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Matrix-metalloproteinase Activity in First Trimester Placental Bed Biopsies in Further Complicated and Uncomplicated Pregnancies
Placenta (2004), 25, 253–258 doi:10.1016/j.placenta.2003.10.006
Matrix-metalloproteinase Activity in First Trimester Placental Bed Biopsies in Further Complicated and Uncomplicated Pregnancies M. A. Huisman a, A. Timmer b, M. Zeinstra b, E. K. Serlier a, R. Hanemaaijer c, H. v. Goor b and J. J. H. M. Erwich a,* a
Department of Obstetrics and Gynaecology, University Hospital Groningen, CMC V, 4th floor, Y4187, PO Box 30001 Hanzeplein 1, 9700 RB Groningen, The Netherlands; b Department of Pathology and Laboratory Medicine, University Hospital Groningen, The Netherlands; c TNO PG, Gaubius Laboratory Leiden, The Netherlands Paper accepted 5 October 2003
Trophoblast invasion is partly regulated by matrix-metalloproteinases (MMPs). Aberrations in MMP-activity in early pregnancy are thought to play a role in the pathophysiology of pregnancy associated conditions like pre-eclampsia and intra-uterine growth restriction (IUGR). A direct relation however, has not been published. We tested the hypothesis that MMP activity in the decidua is compromised in the first trimester of pregnancies, which are complicated by hypertensive disorders or IUGR in later pregnancy. During chorionic villus biopsy, decidua is microscopically separated from the villi and stored. A selection of pregnancies complicated by pre-eclampsia or HELLP-syndrome or IUGR was made, with two matched controls each. Zymography was performed to identify the presence of MMPs, and subsequently immunohistochemistry for MMP-2 and -9 and cytokeratin 7 to examine differences between cases and controls. Next, a specific immuno-capture assay was used to determine the activity of MMP-1, -2, -3, -8, -9, and 13, total as well as active. Although presence of MMP-2 and MMP-9 was found, which corresponded with the immunohistochemistry, no significant differences could be demonstrated between activity of total MMP-2 and total MMP-9 in complicated and uncomplicated pregnancies. Activity of MMP-1, -3, -8 and -13 could not be detected. In conclusion: our study confirms the presence of MMP-2 and -9 in first trimester placental bed biopsies, but does not support the current concept of deranged MMP-activity in early pregnancy in further complicated pregnancies. Placenta (2004), 25, 253–258 2003 Elsevier Ltd. All rights reserved.
INTRODUCTION Placental dysfunction can be partly explained by the maladaptation of uterine spiral arteries through impaired implantation and inadequate trophoblast invasion. This invasion of cytotrophoblasts is partly regulated by the secretion of proteases, in particular matrix-metalloproteinases [1]. Matrix-metalloproteinases (MMPs) are a family of zincrequiring enzymes [2–4]. They play a key role in tissue remodeling in both normal and pathological processes [5,6]. MMPs can be divided in four groups [7], the collagenases [interstitial collagenase (=MMP-1), neutrophil collagenase (=MMP-8), human collagenase (=MMP-13)], type IV collagenases or gelatinases [gelatinase A (=MMP-2), gelatinase B (=MMP-9)], stromelysins [stromelysin-1 (=MMP-3)] and the membrane-type matrixmetalloproteinases (MT-MMP). The extracellular matrix boundary for invasion, the basement membrane, mainly consists of type IV collagen [4,8,9]. Type IV collagen is also present next to the perivascular cells *
To whom correspondence should be addressed. Tel.: +31-503613020; Fax: +31-50-3611806; E-mail: [email protected] 0143-4004/$–see front matter
[10] and is a substrate for MMP-2 and -9. It has been demonstrated that human trophoblast invasiveness in vitro depends on the production of these MMPs and that both MMP-2 and MMP-9 are secreted by human trophoblasts isolated from first trimester placenta. Previous studies indicate that there is production of MMP-2 protein and expression of its mRNA in both cytotrophoblasts from cell columns of anchoring villi and from decidual cells of first trimester human pregnancies [11–13]. Immunohistochemistry of MMP-9 showed labelling of both syncytiotrophoblast and of extravillous cytotrophoblast [14–16]. Trophoblast from third trimester placentae secretes primarily MMP-9 and minimal amounts of MMP-2 [17]. Presence of the collagenase MMP-1 as well as the stromelysin MMP-3 was earlier described in trophoblast, but not in decidua [13]. MMP-8 and MMP-13 have not been described in trophoblast, but have a role in tumour growth and invasion [6]. MMP-13 preferentially cleaves type II collagen, and may act like a gelatinase [18]. It is not known whether placental-bed MMP-activity is compromised in the first trimester of pregnancies which develop pathological conditions like pre-eclampsia or 2003 Elsevier Ltd. All rights reserved.
