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Pregnancy; |
Department of Obstetrics, Gynecology and Reproductive Sciences,3 Yale University School of Medicine, New Haven, Connecticut 06510
Department of Histology and Embryology,4 Trakya University Medical Faculty, 22030 Edirne, Turkey
ABSTRACT
Extravillous trophoblasts (EVTs) invade human decidua via sequential integrin-mediated binding and proteolysis of basement membrane proteins in the extracellular matrix (ECM). In preeclampsia, shallow EVT invasion impairs spiral artery and arteriole remodeling to reduce uteroplacental blood flow. Excess decidual cell-expressed matrix metalloproteinases (MMPs) 2 and 9, in response to preeclampsia-related interleukin 1 beta (IL1B) and tumor necrosis factor alpha (TNF), may inappropriately degrade these basement membrane proteins and impede EVT invasion. This study found significantly higher immunohistochemical MMP9 levels in decidual cells and adjacent interstitial trophoblasts in placental sections of preeclamptic versus gestational age-matched control women. In contrast, immunostaining for MMP2 and tissue inhibitor of matrix metalloproteinases 1 and 2 (TIMP1 and TIMP2) were similar in preeclamptic and control groups. First-trimester decidual cells were incubated with estradiol (E2) or E2 + medroxyprogesterone acetate (MPA), with or without TNF or IL1B. As measured by ELISA, both cytokines elicited concentration-dependent increases in secreted MMP9 levels that were unaffected by MPA. In contrast, secreted levels of MMP2, TIMP1, and TIMP2 were unchanged in all treatment groups. Substrate gel zymography and Western blotting confirmed that each cytokine increased secreted levels of MMP9 but not MMP2. Similarly, quantitative RT-PCR found that TNF and IL1B enhanced MMP9, but not MMP2, mRNA levels. At the implantation site, inflammatory cytokine-enhanced MMP9 may promote preeclampsia by disrupting the decidual ECM to interfere with normal stepwise EVT invasion.
cytokines, decidua, extracellular matrix, matrix metalloproteinase, pregnancy
Progesterone transforms estradiol (E2)-primed human endometrial stromal cells into decidual cells in the luteal phase of the menstrual cycle and during gestation [1, 2]. Decidualization involves conversion of the follicular-phase interstitial extracellular matrix (ECM) to a peridecidual ECM enriched in basement membrane-type components [3–5]. Extravillous trophoblasts (EVTs) penetrate the decidua and then breach its capillaries to provide the embryo with oxygen and nutrients prior to placentation [6]. Subsequently, EVTs traverse the decidua and penetrate into the myometrium. Migrating EVTs enter and remodel spiral arteries and arterioles [7, 8] to create low-resistance, high-capacitance vessels that increase blood flow to the intervillous space required for growth and development of the fetoplacental unit [8].
Invasion of the decidua involves sequential attachment of EVT-expressed adhesion molecules, such as the integrins (ITGs) ITG alpha 1/ITG beta 1 and ITG alpha 5/ITG beta 1, which recognize laminin/collagen IV and fibronectin, respectively, in the decidual ECM [3, 4, 9], as well as vascular endothelial cadherin, an endothelial cell receptor [10]. A dysregulated pattern of decidual adhesion molecule expression is associated with shallow EVT invasion and incomplete vascular transformation [11]. The resulting reduced uteroplacental blood flow produces a placenta that becomes hypoxic [12] and secretes several putative inducers of endothelial cell activation and vascular damage into the maternal circulation (reviewed in Karumanchi and Bdolah [13] and Borzychowki et al. [14]). Ultimately, hypertension and proteinuria, the hallmarks of the maternal syndrome, ensue [15, 16].
The decidual ECM composition reflects a balance between new basement membrane protein synthesis and ECM-degrading protease expression. The matrix metalloproteinases (MMPs) are a family of zinc-dependent proteases that bear the primary responsibility for degrading the bulk of ECM components. Members of this family are generally classified on the basis of substrate specificity and include collagenases, which mediate proteolysis of fibrillar collagens; stromelysins, which can degrade an extremely broad array of ECM components and activate the zymogenic form of other MMPs; and gelatinases, which degrade denatured fibrillar collagens (gelatins) as well as basement membrane-associated proteins [17–19] Members of all three MMP classes, as well as tissue inhibitors of MMPs (TIMPs), are expressed by human endometrial stromal/decidual cells and are implicated in ECM turnover [20–24]. Among the MMPs, gelatinase A (MMP2) and gelatinase B (MMP9) preferentially degrade basement membrane components [17, 18].
