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• W2130880327 abstract "Maternal vascular dysfunction is a hallmark of preeclampsia. A recently described vascular phenotype of preeclampsia involves increased expression of matrix metalloproteinase-1 (MMP-1) in endothelial cells, vascular smooth muscle, and infiltrating neutrophils. In contrast, the expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) and collagen type Iα 1 is either reduced or not changed in the vessels, suggesting an imbalance in vessel collagen degradation and synthesis in preeclampsia. In the present study, we explored the possible contribution of DNA methylation to the altered expression of genes involved in collagen metabolism. We assayed the differences in DNA methylation in omental arteries from normal pregnant and preeclamptic women, and determined whether reduced DNA methylation increases the expression of MMP-1 in cultured vascular smooth muscle cells and a neutrophil-like cell line, HL-60. Several MMP genes, including MMP1 and MMP8, were significantly less methylated in preeclamptic omental arteries, whereas TIMP and COL genes either were significantly more methylated or had no significant change in their DNA methylation status compared with normal pregnancy. Experimentally induced DNA hypomethylation increased MMP-1 expression in cultured vascular smooth muscle cells and MMP-1 cells. Our findings suggest that epigenetic regulation contributes to the imbalance in genes involved in collagen metabolism in blood vessels of preeclamptic women. Maternal vascular dysfunction is a hallmark of preeclampsia. A recently described vascular phenotype of preeclampsia involves increased expression of matrix metalloproteinase-1 (MMP-1) in endothelial cells, vascular smooth muscle, and infiltrating neutrophils. In contrast, the expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) and collagen type Iα 1 is either reduced or not changed in the vessels, suggesting an imbalance in vessel collagen degradation and synthesis in preeclampsia. In the present study, we explored the possible contribution of DNA methylation to the altered expression of genes involved in collagen metabolism. We assayed the differences in DNA methylation in omental arteries from normal pregnant and preeclamptic women, and determined whether reduced DNA methylation increases the expression of MMP-1 in cultured vascular smooth muscle cells and a neutrophil-like cell line, HL-60. Several MMP genes, including MMP1 and MMP8, were significantly less methylated in preeclamptic omental arteries, whereas TIMP and COL genes either were significantly more methylated or had no significant change in their DNA methylation status compared with normal pregnancy. Experimentally induced DNA hypomethylation increased MMP-1 expression in cultured vascular smooth muscle cells and MMP-1 cells. Our findings suggest that epigenetic regulation contributes to the imbalance in genes involved in collagen metabolism in blood vessels of preeclamptic women. Preeclampsia is a multisystemic disorder that is diagnosed by new onset of hypertension and proteinuria, which occur after 20 weeks of gestation in pregnant women who are otherwise normal.1Cunningham F.G. Leveno K.J. Bloom S.L. Gilstrap III, L.C. Hauth J.C. Wenstrom K.D. Williams Obstetrics. McGraw-Hill, New York2005Google Scholar It is also associated with pathological edema due to increased vascular permeability. Preeclampsia and its complications are among the leading causes of obstetrical mortality and morbidity.1Cunningham F.G. Leveno K.J. Bloom S.L. Gilstrap III, L.C. Hauth J.C. Wenstrom K.D. Williams Obstetrics. McGraw-Hill, New York2005Google Scholar In the United States, preeclampsia occurs in approximately 6% to 8% of pregnant women, and is estimated to be responsible for 15% of obstetrical deaths.2Report of the National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy.Am J Obstet Gynecol. 2000; 183: S1-S22Crossref PubMed Google Scholar Recently, neutrophil infiltration associated with marked inflammation of maternal systemic blood vessels was reported as a characteristic phenotype in women with preeclampsia3Shah T.J. Walsh S.W. Activation of NF-kappaB and expression of COX-2 in association with neutrophil infiltration in systemic vascular tissue of women with preeclampsia.Am J Obstet Gynecol. 