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Vascular Smooth Muscle Polyploidization as a Biomarker for Aging and Its Impact on Differential Gene Expression

2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês

10.1074/jbc.m308406200

1083-351X

Matthew R. Jones, Katya Ravid,

CRISPR and Genetic Engineering

Polyploidy is characterized by a greater than diploid content of DNA in a cell. Previous measurements of ploidy level in different organs of humans and rodents, including the aorta, indicated an increase in old versus young. We hypothesized that aortic vascular smooth muscle polyploidy is a biomarker for aging and that the augmented DNA dosage affects selective gene-specific transcript expression. Our results demonstrate that tetraploidy increases exponentially over the life span of the animal, serving as an indicator of age. Approximately 60% of the vascular smooth muscle cells in the thoracic aorta of 36-month-old Brown Norway rats are tetraploid compared with 8% in their 3-month-old counterparts. Microarray analysis and reverse transcriptase-PCR was performed with mRNA isolated from sorted diploid (2N) and tetraploid (4N) vascular smooth muscle cells from old rats to identify differentially expressed transcripts. For the majority of detectable transcripts, an increase in DNA content led to a proportional increase in mRNA. A select group of transcripts, however, were reduced in tetraploid compared with diploid cells. These mRNAs correspond to guanine deaminase, to the matrix proteins rat glypican 3 (OCI-5) and decorin, as well as to the inflammation-associated transcripts, insulin-like growth factor-binding protein 6, macrophage inflammatory protein 2 precursor, macrophage galactose N-acetylgalactoseamine-specific lectin, and complement component C4. Our study is the first to describe aortic ploidy level as a biomarker for aging and to indicate that changes associated with increased DNA content per cell may selectively suppress the expression of specific genes. Polyploidy is characterized by a greater than diploid content of DNA in a cell. Previous measurements of ploidy level in different organs of humans and rodents, including the aorta, indicated an increase in old versus young. We hypothesized that aortic vascular smooth muscle polyploidy is a biomarker for aging and that the augmented DNA dosage affects selective gene-specific transcript expression. Our results demonstrate that tetraploidy increases exponentially over the life span of the animal, serving as an indicator of age. Approximately 60% of the vascular smooth muscle cells in the thoracic aorta of 36-month-old Brown Norway rats are tetraploid compared with 8% in their 3-month-old counterparts. Microarray analysis and reverse transcriptase-PCR was performed with mRNA isolated from sorted diploid (2N) and tetraploid (4N) vascular smooth muscle cells from old rats to identify differentially expressed transcripts. For the majority of detectable transcripts, an increase in DNA content led to a proportional increase in mRNA. A select group of transcripts, however, were reduced in tetraploid compared with diploid cells. These mRNAs correspond to guanine deaminase, to the matrix proteins rat glypican 3 (OCI-5) and decorin, as well as to the inflammation-associated transcripts, insulin-like growth factor-binding protein 6, macrophage inflammatory protein 2 precursor, macrophage galactose N-acetylgalactoseamine-specific lectin, and complement component C4. Our study is the first to describe aortic ploidy level as a biomarker for aging and to indicate that changes associated with increased DNA content per cell may selectively suppress the expression of specific genes. Polyploidy, which is the state of having a greater than diploid content of DNA, has been observed in a variety of mammalian cell types including hepatocytes (1Guidotti J.E. Bregerie O. Robert A. Debey P. Brechot C. Desdouets C. J. Biol. Chem. 2003; 278: 19095-19101Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar), arterial smooth muscle cells (2Owens G.K. Am. J. Physiol. 1989; 257: H1755-H1765PubMed Google Scholar), megakaryocytes (3Ravid K. Lu J. Zimmet J.M. Jones M.R. J. Cell. Physiol. 