By altering turnover of the decidual ECM, aberrant decidual cell-expressed MMP2 and/or MMP9 expression could promote preeclampsia by interfering with adhesion molecule-mediated stepwise EVT invasion. The proinflammatory cytokines interleukin 1 beta (IL1B) and tumor necrosis factor alpha (TNF) are associated with various aspects of preeclampsia [25–27]. Underlying maternal infections in subsets of preeclamptic cases (reviewed in Sibai et al. [16]) and an excess of decidual macrophages in the preeclamptic decidua [28–30] are likely sources of these cytokines [31]. The crucial role played by progesterone in pregnancy maintenance prompted assessment of IL1B and TNF effects with and without a progestin on MMP2 and MMP9 expression in first-trimester decidual cells, the major cell type encountered by invading EVTs at the implantation site [32, 33]. These in vitro studies were complemented by immunohistochemical localization of MMP2 and MMP9 in the decidua of preeclamptic versus gestational age-matched control placental sections.
For immunohistochemical studies, placentas and attached fetal membranes were obtained from uncomplicated pregnancies after cesarean or vaginal idiopathic spontaneous preterm delivery (n = 11) and from cesarean and vaginal preterm deliveries indicated by preeclampsia (n = 11) after receiving written informed consent at the Yale-New Haven Hospital under Human Investigation Committee approval. The mean ± SEM gestational ages were 30.5 ± 1.2 wk and 30.3 ± 1.0 wk for preterm controls and preeclampsia cases, respectively. The gestational ages of the control and preeclamptic patients were not statistically different. Gestational age was estimated by the last menstrual period and ultrasound in the first 20 wk of pregnancy. None of the patients exhibited signs of decidual hemorrhage or chorioamnionitis or chronic villitis or other indications of underlying acute or chronic inflammation as determined from histological examination of each specimen.
Preeclampsia was defined by standard criteria [34] as systolic and diastolic blood pressures of 
140 mm Hg and 90 mm Hg, respectively, and proteinuria defined as 0.3 g protein in a 24-h urine collection occurring after 20 wk of gestation in a woman with previously normal blood pressure. Severe preeclampsia (8 of the 11 patients) was defined as systolic and diastolic blood pressures of 160 mm Hg and 110 mm Hg, respectively, on two occasions at least 6 h apart during bed rest and/or proteinuria of 5 g in a 24-h urine specimen or 300 mg/dl on two random urine samples collected at least 4 h apart.
For cell cultures, decidual specimens from eight uncomplicated, elective terminations between 6 and 12 wk of gestation were obtained under Institutional Review Board approval at Bellevue Hospital (New York, NY). The decidua was separated, and a small portion of the former was formalin fixed and paraffin embedded and then examined histologically for signs of underlying acute and chronic inflammation. The remainder was used for the isolation of decidual cells.
