PLoS ONE

PLoS One. 14(5): e0216218

Epigenetic control of the angiotensin-converting enzyme in endothelial cells during inflammation

Thomas Mudersbach, Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft¹², Daniel Siuda, Conceptualization, Formal analysis, Investigation, Methodology, Supervision¹², Karin Kohlstedt, Funding acquisition¹, http://orcid.org/0000-0003-1881-3635Ingrid Fleming, Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing¹²*

Osman El-Maarri, Editor

1. Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany

2. German Centre for Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany

University of Bonn, Institute of Experimental Hematology and Transfusion Medicine, GERMANY

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: fleming@em.uni-frankfurt.de

© 2019 Mudersbach et al

DOI: 10.1371/journal.pone.0216218

Published online: 1 May 2019

Abstract

The angiotensin-converting enzyme (ACE) plays a central role in the renin-angiotensin system, which is involved in the regulation of blood pressure. Alterations in ACE expression or activity are associated with various pathological phenotypes, particularly cardiovascular diseases. In human endothelial cells, ACE was shown to be negatively regulated by tumor necrosis factor (TNF) α. To examine, whether or not, epigenetic factors were involved in ACE expression regulation, methylated DNA immunoprecipitation and RNA interference experiments directed against regulators of DNA methylation homeostasis i.e., DNA methyltransferases (DNMTs) and ten-eleven translocation methylcytosine dioxygenases (TETs), were performed. TNFα stimulation enhanced DNA methylation in two distinct regions within the ACE promoter via a mechanism linked to DNMT3a and DNMT3b, but not to DNMT1. At the same time, TET1 protein expression was downregulated. In addition, DNA methylation decreased the binding affinity of the transcription factor MYC associated factor X to the ACE promoter. In conclusion, DNA methylation determines the TNFα-dependent regulation of ACE gene transcription and thus protein expression in human endothelial cells.

Introduction

The angiotensin-converting enzyme (ACE or CD143) is an ectoenzyme that plays a central role in the generation of the angiotensin II as well as in the degradation of bradykinin, and thus has consequences on the regulation of blood pressure [1] and vascular remodeling [2]. Alterations in endothelial ACE expression or activity are associated with inflammatory cardiovascular diseases, including diabetes [3] and atherosclerosis [4]. The consequences of ACE inhibition should not be attributed solely to its ability to cleave angiotensin I and bradykinin as ACE can regulate inflammation through mechanisms independent of angiotensin II synthesis and kinin breakdown. For example, the N–and C–catalytic domains of the ACE enzyme are also able to process substrates such as the anti-inflammatory tetrapeptide N-acetyl–seryl-asparyl-lysyl-proline [5]. Given these considerations it is rather intriguing that while ACE inhibitors are used to effectively treat vascular disease, endothelial cell activation in response to the inflammatory cytokine tumor necrosis factor (TNF) α, decreases rather than increases ACE expression [6].

ACE expression has been reported to increase in vascular smooth muscle cells following exposure to fluid shear stress [7] or pulsatile pressure changes [8]. The transcription factor Krüppel-like factor 2, which is activated by shear stress [9], has also been linked to ACE expression in endothelial cells as overexpression of the transcription factor decreases levels of the protein [10]. More recently, the consequences of shear stress on ACE expression were also linked to a mechanism involving the AMP–activated protein kinase, the phosphorylation of p53 and upregulation of miR-143/145 [11]. There is growing evidence that epigenetic alterations are involved in the pathological mechanisms of many chronic disorders. As a general rule, hypermethylation of a gene promoter CpG region tends to silence gene expression, while the CpG island hypomethylation results in the opposite response. Importantly, the human ACE gene promoter has been shown to harbor CpG islands [12], and CpG island hypomethylation of the ACE gene has been linked with fetal programming and the potential development of later disease [13]. Surprisingly little is known about how inflammatory stimuli can influence cellular ACE activity. Given that inflammation leads to global DNA hypermethylation and hence persistent changes in gene expression [14,15], the aim of this study was to assess the role of DNA methylation in the TNFα-dependent regulation of ACE expression in endothelial cells.

Materials and methods

Cell culture

Human umbilical endothelial cells were isolated and purified from veins and arteries of umbilical cords using VE-cadherin (CD144) antibody-coated magnetic beads from Dynal Biotech (Brown Deer/Wisconsin, USA) as described [16] (dx.doi.org/10.17504/protocols.io.ybdfsi6). Endothelial cells were cultured in MCDB131 medium from Gibco (Invitrogen, Karlsruhe, Germany) supplemented with 10 mmol/L L-glutamine, 8% fetal calf serum, 1 g/L NaHCO₃, 50 mg/L penicillin, 50 mg/L streptomycin, 0.1 μg/L epidermal growth factor, 1 μg/L fibroblast growth factor and 2 mL/500 mL endothelial cell growth supplement plus heparin from Promocell (Heidelberg, Germany, #C-30120). For all experiments, cells were used between passages 1 and 4. The use of human material in this study conforms to the principles outlined in the Declaration of Helsinki [17], and the isolation of endothelial cells was approved in written form by the ethic committee of the Goethe University.

Cell treatment

Human endothelial cells were treated with TNFα (PeproTech, Hamburg, Germany, #300-01A) for the times indicated in the results section. Preliminary concentration response experiments revealed 10 ng/mL TNFα as an optimal concentration with a maximum effect on ACE expression and low cell toxicity. For wash out experiments, cells were washed twice with basal MCDB131 medium and further cultured in normal growth medium (without TNFα). The TNFα receptor was inhibited by pretreatment with R-7050 (10 μmol/L) from Santa Cruz (Dallas/Texas, USA, CAS#303997-35-5) 30 minutes prior to stimulation. For the RNA stability assay, cells were pretreated with the RNA polymerase II inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB; 20 μg/mL; Cayman Chemical, Ann Arbor/Michigan, USA, CAS#53-85-0) two hours prior to stimulation. RG-108 (30 μmol/L; Active Motif, La Hulpe, Belgium, #14104) was used to inhibit DNMT. The p38 mitogen activated protein kinase inhibitor; SB202190 was from Calbiochem (Darmstadt, Germany).