2002; 190: 7-20Crossref PubMed Scopus (214) Google Scholar), cardiac myocytes (4Brodsky V. Sarkisov D.S. Arefyeva A.M. Panova N.W. Gvasava I.G. Virchows Arch. 1994; 424: 429-435Crossref PubMed Scopus (77) Google Scholar), and Purkinje cells (5Bohn R.C. Mitchell R.B. J. Neurobiol. 1976; 7: 255-258Crossref PubMed Scopus (22) Google Scholar). Furthermore, polyploid cells may be found in certain tissues under conditions of stress, such as uterine smooth muscle cells during pregnancy, thyroid cells during hyperthyroidism, and in the seminal vesicles with aging (3Ravid K. Lu J. Zimmet J.M. Jones M.R. J. Cell. Physiol. 2002; 190: 7-20Crossref PubMed Scopus (214) Google Scholar). Cellular polyploidization also occurs in cancer but this genomic instability more often leads to the development of intermediate DNA ploidy values, which are indicative of aneuploidization (6Galipeau P.C. Cowan D.S. Sanchez C.A. Barrett M.T. Emond M.J. Levine D.S. Rabinovitch P.S. Reid B.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7081-7084Crossref PubMed Scopus (280) Google Scholar).Arterial vessels thicken during aging and this process is mediated by an increase in smooth muscle cell mass and deposition of connective tissue. The increase in vascular smooth muscle cell (VSMC) 1The abbreviations used are: VSMCvascular smooth muscle cell(s)2Ndiploid4NtetraploidRTreverse transcriptase.1The abbreviations used are: VSMCvascular smooth muscle cell(s)2Ndiploid4NtetraploidRTreverse transcriptase. mass is primarily due to the hypertrophy of preexisting cells, which occurs concomitantly with polyploidization, and not hyperplasia. No information is available, however, on the degree of polyploidization over the lifespan. Hyperplasia, which is characterized by an increase in VSMC proliferation, occurs during atherosclerotic plaque development (7Matturri L. Cazzullo A. Turconi P. Lavezzi A.M. Cardiologia. 1997; 42: 833-836PubMed Google Scholar) and balloon injury (8Fattori R. Piva T. Lancet. 2003; 361: 247-249Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Although chromosomal alterations in VSMC have been demonstrated to occur in atherosclerotic plaques (9Matturri L. Cazzullo A. Turconi P. Lavezzi A.M. Vandone P.L. Gabrielli L. Fernandez Alonso G. Grana D. Milei J. Atherosclerosis. 2001; 154: 755-761Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), the plaques themselves were found to contain less tetraploid nuclei than the corresponding media (10Barrett T.B. Sampson P. Owens G.K. Schwartz S.M. Benditt E.P. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 882-885Crossref PubMed Scopus (73) Google Scholar).Eukaryotic cells are generally prevented from becoming polyploid, due in part to the mitotic spindle checkpoint, and do not exit from mitosis until the mitotic cyclin, cyclin B, is successfully degraded. Recent studies of cultured VSMC have demonstrated that perturbations of the mitotic cell cycle checkpoint by overexpression of Akt/PKB (11Hixon M.L. Muro-Cacho C. Wagner M.W. Obejero-Paz C. Millie E. Fujio Y. Kureishi Y. Hassold T. Walsh K. Gualberto A. J. Clin. Invest. 2000; 106: 1011-1020Crossref PubMed Scopus (65) Google Scholar), a serine/threonine kinase that mediates signaling via the insulin-like growth factor and platelet-derived growth factor receptors, leads to polyploidy. Additionally, overexpression of Cks 1, a Cdc2 kinase adaptor protein that promotes the degradation of cyclin B, also results in the development of polyploidy (12Hixon M.L. Obejero-Paz C. Muro-Cacho C. Wagner M.W. Millie E. Nagy J. Hassold T.J. Gualberto A. J. Biol. Chem. 2000; 275: 40434-40442Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). It was suggested, but not proven by structural examination, that a premature exit from M-phase and cell cycle re-entry is responsible for achieving high ploidy in this cell type.Currently, there are only two studies that have examined differential transcript expression as a function of ploidy. One investigation examined isogenic yeast that differed only in ploidy and the other used cDNA-amplified fragment length polymorphism to examine transcriptional differences in isogenic diploid versus autotetraploid Arabidopsis thaliana plants (13Lee H.S. Chen Z.