Differential Effect of 17-ß-Estradiol on Smooth Muscle Cell and Aortic Explant MMP21

Methods This investigation comprised 3 sets of experiments. Experiment I: Adult male and female rat aortic smooth muscle cells (RASMCs) at passages 4-8 were stimulated in serum-free media for 48 h with interleukin(IL)1ß at doses encountered in human abdominal aortic aneurysms (2 ng/mL). Messenger RNA was extracted from the RASMCs, and gene expression of MMP2 and tissue inhibitor of metalloproteinase 2 (TIMP2), a major MMP2 inhibitor, was measured by real-time polymerase chain reaction. MMP2 protein levels in conditioned media were measured by Western blotting, and MMP2 and TIMP2 activity quantified by standard and reverse gelatin zymography. Experiment II: Male and female RASMCs were incubated for 48 h in Dulbecco's modified Eagler's medium containing IL-1β and 17-β-estradiol at doses from 1 × 10 −10 to 1 × 10 −6 molar. MMP2 activity in the conditioned media was then determined. Experiment III: Male rats underwent sustained 17-β-estradiol exposure for 21 d using extended-release, subcutaneously implanted pellets prior to sacrifice and aortic explantation. Aortas from males, females, and estradiol-treated males were stimulated with IL-1β for 48-h, and MMP2 activity in the conditioned media was determined. Results Experiment I: MMP2 gene expression was 3-fold higher in male compared with female IL-1β stimulated RASMCs ( P < 0.0001). MMP2:TIMP2 gene expression ratio was 7.5-fold greater in male versus female RASMCs. MMP2 protein levels were 3-fold higher (2.68 versus 0.96 o.d./mg total protein, P = 0.003) in male versus female RASMCs. Gelatinolytic activity was more than 6-fold higher (15,010 versus 2,472 o.d./mg total protein, P = 0.002) in male versus female RASMCs. Experiment II: MMP2 activity in male and female RASMCs was not altered by a wide range of 17-β-estradiol concentrations. Experiment III: When pretreated with 17-β-estradiol, MMP2 activity in the media of male rat whole-aortic explants decreased 2-fold ( P = 0.002). This post-17-β-estradiol treatment male level was not different than baseline female aortic explant MMP2 levels. Conclusions MMP2 is higher in male RASMCs compared to female RASMCs. Exogenous 17-β-estradiol did not alter MMP2 activity in vitro , but in vivo 17-β-estradiol exposure greatly decreased male aortic MMP2 production to levels seen in the female aorta. Gender differences in MMP2 are speculated to be associated with phenotypic differences in human abdominal aortic aneurysm formation. Key Words MMP aorta smooth muscle cells estrogen hormones Introduction Matrix metalloproteinases are a family of proteinases that are strongly associated with abdominal aortic aneurysms (AAAs). Specifically, matrix metalloproteinase 2 (MMP2, Gelatinase A, 72 kDa Gelatinase) is produced by vascular smooth muscle cells [1] and is increased in both human [2–4] and experimental murine AAAs [5] . MMP2 appears critical in the formation of experimental murine AAAs [5] ; it has been shown that mice deficient in MMP2 are highly resistant to experimental aneurysm formation [6] . Additionally, inhibitors of MMP2 have been shown to decrease aortic wall elastin degradation [7] . In addition to the long-standing [8] and repeated association of MMP2 and abdominal aortic aneurysm disease, it has long been observed that the incidence and size of human AAAs are greater in males than females [9, 10] , but that the differences tend to decrease in post-menopausal women. While the basis of the gender-related disparity is not known, the same phenomenon is observed in experimental animal models of AAA [11] . Given the association of MMP2 and AAA formation, and the observation of gender-related differences in AAA, this investigation tested the hypothesis that intrinsic gender-related differences exist in aortic smooth muscle cell MMP2. Additionally, we sought to determine if modifications to the