Small interfering RNA (siRNA)

Cells were transfected at approximately 80% of confluence. siRNAs against DNMT1 (Ambion ID:110914; Thermo Fisher Scientific, Massachusetts, USA), DNMT3a (Ambion ID:s200426), DNMT3b (Ambion ID:111746) and TET1 (Ambion ID:147894) were used at a final concentration of 50 μmol/L. Transfection was carried out using Lipofectamine RNAiMAX from Invitrogen (Karlsruhe, Germany) according to the manufacturer’s instructions (dx.doi.org/10.17504/protocols.io.xtqfnmw).

Chromatin immunoprecipitation (ChIP)

DNA was extracted using the DNeasy Blood Mini Kit from Qiagen (Hilden, Germany). DNA was sheared in ice-cold water (20/20 rounds high/low shear of 10 seconds on/off cycles) using the Bioruptor plus from Diagenode (Seraing, Belgium) to achieve fragments of 200-1000 bp in length. ChIP assay was performed using a chromatin immunoprecipitation kit (Merck Millipore #17–10085; Darmstadt, Germany) according to the manufacturer’s instructions. The MYC associated factor X (MAX) antibody used to detect MAX-binding to DNA was from Abcam (Cambridge, UK). Changes in the DNA methylation status were measured by using a methylated DNA immunoprecipitation kit (Diagenode, Seraing, Belgium,). Immunoprecipitated DNA was analyzed by qPCR. ACE specific primers were designed to flank the MAX binding sites upstream of the transcription start site (from -1336 bp to -1135 bp; “upstream”) and within the CpG island (from +27 bp to +318 bp; “CpG”) as listed in Table 1. All samples were normalized to their corresponding input and solvent treated cells.

See full table Table 1. Primer sequences for qPCR.

Real-time quantitative PCR (RT-qPCR)

Total RNA was isolated using peqGOLD TriFast (VWR, Darmstadt, Germany) and 300 ng were reversely transcribed using SuperScript III reverse transcriptase (Life Technologies, Darmstadt, Germany). Real-time qPCR from equal amounts of cDNA was performed using Magnetic Induction Cycler from Biozym (Hessisch Oldendorf, Germany) and a SYBR Green master mix (Thermo Fisher Scientific, Massachusetts, USA). The relative expression levels of the different genes studied were calculated using the ΔΔCt method and normalized to the geometric mean of the housekeeping genes (HKG) glyceraldehyde-3-phosphate dehydrogenase, TATA-box binding protein and 18S ribosomal RNA in each sample. The primers used (Table 1) were from Biospring (Frankfurt, Germany).

Immunoblotting

Cells were lysed in Nonidet lysis buffer: 20 mmol/L Tris (pH 8.0), 1% Nonidet, 137 mmol/L NaCl, 25 mmol/L β-glycerophosphate, 2 mmol/L Na₄P₂O₇, 10% glycerol, freshly enriched with protease and phosphatase inhibitors. Equal amounts of protein were separated on SDS-PAGE and transferred to nitrocellulose membranes. After blocking for one hour in Roti-block from Carl Roth (Karlsruhe, Germany), membranes were incubated in primary antibody (4°C overnight), followed by detection using secondary antibody (1:20.000) coupled to horseradish peroxidase. Proteins were visualized by enhanced chemiluminescence.

Antibodies

For Western blotting all antibodies were diluted in Roti-block. The anti-ACE antibody (1:1000) was a kind gift from Dr. Peter Bünning (Aventis, Frankfurt). Anti-non-muscle myosin heavy chain (NMM) (ab75590, 1:2000; Cambridge, UK), anti-MAX (ab53570, 1:1000) and anti-DNMT1 (ab13537, 1:1000) were purchased from Abcam (Cambridge, UK), anti-TET1 (sc-293186, 1:500) was from Santa Cruz (Heidelberg, Germany), and the β-actin antibody (A5541, 1:5000) was from Sigma (Darmstadt, Germany).

Statistics

Unless otherwise indicated values are expressed as the mean ± SEM, and statistical evaluation was performed using Student’s t test for analysis between two groups containing normally distributed data and one-way ANOVA followed by the Newman-Keuls post-test or two-way ANOVA followed by the Bonferroni post-test for comparisons between multiple groups. All statistical calculations were performed using Prism 7 software (GraphPad Inc., La Jolla, California). Values of P<0.05 were considered statistically significant.

Results

Effect of TNFα on ACE expression in human endothelial cells

The treatment of human umbilical vein endothelial cells with TNFα for up to 72 hours resulted in the significant downregulation of ACE mRNA (Fig 1A) and protein (Fig 1B) levels. The effect was not restricted to venous endothelial cells as similar results were obtained using human umbilical artery endothelial cells (Fig 1C&1D). The observations could be directly attributed to TNFα, rather than an indirect mechanism as the TNFα receptor antagonist R-7050 abrogated the cytokine-induced downregulation of ACE mRNA (S1A Fig).

Fig 1View larger versionFig 1. Effect of TNFα on ACE mRNA and protein expression in human endothelial cells.(A) Time course of the effects of TNFα (10 ng/mL) on ACE mRNA levels in human umbilical vein endothelial cells relative to a triplet of housekeeping genes (HKG); n = 6 independent cell batches (two-way ANOVA/Bonferroni). (B) Time course of the effects of TNFα on ACE protein expression in human umbilical vein endothelial cells relative to non-muscle myosin heavy chain (NMM); n = 4–5 independent cell batches (one-way ANOVA/Newman-Keuls). (C&D) Expression of ACE mRNA (C) and protein (D) in human umbilical artery endothelial cells; n = 3 independent cell batches. ***P<0.001.

