro A.
      • Edwards J.G.
      • Rocic P.
      • Gupte R.
      • Gupte S.A.
      G6PD activity contributes to the regulation of histone acetylation and gene expression in smooth muscle cells and to the pathogenesis of vascular diseases.
      ). We found that the MI was higher in the middle and proximal regions of the Sod2, Abra, and Serpine1 promoters in SU/Hx/Nx than SU/Nx WT rats (Figure 4B, C and D). Very interestingly, the MI decreased in the middle and proximal region of Nos3, Sod2, Abra, and Serpine1 promoters in SU/Hx/Nx G6PDS188F rats (Figures 4A-D).
      Table 1MS-PCR primers for middle and proximal regions of the gene promoter
      Primer NameForwardReverse
      Serpine1-MM5'-TATCGCGTACGTAATTAAAAC-3’5'-AAAATATATACCGCCGTACC-3'
      Serpine1-MU5'-ATGTAAGAAGAAAGTGGGGT-3'5'-TCCAACTCACAAAAATCAAC-3'
      Serpine1-PM5'-CGTTAAGGGTTTAGACGAT-3'5'-CCCAATAACTTACTAAAACACG-3'
      Serpine1-PU5'-GTTAAGGGTTTAGATGATTGAT-3'5'-TCCCAATAACTTACTAAAACACA-3'
      Abra-MM5'-TTAGAGCGTTGGTTTAGTAAGC-3'5'-TTTCCGAAACTAAAAACCGA-3'
      Abra -MU5'-TGGTTAGAGTGTTGGTTTAGTAAGT-3'5'-CAAAACTAAAAACCAAACCCA -3'
      Abra -PM5'-GTTTGTGACGGTGGAAATTAT-3'5'-AAACAAAACACCGTCAAAAC-3'
      Abra -PU5'-GTGTTTGTGATGGTGGAAATTA -3'5'-AAAACACCATCAAAACCATAAC-3'
      Nos3-MM5'-AGATTGTTTCGGTTTGTTGTAT-3'5'-TACAACCTCGACCGATCCT-3'
      Nos3-MU5'-GAAGATTGTTTTGGTTTGTTGTA-3'5'-AACCTCAACCAATCCTCCTC-3'
      Nos3-PM5'-GCGTATTAGTTGGCGTTTTA-3'5'-CCGTATCAATTCAACCATCA-3'
      Nos3-PU5'-TTGTGTAGTGTATTAGTTGGTGTTT-3'5'-CCCCATATCAATTCAACCAT-3'
      Sod2-MM5'-TAAGGCGGTTCGAGAAGAG-3'5'-GCGAACTTACCACGACCACG-3'
      Sod2-MU5'-GGTTAAGGTGGTTTGAGAAGA-3'5'-CACAAACTTACCACAACCACAC-3'
      Sod2-PM5'-AAGTTGGGTCGTTCGTGTC-3'5'-AACCGCGCGTCTACTCTAC-3'
      Sod2-PU5'-GTTGTTTGTGTTGTGGTTTTT-3'5'-ACCACACATCTACTCTACAACATC-3'
      MM= methylated target in middle region of the promoter; MU= unmethylated target in middle region of the promoter; PM = methylated target in proximal region of the promoter; PU= unmethylated target in proximal region of the promoter.
      Figure thumbnail gr4
      Figure 4Methylation and expression of the Nos3, Sod2, Abra, and Serpine1, in lungs of SU/Hx/Nx WT and G6PDS188F rats. Cumulative results showing that the methylation indexes for CpG islands in the promoter (middle promoter and proximal promoter) of the (A) Nos3, (B) Sod2, (C) Abra and (D) Serpine1, in the lungs from SU/Hx/Nx than normoxic WT (left panels; SU/Nx: red bars and SU/Hx/Nx: blue bars) and G6PDS188F (right panels; SU/Nx: white bars and SU/Hx/Nx: green bars) rats. The methylation index increased in SU/Hx/Nx WT but not G6PDS188F rats. Primers were designed to CpG regions in the middle and proximal to the transcription start site in each gene. (E) RT-PCR results showing that pulmonary expression of Nos3, Sod2, Abra, and Serpine1, is lower in SU/Hx/Nx than normoxic WT, but not G6PDS188F rats. A representative Western blot of three separate experiments (F) and summary results (G) showing expression of NOS3, SOD2 and SERPINE1 in lungs of rats that received sugen5416 (SU) kept in hypoxia + normoxia than in normoxia. N (number of rats) = 5 in each group (Panels A to D, and E). Comparisons among all four groups were made using two-way ANOVA and between two groups using Student’s t test. *P<0.05; **P<0.005; and #P<0.05

      SU/Hx/Nx decreases expression of genes encoding NOS3, SOD2, and ABRA in the lungs of wild-type but not G6PDS188F rats

      Further, we wanted to determine whether expression of these genes was affected by the elevated DNMT activity seen in the lungs of SU/Hx/Nx rats. Using RT-PCR, we found that levels of pulmonary Nos3, Sod2, Abra, and Serpine1 expression were decreased in SU/Hx/Nx compared to SU/Nx WT rats (Figure 4E). In contrast, expression of Nos3 and Sod2 was augmented in SU/Hx/Nx compared to SU/Nx G6PDS188F rats (Figure 4E). Next, we performed Western blotting for the expression of NOS3, SOD2, and SERPINE1. As shown in representative blots and summary results, the expression of NOS3 and SOD2 showed an increasing trend in SU/Hx/Nx as compared to SU/Nx G6PDS188F rats (Figures 4F-G), while expression of SERPINE1 showed increasing trend in SU/Hx/Nx as compared with SU/Nx WT and G6PDS188F rats (Figures 4F-G). Interestingly, expression of NOS3 increased in lungs of SU/Hx/Nx-G6PDS188F rats as compared to SU/Hx/Nx-WT rats (Figure 4G). Due to the lack of a specific antibody against rat ABRA, we performed Westerns with antibodies against human and mouse species, but ABRA signals were undetectable (data not shown).

      Proteomic analysis reveals modification of the pulmonary expression of proteins in SU/Hx/Nx rats