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intra-uterine growth restriction in later trimesters. Therefore, our current study is designed to detect MMP-activity in total as well as in latent form in first trimester placental bed biopsies of pregnancies with a known outcome. Activity of MMP-1, -2, -3, -8, -9 and -13 was examined in pregnancies further complicated by hypertensive disorders or intra-uterine growth restriction and uncomplicated control pregnancies, using immunohistochemistry, zymography and immunocapture assays.
20 µm sections, subsequently adding of buffer and homogenization [buffer (pH 7.6): 50 m Tris, 5 m CaCl2, 1 µ ZnCl2, 1 per cent Triton X-100 (100 µl buffer/mg sample)]. Next, the homogenate was centrifuged and 50 µl aliquots of the supernatant were stored. All steps were performed on ice, and slides and supernatants were subsequently kept at 20(C. Protein concentrations were determined using a BCA Protein Assay Kit (23225, Pierce, IL, USA) according to the manufacturers recommendations.
MATERIALS AND METHODS
Zymography
Chorionic villus sampling (CVS) was performed vaginally between 10 and 12 weeks of gestation, largely for maternal age or serumscreening related risk for Down syndrome. Length of gestation was calculated according to the last menstrual period and subsequently confirmed by ultrasound (crown–rump length). Tissue was obtained using a biopsycatheter (Cook, K-CMA-5000). Follow-up of these pregnancies was available by a questionnaire returned by the patient postpartum. During CVS, surplus material was obtained containing decidua with little trophoblast. Under the microscope, this decidua was mechanically separated from the villi using needles and stored immediately at 20(C until further processing. From 1998 onwards, 700 samples with known outcome were collected. Out of these samples, a selection was made of 13 pregnancies complicated by hypertensive disorders like pre-eclampsia (PE) and HELLP-syndrome or intra-uterine growth restriction (IUGR). Pre-eclampsia was defined as a diastolic blood-pressure (BP) of 90 mmHg or a raise of 15 mm Hg compared with first trimester BP, measured twice, combined with proteinuria of at least 300 mg/day. HELLP syndrome was defined by laboratory results indicating haemolysis, a raise in alanin-aminotransferase (ALAT), aspartateaminotransferase (ASAT) and lactatedehydrogenase (LDH), and a platelet count lower than 100109/l. For IUGR, criteria according to Kloosterman [19] were used. Birthweight was corrected for gestational age, and percentiles were computed. IUGR was defined as a corrected birthweight below the 5th percentile. Each case was matched with two controls, matching for maternal age, parity and gestational age at time of sampling. Patients with concurrent morbidity (e.g. diabetes), smoking or on medication were excluded. Fetal karyotyping showed no chromosomal abnormalities. Patients were informed that surplus material could be used for research, according to the ‘Guideline Good Use’ by the FMWV-committee (Federation of Medical Scientific Associations). The placental bed biopsies were divided into parts of which from the center of the sample cryostat sections were cut for histology (H&E, Cytokeratin 7, MMP-2 and MMP-9). The remaining sample was homogenized for further assessment of MMP-activity and protein concentrations. Homogenization was performed, after weighing, by cutting the samples into