local hormone environment would alter MMP2 activity. Methods Cell Culture Reagents were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. All experiments were performed with approval of the University of Michigan Committee on Laboratory Animal Medicine. Rat aortic smooth muscle cells (RASMCs) were cultured from the abdominal aortas of young (190–210 g) male and female Sprague-Dawley rats (Charles River Labs, MA). After animal sacrifice and aortic explantation under general inhalational anesthesia, the aortic tissue was cut into 2 mm 2 pieces and placed in 60 mm diameter plastic tissue culture dishes. Basement membrane Matrigel (Collaborative Research, Bedford, MA) was applied to each section of explanted tissue to prevent floating. Cultures were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 100 units/mL penicillin, 100 μg/mL streptomycin, and glutamine 292 mcg/mL. Tissue culture media and antibiotics were obtained from Gibco (Rockville, MD). Tissues were incubated at 37°C in a humidified, 5% CO 2 atmosphere for 4 to 7 d, until spindle-shaped SMC were observed extending from the tissue. After removing the explant, cells were dispersed by treatment with trypsin (Gibco), centrifuged, resuspended in complete medium, and placed into 75 cm 2 culture flasks. Postconfluent cultures assumed a hill and valley topography characteristic of SMCs grown in vitro . RASMCs were confirmed by staining with a monoclonal antibody against SMC-specific α-actin and determined to nearly 100% SMCs. Experiment I Adult male and female RASMCs were isolated and grown in phenol red free DMEM with 10% fetal bovine serum. Confluent monolayers of RASMCs at passages 4 through 8 were stimulated in serum-free media with interleukin(IL)-1ß at doses encountered in human aortic aneurysms (2 ng/mL) [12] . After 48 h of stimulation, culture media was collected and mRNA was extracted from the RASMCs. Following reverse transcription to cDNA, gene expression of MMP2 and TIMP2 (tissue inhibitor of MMP2; a major MMP2 inhibitor) was measured by real-time polymerase chain reaction. MMP2 protein levels in the conditioned media were measured by Western blotting, while MMP2 activity and TIMP2 activity were quantified by standard and reverse gelatin zymography. The remaining SMCs were lysed in 1% sodium dodecyl sulfate (SDS) and total cellular protein was determined by a bicinchoninic acid protein assay (BCA; Pierce, Rockford, IL). Experiment II Male and female RASMCs ( N = 6 repeated in triplicate) were incubated for 48 h in phenol red free DMEM containing 17-β-estradiol and IL-1β. The 5 increasing concentration levels of 17-β-estradiol (1 × 10 −10 to 1 × 10 −6 molar) included both rat and human female physiologic levels of 17-β-estradiol. MMP2 activity in the conditioned media was determined by standard zymography. Experiment III Male Sprague-Dawley rats (190–210 g) were again obtained from Charles River Laboratories (Wilmington, MA). Under general inhalational anesthesia (isofluorane 1.5%), 21-d sustained-release 17-ß-estradiol pellets (0.1 mg/pellet, Innovative Research of America, Sarasota, FL) were implanted in the interscapular subcutaneous plane. This particular estradiol dose results in serum estradiol levels at 2 to 3 wk of 44.7 ± 6.1 pg/mL compared with 15.0 ± 2.2 pg/mL in control rats ( P < 0.05) [13] . Three weeks after pellet implantation, the infrarenal aorta was explanted under general anesthesia. The aorta was briefly rinsed free of blood and debris with cold 1X phosphate-buffered saline containing antibiotics, cut into 1 mm rings, and incubated for 48 h in 1 mL of serum-free DMEM containing antibiotics and IL-1β at 37 °C. Following the incubation period, the conditioned media was collected for MMP2 activity analysis by standard gelatin zymography as follows. Tissue pieces were incubated for 24 h at 37 °C in 500μL of 1% SDS to extract protein which was subsequently assayed by BCA as follows. Substrate