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6753-6758Crossref PubMed Scopus (271) Google Scholar, 14Galitski T. Saldanha A.J. Styles C.A. Lander E.S. Fink G.R. Science. 1999; 285: 251-254Crossref PubMed Scopus (512) Google Scholar). In both investigations it was concluded that a selected group of genes was not expressed proportionally to DNA content. In the current study, we have refined a cell sorting method to isolate large quantities of aortic VSMC that are fractionated based on their DNA content and have shown that ploidy-dependent mechanisms of gene expression occur within a mammalian system. There are only a few markers that serve as accurate predictors of age. Our study also concluded that aortic VSMC ploidy level can serve as a biomarker for aging.EXPERIMENTAL PROCEDURESExperimental Animals—Inbred Brown Norway rats were obtained from the NIA colony maintained by Harlan Sprague-Dawley (Indianapolis, IN) and were raised under specific pathogen-free conditions. All animals were used under an animal care protocol that was approved by the Boston University Institutional Animal Care and Use Committee. Brown Norway rats were fed ad libitum as specified by the NIA (www.nia.nih.gov/research/rodent.html). Male C57BL/6 mice at 3 months and 24 months of age were also obtained from Harlan Sprague-Dawley and raised under specific pathogen-free conditions.Vascular Smooth Muscle Cell Dispersion—VSMC were isolated from the rat aorta by enzymatic dispersion as previously described (15Devlin A.M. Gordon J.F. Davidson A.O. Clark J.S. Hamilton C.A. Morton J.J. Campbell A.M. Reid J.L. Dominiczak A.F. J. Hypertens. 1995; 13: 211-218Crossref PubMed Scopus (29) Google Scholar). This method was modified to stain the DNA content of individual cells dissociated from the aorta compromising the ascending to thoracic segment. To this end, each aorta was dissected under sterile conditions and immersed in high glucose, Dulbecco's modified Eagle's medium (Invitrogen) on ice. After the adventitia was carefully removed, each vessel was suspended in 1 ml of enzyme dissociation solution (1 mg/ml collagenase, 0.25 mg/ml elastase, and 0.5 mg/ml soybean trypsin inhibitor; Sigma) for 30 min at 37 °C. The aortas were subsequently opened longitudinally and incubated an additional 30 min at 37 °C in fresh enzyme dissociation solution. The vessels were minced into 1–2-mm pieces and suspended in fresh enzyme dissociation solution (1 ml/aorta) for 4 h in a 37 °C, in a humidified cell culture incubator equilibrated at atmospheric conditions of 5% carbon dioxide and 95% air. The digestion was stopped by adding Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and centrifuged. The cell pellet was then suspended in Hoechst 33342 staining solution (12 mm final concentration) at a final concentration of 1–2 × 106 cells/ml and incubated at 37 °C for 10 min. The cells were then placed immediately on ice and were protected from light until they are ready to be sorted. Because the aorta consists of VSMC as well as other cell types, and because ∼28% of the total number of smooth muscle cells isolated from 24-month-old animals were tetraploid, only 1.4–2.0 × 106 of the total number of cells (∼10–14 × 106) isolated from four aortas were successfully collected as tetraploids during each cell sorting procedure. In this regard, pooling of samples was stated as presenting advantages in obtaining statistically significant data (16Miller R.A. Galecki A. Shmookler-Reis R.J. J. Gerontol. A Biol. Sci. Med. Sci. 2001; 56: B52-B57Crossref PubMed Scopus (79) Google Scholar).Cell Sorting—An Epics Altra Flow Cytometer (Beckman Coulter) was equipped with a HyPer Sort System to sort cells with high efficiency. VSMC were sorted directly into tubes chilled at 4 °C. 5 milliwatss of ultraviolet laser light was used to excite the Hoechst 33342-stained nuclei. Cells were sorted at a rate of 1000–2800 cells/second. DNA histograms were generated with Expo32 software and non-overlapping regions were used to separate diploid and tetraploid VSMC. Peak versus integrated Hoechst 33342 fluorescence was used to discriminate between singlet and doublet cells. Dye emission was collected by photomultiplier tubes through a 405-nm (±20 nm) bandpass filter. Forward and right angle light scatter were collected as well.Flow Cytometric Analysis—VSMC were centrifuged and resuspended, at a concentration of about 2 × 106 cells/ml, in DNA staining solution I (solution I: 50 μg/ml propidium iodide, 100 units/ml RNase A, 0.1% Triton X-100, and 4 mm sodium citrate) for 10 min at room temperature (17Tate E.H. Wilder M.E. Cram L.S. Wharton W. Cytometry. 