Presence of MMPs was analysed by zymography, using a 10 per cent polyacrylamide gel (108 cm in SE 245 Mighty Small device of Hoefer, San Fransisco, USA), containing 0.1 per cent gelatin (type A: from procine skin, Sigma) as a substrate. The gel was loaded with 15 µl samples of a 1 : 1 mixture of supernatant and non-reducing loading buffer (4 per cent SDS, 20 per cent Glycerol, 0.005 per cent phenolblue and 0.125 Tris pH 6.8), incubated for 20 min. Electrophoresis was performed in a cold chamber (4(C) with 20 mA per gel, followed by incubation for 30 min with renaturing fluid (2.5 per cent Triton X-100) and a wash with developing buffer (50 m Tris pH 7.6; 5 m CaCl2; 0.2 NaCl; 0.02 per cent Triton X-100). The gels were incubated overnight in fresh developing buffer at 37(C. After staining with Coomassie Blue (10 per cent glacial acetic acid; 30 per cent methanol; 1.25 g Coomassie Brilliant Blue) for 30 min, destaining (10 per cent glacial acetic acid; 30 per cent methanol) took 1 h. Proteolytic activities were visualized by clear zones against a dark blue background, indicating lysis of gelatin. No proteolytic activity was seen when after electrophoresis the gel was incubated in EDTA containing buffers (10 m EDTA) confirming true MMP activity. Moleclular weights (MW) were determined running a standard panel of seven proteins with known MWs ranging from 6.5–200.0 kD (Kaleidoscope Prestained Standards, Bio-Rad, Hercules, CA, USA) together with the samples.
Immunohistochemistry (cytokeratin 7, double-staining MMP-2 and -9/cytokeratin 7) Cryostat sections (4 µm) were dried and fixed in acetone for 10 min. All steps were performed at room temperature and followed by rinsing with Phosphate Buffered Saline, pH 7.4 (PBS) for 5 min. Next, sections were incubated with a monoclonal antibody against cytokeratin 7 (RKC 105, Eurodiagnostics, Apeldoorn, the Netherlands) in PBS/ 1 per cent BSA 1 : 50, for 60 min. Subsequently, endogenous peroxidase was blocked by incubation in 0.075 per cent H2O2 in PBS for 30 min. The secondary antibody, peroxidase conjugated Rabbit anti-Mouse antibody (DAKO, Glostrup, Denmark), was used in a 1 : 50 dilution in PBS/1 per cent BSA, containing 1 per cent AB serum and incubated for
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30 min. Peroxidase activity was visualized using 3-Amino-9Ethyl-Carbazol (AEC). After rinsing with demiwater for 5 min, counterstaining was performed using haematoxylin. Sections were subsequently mounted in Kaiser’s glycerol gelatin. The slides were examined with a 40 objective microscope (Olympus BHC, Japan) and scored 0/+/++ for cytokeratin 7 as determination of the amount of trophoblast in the placental bed biopsy. A score of 0 was given when no trophoblast cells were present, + in case of few and ++ when trophoblast was abundant. For the double-staining with cytokeratin 7 and MMP-2 or MMP-9, first, endogenous peroxidase was blocked with 125 µl H2O2 in 50 ml of PBS for 30 min. Next, slides were incubated with a monoclonal antibody against MMP-2 (Ab-3, Oncogene/Calbiochem, Cambridge, MA, USA) in PBS/1 per cent BSA 1 : 50 or MMP-9 (#56-2A4, Oncogene/ Calbiochem, Cambridge, MA, USA) in PBS/1 per cent BSA 1 : 200 for 60 min. Subsequently the sections were incubated consecutively with peroxidase conjugated Rabbit anti-Mouse, Goat anti-Rabbit and Rabbit anti-Goat antibody (all DAKO, Glostrup, Denmark) with a 1 : 50 and 1 : 100 dilution in PBS/1 per cent BSA with 1 per cent AB serum during 30 min. For visualization of the peroxidase-activity, 3-Amino-9-EthylCarbazol (AEC) was used. To avoid aspecific staining of the second primary antibody 0.1 Glycin, pH=2 for 45 min was applicated. The slides were incubated with the second primary antibody, cytokeratin 7 (RCK 105, Eurodiagnostics, Apeldoorn, the Netherlands) 1 : 50 in PBS/1 per cent BSA for 1 h. As a secondary antibody Alkaline Phosphatase-Conjugated Goat anti-Mouse antibody (DAKO, Glostrup, Denmark) 1 : 50 in PBS/1 per cent BSA containing 1 per cent AB serum was used, and incubated for 30 min. For visualization of the second primary antibody, Fast Blue was used (10 mg Naphtol AS-MX in 50 ml 0.1 Tris/HCl pH=8.2 with 10 per cent MgSO4, 50 mg of Fast Blue BB and 12 mg Levamisol) for 30 min. After rinsing the slides were mounted with Kaisers glycerol gelatin. Slides stained for only cytokeratin 7 and slides that were double-stained were compared to confirm blocking after the first primary antibody.