Gel Zymography and Reverse Zymography Zymography supplies were purchased from Novex (San Diego, CA). MMP distribution after treatment of RASMC with IL-1β and increasing concentrations of 17-β-estradiol was determined. Gelatin substrate zymograms were prepared using precast 10% SDS-polyacrylamide gels containing 1 mg/mL of gelatin. Equal volumes of experimental media samples were diluted into 2× tris-glycine SDS sample buffer and electrophoretically separated under non-reducing conditions. Proteins were renatured in 2.7% Triton X-100 and the gels were developed overnight at 37°C in 50 mM tris-HCl containing 5 mM CaCl 2 and 2% Brij 35. Following overnight staining with Coomassie blue R-250 and de-staining for 4 h with 10% acetic acid and 40% methanol in water, gelatinase activity was evident by clear bands against a dark blue background. Reverse zymography was performed with RASMC-conditioned media samples. Gelatin substrate reverse zymograms were prepared using a 15% acrylamide resolving gel containing 1 mg/mL porcine gelatin and conditioned serum-free medium from separate RASMC cultures as a source of pro-gelatinase A. A standard 5% polyacrylamide stacking gel was used. Experimental samples containing equal volumes were diluted into 2× tris-glycine SDS sample buffer and electrophoretically separated under non-reducing conditions. Proteins were renatured in 2 changes of 2.7% Triton X-100 for 60 min each. The gels were developed for 24 h at 37 °C in 50 mM Tris-HCl, 5 mM CaCl2 and 0.2% Brij 35. Following overnight staining with Coomassie blue R-250 and destaining for 5 h with 10% acetic acid and 40% methanol in water, gelatinase inhibitory activity was evident as a blue band against a clear background. The TIMP2 band was determined by comparison with authentic TIMP2 [22kD] obtained from Calbiochem (San Diego, CA). Semiquantitative measurements of TIMP2 activity were performed by densitometry and corrected to total cellular protein. Western Blot Analysis Electrophoresis and Western blotting supplies were obtained from BioRad (Hercules, CA). Equal volumes of media from IL-1β-stimulated RASMC cultures were electrophoretically separated on a 7.5% acrylamide gel and blotted onto nitrocellulose membranes. Nonspecific binding was blocked by incubating the membrane overnight in 20 mM tris-HCl (pH 7.5) containing 0.5 M NaCl, 0.1% Tween 20 and 5% nonfat milk. The primary antibody was monoclonal mouse anti-rat antibody to MMP2 ( Calbiochem, San Diego, CA ) . Peroxidase-coupled goat anti-mouse antibody was used as a secondary antibody (Calbiochem). Immunoreactive bands were visualized using an ECL chemiluminescence detection kit from Amersham (Piscataway, NJ), and the amount of protein (corrected to total cellular protein) was measured by densitometry. Semiquantitative Reverse Transcriptase-Polymerase Chain Reaction Expression of MMP2 and TIMP2 messenger RNA (mRNA) was determined using semiquantitative real-time polymerase chain reaction (PCR). RASMCs treated with IL-1β were lysed and total cellular RNA was extracted using TRIzol reagent from Life Technologies (Rockville, MD), and mRNA was purified. Messenger RNA samples were reverse transcribed for 60 min at 42°C using an oligo-(dT) primer and Moloney's murine leukemia virus (M-MLV) reverse transcriptase (Life Technologies). Reverse transcriptase products were used as the substrate for PCR amplification of MMP2, TIMP2, and β-actin cDNAs with 5 U/mL Taq DNA polymerase from Promega (Madison, WI). Cycling was performed at 94°C for 2 min, followed by 32 cycles of 94°C for 1 min, annealing at 57°C for 1 min, and extension at 72°C for 1 min, and a final incubation at 72°C for 5 min. Amplification was carried out in a GeneAmp 2400 PCR system from Perkin-Elmer (Foster City, CA). Primers were designed by Primer Premier Software (Premier Biosoft International, Palo Alto, CA), and were obtained from Sigma Genosys (Houston, TX). The