Formalin-fixed, paraffin-embedded 5-µm sections were deparaffinized in xylene and rehydrated in a graded series of ethanol. Immunostaining for MMP2, tissue inhibitor of matrix metalloproteinase 1 (TIMP1), tissue inhibitor of matrix metalloproteinase 2 (TIMP2), vimentin, and cytokeratin involved boiling in citrate buffer (10 mM; pH 6.0) for 20 min, and thereafter sections for vimentin staining were incubated in a humidified chamber with trypsin for 5 min for antigen retrieval. Sections for cytokeratin, MMP2, MMP9, TIMP1, and TIMP2 staining were immersed in 3% hydrogen peroxide (in 50% methanol/50% distilled water) for 10 min to block endogenous peroxidase activity. After washing several times in Tris-buffered saline (TBS; pH 7.6), slides were incubated in a humidified chamber with 5% normal horse or goat serum (Vector Laboratories Inc., Burlingame, CA) in TBS for 30 min at room temperature to block nonspecific binding. After removing excess serum, the sections were incubated with primary antibodies (mouse MMP2 monoclonal antibody [1:140 dilution; Calbiochem Inc., San Diego, CA] at room temperature for 60 min; rabbit MMP9 polyclonal antibody [1:100 dilution; NeoMarkers, Fremont, CA] overnight at 4°C; rabbit TIMP1 or TIMP2 polyclonal antibody [1:400 dilution; NeoMarkers] overnight at 4°C; mouse vimentin monoclonal antibody [1:100 dilution; Abcam, Cambridge, MA] at room temperature for 30 min; and mouse cytokeratin monoclonal antibody [1:100 dilution; DAKO Inc., Carpinteria, CA] at room temperature for 45 min) in a humidified chamber. For negative controls, sections were treated with either normal mouse immunoglobulin G1 (IgG1) isotype monoclonal or normal rabbit IgG antibodies (R&D Systems Inc., Minneapolis, MN). Sections were rinsed in TBS three times for 5 min each, then biotinylated horse anti-mouse or anti-rabbit antibodies (Vector Laboratories) were added at a 1:400 dilution for 30 min at room temperature. After washing three times in TBS for 5 min each, the antigen-antibody complex was detected with an avidin-peroxidase kit or avidin-alkaline phosphatase kit (Vector Laboratories). The immunoreaction was developed using the chromogens diaminobenzidine (3, 3-diaminobenzidine tetrahydrochloride dehydrate; Vector Laboratories) for MMP2, MMP9, TIMP1, and TIMP2, and Fast Red (Vector Laboratories) for vimentin and cytokeratin. Sections were counterstained with hematoxylin (Sigma) and mounted with Permount (Fisher Chemicals, Springfield, NJ) on glass slides.
The intensities of MMP2, MMP9, TIMP1, and TIMP2 immunostaining were evaluated semiquantitatively by the following categories: 0 (no staining), 1+ (weak, but detectable, staining), 2+ (moderate or distinct staining), and 3+ (intense staining). For each tissue, an HSCORE value was derived by taking the sum of the percentage of cells that stained at each intensity category and multiplying that value by the weighted intensity of the staining, using the formula HSCORE = 
Pi (i + l), where i represents the intensity score and Pi the corresponding percentage of cells staining. In each slide, five different areas and 100 cells per field were evaluated microscopically with x40 objective magnification. The average score was obtained after evaluation by two investigators blinded to the samples.
Tissues were minced and digested with 0.1% collagenase type IV and 0.01% DNAse in RPMI-1640 containing 20 µg/ml penicillin/streptomycin, 1 µl/ml fungizone (Gibco, Grand Island, NY) in a 37°C shaking water bath for 30 min. After washing with sterile PBS, the digestate was washed three times and subjected to consecutive filtration through 100-µm, 70-µm, and 40-µm millipore filters. Cells were resuspended in RPMI-1640, grown to confluence on polystyrene tissue culture dishes, harvested using trypsin/EDTA, and analyzed by flow cytometric analysis with anti-CD45 and anti-CD14 mAbs (BD Pharmingen, San Diego, CA) to monitor the presence of leukocytes after each passage. After three to four passages, cell cultures were found to be leukocyte free (<1%). Monolayers of these decidual cells were vimentin positive and cytokeratin negative and displayed decidualization-related morphological and biochemical changes during incubation with a progestin. The latter included enhanced expression of prolactin and of plasminogen activator inhibitor 1 (PAI1) and inhibited expression of MMP1 and MMP3 (data not shown). Cell aliquots were frozen in fetal calf serum/dimethyl sulfoxide (9:1; Sigma-Aldrich) and stored in liquid nitrogen.
Experimental Decidual Cell Incubations
Thawed cells were incubated in basal medium, a phenol red-free 1:1 v/v mix of Dulbecco MEM (Gibco) and Ham F-12 (Flow Labs, Rockville, MD), with 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml fungizone supplemented with 10% charcoal-stripped calf serum (BMS). After two additional passages, confluent cultures were incubated in parallel in BMS containing either 10–8 M E2 or E2 + 10–7 M medroxyprogesterone acetate (MPA; Sigma-Aldrich), which was used in place of progesterone because of its greater stability under culture conditions [35]. After 7 days, the cultures were washed twice with Hanks balanced salt solution to remove residual serum. The cultures then were switched to a defined medium (DM) consisting of basal medium plus ITS+ (Collaborative Research, Waltham, MA), 5 µM FeSO4, 50 µM ZnSO4, 1 nM CuSO, 20 nM Na2SeO3, trace elements (Gibco), 50 µg/ml ascorbic acid (Sigma-Aldrich), and 50 ng/ml epidermal growth factor (Becton-Dickinson, Bedford, MA), with either vehicle control (0.1% ethanol) or steroids added with or without 0.01–10 ng/ml IL1B or TNF (R&D Systems). After the test period, cells were harvested by scraping into ice-cold PBS, pelleted, and extracted in ice-cold lysis buffer. Conditioned medium supernatants and cell lysates were stored at –70°C. In parallel incubations, total RNA was extracted with Tri Reagent (Sigma-Aldrich).