To determine whether or not TNFα destabilized ACE mRNA, cells were treated with TNFα in the absence and presence of the RNA-polymerase II inhibitor DRB. The turnover of ACE mRNA was relatively fast as eight hours after DRB treatment ACE mRNA levels were decreased by 32.7 ± 9.2% (S1B Fig). Stimulation with TNFα in addition to DRB had, however, no additional effect on ACE mRNA levels (decrease of 35.7 ± 7.4%). Given that TNFα was previously reported to attenuate ACE expression by a mechanism involving p38 mitogen-activated protein kinase (MAPK) [6], studies were performed using the p38 MAPK inhibitor, SB202190. However, p38 MAPK inhibition was without effect on the TNFα-induced downregulation of ACE in primary endothelial cells (S1C Fig), indicating that alternative mechanisms play a more dominant role in its regulation.

TNFα-induced changes at the ACE promoter region

To assess the recovery of ACE expression, endothelial cells were incubated with TNFα for 24 hours. Thereafter, the cells were washed and ACE expression was monitored for up to 72 hours. This procedure resulted in a slow recovery of ACE mRNA (Fig 2A) but not protein expression (Fig 2B). This delay in recovery was suggestive of regulation by epigenetic mechanisms.

Fig 2View larger versionFig 2. Involvement of epigenetic factors in TNFα-mediated changes in ACE expression.(A&B) Recovery (rec.) of ACE mRNA (A) and protein (B) in human endothelial cells after 24 hours TNFα (10 ng/mL) stimulation followed by washout for up to 72 hours; n = 6, relative to a triplet of housekeeping genes (HKG) (two-way ANOVA/Bonferroni, A) and n = 9, relative to non-muscle myosin heavy chain (NMM) (one-way ANOVA/Newman-Keuls, B). (C) Effect of TNFα (10 ng/mL, 24 hours) on DNA methylation and MAX binding to three distinct sites within the ACE promoter, quantified by chromatin immunoprecipitation (IP), n = 5–11 independent cell batches (Student’s t-test). *P<0.05, ***P<0.001.

The ACE promoter region (http://genome.ucsc.edu, GRCh37/hg19; ACE: NM_000789; S2A Fig) contains a CpG island, that spans from -612 base pairs (bp) to +605 bp, relative to the transcription start site (TSS), containing 139 CpG-sites, as well as two binding sites for the transcription factor MYC associated factor X (MAX). Chromatin immunoprecipitation (ChIP) experiments were performed to investigate TNFα-induced changes in the DNA methylation status of ACE and the binding of the transcription factor MAX, using appropriate antibodies. The primer pairs used were designed to bind to the precipitated DNA, covering the two MAX binding sites “upstream site” (from -1336 bp to -1135 bp upstream of the TSS) and the “CpG site” (+27 bp to +318 bp) within the ACE promoter region. Another set of primers covered the region from -6008 bp to -5888 bp relative to the TSS of the ACE gene and served as an internal control. TNFα stimulation increased cytosine methylation within the ACE promoter region at the upstream site, as well as in the CpG site, while the internal control was unaffected (Fig 2C). These changes in DNA methylation were associated with a decrease in MAX binding without any effect on global MAX expression (S2B Fig). These observations suggest that the methylation of the CpG islands in the ACE promoter modify ACE expression by directly affecting MAX binding.

Role of DNMTs in TNFα-mediated ACE expression

Since TNFα induced the DNA methylation of the ACE promoter, the consequences of TNFα on the expression of DNA methyltransferases (DNMT) were assessed. TNFα failed to alter the expression of DNMT1 (Fig 3A) or DNMT3a (Fig 3B), but induced a transient decrease in DNMT3b after four hours (Fig 3C). The pan-DNMT inhibitor; RG-108 (30 μmol/L), did not alter either the expression of ACE mRNA (Fig 3D) or protein (Fig 3E). However, RG-108 was designed to bind to the catalytic domain of DNMT1 and was only predicted to bind to the other two isoforms [18,19]. Therefore, to investigate the role of DNMT proteins in more detail the different family members (DNMT1, DNMT3a and DNMT3b) were downregulated using a siRNA approach (S3 Fig). While the downregulation of DNMT1 did not affect the TNFα-induced decrease in ACE expression (Fig 4A), the response was largely prevented by the downregulation of DNMT3a or DNMT3b (Fig 4B&4C). Thus, TNFα-induced changes in ACE expression are at least partly regulated by DNMT3a and DNMT3b.

Fig 3View larger versionFig 3. Expression of DNMTs in TNFα-treated human endothelial cells.(A-C) Time course of the effects of TNFα (10 ng/mL) on DNMT1 (A), DNMT3a (B) and DNMT3b (C) mRNA levels in human endothelial cells relative to a triplet of housekeeping genes (HKG); all n = 6 independent cell batches (two-way ANOVA/Bonferroni). (D-E) Effect of RG-108 (RG, 30 μmol/L) on the expression of ACE in human endothelial cells treated with solvent (Sol) or TNFα (10 ng/mL). Time course of ACE mRNA (D); n = 4 independent cell batches (two-way ANOVA/Bonferroni) and ACE protein (E) relative to non-muscle myosin heavy chain (NMM) 24 hours after treatment; n = 3 independent cell batches. **P<0.01, ***P<0.001.