sequences were as follows: MMP2 sense 5'- CAT CGC TGC ACC ATC GCC CAT CAT C -3'; MMP2 antisense 5'- CCC AGG GTC CAC AGC TCA TCA TCA TCA AAG -3'; TIMP2 sense 5'- TGC CCT ATG ATC CCA TGC TA -3'; TIMP2 antisense 5'- TCT GTG ACC CAG TCC ATC CA -3'; β-actin sense 5'-ATG GGT CAG AAG GAT TCC TAT GTG-3'; β-actin antisense 5'-CTT CAT GAG GTA GTC AGT CAG GTC-3'. The MMP2 primers were purchased from Sigma Genosys; TIMP2 and β-actin primers were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). Densitometry All gel images were acquired using a FOTO/Analyst CCD CAMERA from Fotodyne (Hartland, WI). Band strength was quantified using GEL-Pro Analyzer software version 3.1 from Media Cybernetics (Silver Spring, MD). In cell culture experiments, only pro-MMP2 bands were observed. Data Analysis All experiments were performed in triplicate or quadruplicate. An unpaired, 2-tailed Student's t -test was used to determine differences in MMP2 and TIMP-2, with P < 0.05 considered significant. Statistical calculations were carried out using GraphPad Prism ver. 3.0a for Macintosh (GraphPad Software, San Diego, CA). Results Experiment I—Gender Related Differences Intrinsic gender-related differences were observed between male and female RASMCs with regard to MMP2 gene expression, protein production, and gelatinolytic activity. MMP2 gene expression relative to β-actin was 3-fold higher in male compared with female IL-1β stimulated RASMCs (40.93 ± 4.9 versus 12.54 ± 2.7; P < 0.0001, see Fig. 1 ). TIMP2 gene expression was greater in female than in male RASMCs (12.52 ± 3.6 versus 5.4 ± 0.81; P < 0.05), and the corresponding MMP2:TIMP2 gene expression ratio was greater in male vs. female cells (7.57 versus 1.00, respectively). By Western blotting, MMP2 protein levels (o.d./mg total protein) were 3-fold higher (2.68 ± 0.36 versus 0.96 ± 0.10) in male versus female RASMCs ( P = 0.003), see Fig. 2 . Gelatinolytic activity by standard gelatin zymography (o.d./mg total protein) was more than 6-fold higher (15,010 ± 3318 versus 2,472 ± 363) in male versus female cells ( P = 0.002), see Fig. 2 . Experiment II—Treatment of RASMCs with Exogenous Estradiol After observing apparent intrinsic differences in MMP2 and TIMP2 across gender, the second set of experiments sought to determine if MMP2 levels could be pharmacologically manipulated by co-culture with 17-β-estradiol. However, despite culture conditions that reflected physiologic female levels of estradiol for both rats [14] and humans [15] , significant differences in MMP2 activity (by gelatin zymography) were not observed in male RASMCs. ( Fig. 3 ) Similar results were documented in female RASMCs; namely, that exogenous supplementation of 17-β-estradiol did not alter secretion of pro-MMP2 ( P = 0.9493). Experiment III— In Vivo Estradiol Therapy Given the inability of in vitro hormone alterations to alter MMP2 activity, we wished to determine if 17-β-estradiol may alter MMP2 activity in the more complex in vivo system. After pretreatment with extended-release 17-β-estradiol pellets, male rats had a 2-fold decrease in MMP2 activity (o.d./mg total protein) in the media of whole-aortic explants (2.0 × 10 5 estradiol-treated versus 4.35 × 10 5 control, P = 0.002). By further comparison to controls, MMP2 activity in estradiol-treated males was found to be similar to female MMP2 activity (2.0 × 10 5 versus 2.5 × 10 5 ; P = 0.06, see Fig. 4 ). Discussion MMP2 (also known as gelatinase-A and 72 kDa gelatinase) has been repeatedly associated with AAA pathogenesis. MMP2 is constitutively expressed by vascular smooth muscle cells [1] , and it is increased in human [2–4] and murine AAAs [5] . Additionally, MMP2 knockout mice are highly resistant to experimental aneurysm formation [6] . Despite these repeated and consistent associations, MMP2 is certainly only a part of AAA pathogenesis [16] . Indeed, it is no longer believed that a single matrix metalloproteinase—be