1983; 4: 211-215Crossref PubMed Scopus (66) Google Scholar). DNA staining solution II (solution II: 50 μg/ml propidium iodide, 0.1% Triton X-100, and 400 mm sodium chloride) was subsequently added at a volume equal to that of solution I, and the solution was incubated in the dark at 4 °C for at least 2 h prior to cytometric analysis. Flow cytometry was performed on a BD Biosciences FACScan. Laser excitation at 488 nm was produced by a 15-milliwatt argon-ion laser. Fluorescence collection for fluorescein isothiocyanate (green) and propidium iodide (red) was performed with 530/30 (FL1) and 585/42 (FL2) bandpass filters, respectively. A minimum of 104 events were analyzed per sample, and all experiments were repeated at least twice. The doublet discrimination parameter was employed on integrated and peak DNA signals to exclude cell aggregates. Statistics and graphics were calculated with the CELLQuest™ software (BD Biosciences).Total RNA and Microarray Preparation—Total RNA was isolated from sorted cells with TRIzol Reagent (Invitrogen). RNA isolation was performed as per manufacturer's instructions. For analyzing the integrity of total RNA, 2–5 μg was electrophoresced on a 1% agarose-formaldehyde Northern gel. RNA quantitation was also pursued with spectrophotometric analysis (1 OD = 20 μg of total RNA at 260 nm). The gel was analyzed for ethidium bromide staining and transferred onto a nylon membrane. The transferred RNA was further analyzed by methylene blue staining after UV cross-linking (5 min in 5% acetic acid solution, 5 min in 0.04% methylene blue solution, and washed for 5 min in water). Clontech Atlas rat 1.2 and 1.2 II membranes (catalog number 7854-1 and 7856-1; www.clontech.com) each contain 1176 spotted cDNA fragments in addition to control, housekeeping cDNA spots. 5 μg of total RNA was used to generate cDNA probes as per manufacturer's instructions using [α-32P]dCTP (10 mCi/ml from PerkinElmer Life Sciences; number BLU-513H). The probe was purified as per the manufacturer's instructions for the QIAquick Nucleotide Removal Kit (Qiagen, Valencia, CA). After purification, 200 μl of 10 mg/ml sheared salmon sperm DNA and 5 μl of rat C0t-1 DNA (1 mg/ml) were added to the probe, and the membranes were allowed to hybridize overnight at 67 °C. The membranes were washed according to the manufacturer's instructions and subsequently exposed to a storage phosphor screen for 3–5 days and evaluated with a PhosphorImager (Amersham Biosciences). AtlasImage 2.01 software (Clontech) was used to quantitate transcript expression and for normalization. To determine differential transcript expression, each experiment was normalized based on global gene expression. For each gene represented on the arrays, the ratio of transcript expression was determined by dividing the adjusted signal intensity of the 4N cells by that of the 2N cells. Three separate batches of RNA were prepared for each array hybridization, using at least two different membranes. A mean ratio and S.D. (σ) were calculated for each gene-specific transcript expressed.RT-PCR Analysis—RT-PCR was conducted as follows: 5 μg of total RNA was diluted to 14 μl with dH2O, and 8 μl of oligo(dT)12–18 primer (0.5 μg/μl in dH2O) was added. The mixture was heated at 70 °C for 10 min and then cooled on ice. The following reagents were subsequently added: 12.4 μl of 5 × RT buffer, 2 μl of 0.1 m dithiothreitol, 3 μl of dNTPs (dATP, dCTP, dGTP, and dTTP at 20 mm each), 3 μl of Superscript II RNase H– RT, and 20 μl of dH2O. The mixture was incubated at 42 °C for 90–120 min. Cycle number was varied to ensure that amplicons were below the visual saturation. PCR primers and conditions are listed in Table I.Table IRT-PCR primersPrimerafor, forward; rev, reverse.Sequence (5′-3′)Position and lengthbPosition is defined by the location on the reference sequence. PCR amplicon size is indicated in base pairs (bp).Anneal. temp.cAnnealing temperature.Reference sequence accession no.