Immunocapture-assay MMP Although there is a clear link between zymography and the MMP-assay [20], zymography is semi-quantitative and does not measure true activity of the MMPs. TIMP-MMP complexes dissociate with zymography and seem to increase the amount of active MMP, this in contrast to an immunocaptureassay. After dilution of all the samples to a protein concentration of 80 µg/ml, a specific immunocapture-assay (Amersham Biosciences, Buckinghamshire, UK) was used to determine the activity of the different MMPs [20–23]. The assay is an established method, and is based on a modified pro-urokinase, where the activation sequence, normally recognized by plasmin (Pro–Arg–Phe–Lys [Ile–Ile–Gly–Gly), is replaced by a sequence that is specifically recognized by
Figure 1. Immunohistochemistry with cytokeratin 7 (RCK 105) and Mayers haematoxylin. There is clear staining of cytotrophoblast in red with nuclei staining in blue. Patients scoring 0 (A), ‘+’ (B) or ‘++’ (C), corresponding with the amount of trophoblast cells in the samples. ‘0’ no trophoblast cells, ‘+’ few, and ‘++’ abundant. Scale bar=50 µm.
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3UR003 3UR003
Figure 2. Zymography of one of the homogenates. Two bands appear of 72 and 90 kD, corresponding with proMMP-2 and proMMP-9 respectively. The other bands have higher molecular weights and are believed to be complexes of MMP-9 with other proteins. Lanes 1, 2 and 3 are loaded with 15, 12 and 9 µg protein respectively. Lanes 4–9: serial dilutions of homogenate starting with 6 µg protein loaded in lane 4. Note that MMP-2 and MMP-9 are still visible in lane 7, loaded with less than 1 µg of protein.
MMPs (Arg–Pro–Leu–Gly ?Ile–Ile–Gly–Gly). A chromogenic peptide substrate for urokinase is then added to measure the active urokinase, generated through MMP activation of the modified urokinase. Specific MMP-1, -2, -8, -9 and -13 antibody-coated microtiter plates are used to confirm MMPspecificity. Immobilized latent MMP is then activated using p-aminophenylmercuric acetate (APMA) to measure total MMP. Differential activation with APMA enables the analysis of the activity of both already active and latent forms of MMPs. The activity is expressed as (DeltaA/ to2*1000)/10 µl.