Total cell protein levels were measured by a modified Lowry assay (Bio-Rad Laboratories, Hercules, CA). Commercial ELISA kits were used to measure total, noncomplexed immunoreactive levels each of MMP2, MMP9, TIMP1, and TIMP2 in the cell-conditioned medium according to instructions provided by the manufacturer (Quantikine kits; R&D Systems). Both MMP ELISAs have sensitivities of 0.16 ng/ml. The MMP2 assay coefficients of variation for intraassay and interassay are 4.8% and 7.7%, respectively. The MMP9 assay coefficients of variation for intraassay and interassay are 2.3% and 7.5%, respectively. Both of the TIMP ELISAs have sensitivities less than 0.011 ng/ml. The TIMP1 coefficients of variation for intraassay and interassay are 4.8% and 7.7%, respectively. The TIMP2 coefficients of variation for intraassay and interassay are 5.6% and 6.9%, respectively.
Gel zymography, with gelatin as the substrate in the gel, was used to detect the proteolytic activity of MMP2 and MMP9. Conditioned media from decidual cell cultures were centrifuged, and the supernatants were pretreated with dimethyl sulfoxide at a concentration of less than 7% of the total volume, with and without 2 mM aminophenylmercuric acetate (APMA; Sigma) at 37°C for 2 h. This APMA converts latent (pro) forms of the MMPs to their active forms. This reaction was stopped by mixing the samples 1:1 with nonreducing Zymogram sample buffer (Bio-Rad Laboratories) and then incubating for 10 min at room temperature. Samples were loaded onto a 10% gelatin Zymogram gel (Bio-Rad Laboratories), then electrophoresed for 1.75 h in Tris-glycine SDS running buffer. To enable the enzymes to renature, the gel was incubated for 45 min in 2.5% Triton X-100 (BioRad Laboratories) at room temperature and incubated in Zymogram development buffer (Bio-Rad Laboratories) for 30 min, and then placed in fresh Zymogram development buffer overnight at 37°C. The gel was stained with 0.5% Coomassie Brilliant Blue (Sigma) solution of methanol/acetic acid/water (40:10:50 v/v) for 2 h at room temperature, and then destained with methanol/acetic acid/water (30:10:60 v/v) for 4 h at room temperature. The presence of clear bands in the gels at the appropriate molecular weights reflects gelatonolytic activity of the latent (pro) and active forms of MMP2 and MMP9.
Western blot analysis was carried out on concentrated conditioned DM supernatants, which were diluted 1:6 in Laemmli 6x sample buffer (Boston Bioproducts, Boston, MA) and then boiled for 4 min. The centrifuged media were subjected to SDS-PAGE on a 7.5% Tris-HCl gel (Bio-Rad Laboratories), with subsequent electroblotting transfer onto a 0.2-µm nitrocellulose membrane (Bio-Rad Laboratories). After transfer, the membrane was blocked overnight in Tris-buffered saline (Fisher, Fairlawn, NJ) with 4% nonfat dry milk and then incubated for 2 h with 0.1 µg/ml of a mouse anti-human MMP9 monoclonal antibody (R&D Systems). Membranes were rinsed in PBS and 0.1% Tween 20 prior to and after incubation with horseradish peroxidase-conjugated anti-mouse IgG (ICN Biomedicals, Aurora, OH). Chemiluminescence was detected with ECL reagents (Perkin-Elmer Life Sciences, Boston, MA) and autoradiography film (Amersham Pharmacia) according to the manufacturer's instructions. The same membrane then was rinsed as above and incubated in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) for 30 min at 50°C, with subsequent rinsing and reblocking as indicated above. The membrane then was incubated for 2 h with 1 µg/ml of a mouse anti-human MMP2 monoclonal antibody (Oncogene, San Diego, CA) and processed as indicated above.