2007; 196: 48.e1-48.e8Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 4Leik C.E. Walsh S.W. Neutrophils infiltrate resistance-sized vessels of subcutaneous fat in women with preeclampsia.Hypertension. 2004; 44: 72-77Crossref PubMed Scopus (95) Google Scholar, 5Mishra N. Nugent W.H. Mahavadi S. Walsh S.W. Mechanisms of enhanced vascular reactivity in preeclampsia.Hypertension. 2011; 58: 867-873Crossref PubMed Scopus (49) Google Scholar We recently reported that matrix metalloproteinase (MMP)-1 expression is increased in systemic blood vessels of preeclamptic women compared with normal pregnant women.6Estrada-Gutierrez G. Cappello R.E. Mishra N. Romero R. Strauss 3rd, J.F. Walsh S.W. Increased expression of matrix metalloproteinase-1 in systemic vessels of preeclamptic women: a critical mediator of vascular dysfunction.Am J Pathol. 2011; 178: 451-460Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar The increased expression of MMP-1 in omental arteries was detected in vascular smooth muscle cells (VSMCs), endothelial cells, and infiltrating leukocytes. Plasma levels of MMP-1 were also increased in preeclamptic women. Interestingly, omental arteries collected from preeclamptic women showed no change in the expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) and decreased expression of collagen type Iα 1 (COLIA1), which is the main component of type 1 collagen. Collectively, these observations suggest an imbalance in vascular collagen metabolism that favors degradation in preeclampsia. In addition, MMP-1 was a regulator of arterial vessel tone, implicating this metalloproteinase in the pathogenesis of systemic hypertension associated with preeclampsia.6Estrada-Gutierrez G. Cappello R.E. Mishra N. Romero R. Strauss 3rd, J.F. Walsh S.W. Increased expression of matrix metalloproteinase-1 in systemic vessels of preeclamptic women: a critical mediator of vascular dysfunction.Am J Pathol. 2011; 178: 451-460Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar The expression level of several MMP genes, including MMPs 1, 2, 3, 7, 9, and 14, is regulated by DNA methylation,7Sato N. Maehara N. Su G.H. Goggins M. Effects of 5-aza-2′-deoxycytidine on matrix metalloproteinase expression and pancreatic cancer cell invasiveness.J Natl Cancer Inst. 2003; 95: 327-330Crossref PubMed Scopus (114) Google Scholar and MMP-9 has been shown to be regulated by DNA methylation in placentas of women with preeclampsia.8Wang Z. Lu S. Liu C. Zhao B. Pei K. Tian L. Ma X. Expressional and epigenetic alterations of placental matrix metalloproteinase 9 in preeclampsia.Gynecol Endocrinol. 2009; 26: 96-102Crossref Scopus (44) Google Scholar DNA hypomethylation induced by 5-aza-2-deoxycytidine (5-Aza) treatment resulted in a significant increase in MMP-1 expression in cultured amnion fibroblasts.9Wang H. Ogawa M. Wood J.R. Bartolomei M.S. Sammel M.D. Kusanovic J.P. Walsh S.W. Romero R. Strauss 3rd, J.F. Genetic and epigenetic mechanisms combine to control MMP1 expression and its association with preterm premature rupture of membranes.Hum Mol Genet. 2008; 17: 1087-1096Crossref PubMed Scopus (63) Google Scholar The increase in MMP-1 expression correlated with decreased DNA methylation at a particular location in the MMP1 promoter 1538 bp upstream from the transcription start site. DNA methylation at the same site was significantly reduced in amnion tissues collected from pregnancies with preterm premature rupture of the fetal membranes.9Wang H. Ogawa M. Wood J.R. Bartolomei M.S. Sammel M.D. Kusanovic J.P. Walsh S.W. Romero R. Strauss 3rd, J.F. Genetic and epigenetic mechanisms combine to control MMP1 expression and its association with preterm premature rupture of membranes.Hum Mol Genet. 2008; 17: 1087-1096Crossref PubMed Scopus (63) Google Scholar This evidence for epigenetic regulation of MMP1, and other MMP genes, led us to hypothesize that reduced DNA methylation might contribute to the increased expression of MMPs in the systemic vasculature of preeclamptic women, leading to the imbalance in genes involved in collagen metabolism.6Estrada-Gutierrez G. Cappello R.E. Mishra N. Romero R. Strauss 3rd, J.F. Walsh S.W. Increased expression of matrix metalloproteinase-1 in systemic vessels of preeclamptic women: a critical mediator of vascular dysfunction.Am J Pathol. 