RESULTS Microscopic examination of the tissues (H&E) showed the presence of decidual cells. Staining for cytokeratin 7 revealed a positive reaction in 18 out of 39 samples. Fifteen scored ‘+’, 3 scored ‘++’ for the amount of trophoblast in the tissue (Figure 1). The cytokeratin-positive samples were cases as well as controls, and no significant differences could be seen between the group of hypertensive disorders, IUGR or controls. Using zymography of four random samples, two bands of 72 kD and 90 kD appeared, corresponding with respectively proMMP-2 and proMMP-9. These bands could still be detected with a minimum of 25 µg of substrate, corresponding with 1 µg of protein (Figure 2). The assay for the activity of the different MMPs was first performed on a subset of samples of both groups (complicated (n=4) as well as uncomplicated pregnancies (n=4)), and no activity of MMP-1, -3, -8 or -13 could be measured, nor in the active form nor in the total complex. In case of MMP-2, total MMP-2 was detected in a range of 0.4 to 12.0 U/µl ((DeltaA/to2*1000)/10 µl). However, no significant differences were found between complicated pregnancies and their matched controls. Activity of total MMP-9 was detected in six out of the 39 samples, ranging from 1.4 to 26.7 U/µl ((DeltaA/to2*1000)/10 µl). Comparing the results of the scores for cytokeratin 7 with the activity of total MMP-2 or total MMP-9 for the cases and their controls, no relation between cases and their controls could be found. Immunohistochemistry for MMP-2 and MMP-9 of 2 selected samples showed clear staining for MMP-2 in case of high MMP-2 activity in the assay. Staining of the decidual
cells, as well as of the trophoblasts was seen (Figure 3A and B). MMP-9 activity was low in both samples, but in the sample where trophoblast was abundant, immunostaining for MMP-9 was profound (Figure 3D). Less staining for MMP-2 was related to low activity (Figure 3C). Comparing the complicated pregnancies (hypertensive and IUGR) with their controls, no significant differences could be found. Tables 1a and 1b shows the differences in the means of activity of total MMP-2. No significant differences could be found between cases and controls. For MMP-9, no difference between the medians of MMP-activity in first trimester placental bed biopsies in further complicated or uncomplicated pregnancies could be demonstrated.
DISCUSSION An essential feature of human placentation is the interaction of invading trophoblast with the uterine vasculature [24]. Interaction of trophoblast with the extracellular matrix is one of the main factors in providing a substrate for attachment and growth or migration. In pre-eclamptic patients this trophoblast invasion is impaired, mainly in the spiral arteries. Although many observations regarding impaired early placentation in pregnancies further complicated by pre-eclampsia or IUGR are known [25], the exact regulation is still to be elucidated. The decidualized endometrium has influence on trophoblast function by for example altering the expression of the metalloproteinases. Stromal cell differentiation is associated with loss of collagen VI, which facilitates invasion through the interstitial spaces of the decidua [26]. Regarding the profound changes in early pregnancy mentioned above, we hypothesized that differences in MMP activity exists in first trimester decidua of pregnancies that later develop complications like pre-eclampsia or IUGR. Storage of decidual samples, obtained by cvs, till after the outcome of the pregnancy was known, enabled us to investigate a case-control series. From the results of our current study we may conclude that in week 10 to 12 of pregnancy, MMP-2 and -9 can already be detected in small decidual samples. As shown earlier by Huppertz et al. [13], we found MMP-2 and MMP-9 expression in extravillous trophoblast as well as in decidual cells.
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Figure 3. Immunohistochemical double-staining for cytokeratin 7/MMP-2 or cytokeratin 7/MMP-9. Cytokeratin is stained blue, with the different MMPs stained brownish. Scale bar=50 µm. (A) Patient scoring ‘+’ for cytokeratin 7 with high total MMP-2 activity and low total MMP-9 activity in the assay. Double-staining for cytokeratin 7 and MMP-2. Strong staining for MMP-2 can be seen, of trophoblast cells as well as of decidual cells. (B) Patient scoring ‘+’ for cytokeratin 7 with high total MMP-2 activity and low total MMP-9 activity in the assay. Double-staining for cytokeratin 7 and MMP-9. The sample shows weak staining of MMP-9, corresponding with the low activity in the assay. (C) Patient scoring ‘++’ for cytokeratin 7 with low total MMP-2 and low total MMP-9 activity. Double-staining for cytokeratin 7 and MMP-2. Weak staining of the trophoblast and decidua with MMP-2. (D) Patient scoring ‘++’ for cytokeratin 7 with low total MMP-2 and low total MMP-9 activity. Double-staining for cytokeratin 7 and MMP-9. Strong staining of MMP-9, of the decidua as well as of the trophoblast.