To verify that the MMP2, MMP9, and beta-actin (ACTB) probes yielded the correct bands, extracted RNA from experimental cell incubations was subjected to semiquantitative RT-PCR using a kit from Invitrogen (Carlsbad, CA) and carrying out 35 cycles with the Eppendorf Mastercycler (Eppendorf, Westbury, NY). To perform quantitative real-time RT-PCR, reverse transcription was initially carried out with AMV reverse transcriptase (Invitrogen). A quantitative standard curve was created between 1 and 40 ng cDNA with a Roche Light Cycler (Roche, Indianapolis, IN) by monitoring increasing fluorescence of PCR products during amplification. Upon establishing the standard curve, quantification of the unknowns was determined with the Roche Light Cycler and adjusted to the quantitative expression of ACTB from the corresponding unknowns. Melting curve analysis determined the specificity of the amplified products and the absence of primer-dimer formation. All products obtained yielded correct melting temperatures. The primers listed in Table 1 were synthesized and gel purified at the Yale DNA Synthesis Laboratory, Critical Technologies.
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Comparisons of control and the various treatment groups were performed using the Kruskal-Wallis ANOVA on Ranks test followed by the Student-Newman-Keuls posthoc test, with a P value < 0.05 representing statistical significance. For immunohistochemistry analysis of HSCORES, t-tests were employed to compare control versus preeclamptic specimens, with P < 0.05 representing statistical significance.
Immunostaining of MMP9 and MMP2 in Decidua
Decidual cells in sections from the decidua basalis from preterm controls (Figs. 1B and 2B) and preeclamptic specimens (Figs. 1D and 2D) were distinguished by vimentin immunoreactivity. Interstitial cytotrophoblast cells in sections from the decidua basalis from preterm controls (Figs. 1B and 2B, insets) were distinguished by cytokeratin immunoreactivity.
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Matrix metalloproteinase 9 immunoreactivity (brown stain) is shown in the decidual cells of both preterm control (Fig. 1A) and preeclamptic specimens (Fig. 1C). The HSCORE values of MMP9 levels (Fig. 1F) in the decidual cells of preeclamptic specimens (mean ± SEM: 183 ± 5) were significantly higher than those found in preterm controls (125 ± 9; P < 0.001). Immunostaining for MMP9 in interstitial cytotrophoblasts was not significantly different between preeclamptic (127 ± 7) and preterm control (115 ± 10) specimens. Negative antibody control is seen in Figure 1E.
Likewise, MMP2 immunoreactivity (brown stain) is shown in the decidual cells of both preterm control (Fig. 2A) and preeclamptic specimens (Fig. 2C). There were no significant changes in the HSCORE values for MMP2 immunoreactivity (Fig. 2F) in decidual cells from preeclamptic specimens (mean ± SEM: 162 ± 6) compared with those from preterm controls (139 ± 11). Similarly, there was no significant difference between the HSCORE values of MMP2 immunoreactivity in interstitial cytotrophoblast cells from preterm controls (105 ± 8) and preeclamptic specimens (111 ± 9). Negative antibody control is seen in Figure 2E.
Immunostaining of TIMP1 and TIMP2 in Decidua
Tissue inhibitor of MMP1 showed weak immunoreactivity in both preterm control (Fig. 3A) and preeclamptic (Fig. 3C) specimens. HSCORE values of TIMP1 staining (Fig. 3G) were not significantly different between preterm control and preeclamptic specimens in either decidual cells (mean ± SEM: 32 ± 10 vs. 36 ± 4, respectively) or interstitial cytotrophoblasts (30 ± 9 vs. 22 ± 4, respectively). Tissue inhibitor of MMP2 immunoreactivity was also weak in preterm control (Fig. 3B) and preeclamptic (Fig. 3D) specimens. Tissue inhibitor of MMP2 HSCORE values (Fig. 3H) were not significantly different between preterm control and preeclamptic specimens in either decidual cells (80 ± 24 vs. 102 ± 13, respectively) or interstitial trophoblasts (64 ± 15 vs. 63 ± 12, respectively). Negative antibody control for TIMP1 and TIMP2 revealed no staining, as shown in Figure 3, E and F, respectively.