      Because DNMT expression and activity increased in SU/Hx/Nx-WT rats, we speculated this will alter or modify expression of several other genes and proteins, in addition to NOS3 and SOD2, in the lungs. Using quantitative mass spectrometry (MS), we detected pulmonary expression of 2300 proteins in rat lungs (Supplement Table 1). Pulmonary expression of proteins involved in glycolysis (ALDOA and IDH3B), Ca2+ handling (ITPR2), Gq receptor signaling (ARRB1), solute transporter (SLC44A2), and the complement cascade protein (C9), was higher in SU/Hx/Nx than SU/Nx WT rats, but not G6PDS188F rats (Table 2). By contrast, pulmonary expression of proteins involved in the mitochondrial respiratory chain (ACCA2 and ATP5F1A), protein kinase A signaling (PRKAR1A), Ca2+ handling (SERCA2/ATP2A2 and CACNA2D1), inhibition of neutrophil elastase and cathepsin G (SERPINA1 and SERINA3K), differentiation of smooth muscle cell (SMC) phenotype (CSRP1), and gene transcription (NPM1 and SART1) were all lower in SU/Hx/Nx than SU/Nx WT rats but not G6PDS188F rats (Table 2). Proteomic data was deposited into MassIVE (ftp://[email protected] or ftp://massive.ucsd.edu/MSV000089037/).
      Table 2Total spectrum count of identified proteins in the rat lungs
      Accession NumberAlternate IDG6PDS188F+SuG6PDS188F +Su+HxWT+SuWT +Su+Hx
      Mitochondrial Proteins
      THIM_RATAcaa224.2±3.916.0±3.223.3±1.814.5±1.6
      P<0.05 vs WT+Su;
      ATPA_RATAtp5f1a72.0±0.473.3±5.175.5±3.165.8±1.6
      P<0.05 vs WT+Su;
      M2OM_RATSlc25a115.8±0.48.0±0.5
      P<0.05 vs G6PDS188F+Su;
      6.0±0.69.8±0.5
      P<0.05 vs WT+Su;
      Glycolysis Proteins
      ALDOA_RATAldoa63.0±2.267.8±2.365.0±1.772.0±3.2
      P<0.05 vs WT+Su;
      IDH3B_RATIdh3B23.5±1.323.5±2.121.5±2.628.8±1.9
      P<0.05 vs WT+Su;
      PPP Proteins
      G6PD_RATG6pdx17.3±1.0
      P<0.05 vs WT+Su;
      21.3±1.7
      P<0.05 vs WT+Su+Hx
      29.5±1.033.8±2.6
      TKT_RATTkt96.3±1.5
      P<0.05 vs WT+Su;
      102.3±7.3107.3±4.0108.5±2.8
      Cell Signaling Proteins
      KAP0_RATPrkar1a23.8±2.422.3±1.425.5±2.517.3±1.1
      P<0.05 vs WT+Su;
      ARRB1_RATArrb117.8±1.120.5±0.618.8±1.725.8±2.8
      P<0.05 vs WT+Su;
      PDE5A_RATPde5a25.0±2.0
      P<0.05 vs WT+Su;
      25.0±1.230.5±1.226.0±3.6
      Calcium Handling Proteins
      AT2A2_RATAtp2a242.0±6.145.3±7.047.3±6.134.3±2.0
      P<0.05 vs WT+Su;
      CA2D1_RATCacna2d110.0±1.77.3±1.515.3±2.110.5±0.9
      P<0.05 vs WT+Su;
      ITPR2_RATItpr24.3±2.56.8±2.73.5±2.19.3±1.8
      P<0.05 vs WT+Su;
      Serine Proteases
      A1AT_RATSerpina181.5±8.976.8±5.2
      P<0.05 vs WT+Su+Hx
      94.0±8.262.8±4.0
      P<0.05 vs WT+Su;
      SPA3K_RATSerpina3k107.0±9.3120.0±7.9
      P<0.05 vs WT+Su+Hx
      121.0±10.574.5±8.6
      P<0.05 vs WT+Su;
      Complement Proteins
      CO9_RATC935.3±1.937.8±4.133.3±0.545.8±3.1
      P<0.05 vs WT+Su;
      SMC Proteins
      CSRP1_RATCsrp150.0±2.254.3±2.164.8±3.346.0±3.7
      P<0.05 vs WT+Su;
      Nuclear and Transcription Proteins
      NPM_RATNpm121.0±2.319.5±1.324.0±0.717.8±1.3
      P<0.05 vs WT+Su;
      SNUT1_RATSart15.0±2.02.5±1.58.5±2.42.3±2.3
      P<0.05 vs WT+Su;
      Other Proteins
      DHB11_RATHsd17b116.5±0.66.5±0.6
      P<0.05 vs WT+Su+Hx
      7.5±1.511.5±1.5
      P<0.05 vs WT+Su;
      RTN3_RATRtn315.0±0.917.3±1.512.8±1.120.5±1.3
      P<0.05 vs WT+Su;
      SFTPA_RATSftpa121.8±2.625.3±1.716.5±0.624.5±1.5
      P<0.05 vs WT+Su;
      SFXN3_RATSfxn312.0±2.210.3±0.9
      P<0.05 vs WT+Su+Hx
      10.5±1.46.5±1.0
      P<0.05 vs WT+Su;
      CTL2_RATSlc44a230.0±1.432.3±1.1
      P<0.05 vs WT+Su+Hx
      28.0±0.940.0±2.3
      P<0.05 vs WT+Su;
      VWA5A_RATVwa5a17.3±2.116.0±2.322.3±2.015.3±2.1
      P<0.05 vs WT+Su;
      ST1A1_RATSult1a115.8±1.516.3±2.3
      P<0.05 vs WT+Su+Hx
      16.8±1.322.8±1.0
      P<0.05 vs WT+Su;
      Mean±SEM; N=4 in each group;
      P<0.05 vs WT+Su;
      $ P<0.05 vs G6PDS188F+Su;
      # P<0.05 vs WT+Su+Hx

      SU/Hx/Nx alters linker histone1H1 expression in G6PDS188F rats

      Next, in quantitative MS-proteomic analysis (Supplement Table 1), we found expression of linker histone1H1A (H1.1) and histone1H1E (H1.4), which binds DNMT1 and DNMT3B and reduces methylation of genes (
      • Kalashnikova A.A.
      • Rogge R.A.
      • Hansen J.C.
      Linker histone H1 and protein-protein interactions.
      ), respectively, increased and decreased, in SU/Hx/Nx than SU/Nx G6PDS188F rats (Figure 5A). While histone1H1A and histone1H1E showed decreasing trends in SU/Hx/Nx than SU/Nx WT rats (Figure 5A).
      Figure thumbnail gr5
      Figure 5Linker histone1H1 and acetylation and methylation of histone3 was increased SU/Hx/Nx G6PDS188F rats. Mass spectrometry-based proteomic analysis shows (A) linker histone1H1 and (B-C) acetylation and methylation of Lys residues in H3 variants, H3.1, in rat lungs. Cumulative analysis of the (A) total spectrum count showing higher histone1H1A and lower histone1H1E, and (D) post-translation modification (PTM) spectrum counts showing higher H3.1-K24ac and H3.1-K80me levels, in the lungs of SU/Hx/Nx and normoxic G6PDS188F but not WT rats. N = 5 in each group. Comparisons between two groups were made using Student’s t test. *P<0.05.

      Histone acetylation and methylation increases in lungs of SU/Hx/Nx and normoxic G6PDS188F rats but not wild-type rats

      Because it was recently proposed that post-translation histone modification (acetylation and methylation) contributes to gene regulation and to the pathogenesis of pulmonary arterial hypertension (
      • Hu C.J.
      • Zhang H.
      • Laux A.
      • Pullamsetti S.S.
      • Stenmark K.R.
      Mechanisms contributing to persistently activated cell phenotypes in pulmonary hypertension.
      ), we speculated that histone modifications occur in the lungs of SU/Hx/Nx rats. Using mass spectrometry, we detected acetylation and methylation of H3 variant (H3.1) at K24 and K80 (Figure 5B and C) and Supplement Table 2 and 3. Interestingly, post-translation modification (PTM) spectrum counts of H3.1-K24ac and H3.1-K80me were higher (p<0.05) in the lungs of SU/Hx/Nx compared to SU/Nx G6PDS188F but not WT rats (Figure 5D). While there was a decreasing trend in PTM spectrum counts between SU/Nx G6PDS188F and WT rats (Figure 5D). Thus, acetylation and methylation of histones was increased in SU/Hx/Nx G6PDS188F rats.