2011; 178: 451-460Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar In this study, we examined DNA methylation in the promoter regions of MMP, TIMP, and COL genes in omental arteries obtained from normal pregnant and preeclamptic women. We also studied the effect of DNA hypomethylation on the expression of MMP-1, TIMP-1, and COLIA1 in primary cultures of human VSMCs and the expression of MMP-1 and the neutrophil collagenase, MMP-8, in a neutrophil-like cell line.10Gallagher R. Collins S. Trujillo J. McCredie K. Ahearn M. Tsai S. Metzgar R. Aulakh G. Ting R. Ruscetti F. Gallo R. Characterization of the continuous, differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia.Blood. 1979; 54: 713-733Crossref PubMed Google Scholar We examined the effect of hypomethylation in a neutrophil cell line because of the extensive vascular infiltration of neutrophils that occurs in women with preeclampsia.3Shah T.J. Walsh S.W. Activation of NF-kappaB and expression of COX-2 in association with neutrophil infiltration in systemic vascular tissue of women with preeclampsia.Am J Obstet Gynecol. 2007; 196: 48.e1-48.e8Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 4Leik C.E. Walsh S.W. Neutrophils infiltrate resistance-sized vessels of subcutaneous fat in women with preeclampsia.Hypertension. 2004; 44: 72-77Crossref PubMed Scopus (95) Google Scholar, 5Mishra N. Nugent W.H. Mahavadi S. Walsh S.W. Mechanisms of enhanced vascular reactivity in preeclampsia.Hypertension. 2011; 58: 867-873Crossref PubMed Scopus (49) Google Scholar Omental and s.c. fat samples of approximately 2 × 2 × 0.5 cm were obtained at cesarean section from normal pregnant women (n = 18) and women clinically diagnosed as having mild or severe preeclampsia (n = 21) at the Medical College of Virginia Hospital, Virginia Commonwealth University Medical Center (Richmond, VA). Mild preeclampsia was diagnosed when the blood pressure was >140 mmHg systolic or >90 mmHg diastolic and there was 0.3 g of urinary protein per 24 hours after 20 weeks of gestation. Severe preeclampsia was diagnosed when the blood pressure was >160 mmHg systolic or >110 mmHg diastolic, with 5 g of protein in the urine within 24 hours after 20 weeks of gestation.1Cunningham F.G. Leveno K.J. Bloom S.L. Gilstrap III, L.C. Hauth J.C. Wenstrom K.D. Williams Obstetrics. McGraw-Hill, New York2005Google Scholar Omental fat arteries were used because they are a component of the maternal systemic vasculature, and they play a role in blood pressure regulation by contributing to the total peripheral vascular resistance. Women with chorioamnionitis, infections, active sexually transmitted diseases, lupus, or diabetes and women who were smokers or in labor were excluded because these conditions are associated with inflammatory changes. Patient clinical data are shown in Table 1. This study was approved by the Office of Research Subjects Protection, Virginia Commonwealth University. All subjects gave informed consent.Table 1Clinical Characteristics of Patient GroupsVariableNormal pregnant women (n = 18)Preeclamptic women (n = 21)Maternal age (years)26.1 ± 1.324.6 ± 1.0Prepregnancy BMI (kg/m2)26.2 ± 1.230.7 ± 1.6⁎P < 0.05,Systolic blood pressure (mmHg)117.1 ± 2.3166.8 ± 2.4⁎⁎P < 0.001 by t-test.Diastolic blood pressure (mmHg)70.18 ± 1.893.5 ± 2.6⁎⁎P < 0.001 by t-test.Proteinuria (mg/24 hours)ND286.8 ± 21.6 (n = 8)DipstickND2.7 ± 0.3 (n = 13)Parity Primiparous315 Multiparous156Gestational age (weeks)39.2 ± 0.235.4 ± 0.9⁎⁎P < 0.001 by t-test.Infant birth weight (g)3503 ± 1002452 ± 248⁎⁎P < 0.001 by t-test.Values are presented as mean ± SEM unless otherwise indicated.BMI, body mass index; ND, not determined. P < 0.05, P < 0.001 by t-test. Open table in a new tab Values are presented as mean ± SEM unless otherwise indicated. BMI, body mass index; ND, not determined. Omental arteries from nine normal pregnant women and nine preeclamptic women (five mild and four severe) were dissected and cleaned of adhering fat. DNA was extracted from the arteries (approximately 10 mg by weight) using a QuickGene DNA tissue kit and a QuickGene-Mini80 system (AutoGen, Holliston, MA). DNA was treated with RNase A (Qiagen, Valencia, CA). DNA (1 μg) was bisulfite treated using a MethylEasy Exceed kit (Human Genetic Signatures, Randwick, Australia), and DNA sequencing was performed as previously described.9Wang H. Ogawa M. Wood J.R. Bartolomei M.S. Sammel M.D. Kusanovic J.P. Walsh S.W. Romero R. Strauss 3rd, J.F. Genetic and epigenetic mechanisms combine to control MMP1 expression and its association with preterm premature rupture of membranes.Hum Mol Genet. 