Fig 4View larger versionFig 4. Involvement of DNMTs in TNFα-mediated alterations in ACE expression in human endothelial cells.(A) Consequences of DNMT1 downregulation (si1) on solvent (Sol) and TNFα (10 ng/mL, 24 hours)-induced changes in ACE mRNA relative to a triplet of housekeeping genes (HKG). A scrambled (Scr) oligonucleotide was used as control, n = 4–6 independent cell batches (two-way ANOVA/Bonferroni). (B&C) Consequences of DNMT3a (si3a) and 3b (si3b) downregulation on solvent (Sol) and TNFα (10 ng/mL, 24 hours)-induced changes in ACE mRNA (B) and protein (C) expression. A scrambled (Scr) oligonucleotide was used as control, n = 5–9 independent cell batches (two-way ANOVA/Bonferroni). *P<0.05, **P<0.01, ***P<0.001.

Involvement of TET enzymes in TNFα-dependent ACE expression

The process of DNA methylation is catalyzed by DNMT enzymes [20] but no single enzyme is able to directly remove the methyl group from the 5’-methylcytosine (5mC) [21]. Rather, demethylation is achieved via several intermediate steps and members of ten-eleven translocation methylcytosine dioxygenase (TET) family of proteins oxidize 5mC to 5’-hydroxymethylcytosine (5hmC) [22,23], which is genomically stable and opposes the actions of 5mC [24].

TNFα had marked effects on TET mRNA expression in endothelial cells and induced a decrease in TET1 (Fig 5A), was largely without effect on TET2 (Fig 5B), but increased TET3 (Fig 5C). Because TET enzymes reduce 5mC levels, a decrease in TET1 expression can stabilize 5mC. Indeed, the downregulation of TET1 in endothelial cells elicited a decrease in ACE protein levels (Fig 5D). Consistent with a previous report that decreased TET1 activity attenuated 5hmC in cyclooxygenase 2 and interleukin 6 [25], the expression on both genes increased following stimulation with TNFα or siRNA directed against TET1 (Fig 5E&5F).

Fig 5View larger versionFig 5. TET1-dependency of TNFα-mediated ACE downregulation in human endothelial cells.(A-C) Time course of the effects of TNFα (10 ng/mL) on TET1 (A), TET2 (B) and TET3 (C) mRNA levels in human endothelial cells relative to a triplet of housekeeping genes (HKG); all n = 5–6 independent cell batches (two-way ANOVA/Bonferroni). (D) RNA interference experiments (50 μmol/L) against TET1 (siT1). A scrambled (Scr) oligonucleotide was used as control. Protein expression levels of TET1 (left panel) and ACE (right panel) versus non-muscle myosin heavy chain (NMM); n = 4-5 independent cell batches (Student’s t-test). (E-F) Effect of TNFα (10 ng/mL, 24 hours, left panel) and siRNA against TET1 (50 μmol/L, right panel) on the expression of cyclooxygenase-2 (COX-2) mRNA (E) and interleukin-6 (IL-6) mRNA (F) versus HKG; n = 4–5 independent cell batches (Student’s t-test). *P<0.05, **P<0.01, ***P<0.001.

Discussion

ACE inhibitors have potent clinical effects and anti-inflammatory effects on the vascular wall [26]. Indeed, while endothelial cell activation is generally associated with enhanced ACE expression it is not generally appreciated that cytokines, such as TNFα, actually decrease ACE expression [6]. The results of the present study revealed that TNFα elicited the downregulation of ACE mRNA and protein in human endothelial cells by a mechanism at least partially regulated by DNMT3a, DNMT3b and TET1, and linked to increased methylation of the ACE promoter and decreased binding of the transcription factor MAX.

Previous reports have implicated nuclear factor κB and the p38 MAPK signaling pathway, in the regulation of endothelial cell ACE expression by TNFα [6]. However, by closely studying the time course of the TNFα-induced decrease in ACE expression as well as its recovery over 72 hours, it was evident that neither p38 MAPK nor mRNA destabilization processes played a role in the phenomenon observed. Rather, epigenetic regulatory mechanisms determined ACE levels in endothelial cells. Methylated DNA immunoprecipitation experiments confirmed the TNFα-dependent increase in 5mC content within the ACE promoter.

CpG islands are CG-rich regions with a CG content over 50% and an expected to observed CG ratio over 60%, covering a distance of at least 200 bp [27,28], and are often enriched at promoter sites and 5’-untranslated regions [26,29]. In general, promoter hypermethylation results in condensed chromatin structures and hence reduced transcription rates [30], largely due to hindered transcription factor binding. In this study the transcription factor MAX was the main focus as the ACE promoter region contains a CpG island that harbors two MAX binding sites. The latter transcription factor functions as an adapter protein for MAX dimerization protein (MAD) family members and binds the E-box sequence CANNTG [31,32]. Given that the binding affinity of MAX to DNA is highly sensitive to alterations in 5mC [32], and some MAD proteins, e.g. MAD1 and MAX gene-associated protein, are known to be regulated by short term (<24 hours) TNFα stimulation in human endothelial cells [33–35], it was tempting to speculate that the methylation of CpG-sites in the ACE promoter directly affected MAX binding. This turned out to be the case, as ChIP experiments revealed a decrease in the binding of MAX to those portions of the ACE promoter in which DNA methylation increased in response to TNFα stimulation. Ours is not the first study to suggest a role for DNA methylation in the regulation of ACE expression as DNA hypomethylation in fetal murine brains exposed to protein malnutrition in utero was previously linked with increased ACE mRNA expression [36]. Also, low birth weight in humans was linked to a decrease in the ACE methylation levels coupled with higher ACE activity [13]. Indeed, a significant inverse correlation was evident between the degree of DNA methylation and both ACE activity and systolic blood pressure levels [13]. ACE promoter hypermethylation in patients with clinical depression (which has been associated with cardiovascular disease), on the other hand, was linked with decreased ACE mRNA levels [37]. Although the latter observation has recently been questioned, it now seems that ACE genetic variants (seven polymorphisms across the ACE gene were studied) influence methylation and that ACE methylation is inversely correlated with cortisol levels [38]. The DNMT family of proteins (DNMT1, DNMT3a and DNMT3b), catalyze the transfer of a methyl group to a CpG-site. While DNMT1 preferably binds hemi-methylated DNA, DNMT3a and DNMT3b generally bind to non-methylated DNA and lead to de novo DNA methylation (for review see [26]). The siRNA-mediated downregulation of DNMT3a and DNMT3b largely prevented the TNFα-induced decrease in ACE mRNA and protein, while DNMT1, which is mainly responsible for the maintenance of DNA methylation, failed to affect ACE levels. Methylated CpG islands are very stable epigenetic marks and can be counterbalanced by the TET enzymes so that a decrease of TET expression can stabilize 5mC levels. Not only were DNMT3a and DNMT3b implicated in the response to TNFα, but cell stimulation also elicited a decrease in TET1, indicating that ACE levels were regulated by the reciprocal regulation of DNMT3a/b and TET1. While detailed data is not available for endothelial cells, a direct link between cytokine treatment, decreased TET1 activity and the loss of 5hmC marks has been described for a subset of genes in human chondrocytes [25], including cyclooxygenase 2 and interleukin 6. Importantly, the expression of both of these proteins was also increased in human endothelial cells following TET1 downregulation.