      SU/Hx/Nx increases RV pressure and hypertrophy in wild-type but not G6PDS188F rats

      As in previous studies (
      • Abe K.
      • Toba M.
      • Alzoubi A.
      • Ito M.
      • Fagan K.A.
      • Cool C.D.
      • Voelkel N.F.
      • McMurtry I.F.
      • Oka M.
      Formation of plexiform lesions in experimental severe pulmonary arterial hypertension.
      ,
      • Jiang B.
      • Deng Y.
      • Suen C.
      • Taha M.
      • Chaudhary K.R.
      • Courtman D.W.
      • Stewart D.J.
      Marked Strain-Specific Differences in the SU5416 Rat Model of Severe Pulmonary Arterial Hypertension.
      ), exposing WT rats to SU/Hx/Nx, as illustrated in the schematic in Figure 6A, increased RVSP by 69.9% (p<0.05; Figure 6B), RV end diastolic pressure (RVDP) by 52.1% (p<0.05; Figure 6C), and RV hypertrophy by 32.2% (p<0.05; Figure 6D). Next, we determined whether G6PD contributes to increase RV pressure and hypertrophy. In G6PDS188F rats, SU/Hx/Nx raised RVSP by 25.1% (p<0.05). Intriguingly, RVSP increased (44.8%) more in WT than G6PDS188F rats. Moreover, while SU/Hx/Nx did not increase RVDP (Figure 6C) or RV hypertrophy (Figure 6D) as compared to SU/Nx in G6PDS188F rats, it significantly increased (p<0.05) in WT rats. The effects of SU/Hx/Nx on RVDP and RV hypertrophy were significantly greater in WT than G6PDS188F rats.
      Figure thumbnail gr6
      Figure 6Experimental schema and hemodynamic measurements in wild-type and Mediterranean G6PDS188F variant rats. (A) Schematic showing that WT and G6PDS188F rats injected with sugen5416 (20 mg/kg subcutaneously) were kept in normobaric hypoxia (10% O2) for 3 weeks (21 days) and then in normoxia (21% O2) for 5 weeks (56 days). Rats injected with sugen5416 (20 mg/kg subcutaneously) and kept under normoxia for 8 weeks (56 days) were used as controls. At the end of day 56, right ventricular pressure was measured through catheterization. (B-D) Right ventricular pressure (RVSP and RVDP) and hypertrophy in SU/Hx/Nx WT and G6PDS188F rats. N=5 in each group. Comparisons among all four groups were made using two-way ANOVA. *P<0.05; **P<0.005; ***P<0.0005; and ****P<0.0001