2008; 17: 1087-1096Crossref PubMed Scopus (63) Google Scholar Briefly, 100 ng of bisulfite-treated DNA was PCR amplified for two rounds using our previously published primers for the −1538 site in the MMP1 promoter. PCR products were purified with a QIAquick PCR Purification kit (Qiagen), and 1 μL of each product was cloned into pCR 2.1-TOPO vectors using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). After bacteria amplification, eight cloned PCR segments for each patient were sequenced in the Nucleic Acids Research Facilities at Virginia Commonwealth University. The −1538 site was selected because we previously demonstrated that its methylation correlates with the expression of MMP-1.9Wang H. Ogawa M. Wood J.R. Bartolomei M.S. Sammel M.D. Kusanovic J.P. Walsh S.W. Romero R. Strauss 3rd, J.F. Genetic and epigenetic mechanisms combine to control MMP1 expression and its association with preterm premature rupture of membranes.Hum Mol Genet. 2008; 17: 1087-1096Crossref PubMed Scopus (63) Google Scholar Omental arteries from five normal pregnant and seven severe preeclamptic women were processed for DNA extraction. DNA was bisulfite treated and used in the high-throughput Illumina Infinium HumanMethylation27 BeadChip assay (Illumina, San Diego, CA) for global DNA methylation analysis. The BeadChip was run by the Nucleic Acids Research Facilities at Virginia Commonwealth University using the protocol provided by Illumina. Data analysis was performed using the beadarray package in the R programming environment.11Dunning M.J. Smith M.L. Ritchie M.E. Tavaré S. beadarray: R classes and methods for Illumina bead-based data.Bioinformatics. 2007; 23: 2183-2184Crossref PubMed Scopus (380) Google Scholar Methylation (β) values are expressed as a range from 0 to 1, where 0 means not methylated and 1 means fully methylated. Differences in methylation between normal pregnant and preeclamptic women were indicated by Δβ values. Omental and s.c. fat samples from six normal pregnant and six preeclamptic women were cut into small pieces, fixed immediately in 10% neutral-buffered formalin, and embedded in paraffin. Tissues were cut into sections (8 μm thick), which were stained for rabbit anti-human polyclonal antibody specific for MMP-8, neutrophil collagenase (1:100 titer; catalog number 17874-1-AP; Proteintech, Chicago, IL), and mouse monoclonal IgM anti-human CD66b, a neutrophil antigen (1:50; catalog number 555723; BD BioSciences, San Diego, CA). Rabbit primary antibody isotype control (Invitrogen) was used as a negative control. A kit was used for immunohistochemical (IHC) staining [SuperPicTure Polymer Detection Kit Broad Spectrum (diaminobenzidine); Invitrogen]. To quench endogenous tissue peroxidase activity, slides were incubated in 3% hydrogen peroxide in methanol for 30 minutes. For antigen retrieval, slides were heat treated in 10 mmol/L citrate buffer for 5 minutes with a pressure cooker. Tissue slides were counterstained with a 1:5 dilution of Hematoxylin QS (Vector Laboratories, Burlingame, CA). Vessel staining for MMP-8 was evaluated using an intensity scale ranging from 0 to 4, where 0 was assigned for no staining and 4 was dark and extensive staining. Scoring was verified by a second investigator (S.W.W.) and by measuring the OD of staining using image analysis software (cellSens Imaging Software; Olympus America, Center Valley, PA), as previously described.3Shah T.J. Walsh S.W. Activation of NF-kappaB and expression of COX-2 in association with neutrophil infiltration in systemic vascular tissue of women with preeclampsia.Am J Obstet Gynecol. 2007; 196: 48.e1-48.e8Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 4Leik C.E. Walsh S.W. Neutrophils infiltrate resistance-sized vessels of subcutaneous fat in women with preeclampsia.Hypertension. 