Taken together, the present study highlights the role of DNA methylation in the regulation of ACE expression in human endothelial cells following inflammatory activation. Given the positive effects of ACE inhibitors in the prevention and treatment of cardiovascular disease, it seems counterintuitive that ACE expression decreases in endothelial cells activated by inflammatory stimuli. However, ACE downregulation may, at least initially, help to dampen the inflammatory response by attenuating the metabolism of vasoprotective/anti-inflammatory ACE substrates, such as bradykinin and N-acetyl–seryl-asparyl-lysyl-proline at the same time as preventing the generation of angiotensin II. However, a similar response in the bone marrow would be expected to increase the mobilization of bone marrow-derived hematopoietic progenitor cells [39], may elicit the opposite effect and accelerate vascular inflammation and cardiovascular disease.

While ACE expression and activity is concentrated in endothelial cells in the lung and kidney, it is important to note that the ACE protein can be detected in several different cell types. These include, vascular smooth muscle cells, adipose tissue, leukocytes, macrophages and monocytes [40] as well as in cells lining the endosteal bone [39], primitive human embryonic hemangioblasts [41] and hematopoietic stem cells in both fetal and adult hematopoietic tissues [42]. In many cases, particularly in monocyte-derived macrophages [43] and vascular smooth muscle cells [44], it seems that the degree of cell differentiation/dedifferentiation markedly affects ACE levels. Although monocytes express ACE, and ACE expression increases during differentiation to macrophages [43], the functional consequences of enzyme regulation are unclear. Even though murine macrophages (in contrast to human macrophages) express only low levels of ACE [45], studies with transgenic mice genetically engineered to express high levels of the enzyme in macrophages, revealed an exaggerated inflammatory response that can limit melanoma growth [46]. A recent study in which the N- and C-catalytic domains of ACE were deleted revealed that TNFα blockade reverted the phenotype of ACE overexpressing macrophages lacking the N-terminal catalytic site to that of wild-type macrophages [47]. Given that TNFα is important for the classical activation of monocytes it would be interesting to compare the role of methylation and MAX in regulating ACE expression in endothelial cells and monocytes. It is certainly tempting to speculate that the TNFα generated during the classical activation of monocyte-derived macrophages may act to curtail the inflammatory response the promote cell repolarization to the alternatively activated phenotype that is associated with the resolution of inflammation.

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Acknowledgments

The authors are indebted to Isabel Winter and Marie von Reutern for expert technical assistance.

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References

Paul M, Mehr AP, Kreutz R (2006) Physiology of local renin-angiotensin systems. Physiol Rev 86: 747–803. 10.1152/physrev.00036.2005 16816138

Heeneman S, Sluimer JC, Daemen Mat JAP (2007) Angiotensin-converting enzyme and vascular remodeling. Circ Res 101: 441–454. 10.1161/CIRCRESAHA.107.148338 17761934

Mercier K, Smith H, Biederman J (2014) Renin-angiotensin-aldosterone system inhibition: overview of the therapeutic use of angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, mineralocorticoid receptor antagonists, and direct renin inhibitors. Prim Care 41: 765–778. 10.1016/j.pop.2014.08.002 25439533

Husain K, Hernandez W, Ansari RA, Ferder L (2015) Inflammation, oxidative stress and renin angiotensin system in atherosclerosis. World J Biol Chem 6: 209–217. 10.4331/wjbc.v6.i3.209 26322175

Eriguchi M, Bernstein EA, Veiras LC, Khan Z, Cao DY, Fuchs S et al (2018) The absence of the ACE N-domain decreases renal inflammation and facilitates sodium excretion during diabetic kidney disease. J Am Soc Nephrol 29: 2546–2561. 10.1681/ASN.2018030323 30185469

Saijonmaa O, Nyman T, Fyhrquist F (2001) Downregulation of angiotensin-converting enzyme by tumor necrosis factor-α and interleukin-1β in cultured human endothelial cells. J Vasc Res 38: 370–378. 10.1159/000051068 11455208

Gosgnach W, Challah M, Coulet F, Michel JB, Battle T (2000) Shear stress induces angiotensin converting enzyme expression in cultured smooth muscle cells: possible involvement of bFGF. Cardiovasc Res 45: 486–492. 10.1016/S0008-6363(99)00269-2 10728370

Iizuka K, Machida T, Kawaguchi H, Hirafuji M (2008) Pulsatile mechanical pressure promotes angiotensin-converting enzyme expression in aortic smooth muscle cells. Cardiovasc Drugs Ther 22: 383–390. 10.1007/s10557-008-6118-7 18679784

Dekker RJ, van Soest S, Fontijn RD, Salamanca S, de Groot PG, VanBavel E et al (2002) Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Krüppel-like factor (KLF2). Blood 100: 1689–1698. 10.1182/blood-2002-01-0046 12176889