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MMP9 and MMP2 Protein Expression in Decidual Cell Monolayer Cultures
Figure 4 describes the separate and interactive effects of MPA and cytokines on MMP2 (Fig. 4A) and MMP9 (Fig. 4B) output by leukocyte-free, first-trimester decidual cells. Circulating levels of E2 and progesterone are elevated during the first trimester, prompting use of E2 as the control incubation for E2 + MPA. Basal output of MMP2 was several-fold higher than that of MMP9 and was unaffected by exposure to either MPA or inflammatory cytokines (Fig. 4A). Although basal MMP9 output was also unaffected by MPA, it was markedly upregulated during incubations with the proinflammatory cytokines (Fig. 4B). Compared with the basal output in cultures incubated with E2 alone (0.29 ± 0.02 pg/ml per microgram of cell protein, n = 8), MMP9 output was elevated to 42.5 ± 19.7 and 264.7 ± 151.5 (P < 0.05) by 1 ng/ml of IL1B and TNF, respectively. Similarly, in cultures incubated in parallel with E2 + MPA, IL1B and TNF elevated MMP9 output to 51.0 ± 27.1 and 495.1 ± 269.6, respectively, compared with 0.44 ± 0.16 pg/ml per microgram of cell protein in cultures maintained in E2 + MPA alone (P < 0.05). Moreover, E2 + MPA neither affected MMP9 output compared with E2 alone nor did it alter the response to IL1B and TNF. Figure 4C displays the results of Figure 4, A and B as fold-changes from E2 for each experiment to contrast constitutive MMP2 output with the marked enhancement of MMP9 output elicited by IL1B (214 ± 128-fold) and by TNF (1977 ± 1125-fold), P < 0.05.
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The zymograms depicted in Figure 5 demonstrate that decidual cell-conditioned medium contains discrete zones of gelatinolytic activity that correspond to MMP2 and MMP9 at 72 kDa and 92 kDa, respectively; that is, the pro-forms of the MMPs. [36]. As evidenced by the zymograms, pretreatment of the decidual cell-conditioned medium with the organomercurial APMA shifts virtually all of the pro-MMP forms to the active MMP forms (66 kDa for MMP2 and 86 kDa for MMP9). Consistent with the differential regulation of MMP2 and MMP9 indicated by the ELISA results of Figure 4, neither the cytokines nor MPA affected the magnitude of MMP2, whereas that of MMP9 was markedly increased by IL1B and TNF but unaltered by MPA. The Western blot depicted in Figure 6 confirms the ELISA and zymography results by demonstrating that the decidual cells secrete MMP2 and MMP9 in their pro-forms that correspond to the molecular weights of pro-form positive controls.
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As the addition of a progestin did not modulate either basal or cytokine-enhanced MMP9 output, the dose-response effects of IL1B and TNF on MMP9 output by decidual cells that were performed after priming with E2 + MPA are shown in Figure 7. These results indicate that secreted MMP9 levels were enhanced by IL1B (Fig. 7A) and by TNF (Fig. 7B) in a concentration-dependent manner.
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MMP9 and MMP2 mRNA Expression in Decidual Cell Monolayer Cultures
Figure 8 displays steady-state MMP2 (Fig. 8A) and MMP9 (Fig. 8B) mRNA levels as determined by quantitative real-time RT-PCR in parallel cultures primed with either E2 or with E2 + MPA. Consistent with the results for secreted MMP2 and MMP9 levels shown in Figure 4, neither MPA, IL1B, or TNF exerted significant effects on MMP2 mRNA levels, whereas MMP9 mRNA levels were markedly upregulated by IL1B and TNF. As shown in Figure 8C, the fold changes compared with E2 treatment alone indicate the absence of a statistically significant effect of MPA on MMP9 mRNA levels, whereas IL1B and TNF enhanced MMP9 mRNA levels by 54.3 ± 13.7-fold and 103.9 ± 40.0-fold, respectively.
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TIMP1 and TIMP2 Protein Expression in Decidual Cell Monolayer Cultures
Figure 9 displays the ELISA results of the addition of IL1B and TNF on secreted levels of TIMP1 and TIMP2 by first-trimester human decidual cell cultures. Note that in contrast to the marked increased MMP9 responses, there were no significant effects of either cytokine on either TIMP1 or TIMP2 levels.