      Discussion

      Evidence that alteration of DNA and histone methylation or acetylation (epigenetic modifications) associate with the development of PH has started to accumulate (
      • Napoli C.
      • Benincasa G.
      • Loscalzo J.
      Epigenetic Inheritance Underlying Pulmonary Arterial Hypertension.
      ). It has been proposed, for example, that altered methylation and acetylation marks on histones produce a persistent inflammatory phenotype in fibroblasts and an apoptosis-resistant and hyperproliferative phenotype of endothelial cells and SMCs from hypertensive patients and animal models (
      • Hu C.J.
      • Zhang H.
      • Laux A.
      • Pullamsetti S.S.
      • Stenmark K.R.
      Mechanisms contributing to persistently activated cell phenotypes in pulmonary hypertension.
      ). However, detailed information regarding the role of DNA methylation and aberrant gene/protein expression on the development and progression of PH remains to be determined.
      Because methyltransferases and demethylases regulate the level of DNA and histone methylation and can thus alter gene transcription (
      • Peixoto P.
      • Cartron P.F.
      • Serandour A.A.
      • Hervouet E.
      From 1957 to Nowadays: A Brief History of Epigenetics.
      ), we investigated DNMT expression and total activity in the lungs of SU/Hx/Nx rats, an experimental model of PH. While Dnmt1/DNMT1 and Dnmt3b/DNMT3B expression either increased or showed increasing trend in SU/Hx/Nx-WT rats, Dnmt3a and Dnmt3b expression was upregulated in Hx-WT rats. Consistently, methylation of H19ICR sequence, determined by MeDIP that linearly measures methylated cytosines (
      • Weber M.
      • Davies J.J.
      • Wittig D.
      • Oakeley E.J.
      • Haase M.
      • Lam W.L.
      • Schubeler D.
      Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells.
      ), increased in lungs of SU/Hx/Nx-WT rats but not G6PDS188F rats. Because DNMT1 and DNMT3B expression tend to increase in arteries and alveoli, we anticipate total differential methylation occurred in the lungs (including arteries, airway, and alveoli) contributed to the pathogenesis of pulmonary disease. Further, since expression of all three DNMT mRNA and total DNMT activity increases in leukocytes and in extracellular vesicles of scleroderma-associated and idiopathic PH patients (
      • D'Addario C.A.
      • Lanier G.M.
      • Jacob C.
      • Bauer N.
      • Hewes J.L.
      • Bhadra A.
      • Gupte S.A.
      Differences in the expression of DNA methyltransferases and demethylases in leukocytes and the severity of pulmonary arterial hypertension between ethnic groups.
      ), we propose that release of increased arterial and alveolar DNMT1 and DNMT3B in extracellular vesicles perhaps has paracrine-like action. One recent study suggests DNMT3B protein increases in lungs of congenital heart disease-associated PH patients and rats exposed to monocrotaline or Hx, and knockdown of Dnmt3b in rats exacerbates monocrotaline- and Hx-induced PH (
      • Yan Y.
      • He Y.Y.
      • Jiang X.
      • Wang Y.
      • Chen J.W.
      • Zhao J.H.
      • Ye J.
      • Lian T.Y.
      • Zhang X.
      • Zhang R.J.
      • Lu D.
      • Guo S.S.
      • Xu X.Q.
      • Sun K.
      • Li S.Q.
      • Zhang L.F.
      • Zhang X.
      • Zhang S.Y.
      • Jing Z.C.
      DNA methyltransferase 3B deficiency unveils a new pathological mechanism of pulmonary hypertension.
      ).
      It is now well established that increased DNMT-catalyzed methylation of CpG islands within gene promoter silences expression of those genes. Altered DNA methylation in the lungs of hypoxic mice was previously reported to reduce expression of several genes encoding proteins implicated in the pathogenesis of PH (
      • Joshi S.R.
      • Kitagawa A.
      • Jacob C.
      • Hashimoto R.
      • Dhagia V.
      • Ramesh A.
      • Zheng C.
      • Zhang H.
      • Jordan A.
      • Waddell I.
      • Leopold J.
      • Hu C.J.
      • McMurtry I.F.
      • D'Alessandro A.
      • Stenmark K.R.
      • Gupte S.A.
      Hypoxic activation of glucose-6-phosphate dehydrogenase controls the expression of genes involved in the pathogenesis of pulmonary hypertension through the regulation of DNA methylation.
      ). Other important gene (for example, Sod2 and Nos3) promoters are hypermethylated in both adult and neonatal PH (
      • Archer S.L.
      • Marsboom G.
      • Kim G.H.
      • Zhang H.J.
      • Toth P.T.
      • Svensson E.C.
      • Dyck J.R.
      • Gomberg-Maitland M.
      • Thebaud B.
      • Husain A.N.
      • Cipriani N.
      • Rehman J.
      Epigenetic attenuation of mitochondrial superoxide dismutase 2 in pulmonary arterial hypertension: a basis for excessive cell proliferation and a new therapeutic target.
      ,
      • Ke X.
      • Johnson H.
      • Jing X.
      • Michalkiewicz T.
      • Huang Y.W.
      • Lane R.H.
      • Konduri G.G.
      Persistent pulmonary hypertension alters the epigenetic characteristics of endothelial nitric oxide synthase gene in pulmonary artery endothelial cells in a fetal lamb model.
      ). Consistently, Sod2, Abra, and Serpine1 gene promoters were hypermethylated in SU/Hx/Nx-WT rats. Along the line of DNA methylation results, we found that expression of [1] Nos3, which encodes the endothelial nitric oxide synthase that generates vasodilatory nitric oxide (
      • Fischmann T.O.
      • Hruza A.
      • Niu X.D.
      • Fossetta J.D.
      • Lunn C.A.
      • Dolphin E.
      • Prongay A.J.
      • Reichert P.
      • Lundell D.J.
      • Narula S.K.
      • Weber P.C.
      Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation.
      ), [2] Sod2, which encodes mitochondrial superoxide dismutase that scavenges superoxide anion produced in the respiratory chain (
      • Luk E.
      • Yang M.
      • Jensen L.T.
      • Bourbonnais Y.
      • Culotta V.C.
      Manganese activation of superoxide dismutase 2 in the mitochondria of Saccharomyces cerevisiae.
      ), [3] Abra, which encodes actin-binding Rho-activating protein that regulates vascular smooth muscle phenotype (
      • Wallace M.A.
      • Lamon S.
      • Russell A.P.
      The regulation and function of the striated muscle activator of rho signaling (STARS) protein.
      ), and [4] Serpine1, which encodes the prothrombotic protein PAI-1 (
      • Law R.H.
      • Zhang Q.
      • McGowan S.
      • Buckle A.M.
      • Silverman G.A.
      • Wong W.
      • Rosado C.J.
      • Langendorf C.G.
      • Pike R.N.
      • Bird P.I.
      • Whisstock J.C.
      An overview of the serpin superfamily.
      ), either decreased or showed decreasing trends in SU/Hx/Nx-WT rats. Conversely, NOS3 and SOD2 mRNA and protein either increased or showed increasing trend in SU/Hx/Nx-G6PDS188F variant but not WT rats. DNA hypermethylation contributes to the development of vascular diseases (
      • Lacey M.
      • Baribault C.
      • Ehrlich K.C.
      • Ehrlich M.
      Atherosclerosis-associated differentially methylated regions can reflect the disease phenotype and are often at enhancers.
      ,
      • Dong C.
      • Yoon W.
      • Goldschmidt-Clermont P.J.
      DNA methylation and atherosclerosis.
      ) and to right heart failure in PH (
      • Potus F.
      • Ruffenach G.
      • Dahou A.
      • Thebault C.
      • Breuils-Bonnet S.
      • Tremblay E.
      • Nadeau V.
      • Paradis R.
      • Graydon C.
      • Wong R.
      • Johnson I.
      • Paulin R.
      • Lajoie A.C.
      • Perron J.
      • Charbonneau E.
      • Joubert P.
      • Pibarot P.
      • Michelakis E.D.
      • Provencher S.
      • Bonnet S.
      Downregulation of MicroRNA-126 Contributes to the Failing Right Ventricle in Pulmonary Arterial Hypertension.
      ). Our results suggest increased DNMT1, a maintenance methyltransferase, and DNMT3B, which elicits de novo genome-wide methylation, expression and activity methylates and suppresses genes encoding protective as well as malevolent proteins that, respectively, contribute to the pathogenesis of experimental PH and perhaps simultaneously reduce the severity of PH as seen before (
      • Yan Y.
      • He Y.Y.
      • Jiang X.
      • Wang Y.
      • Chen J.W.
      • Zhao J.H.
      • Ye J.
      • Lian T.Y.
      • Zhang X.
      • Zhang R.J.
      • Lu D.
      • Guo S.S.
      • Xu X.Q.
      • Sun K.
      • Li S.Q.
      • Zhang L.F.
      • Zhang X.
      • Zhang S.Y.
      • Jing Z.C.
      DNA methyltransferase 3B deficiency unveils a new pathological mechanism of pulmonary hypertension.
      ).
      DNMT activity is driven by metabolites of one-carbon metabolism and the polyamine pathway (
      • Solary E.
      • Bernard O.A.
      • Tefferi A.
      • Fuks F.
      • Vainchenker W.
      The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases.
      ,
      • Puleston D.J.
      • Villa M.
      • Pearce E.L.
      Ancillary Activity: Beyond Core Metabolism in Immune Cells.