Dekker RJ, van Thienen JV, Rohlena J, de Jager SC, Elderkamp YW, Seppen J et al (2005) Endothelial KLF2 links local arterial shear stress levels to the expression of vascular tone-regulating genes. Am J Pathol 167: 609–618. 10.1016/S0002-9440(10)63002-7 16049344

Kohlstedt K, Trouvain C, Boettger T, Shi L, Fisslthaler B, Fleming I (2013) AMP-activated protein kinase regulates endothelial cell angiotensin-converting enzyme expression via p53 and the post-transcriptional regulation of microRNA-143/145. Circ Res 112: 1150–1158. 10.1161/CIRCRESAHA.113.301282 23476055

Rivière G, Lienhard D, Andrieu T, Vieau D, Frey BM, Frey FJ (2011) Epigenetic regulation of somatic angiotensin-converting enzyme by DNA methylation and histone acetylation. Epigenetics 6: 478–489. 10.4161/epi.6.4.14961 21364323

Rangel M, dos Santos JC, Ortiz PH, Hirata M, Jasiulionis MG, Araujo RC et al (2014) Modification of epigenetic patterns in low birth weight children: importance of hypomethylation of the ACE gene promoter. PLoS ONE 9: e106138 10.1371/journal.pone.0106138 25170764

Kang GH, Lee HJ, Hwang KS, Lee S, Kim JH, Kim JS (2003) Aberrant CpG island hypermethylation of chronic gastritis, in relation to aging, gender, intestinal metaplasia, and chronic inflammation. Am J Pathol 163: 1551–1556. 10.1016/S0002-9440(10)63511-0 14507661

Jang TJ, Kim DI, Shin YM, Chang HK, Yang CH (2001) p16(INK4a) Promoter hypermethylation of non-tumorous tissue adjacent to gastric cancer is correlated with glandular atrophy and chronic inflammation. Int J Cancer 93: 629–634. 10.1002/ijc.1394 11477571

Bess E, Fisslthaler B, Frömel T, Fleming I (2011) Nitric oxide-induced activation of the AMP-activated protein kinase α2 subunit attenuates IκB kinase activity and inflammatory responses in endothelial cells. PLoS ONE 6: e20848 10.1371/journal.pone.0020848 21673972

World Medical Association (2013) World Medical Association Declaration of Helsinki. Ethical principles for medical research involving human subjects. JAMA 310: 2191–2194. 10.1001/jama.2013.281053 24141714

Holloway AF, Oakford PC (2007) Targeting epigenetic modifiers in cancer. Curr Med Chem 14: 2540–2547. 10.2174/092986707782023271 17979706

Brueckner B, Garcia BR, Siedlecki P, Musch T, Kliem HC, Zielenkiewicz P et al (2005) Epigenetic reactivation of tumor suppressor genes by a novel small-molecule inhibitor of human DNA methyltransferases. Cancer Res 65: 6305–6311. 10.1158/0008-5472.CAN-04-2957 16024632

Hermann A, Gowher H, Jeltsch A (2004) Biochemistry and biology of mammalian DNA methyltransferases. Cell Mol Life Sci 61: 2571–2587. 10.1007/s00018-004-4201-1 15526163

Bhutani N, Burns DM, Blau HM (2011) DNA demethylation dynamics. Cell 146: 866–872. 10.1016/j.cell.2011.08.042 21925312

He YF, Li BZ, Li Z, Liu P, Wang Y, Tang Q et al (2011) Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333: 1303–1307. 10.1126/science.1210944 21817016

Ito S, Shen L, Dai Q, Wu SC, Collins LB, Swenberg JA et al (2011) Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333: 1300–1303. 10.1126/science.1210597 21778364

Bachman M, Uribe-Lewis S, Yang X, Williams M, Murrell A, Balasubramanian S (2014) 5-Hydroxymethylcytosine is a predominantly stable DNA modification. Nat Chem 6: 1049–1055. 10.1038/nchem.2064 25411882

Haseeb A, Makki MS, Haqqi TM (2014) Modulation of ten-eleven translocation 1 (TET1), Isocitrate dehydrogenase (IDH) expression, α-ketoglutarate (α-KG), and DNA hydroxymethylation levels by interleukin-1β in primary human chondrocytes. J Biol Chem 289: 6877–6885. 10.1074/jbc.M113.512269 24469454

Maunakea AK, Chepelev I, Zhao K (2010) Epigenome mapping in normal and disease states. Circ Res 107: 327–339. 10.1161/CIRCRESAHA.110.222463 20689072

Gardiner-Garden M, Frommer M (1987) CpG Islands in vertebrate genomes. J Mol Biol 196: 261–282. 10.1016/0022-2836(87)90689-9 3656447

Simmons R (2011) Epigenetics and maternal nutrition: nature v. nurture. 2010/11/29: 73–81. 10.1017/S0029665110003988 21110912

Suzuki MM, Bird A (2008) DNA methylation landscapes: provocative insights from epigenomics. Nat Rev Genet 9: 465–476. 10.1038/nrg2341 18463664

Hardwick JS, Lane AN, Brown T (2018) Epigenetic modifications of cytosine: biophysical properties, regulation, and function in mammalian DNA. BioEssays 40: 1700199 10.1002/bies.201700199 29369386

Blackwood EM, Eisenman RN (1991) Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science 251: 1211–1217. 10.1126/science.2006410 2006410

Wang D, Hashimoto H, Zhang X, Barwick BG, Lonial S, Boise LH et al (2016) MAX is an epigenetic sensor of 5-carboxylcytosine and is altered in multiple myeloma. Nucleic Acids Res. 10.1093/nar/gkw1184 27903915