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The pivotal role played by the depth of EVT invasion of human decidua in remodeling spiral arteries and arterioles into low-resistance, high-capacitance vessels, taken together with the impact that this transformation exerts on uteroplacental blood flow have provoked numerous investigations into the invasiveness of normal and immortalized first-trimester EVTs. Such studies indicate that EVT migration and invasion can be modulated by several factors expressed by trophoblasts and decidual cells at the maternal-fetal interface. These include insulin-like growth factor 2 (IGF2), its IGF2 mannose-6 phosphate receptor [37] and IGF-binding protein 1 (IGFBP1) [38, 39], prostaglandin E2 and its EP1 receptor (PTGER1) [40], and transforming growth factor beta (TGFB) isoforms and decorin, which stores TGFB in the decidual ECM [41–43].
Migration and invasion of human EVTs exhibit similarities with the extensively studied processes in tumor cells. Tumor cell invasion also involves sequential integrin-mediated adherence to, then proteolysis of, ECM components of the host tissue [18, 44]. Numerous observations from studies on tumor cells emphasize the important roles played by urokinase [45] and MMPs, particularly MMP2 and MMP9 [17, 46], and the respective modulating actions of plasminogen activator inhibitor 1 (PAI1) [45] and TIMPs [46]. Use of immunohistochemistry and in situ hybridization in human tissues, taken together with studies in transgenic cancer mouse models indicate that during tumor growth, invasion, and metastasis, the host stromal cells are the major contributors of urokinase and MMPs, with the cancer cells relegated primarily to stimulating neighboring stromal cells to synthesize key components that regulate ECM turnover [47, 48].
Compared with invasion of host tissues by tumor cells, EVT invasion is under far more stringent spatial and temporal control, as it is limited to the endometrium and proximal myometrium and is terminated by midgestation [18, 44, 49]. The decisive role played by the decidua in restraining intrinsic EVT invasiveness is evident from the uncontrolled invasion, with often life-threatening consequences associated with implantation at sites where the decidua is absent, as in ectopic pregnancies, including cesarean scar pregnancies, or deficient, as in placenta accreta [50]. Despite the major responsibility borne by host stromal cell-expressed proteases in tumor cell invasion, studies of EVT invasion of the decidua have primarily focused on MMP2 and MMP9 expression by the trophoblasts [18, 23, 49, 51]. In this regard, trophoblasts from preeclamptic pregnancies were recently found to express lower levels of MMP2 and MMP9 than their normal counterparts [52]. By contrast, there is a dearth of studies evaluating the potential contribution that stromal/decidual-derived proteases could contribute to this invasion.
The current study assessed immunostaining for MMP9 and MMP2 in preeclamptic and control decidual sections. Statistically significant augmentation of staining for MMP9 was observed in the decidual cells of preeclamptic versus control specimens, with no significant differences in MMP2 decidual cell immunostaining in evidence. In contrast, immunostaining for MMPs was not different in the adjacent interstitial trophoblasts of preeclamptic versus control placentas. Consistent with our observations of a marked preeclampsia-related increase in MMP9 immunostaining in the decidua, this study found that IL1B and TNF markedly upregulated MMP9 mRNA and protein expression in cultured leukocyte-free first-trimester decidual cells, whereas MPA affected neither basal nor cytokine-enhanced MMP9 expression. Unlike MMP9, MMP2 expression appears to be constitutive, since it was unaffected by MPA or inflammatory cytokines added separately or together.
Zymography results revealed that, as in other cell systems, first-trimester decidual cells secrete MMP2 and MMP9 as zymogens [19]. Activation of the secreted MMP2 zymogen is usually mediated by membrane type 1 MMP [53, 54]. However, during early human pregnancy several studies suggest that urokinase-type plasminogen activator (uPA) bound to trophoblast surface receptors generates plasmin, which activates ECM-degrading pro-MMP2 and pro-MMP9 to promote EVT invasion (reviewed in Ferretti et al. [44]). Although trophoblasts are generally thought to secrete the pro-MMP2 and pro-MMP9 that drive invasion, the current results suggest that: 1) expression of both MMPs by decidual cells, the predominant cell type encountered by invading EVTs [32, 33], may regulate EVT invasion; 2) overexpression of MMP9 by decidual cells contributes to shallow placentation by interfering with the normal stepwise integrin-mediated EVT invasion of the decidua. Such dysregulation is likely intensified by decidual cell-expressed stromelysin 1 (MMP3), a documented pro-MMP9 activator [55]. We recently found MMP3 to be markedly enhanced by TNF and IL1B in first-trimester human decidual cells with significantly higher immunoreactive MMP3 levels in decidual cells of preeclamptic versus gestational age-matched control placental sections (W. Murk et al., unpublished results).