      ,
      • Stover P.J.
      One-carbon metabolism-genome interactions in folate-associated pathologies.
      ). Each of those pathways is reprogrammed in various cell types residing in the pulmonary artery and lungs of PH patients and animal models (
      • D'Alessandro A.
      • El Kasmi K.C.
      • Plecita-Hlavata L.
      • Jezek P.
      • Li M.
      • Zhang H.
      • Gupte S.A.
      • Stenmark K.R.
      Hallmarks of Pulmonary Hypertension: Mesenchymal and Inflammatory Cell Metabolic Reprogramming.
      ). Further, polyamine pathway metabolite has been associated with the development of monocrotaline-induced PH (
      • Olson J.W.
      • Hacker A.D.
      • Altiere R.J.
      • Gillespie M.N.
      Polyamines and the development of monocrotaline-induced pulmonary hypertension.
      ). Because these metabolic pathways are subject to G6PD/NADPH-dependent regulation (
      • Joshi S.R.
      • Kitagawa A.
      • Jacob C.
      • Hashimoto R.
      • Dhagia V.
      • Ramesh A.
      • Zheng C.
      • Zhang H.
      • Jordan A.
      • Waddell I.
      • Leopold J.
      • Hu C.J.
      • McMurtry I.F.
      • D'Alessandro A.
      • Stenmark K.R.
      • Gupte S.A.
      Hypoxic activation of glucose-6-phosphate dehydrogenase controls the expression of genes involved in the pathogenesis of pulmonary hypertension through the regulation of DNA methylation.
      ,
      • Kitagawa A.
      • Kizub I.
      • Jacob C.
      • Michael K.
      • D'Alessandro A.
      • Reisz J.A.
      • Grzybowski M.
      • Geurts A.M.
      • Rocic P.
      • Gupte R.
      • Miano J.M.
      • Gupte S.A.
      CRISPR-Mediated Single Nucleotide Polymorphism Modeling in Rats Reveals Insight Into Reduced Cardiovascular Risk Associated With Mediterranean G6PD Variant.
      ,
      • Reisz J.A.
      • Tzounakas V.L.
      • Nemkov T.
      • Voulgaridou A.I.
      • Papassideri I.S.
      • Kriebardis A.G.
      • D'Alessandro A.
      • Antonelou M.H.
      Metabolic Linkage and Correlations to Storage Capacity in Erythrocytes from Glucose 6-Phosphate Dehydrogenase-Deficient Donors.
      ) and expression of SAHH/SAHH2 and MAT2A is increased or showed increasing trend in WT but not in G6PDS188F variant rats, we assessed the activity of DNMT in the lungs of normoxic and SU/Hx/Nx rats. While there were no differences in the pulmonary DNMT activity in normoxic WT and G6PDS188F rats, DNMT activity was higher in the lungs of SU/Hx/Nx than normoxic WT rats but not normoxic G6PDS188F rats. This may be attributable to there being less dimethylglycine and spermine, metabolites of one carbon and polyamine pathway which produces substrate required for DNMT activity and DNA methylation, in the vascular tissue of G6PDS188F than WT rats (
      • Kitagawa A.
      • Kizub I.
      • Jacob C.
      • Michael K.
      • D'Alessandro A.
      • Reisz J.A.
      • Grzybowski M.
      • Geurts A.M.
      • Rocic P.
      • Gupte R.
      • Miano J.M.
      • Gupte S.A.
      CRISPR-Mediated Single Nucleotide Polymorphism Modeling in Rats Reveals Insight Into Reduced Cardiovascular Risk Associated With Mediterranean G6PD Variant.
      ), or pulmonary Dnmt1/DNMT1 and Dnmt3b/DNMT3B expression does not differ between SU/Hx/Nx and normoxic G6PDS188F rats. Although the mechanism by which G6PD regulates expression of Dnmts remains unclear, our results demonstrate that G6PD, which regulates one-carbon metabolism and polyamine pathway (
      • Joshi S.R.
      • Kitagawa A.
      • Jacob C.
      • Hashimoto R.
      • Dhagia V.
      • Ramesh A.
      • Zheng C.
      • Zhang H.
      • Jordan A.
      • Waddell I.
      • Leopold J.
      • Hu C.J.
      • McMurtry I.F.
      • D'Alessandro A.
      • Stenmark K.R.
      • Gupte S.A.
      Hypoxic activation of glucose-6-phosphate dehydrogenase controls the expression of genes involved in the pathogenesis of pulmonary hypertension through the regulation of DNA methylation.
      ,
      • Reisz J.A.
      • Tzounakas V.L.
      • Nemkov T.
      • Voulgaridou A.I.
      • Papassideri I.S.
      • Kriebardis A.G.
      • D'Alessandro A.
      • Antonelou M.H.
      Metabolic Linkage and Correlations to Storage Capacity in Erythrocytes from Glucose 6-Phosphate Dehydrogenase-Deficient Donors.
      ), controls the activity of metabolically driven DNMT enzymes. Undoubtedly, the loss-of-function G6PDS188F variant prevented SU/Hx/Nx-induced increases in Dnmt expression/activity and consequentially hypomethylated and, at least partially, contributed to the increased expression of Nos3 and Sod2 genes, and decreased methylation of Abra and Serpine1 genes, in SU/Hx/Nx-G6PDS188F rats as compared with SU/Hx/Nx-WT rats.
      Linker histone1H1A, which is found to be more prevalent in euchromatin regions, and histone1H1E, which is abundant in heterochromatin regions, of the nucleus increased and decreased, respectively, in SU/Hx/Nx-G6PDS188F rats. Since histone1H1E binds DNMT1 and DNMT3B and silencing of histone1H1E reduces DNA methylation (
      • Kalashnikova A.A.
      • Rogge R.A.
      • Hansen J.C.
      Linker histone H1 and protein-protein interactions.
      ), we suggest decreased histone1H1E expression likely contributed to DNA hypomethylation and increased transcription of Nos3 and Sod2 in SU/Hx/Nx-G6PDS188F rats. Chromatin modifications (histone acetylation and methylation) are emerging contributors to gene regulation that persistently activate cell phenotypes in PH (
      • Hu C.J.
      • Zhang H.
      • Laux A.
      • Pullamsetti S.S.
      • Stenmark K.R.
      Mechanisms contributing to persistently activated cell phenotypes in pulmonary hypertension.
      ). Expression of histone deacetylases, which remove acetylation from histones, appears to increase in PH (
      • Chelladurai P.
      • Boucherat O.
      • Stenmark K.
      • Kracht M.
      • Seeger W.
      • Bauer U.M.
      • Bonnet S.
      • Pullamsetti S.S.
      Targeting histone acetylation in pulmonary hypertension and right ventricular hypertrophy.
      ). We detected more acetylation of H3.1-K24 and methylation of H3.1-K80 in the lungs of SU/Hx/Nx-G6PDS188F rats. This indicates that, in addition to mediating modification of DNA methylation, decreased G6PD activity played a role in increasing histone acetylation and methylation, which opens the chromatin structure and activates transcription. Along those lines, we recently found inhibition of G6PD activity or silencing G6pd expression reduces HDAC5 expression and HDAC activity in vascular tissue (
      • Dhagia V.
      • Kitagawa A.
      • Jacob C.
      • Zheng C.
      • D'Alessandro A.
      • Edwards J.G.
      • Rocic P.
      • Gupte R.
      • Gupte S.A.
      G6PD activity contributes to the regulation of histone acetylation and gene expression in smooth muscle cells and to the pathogenesis of vascular diseases.
      ). Since methylation at Lys-80 is linked to gene activation (
      • Hake S.B.
      • Garcia B.A.
      • Duncan E.M.
      • Kauer M.
      • Dellaire G.
      • Shabanowitz J.
      • Bazett-Jones D.P.
      • Allis C.D.
      • Hunt D.F.
      Expression patterns and post-translational modifications associated with mammalian histone H3 variants.
      ,
      • Hyun K.
      • Jeon J.
      • Park K.
      • Kim J.
      Writing, erasing and reading histone lysine methylations.
      ), we suggest along with DNA hypomethylation, increased H3.1-K80me in the lungs of G6PDS188F rats, at least partially, contributed to prevent SU/Hx/Nx-induced decrease of proteins in the mitochondrial respiratory chain, cAMP-PKA related signaling, and SERCA pump. Additionally, several proteins that were upregulated (e.g., ARRB1 and ITPR2) in SU/Hx/Nx as compared with normoxic WT rats remained unchanged in SU/Hx/Nx as compared with normoxic G6PDS188F rats. Our proteomic approach confirmed proteins that are known to increase (e.g., glycolytic pathway proteins and complement proteins) or decrease (e.g., mitochondrial function proteins and SMC phenotype proteins) in PH and additionally identified changes, that were hitherto unknown, in the expression of proteins (cell signaling, serine proteases, RNA transcription and translation, and other pathways) in lungs of SU/Hx/Nx WT but not G6PDS188F rats. Changes in the expression of those proteins from the control levels likely contribute to the pulmonary artery contraction and remodeling associated with PH (
      • Culley M.K.
      • Chan S.Y.
      Mitochondrial metabolism in pulmonary hypertension: beyond mountains there are mountains.
      ,
      • Zhang S.
      • Patel H.H.
      • Murray F.
      • Remillard C.V.
      • Schach C.
      • Thistlethwaite P.A.
      • Insel P.A.
      • Yuan J.X.
      Pulmonary artery smooth muscle cells from normal subjects and IPAH patients show divergent cAMP-mediated effects on TRPC expression and capacitative Ca2+ entry.
      ,
      • Seeger W.
      • Hartmann R.
      • Neuhof H.
      • Bhakdi S.