Wada Y, Ohta Y, Xu M, Tsutsumi S, Minami T, Inoue K et al (2009) A wave of nascent transcription on activated human genes. Proc Natl Acad Sci USA 106: 18357–18361. 10.1073/pnas.0902573106 19826084

Rajashekhar G, Grow M, Willuweit A, Patterson CE, Clauss M (2007) Divergent and convergent effects on gene expression and function in acute versus chronic endothelial activation. Physiol Genomics 31: 104–113. 10.1152/physiolgenomics.00157.2006 17566077

Garcia-Conesa MT, Tribolo S, Guyot S, Tomas-Barberan FA, Kroon PA (2009) Oligomeric procyanidins inhibit cell migration and modulate the expression of migration and proliferation associated genes in human umbilical vascular endothelial cells. Mol Nutr Food Res 53: 266–276. 10.1002/mnfr.200800134 18979505

Goyal R, Goyal D, Leitzke A, Gheorghe CP, Longo LD (2010) Brain renin-angiotensin system: fetal epigenetic programming by maternal protein restriction during pregnancy. Reprod Sci 17: 227–238. 10.1177/1933719109351935 19923380

Zill P, Baghai TC, Schule C, Born C, Frustuck C, Buttner A et al (2012) DNA methylation analysis of the angiotensin converting enzyme (ACE) gene in major depression. PLoS ONE 7: e40479 10.1371/journal.pone.0040479 22808171

Lam D, Ancelin ML, Ritchie K, Saffery R, Ryan J (2018) DNA methylation and genetic variation of the angiotensin converting enzyme (ACE) in depression. Psychoneuroendocrinology 88: 1–8. 10.1016/j.psyneuen.2017.11.003 29132028

Kohlstedt K, Trouvain C, Frömel T, Mudersbach T, Henschler R, Fleming I (2018) Role of the angiotensin-converting enzyme in the G-CSF-induced mobilization of progenitor cells. Basic Res Cardiol 113: 18 10.1007/s00395-018-0677-y 29549541

Das UN (2005) Is angiotensin-II an endogenous pro-inflammatory molecule? Med Sci Monit 11: RA155–RA162. 15874906

Zambidis ET, Soon Park T, Yu W, Tam A, Levine M, Yuan X et al (2008) Expression of angiotensin-converting enzyme (CD143) identifies and regulates primitive hemangioblasts derived from human pluripotent stem cells. Blood 112: 3601–3614. 10.1182/blood-2008-03-144766 18728246

Jokubaitis VJ, Sinka L, Driessen R, Whitty G, Haylock DN, Bertoncello I et al (2008) Angiotensin-converting enzyme (CD143) marks hematopoietic stem cells in human embryonic, fetal and adult hematopoietic tissues. Blood 111: 4055–4063. 10.1182/blood-2007-05-091710 17993616

Danilov SM, Sadovnikova E, Scharenborg N, Balyasnikova IV, Svinareva DA, Semikina EL et al (2003) Angiotensin-converting enzyme (CD143) is abundantly expressed by dendritic cells and discriminates human monocyte-derived dendritic cells from acute myeloid leukemia-derived dendritic cells. Exp Hematol 31: 1301–1309. 10.1016/j.exphem.2003.08.018 14662338

Boettger T, Beetz N, Kostin S, Schneider J, Kruger M, Hein L et al (2009) Acquisition of the contractile phenotype by murine arterial smooth muscle cells depends on the miR143/145 gene cluster. J Clin Invest 119: 2634–2647. 10.1172/JCI38864 19690389

Balyasnikova IV, Sun ZL, Metzger R, Taylor PR, Vicini E, Muciaccia B et al (2006) Monoclonal antibodies to native mouse angiotensin-converting enzyme (CD143): ACE expression quantification, lung endothelial cell targeting and gene delivery. Tissue Antigens 67: 10–29. 10.1111/j.1399-0039.2005.00516.x 16451197

Shen XZ, Li P, Weiss D, Fuchs S, Xiao HD, Adams JA et al (2007) Mice with enhanced macrophage angiotensin-converting enzyme are resistant to melanoma. Am J Pathol 170: 2122–2134. 10.2353/ajpath.2007.061205 17525278

Khan Z, Cao D-Y, Giani JF, Bernstein EA, Veiras LC, Fuchs S et al (2019) Overexpression of the C-domain of angiotensin-converting enzyme reduces melanoma growth by stimulating M1 macrophage polarization. J Biol Chem 294: 4368–4380. 10.1074/jbc.RA118.006275 30670595

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Table 1. Primer sequences for qPCR.