°CCD94 forCCTGGAAAAGTGGATTGGGC445-77258NM_012745CD94 revGGCAGTTGCTTACAGATAAAACGG351 bpCOMP. C4 forTGCCGCCTTCGTTTGGAGCC31-26262U42719COMP. C4 revAGAGTCCGACGCCCTCACAC232 bpDCN forGGAAAATGGCAGTCTGGCTAATG735-118152X59859DCN revAAGTCATACGGGAAAGGCATCC447 bpGAPDH forTCACCATCTTCCAGGAG1064-163356-58AF106860GAPDH revGCTTCACCACCTTCTTG570 bpGDA forAGGTCGGCAAGGATTTTGATG1272-142058NM_031776GDA revCTGCTTTCCACCCACATAAACC170 bpIGFBP-6 forGCCAGAGGGCCGTCGGAAG300-44860M69055IGFBP-6 revCAGGGGCCCATTTCACCATC149 bpMGL forGAGAGCACAGTGGAGAAGAAGGAG550-115158NM_022393MGL revTTGAAATGGGATAGGAGGAGTCC624 bpMIP-2 forATGCTGTACTGGTCCTGCTCCT62-28060U45965MIP-2 revCTTCAGGGTTGAGACAAACTTCA219 bpOCI-5 forTGCCAAGAACTACACCAATGCC474-93160M22400OCI-5 revAAGGTTTGACCATCATCAGTCCC458 bpa for, forward; rev, reverse.b Position is defined by the location on the reference sequence. PCR amplicon size is indicated in base pairs (bp).c Annealing temperature. Open table in a new tab Statistical Analysis—The exponential function and R2 value that were determined in Fig. 1B correlating ploidy level with the age of Brown Norway rats were calculated with Microsoft EXCEL (six data points). The criteria used for differential expression was defined by a ≥2-fold difference (18Levy L. Neuveut C. Renard C.A. Charneau P. Branchereau S. Gauthier F. Van Nhieu J.T. Cherqui D. Petit-Bertron A.F. Mathieu D. Buendia M.A. J. Biol. Chem. 2002; 277: 42386-42393Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 19Denton C.P. Zheng B. Evans L.A. Shi-wen X. Ong V.H. Fisher I. Lazaridis K. Abraham D.J. Black C.M. de Crombrugghe B. J. Biol. Chem. 2003; 278: 25109-25119Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 20Jiang C.H. Tsien J.Z. Schultz P.G. Hu Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1930-1934Crossref PubMed Scopus (264) Google Scholar, 21Leonard M.O. Cottell D.C. Godson C. Brady H.R. Taylor C.T. J. Biol. Chem. 2003; 278: 40296-40304Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 22Tung W.S. Lee J.K. Thompson R.W. J. Vasc. Surg. 2001; 34: 143-150Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). This is a reasonable criteria because the p values of the genes that met this level are <0.05, a level associated with increased false negatives (23Weindruch R. Kayo T. Lee C.K. Prolla T.A. Mech. Ageing Dev. 2002; 123: 177-193Crossref PubMed Scopus (136) Google Scholar), and all differences were confirmed by RT-PCR with the exception of CD94. Microsoft EXCEL was used to calculate individual t tests (assuming homoscedasticity and type II) between individual transcript expression levels in addition to those of the control set of housekeeping genes (Table II).Table IIRelative expression ratios of housekeeping transcriptsGeneGenBank™ accession no.Mean ratioS.D. (σ)40 S ribosomal protein S29X590511.120.21β-ActinV012171.050.09Glyceraldehyde-3-phosphate dehydrogenaseM177011.110.31Hypoxanthine-guanine phosphoribosyltransferaseM639830.890.16PolyubiquitinD165541.050.15Tubulin α-1V012271.170.22 Open table in a new tab RESULTSAortic VSMC Polyploidization Increases Exponentially with Aging—Age-dependent development of polyploidy was determined in both Rattus norvegicus and C57BL/6 mice. As shown in Fig. 1A, induction of tetraploidy in VSMC is significantly increased with aging in these two models. Both increases in ploidy profiles of 24-month-old animals compared with that of 3-month-old animals are statistically significant with a p value less than 0.001.Given these results, we hypothesized that the degree of vascular polyploidization might serve as a biomarker for aging, namely, that this property is precisely programmed, allowing for the reliable prediction of age based on ploidy analysis. To examine this hypothesis, we resorted to the rat model for which different age groups were readily available. Fig. 1B exhibits the percentage of tetraploid VSMC that were freshly dispersed from the BN rat aorta over the following six time points: 3 months (n = 5), 11 months (n = 3), 17 months (n = 3), 24 months (n = 22), 31 months (n = 3), and 36 months (n = 3). An exponential curve function was plotted to fit the six points, and an R2 