2004; 44: 72-77Crossref PubMed Scopus (95) Google Scholar The OD of staining for MMP-8 in vessels was normalized to the OD of the background. Slides were also analyzed for vessels with diffuse staining and leukocytes staining for MMP-8. Images were captured with cellSens Imaging Software. Human VSMCs were cultured from chorionic plate arteries of placentas collected at cesarean section from healthy pregnant women at term deliveries, as previously described.12Leik C.E. Willey A. Graham M.F. Walsh S.W. Isolation and culture of arterial smooth muscle cells from human placenta.Hypertension. 2004; 43: 837-840Crossref PubMed Scopus (51) Google Scholar Cells were cultured in T-25 flasks using Medium-199 (Gibco, Invitrogen) with 10% fetal bovine serum (Gibco, Invitrogen), 1% antibiotics, and antimycotics (100 U/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL amphotericin B; Gibco, Invitrogen). Cells were treated with 10 μmol/L of 5-Aza (Sigma-Aldrich, St. Louis, MO), an agent that inhibits DNA methylation when incorporated into DNA during cell division,13Kendrew J.C. The Encyclopedia of Molecular Biology. Blackwell Science, Cambridge1994Crossref Google Scholar for 48 hours. Treatments were refreshed daily. Cells were 50% confluent at treatment and nearly 100% confluent at harvesting. Conditioned media were collected and centrifuged to remove cell debris. Cells were rinsed in PBS and harvested for RNA or protein extraction. To examine the role of DNA methylation in regulating the expression of MMP-1 and MMP-8 in neutrophils, it was necessary to use a neutrophil-like cell line (HL-60) because neutrophils isolated from patients do not divide and, therefore, 5-Aza cannot be incorporated into the genomic DNA to induce hypomethylation. HL-60 cells (ATCC, Manassas, VA) were cultured in Iscove's modified Dulbecco's medium (ATCC) supplemented with 10% fetal bovine serum (Gibco, Invitrogen), 1% antibiotics, and antimycotics (100 U/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL amphotericin B; Gibco, Invitrogen), as recommended by ATCC. Approximately 500,000 cells/mL were seeded in 5 mL of media in a T-25 flask for treatments. Cell treatments were as follows: i) 10 μmol/L 5-Aza for 48 hours, followed by 10−8 mol/L of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator (Sigma-Aldrich) for 24 hours; ii) 10 μmol/L 5-Aza for 48 hours, followed by no treatment for 24 hours; iii) no treatment for 48 hours, followed by 10−8 mol/L PMA for 24 hours; or iv) no treatment for 72 hours to serve as a control. Cells were collected for RNA and protein extraction. RNA was extracted using a QuickGene RNA cultured cell kit with a QuickGene Mini-80 system (AutoGen, Holliston, MA). DNase treatment was performed using a Turbo DNase kit (Ambion, Austin, TX). RNA (1 μg) was reverse transcribed to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). Quantitative real-time PCRs were performed with RT2 SYBR Green qPCR Mastermix (SABiosciences, Frederick, MD), as recommended by the user manual, and 8 ng of cDNA was used per reaction. GAPDH was used as a housekeeping gene. Commercial primers were used for the MMP1 and MMP8 genes (SABiosciences). Primers for the GAPDH, TIMP1, and COLIA1 genes, shown in Table 2, were synthesized by Integrated DNA Technologies (Coralville, IA). Fold changes were calculated by the ΔΔCT method.Table 2Quantitative RT-PCR Primers Used for Gene ExpressionGeneForward primerReverse primerGAPDH5′-GATTCCACCCATGGCAAATT-3′5′-AGATGGTGATGGGATTTCCATT-3′TIMP15′-CTGTGGCTCCCTGGAACA-3′5′-CCAACAGTGTAGGTCTTGGTGAAG-3′COL1A15′-ACGAAGACATCCCACCAATCAC-3′5′-CGTTGTCGCAGACGCAGAT-3′ Open table in a new tab Cells were lysed in radioimmunoprecipitation assay buffer containing 150 mmol/L sodium chloride, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mmol/L Tris (pH 8.0), and one times Halt protease inhibitor (Thermo Scientific, Pittsburgh, PA) using a tip sonicator (Active Motif, Carlsbad, CA). Protein concentrations were measured using the bicinchoninic acid assay (Thermo Fisher Scientific, Rockford, IL). Denatured protein lysates (100 μg) were resolved by electrophoresis using 10% Tris-glycine SDS-PAGE