      In-situ complement activation, pulmonary hypertension and vascular leakage in rabbit lungs--the role of the terminal complement complex.
      ,
      • Yadav V.R.
      • Song T.
      • Mei L.
      • Joseph L.
      • Zheng Y.M.
      • Wang Y.X.
      PLCgamma1-PKCepsilon-IP3R1 signaling plays an important role in hypoxia-induced calcium response in pulmonary artery smooth muscle cells.
      ). What’s more, SU/Hx/Nx-induced increases in RVSP, RVDP, and RV hypertrophy were less in G6PDS188F than WT rats. We therefore suggest that G6PDS188F through reducing DNA methylation, augmenting histone acetylation and methylation, and a yet unknown mechanism modifies the epigenomes of genome-wide genes to balance expression of genes encoding beneficial and pathogenic proteins, and make these rats less susceptible to SU/Hx/Nx-induced experimental PH.
      PH is a heterogenous and complex disease. It exhibits a dysregulated metabolic program, thrombosis, inflammation, and fibrosis. PH also exhibits cancer-like physiognomies, and hence some studies have characterized PH as cancer-like disease (
      • Culley M.K.
      • Chan S.Y.
      Mitochondrial metabolism in pulmonary hypertension: beyond mountains there are mountains.
      ,
      • Leopold J.A.
      • Maron B.A.
      Molecular Mechanisms of Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension.
      ). One of the hallmarks of cancer is alteration of DNA methylation resulting in increased expression of oncogenes and/or decreased expression of tumor suppressor genes (
      • Ehrlich M.
      DNA hypermethylation in disease: mechanisms and clinical relevance.
      ,
      • Samudio-Ruiz S.L.
      • Hudson L.G.
      Increased DNA methyltransferase activity and DNA methylation following Epidermal Growth Factor stimulation in ovarian cancer cells.
      ). Increased methylation frequently silences DNA repair genes and miRs involved in cancer biology (
      • Lakshminarasimhan R.
      • Liang G.
      The Role of DNA Methylation in Cancer.
      ). As in cancer, differential DNA methylation is associated with PH in experimental models and humans (
      • Joshi S.R.
      • Kitagawa A.
      • Jacob C.
      • Hashimoto R.
      • Dhagia V.
      • Ramesh A.
      • Zheng C.
      • Zhang H.
      • Jordan A.
      • Waddell I.
      • Leopold J.
      • Hu C.J.
      • McMurtry I.F.
      • D'Alessandro A.
      • Stenmark K.R.
      • Gupte S.A.
      Hypoxic activation of glucose-6-phosphate dehydrogenase controls the expression of genes involved in the pathogenesis of pulmonary hypertension through the regulation of DNA methylation.
      ,
      • Potus F.
      • Pauciulo M.W.
      • Cook E.K.
      • Zhu N.
      • Hsieh A.
      • Welch C.L.
      • Shen Y.
      • Tian L.
      • Lima P.
      • Mewburn J.
      • D'Arsigny C.L.
      • Lutz K.A.
      • Coleman A.W.
      • Damico R.
      • Snetsinger B.
      • Martin A.Y.
      • Hassoun P.M.
      • Nichols W.C.
      • Chung W.K.
      • Rauh M.J.
      • Archer S.L.
      Novel Mutations and Decreased Expression of the Epigenetic Regulator TET2 in Pulmonary Arterial Hypertension.
      ,
      • Archer S.L.
      • Marsboom G.
      • Kim G.H.
      • Zhang H.J.
      • Toth P.T.
      • Svensson E.C.
      • Dyck J.R.
      • Gomberg-Maitland M.
      • Thebaud B.
      • Husain A.N.
      • Cipriani N.
      • Rehman J.
      Epigenetic attenuation of mitochondrial superoxide dismutase 2 in pulmonary arterial hypertension: a basis for excessive cell proliferation and a new therapeutic target.
      ,
      • Ke X.
      • Johnson H.
      • Jing X.
      • Michalkiewicz T.
      • Huang Y.W.
      • Lane R.H.
      • Konduri G.G.
      Persistent pulmonary hypertension alters the epigenetic characteristics of endothelial nitric oxide synthase gene in pulmonary artery endothelial cells in a fetal lamb model.
      ,
      • Napoli C.
      • Benincasa G.
      • Loscalzo J.
      Epigenetic Inheritance Underlying Pulmonary Arterial Hypertension.
      ,
      • Potus F.
      • Ruffenach G.
      • Dahou A.
      • Thebault C.
      • Breuils-Bonnet S.
      • Tremblay E.
      • Nadeau V.
      • Paradis R.
      • Graydon C.
      • Wong R.
      • Johnson I.
      • Paulin R.
      • Lajoie A.C.
      • Perron J.
      • Charbonneau E.
      • Joubert P.
      • Pibarot P.
      • Michelakis E.D.
      • Provencher S.
      • Bonnet S.
      Downregulation of MicroRNA-126 Contributes to the Failing Right Ventricle in Pulmonary Arterial Hypertension.
      ,
      • Kitagawa A.
      • Jacob C.
      • Jordan A.
      • Waddell I.
      • McMurtry I.F.
      • Gupte S.A.
      Inhibition of Glucose-6-Phosphate Dehydrogenase Activity Attenuates Right Ventricle Pressure and Hypertrophy Elicited by VEGFR Inhibitor + Hypoxia.
      ). Our data which is consistent with the recent findings (
      • Joshi S.R.
      • Kitagawa A.
      • Jacob C.
      • Hashimoto R.
      • Dhagia V.
      • Ramesh A.
      • Zheng C.
      • Zhang H.
      • Jordan A.
      • Waddell I.
      • Leopold J.
      • Hu C.J.
      • McMurtry I.F.
      • D'Alessandro A.
      • Stenmark K.R.
      • Gupte S.A.
      Hypoxic activation of glucose-6-phosphate dehydrogenase controls the expression of genes involved in the pathogenesis of pulmonary hypertension through the regulation of DNA methylation.
      ,
      • Potus F.
      • Pauciulo M.W.
      • Cook E.K.
      • Zhu N.
      • Hsieh A.
      • Welch C.L.
      • Shen Y.
      • Tian L.
      • Lima P.
      • Mewburn J.
      • D'Arsigny C.L.
      • Lutz K.A.
      • Coleman A.W.
      • Damico R.
      • Snetsinger B.
      • Martin A.Y.
      • Hassoun P.M.
      • Nichols W.C.
      • Chung W.K.
      • Rauh M.J.
      • Archer S.L.
      Novel Mutations and Decreased Expression of the Epigenetic Regulator TET2 in Pulmonary Arterial Hypertension.
      ,
      • Kitagawa A.
      • Jacob C.
      • Jordan A.
      • Waddell I.
      • McMurtry I.F.
      • Gupte S.A.
      Inhibition of Glucose-6-Phosphate Dehydrogenase Activity Attenuates Right Ventricle Pressure and Hypertrophy Elicited by VEGFR Inhibitor + Hypoxia.
      ), suggest DNMT-catalyzed DNA methylation appears to be a critical regulator of genes involved in the development and progression of PH pathology. While previous studies suggest G6PDS188F variant makes no contribution to sickle cell anemia-associated PH, others suggest the frequency of G6PD mutation is higher among idiopathic PH patients than the general population (
      • Chen L.Y.
      • Lai E.J.
      • Collins D.R.
      • Ostrow D.N.
      • Sreenivasan G.M.
      A young woman with episodic angioedema, papilledema, and eosinophilia.
      ,
      • Dahoui H.A.
      • Hayek M.N.
      • Nietert P.J.
      • Arabi M.T.
      • Muwakkit S.A.
      • Saab R.H.
      • Bissar A.N.
      • Jumaa N.M.
      • Farhat F.S.
      • Dabbous I.A.
      • Bitar F.F.
      • Abboud M.R.
      Pulmonary hypertension in children and young adults with sickle cell disease: evidence for familial clustering.
      ,
      • Kurdyukov S.
      • Eccles C.A.
      • Desai A.A.
      • Gonzalez-Garay M.
      • Yuan J.X.
      • Garcia J.G.N.
      • Rafikova O.
      • Rafikov R.
      New cases of Glucose-6-Phosphate Dehydrogenase deficiency in Pulmonary Arterial Hypertension.
      ). A more recent study shows G6PD-deficient mice spontaneously develop a PH phenotype (
      • Varghese M.V.
      • James J.
      • Rafikova O.
      • Rafikov R.
      Glucose-6-phosphate dehydrogenase deficiency contributes to metabolic abnormality and pulmonary hypertension.
      ), while these G6PD-deficient mice develop less Hx-induced PH (
      • Joshi S.R.
      • Kitagawa A.
      • Jacob C.
      • Hashimoto R.
      • Dhagia V.
      • Ramesh A.
      • Zheng C.
      • Zhang H.
      • Jordan A.
      • Waddell I.
      • Leopold J.
      • Hu C.J.
      • McMurtry I.F.
      • D'Alessandro A.
      • Stenmark K.R.
      • Gupte S.A.
      Hypoxic activation of glucose-6-phosphate dehydrogenase controls the expression of genes involved in the pathogenesis of pulmonary hypertension through the regulation of DNA methylation.
      ). Our novel findings suggest that the loss-of-function Mediterranean G6PDS188F variant, which has 60-80% less activity in lungs (Fig. 2B) but not protein expression and develop less systemic hypertension (
      • Kitagawa A.
      • Kizub I.
      • Jacob C.
      • Michael K.
      • D'Alessandro A.
      • Reisz J.A.
      • Grzybowski M.
      • Geurts A.M.
      • Rocic P.
      • Gupte R.
      • Miano J.M.
      • Gupte S.A.
      CRISPR-Mediated Single Nucleotide Polymorphism Modeling in Rats Reveals Insight Into Reduced Cardiovascular Risk Associated With Mediterranean G6PD Variant.
      ), antagonize DNMT-catalyzed DNA methylation and pathogenic gene expression and, in the process, potentially reduce RV pressure overload/PH in SU/Hx/Nx rats.