value of 0.996 was calculated. There is a similar increase in the percentage of 4N cells as well as in the ratio of 4N/2N cells (Fig. 1C), even when the fraction of 8N cells is augmented (age ≥ 33 months). The R value (correlation coefficient squared or coefficient of determination) exhibits a very low total variation. Such correlations contain an inherent predictive value and would allow for the reliable prediction of the age of an animal based on aortic ploidy content. Indeed, a blind examination of aortic VSMC ploidy level enabled us to predict animal age with precision.DNA-dependent VSMC Sorting—To examine the profile of gene-specific transcripts expressed in tetraploid VSMC compared with diploid cells, we adapted a cell sorting method for VSMC. We confirmed that the sorting procedure was successful by examining sorted cells with a separate nuclear staining technique and flow cytometer. A post-sort quality control analysis was conducted after each isolation and contaminating cells were consistently ≤10% of the total population of cells obtained (Fig. 2A). Hoechst 33342-stained, sorted cells (DNA stained) were subsequently analyzed by light microscopy and under ultraviolet illumination as exhibited in Fig. 2B. Visual examination further confirmed that the sorted VSMC were well disaggregated and fractionated into diploid and tetraploid cells. It was also confirmed that total RNA isolated from the sorted cells was intact (Fig. 2C).Fig. 2Analysis of sorted aortic VSMC.A, aortic VSMC derived from 24-month-old Brown Norway rats were sorted into 2N and 4N cell populations. Unsorted and sorted cells were re-analyzed by flow cytometry. Contaminating cells were quantitated and determined to be less than or equal to 10% of the total sorted population. B, sorted 2N and 4N cells were observed by phase contrast microscopy after sorting. Hoechst 33342 staining was visualized by ultraviolet illumination. Magnification was ×200. C, total RNA was isolated from sorted 2N and 4N cells and electrophoresced on an agarose gel. RNA was capillary transferred onto a nylon membrane which was subsequently stained with methylene blue. 28 S, 18 S, and 5 S ribosomal bands are indicated. Lanes 1, 2, and 3 are total RNA isolated from liquid nitrogen snap-frozen 3-month-old aortic tissue, 24-month-old aortic tissue, and brain homogenate, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Ploidy-dependent Differential Transcript Expression—In yeast and plants, an increase in ploidy induces differential expression of few selected genes (13Lee H.S. Chen Z.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6753-6758Crossref PubMed Scopus (271) Google Scholar, 14Galitski T. Saldanha A.J. Styles C.A. Lander E.S. Fink G.R. Science. 1999; 285: 251-254Crossref PubMed Scopus (512) Google Scholar). RNA isolated from diploid and tetraploid aortic VSMC from 24-month-old Brown Norway rats was analyzed and subjected to microarray analysis of transcripts. Analysis of total RNA isolated from equal numbers of diploid and tetraploid cells indicated that the amount of RNA in the tetraploid cells was ∼2-fold greater than that in the diploid cells. This was statistically significant, p < 0.05 (Fig. 3A). Similarly, a separate study demonstrated that tetraploid and octaploid VSMC exhibit a 2.4- and 4.8-fold increase in mass, respectively, compared with that of a diploid cell (24Owens G.K. Schwartz S.M. Circ. Res. 1982; 51: 280-289Crossref PubMed Scopus (277) Google Scholar).Fig. 3Increased RNA expression in non-proliferating aortic VSMC is dependent on DNA dosage.A, total RNA isolated from diploid and tetraploid VSMC was quantitated by UV/visible spectrophotometry. This method primarily detects the total amount of ribosomal RNA. Tetraploid cells contained twice the amount of RNA per cell compared with their diploid counterparts. This 2-fold increase was statistically significant (p < 0.05). B, total protein was isolated from sorted 2N and 4N aortic VSMC and subjected to Western blot analysis to profile proliferating cell nuclear antigen. 15 μg of protein was loaded per each lane. Cultured murine VSMC were used as a positive control for actively proliferating cells. Equal loading was evaluated by determination of actin content.