electrotransferred to a polyvinylidene fluoride membrane (Immobilon-FL; Millipore, Billerica, MA). The primary antibodies used were as follows: goat anti-human MMP-1 (0.2 μg/mL; R&D Systems, Minneapolis, MN), goat anti-human COLIA1 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-human MMP-8 (1:250; Proteintech, Chicago, IL), rabbit anti-human β-actin (1:1000; Sigma-Aldrich), and mouse anti-human β-actin (1:1000; Sigma-Aldrich). Secondary antibodies used included Alexa Fluor 680 donkey anti-rabbit (1:20,000; Invitrogen) for the detection of primary antibodies raised in rabbit for β-actin or MMP-8; IRDye800 donkey anti-goat (1:20,000; Rockland Immunochemicals, Gilbertsville, PA) for the detection of primary antibodies raised in goat for MMP-1 or COLIA1; and IRDye800 goat anti-mouse (1:20,000; Rockland Immunochemicals) for the detection of primary antibody raised in mouse for β-actin. The LI-COR Odyssey Infrared Imaging System (Thermo Scientific, Pittsburgh) was used to detect the immunoreactive proteins. Commercial enzyme-linked immunosorbent assay (ELISA) kits were used to quantify total MMP-1 (GE Healthcare, Piscataway, NJ) and TIMP-1 (GE Healthcare) in VSMC culture medium. For normalization, the total amount of MMP-1 or TIMP-1 protein in 5 mL (volume of medium used in a T-25 flask) was divided by the total amount of DNA (μg) extracted from the cells of the same flask. The Mann-Whitney U-test was used to analyze the data from bisulfite sequencing and for visual intensity score data. A Student's t-test was used to make comparisons of parameters between two groups, and a one-way analysis of variance with a Newman-Keuls test was used to make comparisons of parameters between more than two groups for normally distributed data. Quantitative results were presented as mean ± SEM. We considered P < 0.05 as statistically significant. A statistical software program was used for data analysis (Prism 4 for Macintosh version 4.0a; GraphPad Software, Inc., San Diego, CA). The Illumina BeadChip assay revealed a significant decrease in DNA methylation in the promoter regions of MMP1 and MMP8 genes in omental arteries from preeclamptic women compared with normal pregnant women. However, there were no significant changes in DNA methylation in the promoter regions of TIMP1 or COLIA1 genes. Other MMP, TIMP, and COL genes that were differentially methylated in preeclamptic arteries are shown in Table 3. All MMP genes, except two, were less methylated, whereas TIMP and COL genes were, in general, relatively more methylated. Bisulfite DNA sequencing revealed a significant decrease in methylation at the −1538 site in the promoter region of the MMP1 gene in omental arteries from women with severe preeclampsia compared with normal pregnant women (P < 0.01, Figure 1). Methylation at the same site was not significantly different in arteries from women with mild preeclampsia compared with arteries from normal pregnant women.Table 3List of COL, MMP, and TIMP Genes that Were Significantly Differentially Methylated in Preeclamptic Omental ArteriesSymbolNameGene IDMean β⁎β indicates methylation values where 0 is no methylation and 1 is full methylation, and Δβ indicates the difference in methylation. Negative values indicate less methylation, and positive values indicate more methylation in PE arteries compared with NP arteries.Δβ⁎β indicates methylation values where 0 is no methylation and 1 is full methylation, and Δβ indicates the difference in methylation. Negative values indicate less methylation, and positive values indicate more methylation in PE arteries compared with NP arteries.P valuePENPCOL8A1Collagen, type VIII, α 112950.0780.0540.0240.02COL9A3Collagen, type IX, α 312990.5360.4800.0560.03COL19A1Collagen, type XIX, α 113100.2020.1540.0470.02MMP1Matrix metalloproteinase 143120.7360.816−0.0800.02MMP8Matrix metalloproteinase 843170.8070.882−0.0750.03MMP12Matrix metalloproteinase 1243210.6830.740−0.0570.01MMP13Matrix metalloproteinase 1343220.7450.830−0.0850.01MMP16Matrix metalloproteinase 1643250.0250.0140.0110.02MMP21Matrix metalloproteinase 211188560.4110.543−0.1320.02MMP26Matrix metalloproteinase 26565470.7620.862−0.1000.02MMP27Matrix metalloproteinase 27640660.7690.7260.0430.04TIMP3Tissue inhibitor of