it MMP2, 9, 12, 14, or others—is the sole agent responsible for AAA formation [16, 17] . As suggested by Longo et a l. [6] , it is possible that multiple MMPs work in concert when remodeling (appropriately or pathologically) the aortic wall. Further, it is no longer believed that MMPs are necessarily the proximate damaging agent to the aortic wall. More likely, MMPs are involved in a complicated system of matrix recognition, immune modulation [18] , and vascular remodeling. A recent study from our laboratories demonstrated that male rats that underwent neutrophil depletion were protected from AAA formation despite no difference in (single-gender) MMP2 levels [19] . The present study is the first to characterize fundamental gender differences in MMP2 expression, protein production and protein activity in rat aortic smooth muscle cells. It has been long recognized that men suffer an increased incidence of aneurysm formation [11, 20] , but the reasons for this gender-related disparity are not known. Paralleling differences in AAA susceptibility, this investigation demonstrates that male aortic mesenchymal cells produce significantly greater amounts of MMP2 than do their female counterparts. In addition, the ratio of MMPs and their inhibitors, the TIMPs, have been implicated in aortic disease. Manabe et al. [21] reported that the TIMP2:MMP2 ratio is decreased in the acute phase of aortic dissections, which is consistent with our observations of increased MMP2:TIMP2 ratio in the aneurysm-susceptible male gender. This investigation provides another molecular example to support the observation that certain subsets of the general population are more susceptible to diseases than others. At baseline, MMP2 levels in female RASMCs is less than in male counterparts, and these differences are alterable by in vivo hormone administration. This is a highly localized phenomenon (in abdominal aortic smooth muscle cells); it does not necessarily stand in contradiction to recent reports of equal serum concentrations of MMP2 across human gender [22] . Regardless, it is doubtful that MMP2 will be used in screening for AAA, as serum levels are an insensitive marker for AAA presence or expansion [23, 24] . It was observed in this investigation that estradiol influences MMP2 levels in vivo , and that baseline gender differences may be ameliorated by hormone modulation. This relationship has been observed in other areas of cardiovascular investigation. Chancey et al. [25] report increased susceptibility to cardiac dysfunction in estrogen-deficient (ovariectomized) female animals, which corresponded to increased MMP2 levels. In corresponding randomized clinical trials, however, transdermal estrogen supplementation failed to alter serum MMP2 levels [26] . Conclusions MMP2 gene expression, protein levels, and gelatinolytic activity were higher in male compared with female RASMCs. Exogenous 17-β-estradiol did not alter MMP2 activity in vitro in male or female SMCs, but in vivo 17-β-estradiol exposure greatly decreased male aortic MMP2 production. Gender differences in MMP2 are speculated to be associated with phenotypic differences in human and rat AAA formation. Future investigations in AAA formation will certainly reveal many correlations between gender, molecular-level pathogenesis, and susceptibility to AAA. Acknowledgments The authors acknowledge support for this study by NIH T32 (HL076123) (D.T.W.), NIH KO8 (HL67885-02) (G.R.U.), Von Leibig Award-Lifeline Foundation (G.R.U.), 5R01 (HL081629-02) (G.R.U.). References 1 M. Crowther S. Goodall JL. Jones Increased matrix metalloproteinase 2 expression in vascular smooth muscle cells cultured from abdominal aortic aneurysms J Vasc Surg 32 2000 575 2 S. Goodall M. Crowther DM. Hemingway Ubiquitous elevation of matrix metalloproteinase-2 expression in the vasculature of patients with abdominal aneurysms Circulation 104 2001 304 3 M. Crowther