Primer |  | Sequences
ChIP_ACE-upstream | forward | 5‘-CGGCCAGTGTTTAACAAGGC-3‘
reverse | 5‘-GCTCCAAGTCACCTCAGGTC-3‘
ChIP_ACE-CpG | forward | 5‘-GCCTGCAAGACACCTAAGGTC-3‘
reverse | 5‘-TTCCTCCTCCGCTCCAGAG-3‘
ChIP_ACE-6kb up control | forward | 5‘-GATCACACCACTGCACTCCA-3‘
reverse | 5‘-GGAAACTAGGGCTGCTTCGT-3‘
18S | forward | 5‘-CTTTGGTCGCTCGCTCCTC-3‘
reverse | 5‘-CTGACCGGGTTGGTTTTGAT-3‘
ACE | forward | 5‘-AATTGCCTTCCTGCCCTTTG-3‘
reverse | 5‘-TACCACCAGTCGAAGTTGTAGC-3‘
COX-2 | forward | 5‘-GCCCGACTCCCTTGGGTGTC-3‘
reverse | 5‘-CTGATGCGTGAAGTGCTGGGCA-3‘
DNMT1 | forward | 5‘-TGCCAGCTGAGCGTGGTGGT-3‘
reverse | 5‘-GCATGCGGGCAGCCACCAAT-3‘
DNMT3a | forward | 5‘-CCGGAACATTGAGGACATCT-3‘
reverse | 5‘-CAGCAGATGGTGCAGTAGGA-3‘
DNMT3b | forward | 5‘-GCAAAGACCGAGGGGATGAA-3‘
reverse | 5‘-CTGCCACAAGACAAACAGCC-3‘
GAPDH | forward | 5‘-ATGACATCAAGAAGGTGGTG-3‘
reverse | 5‘-CATACCAGGAAATGAGCTTG-3‘
IL-6 | forward | 5‘-GCCTTCGGTCCAGTTGCCTT-3‘
reverse | 5‘-GCAGAATGAGATGAGTTGTC-3‘
MAX | forward | 5‘-TCAGCAAAATGGTCCTGTGG-3‘
reverse | 5‘-AAGGTTGTAAGGCCAGAGTCAG-3‘
TATA-box binding protein | forward | 5‘-CACGAACCACGGCACTGATT-3‘
reverse | 5‘-TTTTCTTGCTGCCAGTCTGGAC-3‘
TET1 | forward | 5‘-TCTGTTGTTGTGCCTCTGGA-3‘
reverse | 5‘-CCCATGACCACATCTACTGT-3‘
TET2 | forward | 5‘-AGCAATAGGACATCCCTGAG-3‘
reverse | 5‘-CATCTAGGAGCAGGTCCTAA-3‘
TET3 | forward | 5‘-CGGATCGAGAAGGTCATCTA-3‘
reverse | 5‘-ATGACGATCACAGCGTTCTG-3‘

DOI: 10.1371/journal.pone.0216218.t001

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Fig 1. Effect of TNFα on ACE mRNA and protein expression in human endothelial cells.

(A) Time course of the effects of TNFα (10 ng/mL) on ACE mRNA levels in human umbilical vein endothelial cells relative to a triplet of housekeeping genes (HKG); n = 6 independent cell batches (two-way ANOVA/Bonferroni). (B) Time course of the effects of TNFα on ACE protein expression in human umbilical vein endothelial cells relative to non-muscle myosin heavy chain (NMM); n = 4–5 independent cell batches (one-way ANOVA/Newman-Keuls). (C&D) Expression of ACE mRNA (C) and protein (D) in human umbilical artery endothelial cells; n = 3 independent cell batches. ***P<0.001.

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Fig 2. Involvement of epigenetic factors in TNFα-mediated changes in ACE expression.

(A&B) Recovery (rec.) of ACE mRNA (A) and protein (B) in human endothelial cells after 24 hours TNFα (10 ng/mL) stimulation followed by washout for up to 72 hours; n = 6, relative to a triplet of housekeeping genes (HKG) (two-way ANOVA/Bonferroni, A) and n = 9, relative to non-muscle myosin heavy chain (NMM) (one-way ANOVA/Newman-Keuls, B). (C) Effect of TNFα (10 ng/mL, 24 hours) on DNA methylation and MAX binding to three distinct sites within the ACE promoter, quantified by chromatin immunoprecipitation (IP), n = 5–11 independent cell batches (Student’s t-test). *P<0.05, ***P<0.001.

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Fig 3. Expression of DNMTs in TNFα-treated human endothelial cells.

(A-C) Time course of the effects of TNFα (10 ng/mL) on DNMT1 (A), DNMT3a (B) and DNMT3b (C) mRNA levels in human endothelial cells relative to a triplet of housekeeping genes (HKG); all n = 6 independent cell batches (two-way ANOVA/Bonferroni). (D-E) Effect of RG-108 (RG, 30 μmol/L) on the expression of ACE in human endothelial cells treated with solvent (Sol) or TNFα (10 ng/mL). Time course of ACE mRNA (D); n = 4 independent cell batches (two-way ANOVA/Bonferroni) and ACE protein (E) relative to non-muscle myosin heavy chain (NMM) 24 hours after treatment; n = 3 independent cell batches. **P<0.01, ***P<0.001.

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Fig 4. Involvement of DNMTs in TNFα-mediated alterations in ACE expression in human endothelial cells.

(A) Consequences of DNMT1 downregulation (si1) on solvent (Sol) and TNFα (10 ng/mL, 24 hours)-induced changes in ACE mRNA relative to a triplet of housekeeping genes (HKG). A scrambled (Scr) oligonucleotide was used as control, n = 4–6 independent cell batches (two-way ANOVA/Bonferroni). (B&C) Consequences of DNMT3a (si3a) and 3b (si3b) downregulation on solvent (Sol) and TNFα (10 ng/mL, 24 hours)-induced changes in ACE mRNA (B) and protein (C) expression. A scrambled (Scr) oligonucleotide was used as control, n = 5–9 independent cell batches (two-way ANOVA/Bonferroni). *P<0.05, **P<0.01, ***P<0.001.

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Fig 5. TET1-dependency of TNFα-mediated ACE downregulation in human endothelial cells.

(A-C) Time course of the effects of TNFα (10 ng/mL) on TET1 (A), TET2 (B) and TET3 (C) mRNA levels in human endothelial cells relative to a triplet of housekeeping genes (HKG); all n = 5–6 independent cell batches (two-way ANOVA/Bonferroni). (D) RNA interference experiments (50 μmol/L) against TET1 (siT1). A scrambled (Scr) oligonucleotide was used as control. Protein expression levels of TET1 (left panel) and ACE (right panel) versus non-muscle myosin heavy chain (NMM); n = 4-5 independent cell batches (Student’s t-test). (E-F) Effect of TNFα (10 ng/mL, 24 hours, left panel) and siRNA against TET1 (50 μmol/L, right panel) on the expression of cyclooxygenase-2 (COX-2) mRNA (E) and interleukin-6 (IL-6) mRNA (F) versus HKG; n = 4–5 independent cell batches (Student’s t-test). *P<0.05, **P<0.01, ***P<0.001.

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