Studies in several tissues suggest that the activity of secreted pro-MMPs can be blocked by extracellular TIMPs [19]. While all of the TIMPs can bind to some degree to all the MMPs, TIMP2 is generally constitutively expressed and specific for MMP2 [56], whereas expression of TIMP1 binds with high affinity to MMP9 and can be regulated by proinflammatory cytokines [57–59]. The current study in first-trimester decidual cell monolayers observed the expected lack of regulation in MMP2 and TIMP2 expression. However, it found a striking discordance between MMP9 and TIMP1 expression with the multi-fold elevation in MMP9 protein expression elicited by IL1B and TNF, respectively, that was unaccompanied by a significant increase in secreted TIMP1 levels. Immunostaining revealed no differences between preeclamptic versus gestational age-matched control decidual specimens for TIMP1 or TIMP2 levels, with TIMP1 being present at low levels.
Previous studies in specimens of human endometrium obtained during the menstrual cycle found that mRNA levels for several MMPs are downregulated during the progesterone-dominated luteal phase and markedly upregulated in the progesterone withdrawal-initiated menstrual phase. By contrast, mRNAs for TIMP1 and TIMP2 are essentially unchanged over this period [22, 24]. In cultured stromal cells obtained from predecidualized human endometrium, incubation with progestin inhibited expression of MMP3 [60] (and interstitial collagenase [MMP1]), but not that of MMP2 or TIMP1 [20]. Moreover, progestin withdrawal enhanced expression of MMP3 and MMP1 but did not alter MMP2, TIMP1, or TIMP2 expression [20, 61]. Incubation of such stromal cells with IL1B and TNF elevated the expression of MMP1 and MMP9, whereas the expression of MMP2, TIMP1, and TIMP2 was unaffected by the cytokines [62].
While prior reports indicate that IL1B and TNF enhance EVT invasiveness by enhancing levels of MMP9 mRNA, immunoreactive protein, and/or activity [63–65], these increases are orders of magnitude lower than the hundred- to thousand-fold enhancement of MMP9 protein expression by the decidual cells elicited by IL1B and TNF, respectively. Indeed, the hypothesis that IL1B and TNF would induce a net increase in trophoblast invasion of first-trimester decidua is disputed by: 1) the documented association between these cytokines and preeclampsia [25–27]; 2) the established involvement between preeclampsia and impaired EVT invasion of the decidua [7–12]; 3) reports that TNF inhibits trophoblast invasiveness [27, 66]; and 4) the recent observation that inhibition of trophoblast invasiveness by lipopolysaccharide-activated macrophages was relieved by a TNF neutralizing antibody [67]. In summary, our results suggest that enhancement of decidual cell-expressed MMP9 by IL1B and TNF may disrupt the integrity of the decidual ECM composition. The consequent interference with the normal stepwise integrin-mediated invasion of the decidua by EVT would promote the shallow placentation found in preeclampsia.
FOOTNOTES
1Supported by National Institutes of Health grants 2 R01HD33937-05 and 1 R01 HL070004-04 to C.J.L. 
Correspondence: 2Frederick Schatz, Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, 333 Cedar St., Rm 335 FMB, PO Box 208063, New Haven, CT 06520-8063. FAX: 203 737 2327; e-mail: Frederick.Schatz{at}yale.edu
Received: 27 June 2007.
First decision: 24 July 2007.
Accepted: 8 February 2008.
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 Y.-H. Kim, H.-S. Hwang, Y.-T. Kim, H.-S. Kim, and Y.-W. Park Modulation of Matrix Metalloproteinase Secretion by Adenosine A3 Receptor in Preeclamptic Villous Explants Reproductive Sciences, November 1, 2008; 15(9): 939 - 949. [Abstract] [PDF]
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