      Experimental Procedures

      Animal models and experimental protocols

      All animal experiments were approved by the New York Medical College Animal Care and Use Committee, and all procedures conformed to the guidelines from the NIH Guide for the Care and Use of Laboratory Animals. To determine the expression and activity of DNA methylation writers and erasers in pulmonary hypertension samples, male Sprague-Dawley (SD) rats (350-750 gm) were randomly divided into normoxia (Nx; 21% O2; control); hypoxia (Hx: 10% O2; control); sugen5416+normoxia (Nx+SU) and sugen5416+hypoxia+normoxia (SU/Hx/Nx) groups. Rats in the SU+Nx group were placed in an ambient (21% O2) environment. SU/Hx/Nx rats received one subcutaneous injection of SU (20 mg/kg) and were then exposed to 3 weeks of Hx (10% O2) followed by 5 weeks of Nx (21% O2), as described previously (
      • Abe K.
      • Toba M.
      • Alzoubi A.
      • Ito M.
      • Fagan K.A.
      • Cool C.D.
      • Voelkel N.F.
      • McMurtry I.F.
      • Oka M.
      Formation of plexiform lesions in experimental severe pulmonary arterial hypertension.
      ,
      • Jiang B.
      • Deng Y.
      • Suen C.
      • Taha M.
      • Chaudhary K.R.
      • Courtman D.W.
      • Stewart D.J.
      Marked Strain-Specific Differences in the SU5416 Rat Model of Severe Pulmonary Arterial Hypertension.
      ). SU/Hx/Nx rats are preclinical models of PH (
      • Stenmark K.R.
      • Meyrick B.
      • Galie N.
      • Mooi W.J.
      • McMurtry I.F.
      Animal models of pulmonary arterial hypertension: the hope for etiological discovery and pharmacological cure.
      ). In addition, to determine the role of G6PD in the pathogenesis of SU/Hx/Nx-induced experimental PH in rats, we used loss-of-function Mediterranean G6PDS188F variant rats (350-750 g) recently generated in our laboratory and their WT littermates (
      • Kitagawa A.
      • Kizub I.
      • Jacob C.
      • Michael K.
      • D'Alessandro A.
      • Reisz J.A.
      • Grzybowski M.
      • Geurts A.M.
      • Rocic P.
      • Gupte R.
      • Miano J.M.
      • Gupte S.A.
      CRISPR-Mediated Single Nucleotide Polymorphism Modeling in Rats Reveals Insight Into Reduced Cardiovascular Risk Associated With Mediterranean G6PD Variant.
      ). Hemodynamic measurements were made, lungs were harvested, and blood samples were collected. Data analysis was performed in blinded fashion.

      Hemodynamic measurements

      All rats were anesthetized through inhalation of isoflurane (isoflurane, USP; 1-chloro-2,2,2-trifluoroethyl difluoro methyl ether; induced at 3% and maintained at 1.5%) and placed on a heated table. Closed-chest cardiac catheterization was performed using an MPVS Ultra Single Segment Pressure-Volume Unit (Millar Instruments, US) in combination with a cardiac catheter. RV systolic pressure (RVSP) and RV end-diastolic pressure (RVEDP) were measured after catheterization of the right ventricle via the right external jugular vein using a Millar Mikro-Tip catheter (Model SPR-671, tip size of 1.4F, Millar Instruments, US). Once steady-state hemodynamics were achieved, RV ventricular pressure-volume (P-V) loops were recorded and analyzed using LabChart 8 software (ADInstruments, US).

      Hematocrit measurements

      After hemodynamic measurements were completed, blood was collected from the cardiac chambers into a heparinized syringe. The heparinized blood was placed in capillary tubes, and hematocrit (%) was calculated as the length of the erythrocyte layer divided by the length of the entire blood sample.

      Assessment of RV hypertrophy

      Following the cardiac catheterization, the animals were euthanized by cervical dislocation, whole hearts were excised, and the RV free wall and left ventricle, including the ventricular septum (S), were separated, and weighed independently. Fulton’s index (right ventricle/left ventricle + S ratio) was calculated as an index of RV hypertrophy.

      Measurement of DNMT activity

      DNMT activity was measured using kits from Epigentek (USA). DNMT activity was measured in lysates prepared from lungs collected from rats.

      Quantitative real-time PCR

      Real-time PCR(RT-PCR) was used to analyze mRNA expression. Briefly, total RNA was extracted from lungs and human leukocytes using a Qiagen miRNEasy kit (Cat # 217004). The quality and concentration of the input RNA were measured on the Synergy HT Take3 Microplate Reader (BioTek, Winooski, VT) and cDNA was prepared using SuperScript IV. VILO Master Mix (Cat # 11756500, Invitrogen) for mRNA. Quantitative PCR (qPCR) was performed in duplicate using TaqManTM Fast Advanced Master Mix (Cat # 44-445-57) for mRNA using a Mx3000p Real-Time PCR System (Stratagene, Santa Clara, CA). The primers for the qPCR were purchased from Thermo Fisher Scientific/TaqMan. mRNA levels were normalized to internal control Tuba1a, and relative mRNA expression was reported.

      MS-PCR

      DNA methylation was analyzed using methylation-specific PCR (MS-PCR). Briefly, DNA was isolated from lungs (5 μg) using a FitAmpTM General Tissue Section DNA Isolation Kit (EpiGentek Cat. # Kit P-1003), after which the collected DNA was eluted in TE buffer in a total volume of 30 μl. The DNA was then quantified using a fluorescence quantification method, and 150-ng samples of the DNA were used to perform bisulfite conversion using the Methylamp DNA Bisulfite Conversion Kit (EpiGentek Cat. # P-1001). The conversion efficiency of bisulfite-treated DNA was determined with real time PCR using two primer pairs and control DNA. The first primer pair was against bisulfite-converted DNA (β-actin), while the second was against unconverted DNA (GAPDH) in the same bisulfite-treated DNA sample. The DNA was shown to be >98% converted. qPCR was performed in duplicate using 1-μl aliquots of modified DNA and gene-specific primers designed rat genes. Data analysis was performed using qPCR Ct values to calculate the % methylation index (MI) for each target region: % methylation index (MI) = [1 – (2(Ct M-Ct uM)/2(Ct M-Ct uM+1))]x100%. The MI measures the degree of methylation at a specific gene site; a higher MI indicates a higher degree of methylation. The MI for fully methylated DNA is defined as 100%. A relative comparison of the samples can be made based on the MI.