In view of earlier described differences in the third trimester, and the presumed role of MMPs in early pregnancy, it is remarkable that we did not find any differences between cases and controls. However, several factors can be taken into account. First, it may be that the most important area for MMP action is at the cell surface and activities in the tissue homogenates are not informative. Also possible is that, though proMMP-2 and -9 are present and total activity can be measured, they are not discriminating between further complicated and normal pregnancies. This is in contrast to studies in third trimester pregnancies, where differences were found. It may be too early in pregnancy to detect a difference in MMP-activity in decidua.
A third possibility is that the activity of the MMPs in decidual cells is not compromised in complicated pregnancies, but the activity in trophoblast cells is, as shown in placentae at term [27]. However, there is no difference in the relation between the presence of trophoblast and MMP-activity in our cases compared to controls, although we do see profound expression of MMP-9 immunohistochemistry in case of abundant trophoblast. Finally, MMP activity is a balance between proMMP with their inhibitors (TIMPs) and activators. Though measurement of total MMP-activity may not differ, it is still possible that a shift in balance has taken place in compromised pregnancies from activity in trophoblastic to decidual cells or vise versa.
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Table 1a. Means of total MMP-2 activity for cases and their matched controls grouped for pre-eclampsia or HELLP syndrome and for IUGR (n.s.: not significant. Student’s t test) Complication
IUGR PE/HELLP
Total MMP-2 activity (DeltaA/to2*1000/10 µl)
Difference
Mean of cases (SD)
Mean of matched controls (SD)
5.54 (2.7) (n=9) 5.02 (0.6) (n=4)
5.32 (2.6) (n=18) 5.72 (2.6) (n=8)
n.s. n.s.
Table 1b. Medians of total MMP-9 activity for cases and their matched controls (n.s.: not significant. Wilcoxon test) Complication
IUGR PE/HELLP
Total MMP-9 activity (DeltaA/to2*1000/10 µl)
Difference
Median of cases (ranges)
Median of matched controls (ranges)
0.0 (0.0–26.1) (n=9) 0.0 (0.0–0.0) (n=4)
0.0 (0.0–7.4) (n=18) 0.0 (0.0–1.4) (n=8)
The necessary degradation of the extracellular matrixcomponents for the invasion of trophoblast in the maternal decidua seems a complex interaction. MMPs might act from both sides: Trophoblasts producing MMPs to enable invasion, and decidual cells allowing invasion. However, in conclusion, our study does not support the currently held concept of deranged MMP-activity in early pregnancy in further complicated pregnancies.