metalloproteinases 370780.4370.3380.0980.02NP, normal pregnant; PE, preeclamptic. β indicates methylation values where 0 is no methylation and 1 is full methylation, and Δβ indicates the difference in methylation. Negative values indicate less methylation, and positive values indicate more methylation in PE arteries compared with NP arteries. Open table in a new tab NP, normal pregnant; PE, preeclamptic. The IHC staining revealed increased expression of MMP-8 in omental and s.c. blood vessels of preeclamptic women compared with normal pregnant women (Figure 2, A–D), similar to what we previously showed for MMP-1.6Estrada-Gutierrez G. Cappello R.E. Mishra N. Romero R. Strauss 3rd, J.F. Walsh S.W. Increased expression of matrix metalloproteinase-1 in systemic vessels of preeclamptic women: a critical mediator of vascular dysfunction.Am J Pathol. 2011; 178: 451-460Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar MMP-8 was mainly expressed in infiltrating neutrophils, as evidenced by similarity of staining for CD66b in serial sections, although diffuse staining was also observed in the vessels. Scoring for MMP-8 staining intensity was significantly higher in preeclamptic women compared with normal pregnant women (2.8 ± 0.1 versus 1.2 ± 0.2; P < 0.001; Figure 3A), as was the density of staining for MMP-8 (121.6 ± 5.2 versus 66.1 ± 7.0; P < 0.001; Figure 3B). Staining intensity scores and optical density of staining were highly correlated (r = 0.99). The percentage of vessels with neutrophils stained for MMP-8 was significantly higher in preeclamptic women compared with normal pregnant women (87.8% ± 1.5% versus 54.0% ± 7.2%; P < 0.001; Figure 3C). The percentage of vessels with diffuse staining for MMP-8 was also significantly higher in preeclamptic women compared with normal pregnant women (79.8% ± 5.7% versus 38.4% ± 6.1%; P < 0.001; Figure 3D).Figure 3Quantitation of IHC staining for MMP-8 in omental and s.c. blood vessels from normal pregnant (NP; n = 5) and preeclamptic (PE; n = 5) women. A: The staining intensity score for MMP-8 is significantly higher in blood vessels of preeclamptic women compared with normal pregnant women. B: The OD of staining for MMP-8 is significantly higher in preeclamptic blood vessels. C and D: Preeclamptic women have a significantly higher percentage of blood vessels with neutrophils stained for MMP-8 and diffuse staining for MMP-8 compared with normal pregnant women. Data are presented as mean ± SEM. ***P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether DNA methylation could be involved in the regulation of MMP1 gene expression in VSMCs, 5-Aza was used to induce DNA hypomethylation in the cultured cells. Treatment with 5-Aza provoked a significant increase in MMP1 gene expression compared with control cells (5.0 ± 0.6-fold, P < 0.001, Figure 4A). To confirm that increased MMP1 gene expression was associated with an increase in protein production, ELISA was used to quantify MMP-1 secretion into the media. Treatment with 5-Aza significantly increased the production of MMP-1 protein (3.4 ± 0.3 versus 1.2 ± 0.3 ng/μg DNA; P < 0.01; Figure 4B). In contrast to MMP-1, 5-Aza treatment caused no significant change in gene expression of TIMP1 or COLIA1 in cultured VSMCs (Figure 5A) and did not affect protein levels of TIMP-1 in the media (Figure 5B) or COLIA1 content in cells (Figure 5C).Figure 5TIMP-1 and COLIA1 expression in cultured human VSMCs treated with 5-Aza for 48 hours. A: The fold change in the expression for TIMP1 and COLIA1 is not significantly changed by 5-Aza treatment compared with control (n = 8). B: TIMP-1 protein levels in media are not significantly changed compared with controls (n = 4). C: Western blot analysis for pro-COLIA1, and COLIA1 demonstrates no effect of 5-Aza treatment on the protein expression of COLIA1 (n = 3). Data are shown as mean ±" @default.
• W2130880327 created "2016-06-24" @default.
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• W2130880327 date "2012-10-01" @default.
• W2130880327 modified "2023-10-15" @default.
• W2130880327 title "Preeclampsia Is Associated with Alterations in DNA Methylation of Genes Involved in Collagen Metabolism" @default.
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