S. Goodall JL. Jones Localization of matrix metalloproteinase 2 within the aneurysmal and normal aortic wall Br J Surg 87 2000 1391 4 V. Davis R. Persidskaia L. Baca-Regen Matrix metalloproteinase-2 production and its binding to the matrix are increased in abdominal aortic aneurysms Arterioscler Thromb Vasc Biol 18 1998 1625 5 I. Sinha KK. Hannawa JL. Eliason Early MT-1 MMP expression following elastase exposure is associated with increased cleaved MMP2 activity in experimental rodent aortic aneurysms Surgery 136 2004 176 6 G.M. Longo W. Xiong TC. Greiner Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms J Clin Invest 110 2002 625 7 G.D. Treharne Jr. Boyle S. Goodall Marimastat inhibits elastin degradation and matrix metalloproteinase 2 activity in a model of aneurysm disease Br J Surg 86 1999 1053 8 M.I. Patel J. Melrose P. Ghosh Increased synthesis of matrix metalloproteinases by aortic smooth muscle cells is implicated in the etiopathogenesis of abdominal aortic aneurysms J Vasc Surg 24 1996 82 9 D.J. Katz J.C. Stanley G.B. Zelenock Abdominal aortic aneurysms Semin Vasc Surg 8 1995 289 10 D.J. Katz J.C. Stanley G.B. Zelenock Gender differences in abdominal aortic aneurysm prevalence, treatment, and outcome J Vasc Surg 25 1997 561 11 G. Ailawadi JL. Eliason KJ. Roelofs Gender differences in experimental aortic aneurysm formation Arterioscler Thromb Vasc Biol 24 2004 2116 12 V.A. Davis RN. Persidskaia LM. Baca-Regen Cytokine pattern in aneurysmal and occlusive disease of the aorta J Surg Res 101 2001 152 13 E.M. Brooks-Asplund AA. Shoukas SY. Kim Estrogen has opposing effects on vascular reactivity in obese, insulin-resistant male Zucker rats J Appl Physiol 92 2002 2035 14 J.C. Tou SB. Arnaud R. Grindeland The effect of purified compared with nonpurified diet on bone changes induced by hindlimb suspension of female rats Exp Biol Med (Maywood) 230 2005 31 15 L. Holmberg T. Norden A. Lindgren Preoperative oestradiol levels—relation to survival in breast cancer Eur J Surg Oncol 27 2001 152 16 G. Ailawadi J.L. Eliason G.R. Upchurch Jr. Current concepts in the pathogenesis of abdominal aortic aneurysm J Vasc Surg 38 2003 584 17 W.C. Parks A confederacy of proteinases J Clin Invest 110 2002 613 18 K.K. Hannawa JL. Eliason DT. Woodrum L-selectin-mediated neutrophil recruitment in experimental rodent aneurysm formation Circulation 112 2005 241 19 J.L. Eliason KK. Hannawa G. Ailawadi Neutrophil depletion inhibits experimental abdominal aortic aneurysm formation Circulation 112 2005 232 20 National Hospital Discharge Survey Data, in National Center for Health Statistics, November 2002.Available at: http://www.cdc.gov/nchs/data/series/sr_13/sr13_153.pdf . Accessed: May 15, 2005. 21 T. Manabe K. Imoto K. Uchida Decreased tissue inhibitor of metalloproteinase-2/matrix metalloproteinase ratio in the acute phase of aortic dissection Surg Today 34 2004 2205 22 M.H. Tayebjee GY. Lip AD. Blann Effects of age, gender, ethnicity, diurnal variation, and exercise on circulating levels of matrix metalloproteinases (MMP)-2 and -9, and their inhibitors, tissue inhibitors of matrix metalloproteinases (TIMP)-1 and -2 Thromb Res 115 2005 205 23 P. Eriksson S. Jormsjo-Pettersson AR. Brady Genotype–phenotype relationships in an investigation of the role of proteases in abdominal aortic aneurysm expansion Br J Surg 92 2005 1372 24 T. Eugster A. Huber T. Obeid Aminoterminal propeptide of Type III procollagen and matrix metalloproteinases-2 and -9 failed to serve as serum markers for abdominal aortic aneurysm Eur J Vasc Endovasc Surg 29 2005 378 25 A.L. Chancey JD. Gardner DB. Murray Modulation of cardiac mast cell-mediated extracellular matrix degradation by estrogen Am J Physiol Heart Circ Physiol 289 2005 H316 26 J.C. Stevenson A. Oladipo N. Manassiev Randomized trial of effect of transdermal continuous combined hormone replacement therapy on cardiovascular risk markers Br J Haematol 124 2004 802