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Previous investigations have demonstrated that limited proliferation occurred within the aorta during aging (24Owens G.K. Schwartz S.M. Circ. Res. 1982; 51: 280-289Crossref PubMed Scopus (277) Google Scholar, 25Owens G.K. Schwartz S.M. Circ. Res. 1983; 53: 491-501Crossref PubMed Scopus (130) Google Scholar). This suggested that the number of cells recruited to engage the cell cycle is small at any time point but gradually accumulates over a lifespan. We performed Western blot analysis for proliferating cell nuclear antigen, which is a polymerase δ accessory protein and a marker of S phase (26Bravo R. Frank R. Blundell P.A. Macdonald-Bravo H. Nature. 1987; 326: 515-517Crossref PubMed Scopus (1636) Google Scholar). As shown in Fig. 3B, proliferating cell nuclear antigen was not detectable in diploid or tetraploid cells, whereas it was prominently expressed in actively proliferating VSMC in culture. With this observation, we proceeded to explore differential cDNA expression in diploid versus tetraploid cells and related this to DNA content and not to the proliferative state of the cell.The results of the microarray experiment, in which sorted cells were profiled, showed that, in general, the levels of RNA detected were proportional to the total RNA or DNA content in VSMC. Namely, in equal amounts of RNA analyzed in the diploid or tetraploid cells, the majority of transcripts displayed a relative expression ratio of approximately one (Fig. 4A), which includes those belonging to standard housekeeping genes (Table II). Furthermore, relative expression levels of each transcript were plotted (Fig. 4B) and indicate a very similar expression profile in the 2N and 4N cells. Out of 2352 genes surveyed, 322 gene-specific transcripts were found to be expressed, of which 7 mRNAs displayed a ≥2-fold decrease in expression in tetraploid versus diploid cells. Table III lists displayed transcripts in aortic VSMC by accession number in GenBank™, the mean expression ratio in tetraploid versus diploid cells, S.D., and associated p values. The criteria used for differential expression is defined by a ≥2-fold difference in groups analyzed (4N versus 2N), as also applied in other studies (18Levy L. Neuveut C. Renard C.A. Charneau P. Branchereau S. Gauthier F. Van Nhieu J.T. Cherqui D. Petit-Bertron A.F. Mathieu D. Buendia M.A. J. Biol. Chem. 2002; 277: 42386-42393Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 19Denton C.P. Zheng B. Evans L.A. Shi-wen X. Ong V.H. Fisher I. Lazaridis K. Abraham D.J. Black C.M. de Crombrugghe B. J. Biol. Chem. 2003; 278: 25109-25119Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 20Jiang C.H. Tsien J.Z. Schultz P.G. Hu Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1930-1934Crossref PubMed Scopus (264) Google Scholar, 21Leonard M.O. Cottell D.C. Godson C. Brady H.R. Taylor C.T. J. Biol. Chem. 2003; 278: 40296-40304Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 22Tung W.S. Lee J.K. Thompson R.W. J. Vasc. Surg. 2001; 34: 143-150Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), and p values of <0.05, a level associated with increased false negatives (23Weindruch R. Kayo T. Lee C.K. Prolla T.A. Mech. Ageing Dev. 2002; 123: 177-193Crossref PubMed Scopus (136) Google Scholar). A list of all expressed transcripts is exhibited under Supplemental Material. The expression trend for each of the transcripts listed in Table III, except for CD94, was further confirmed by semiquantitative RT-PCR. It is possible that false positive results could be generated from cross-hybridization of one member of the C-type lectin family (CD94) (27Borrego F. Kabat J. Kim D.K. Lieto L. Maasho K. Pena J. Solana R. Coligan J.E. Mol. Immunol. 2002; 38: 637-660Crossref PubMed Scopus (227) Google Scholar). In addition to the list of cDNAs shown in Table III (selected based on the above criteria), we focused on cDNAs in which the p value was less than 0.05, such as decorin. Decorin was repeatedly confirmed by RT-PCR as being significantly reduced in 4N cells (based on Kodak Imaging). All amplicons were single bands and were titrated within the linear range of PCR amplification. Representative example