[14] [15] [16] [17]
REFERENCES [1] Fisher SJ, Leitch MS, Kantor MS, Basbaum CB, Kramer RH. Degradation of extracellular matrix by the trophoblastic cells of first-trimester human placentas. J Cell Biochem 1985;27:31–41. [2] Woessner J-FJ. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 1991;5:2145–54. [3] Jiang W, Bond JS. Families of metalloendopeptidases and their relationships. FEBS Lett 1992;312:110–4. [4] Nagase H, Woessner J-FJ. Matrix metalloproteinases. J Biol Chem 1999; 274:21491–4. [5] Stetler-Stevenson WG, Liotta LA, Kleiner D-EJ. Extracellular matrix 6: role of matrix metalloproteinases in tumor invasion and metastasis. FASEB J 1993;7:1434–41. [6] Mignatti P, Rifkin DB. Biology and biochemistry of proteinases in tumor invasion. Physiol Rev 1993;73:161–95. [7] Bischof P, Meisser A, Campana A. Paracrine and autocrine regulators of trophoblast invasion—a review. Placenta 2000;21:S55–60. [8] Docherty AJ, Murphy G. The tissue metalloproteinase family and the inhibitor TIMP: a study using cDNAs and recombinant proteins. Ann Rheum Dis 1990;49:469–79. [9] Cross JC, Werb Z, Fisher SJ. Implantation and the placenta: key pieces of the development puzzle. Science 1994;266:1508–18. [10] Autio-Harmainen H, Sandberg M, Pihlajaniemi T, Vuorio E. Synthesis of laminin and type IV collagen by trophoblastic cells and fibroblastic stromal cells in the early human placenta. Lab Invest 1991;64:483–91. [11] Fernandez PL, Merino MJ, Nogales FF, Charonis AS, Stetler-Stevenson W, Liotta L. Immunohistochemical profile of basement membrane proteins and 72 kilodalton type IV collagenase in the implantation placental site. An integrated view. Lab Invest 1992;66:572–9. [12] Polette M, Gilbert N, Stas I, Nawrocki B, Noel A, Remacle A et al. Gelatinase A expression and localization in human breast cancers. An in situ hybridization study and immunohistochemical detection using confocal microscopy. Virchows Arch 1994;424:641–5. [13] Huppertz B, Kertschanska S, Demir AY, Frank HG, Kaufmann P. Immunohistochemistry of matrix metalloproteinases (MMP), their
[18] [19] [20]
[21]
[22] [23]
[24] [25] [26] [27]
n.s. n.s.
substrates, and their inhibitors (TIMP) during trophoblast invasion in the human placenta. Cell Tissue Res 1998;291:133–48. Sawicki G, Radomski MW, Winkler-Lowen B, Krzymien A, Guilbert LJ. Polarized release of matrix metalloproteinase-2 and -9 from cultured human placental syncytiotrophoblasts. Biol Reprod 2000;63:1390–5. Isaka K, Usuda S, Ito H, Sagawa Y, Nakamura H, Nishi H et al. Expression and activity of matrix metalloproteinase 2 and 9 in human trophoblasts. Placenta 2003;24:53–64. de Jager CA, Linton EA, Spyropoulou I, Sargent IL, Redman CW. Matrix metalloprotease-9, placental syncytiotrophoblast and the endothelial dysfunction of pre-eclampsia. Placenta 2003;24:84–91. Shimonovitz S, Hurwitz A, Dushnik M, Anteby E, Geva-Eldar T, Yagel S. Developmental regulation of the expression of 72 and 92 kd type IV collagenases in human trophoblasts: a possible mechanism for control of trophoblast invasion. Am J Obstet Gynecol 1994;171:832–8. Pendas AM, Balbin M, Llano E, Jimenez MG, Lopez-Otin C. Structural analysis and promoter characterization of the human collagenase-3 gene (MMP13). Genomics 1997;40:222–33. Kloosterman GJ. Over intra-uteriene groei en de intra-uteriene groeicurve. [Intrauterine growth and intrauterine growth curves]. Ned Tijdschr Verloskd Gynaecol 1969;69:349–65. Sier CF, Casetta G, Verheijen JH, Tizzani A, Agape V, Kos J et al. Enhanced urinary gelatinase activities (matrix metalloproteinases 2 and 9) are associated with early-stage bladder carcinoma: a comparison with clinically used tumor markers. Clin Cancer Res 2000;6:2333–40. Hanemaaijer R, Visser H, Konttinen YT, Koolwijk P, Verheijen JH. A novel and simple immunocapture assay for determination of gelatinase-B (MMP-9) activities in biological fluids: saliva from patients with Sjogren’s syndrome contain increased latent and active gelatinase-B levels. Matrix Biol 1998;17:657–65. Capper SJ, Verheijen J, Smith L, Sully M, Visser H, Hanemaaijer R. Determination of gelatinase-A (MMP-2) activity using a novel immunocapture assay. Ann N Y Acad Sci 1999;878:487–90. Hanemaaijer R, Sier CF, Visser H, Scholte L, van-Lent N, Toet K et al. MMP-9 activity in urine from patients with various tumors, as measured by a novel MMP activity assay using modified urokinase as a substrate. Ann N Y Acad Sci 1999;878:141–9. Pijnenborg R, Vercruysse L, Verbist L, Van AF. Interaction of interstitial trophoblast with placental bed capillaries and venules of normotensive and pre-eclamptic pregnancies. Placenta 1998;19:569–75. Brosens JJ, Pijnenborg R, Brosens IA. The myometrial junctional zone spiral arteries in normal and abnormal pregnancies: a review of the literature. Am J Obstet Gynecol 2002;187:1416–23. Aplin JD, Charlton AK, Ayad S. An immunohistochemical study of human endometrial extracellular matrix during the menstrual cycle and first trimester of pregnancy. Cell Tissue Res 1988;253:231–40. Kolben M, Lopens A, Blaser J, Ulm K, Schmitt M, Schneider KT et al. Proteases and their inhibitors are indicative in gestational disease. Eur J Obstet Gynecol Reprod Biol 1996;68:59–65.