      Methylated DNA immunoprecipitation (MeDIP)

      In addition to MS-PCR, we performed methylated DNA immunoprecipitation-PCR to determine total DNA methylation status. We used quick EpiQuik Tissue Methylated DNA immunoprecipitation kit (Cat # P-2020) from EpiGentek, NY (USA) followed by PCR, according to the manufacturer’s protocol, and determined methylation of DNA.

      Western Blotting

      Rat lungs were homogenized, and cells lysed in lysis buffer (50 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 0.5% Nonidet P-40) containing EDTA-free complete protease and phosphatase inhibitor (Sigma-Aldrich/Roche). Protein levels were measured using Bradford assays (Bio-Rad), after which 35 μg samples were run on SDS-PAGE gels, transferred to nitrocellulose membranes, blocked with either 5% bovine serum albumin (BSA) or 4% milk and subsequently incubated with primary and secondary antibodies and detected using SuperSignal™ West Pico Chemiluminescent Substrate (cat# 34080, ThermoFisher Scientific) on iBright. Protein levels were determined through densitometric analysis using ImageJ software. The following previously validated primary antibodies were used: DNMT1 (Cell Signaling), DNMT3A (Cell Signaling), DNMT3B (Epigentek), MAT2A (Novus Biologicals), SAHH (Santacruz), NOS3 (Novus Biologicals), SOD2 (Novus Biologicals), SERPINE1 (Novus Biologicals), and ABRA (Novus biologicals). ACTB (Cell Signaling) was used as a loading control.

      Immunohistochemistry

      The superior and inferior lobe of the left lung was inflated with 1% neutral buffered formalin (NBF) in 0.5% agarose at 20 cm H2O pressure and fixed in 10% NBF as described previously (
      • Abe K.
      • Toba M.
      • Alzoubi A.
      • Ito M.
      • Fagan K.A.
      • Cool C.D.
      • Voelkel N.F.
      • McMurtry I.F.
      • Oka M.
      Formation of plexiform lesions in experimental severe pulmonary arterial hypertension.
      ). The fixed lung was blocked and embedded in paraffin, after which 5-μm sections were cut and probed for DNMT1 (Novus), DNMT3A (Epigentek), DNMT3B (Thermo Fisher) by immunohistochemistry and images were acquired on Nikon Eclipse 90-i microscope.

      Mass spectrometry-based proteomics

      Sample preparation

      Frozen samples were powdered in liquid nitrogen using a ceramic mortar and pestle and lyophilized. Aliquots of tissue (approximately 2 mg of each) were homogenized in freshly prepared high-salt buffer (50 mM Tris-HCl, 3 M NaCl, 25 mM EDTA, 0.25% w/v CHAPS, pH 7.5) containing 1x protease inhibitor (Halt Protease Inhibitor, Thermo Scientific) at a concentration of 10 mg/mL. Homogenization was accomplished in a bead beater (Bullet Blender Storm 24, Next Advance, 1 mm glass beads) for 3 min at 4 °C. Samples were then spun for 20 min at 18,000 x g at 4 °C, after which the supernatant removed and stored as Fraction 1. A fresh aliquot of high-salt buffer was added to the remaining pellet at 10 mg/mL of the starting weight, vortexed at 4 °C for 15 min, and spun for 15 min. That supernatant was removed and stored as Fraction 2. This high-salt extraction was repeated once more to generate Fraction 3, after which freshly prepared guanidine extraction buffer (6 M guanidinium chloride adjusted to pH 9.0 with NaOH) was added at 10 mg/mL and vortexed for 1 hr at room temperature. The samples were then spun for 15 min, after which the supernatant was removed and stored as Fraction 4. Fractions 1, 2, and 3 were combined, and all fractions were stored at -20 °C until further analysis.

      Sample digestion

      The samples were digested according to the filter aided sample preparation (FASP) protocol using a 10-kDa molecular weight cutoff filter. In brief, 200-μl samples were mixed in the filter unit with 8 M urea, 0.1 M ammonium bicarbonate (AB) pH 8.0, and centrifuged at 14,000 g for 15 min. The proteins were reduced with 10 mM DTT for 30 min at room temperature, centrifuged, and alkylated with 55 mM iodoacetamide for 30 min at room temperature in the dark. Following centrifugation, samples were washed 3 times with urea solution and 3 times with 50 mM AB, pH 8.0. Protein digestion was carried out with sequencing grade modified trypsin (Promega) at 1/50 protease/protein (wt/wt) ratio overnight at 37°C. Peptides were recovered from the filter using 50 mM AB. Samples were dried in a Speed-Vac and then desalted and concentrated on a Thermo Scientific Pierce C18 Tip.

      Mass-spectrometry

      Twenty microliters of each sample were loaded onto individual Evotips for desalting and then washed with 20 μl 0.1% FA followed by addition of 100 μl of storage solvent (0.1% FA) to keep the Evotips wet until analysis. An Evosep One system (Evosep, Odense, Denmark) was used to separate the peptides on a Pepsep column, (150 μm inner diameter, 15 cm) packed with ReproSil C18 1.9 μm, 120 Å resin. The system was coupled to a timsTOF Pro mass spectrometer (Bruker Daltonics, Bremen, Germany) via a nano-electrospray ion source (Captive Spray, Bruker Daltonics). The mass spectrometer was operated in PASEF mode. The ramp time was set to 100 ms and 10 PASEF MS/MS scans per topN acquisition cycle were acquired. MS and MS/MS spectra were recorded from m/z 100 to 1700. The ion mobility was scanned from 0.7 to 1.50 Vs/cm2. Precursors for data-dependent acquisition were isolated within ± 1 Th and fragmented with ion mobility-dependent collision energy, which was linearly increased from 20 to 59 eV in positive mode. Low-abundance precursor ions with an intensity above a threshold of 500 counts but below a target value of 20,000 counts were repeatedly scheduled and otherwise dynamically excluded for 0.4 min.

      Database Searching and Protein Identification

      MS/MS spectra were extracted from raw data files and converted into .mgf files using MS Convert (ProteoWizard, Ver. 3.0). Peptide spectral matching was performed with Mascot (Ver. 2.5) against the Uniprot rat database. Mass tolerances were ±15 ppm for parent ions, and ± 0.4 Da for fragmented ions. Trypsin specificity was used, allowing for 1 missed cleavage. Met oxidation, mono- and dimethylation (KR), trimethylation (K), protein N-terminal acetylation, acetylation (K) and peptide N-terminal pyroglutamic acid formation were set as variable modifications with Cys carbamidomethylation set as a fixed modification. Scaffold (version 4.8, Proteome Software, Portland, OR, USA) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least two identified unique peptides.

      Statistical analysis

      Statistical analysis was performed using GraphPad Prism 9 software. Values are presented as mean ± standard error (SE). Statistical comparisons of samples were made for two groups with Student’s t test. To make comparisons among more than two groups, two-way ANOVA followed by Sidak’s post hoc test for multiple comparisons was used. Values of P<0.05 were considered significant.

      Data Availability

      All data are contained within the manuscript and will be available upon written request to the corresponding author

      Conflict of Interest

      None

      Acknowledgements

      This study was supported by NIH RO1HL132574 (SAG) and AHA Grant-in-Aid 17GRNT33670454 (SAG).

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