Matrix-metalloproteinase Activity in First Trimester Placental Bed Biopsies in Further Complicated and Uncomplicated Pregnancies M. A. Huisman a, A. Timmer b, M. Zeinstra b, E. K. Serlier a, R. Hanemaaijer c, H. v. Goor b and J. J. H. M. Erwich a,* a
Department of Obstetrics and Gynaecology, University Hospital Groningen, CMC V, 4th floor, Y4187, PO Box 30001 Hanzeplein 1, 9700 RB Groningen, The Netherlands; b Department of Pathology and Laboratory Medicine, University Hospital Groningen, The Netherlands; c TNO PG, Gaubius Laboratory Leiden, The Netherlands Paper accepted 5 October 2003
Trophoblast invasion is partly regulated by matrix-metalloproteinases (MMPs). Aberrations in MMP-activity in early pregnancy are thought to play a role in the pathophysiology of pregnancy associated conditions like pre-eclampsia and intra-uterine growth restriction (IUGR). A direct relation however, has not been published. We tested the hypothesis that MMP activity in the decidua is compromised in the first trimester of pregnancies, which are complicated by hypertensive disorders or IUGR in later pregnancy. During chorionic villus biopsy, decidua is microscopically separated from the villi and stored. A selection of pregnancies complicated by pre-eclampsia or HELLP-syndrome or IUGR was made, with two matched controls each. Zymography was performed to identify the presence of MMPs, and subsequently immunohistochemistry for MMP-2 and -9 and cytokeratin 7 to examine differences between cases and controls. Next, a specific immuno-capture assay was used to determine the activity of MMP-1, -2, -3, -8, -9, and 13, total as well as active. Although presence of MMP-2 and MMP-9 was found, which corresponded with the immunohistochemistry, no significant differences could be demonstrated between activity of total MMP-2 and total MMP-9 in complicated and uncomplicated pregnancies. Activity of MMP-1, -3, -8 and -13 could not be detected. In conclusion: our study confirms the presence of MMP-2 and -9 in first trimester placental bed biopsies, but does not support the current concept of deranged MMP-activity in early pregnancy in further complicated pregnancies. Placenta (2004), 25, 253–258 2003 Elsevier Ltd. All rights reserved.
INTRODUCTION Placental dysfunction can be partly explained by the maladaptation of uterine spiral arteries through impaired implantation and inadequate trophoblast invasion. This invasion of cytotrophoblasts is partly regulated by the secretion of proteases, in particular matrix-metalloproteinases [1]. Matrix-metalloproteinases (MMPs) are a family of zincrequiring enzymes [2–4]. They play a key role in tissue remodeling in both normal and pathological processes [5,6]. MMPs can be divided in four groups [7], the collagenases [interstitial collagenase (=MMP-1), neutrophil collagenase (=MMP-8), human collagenase (=MMP-13)], type IV collagenases or gelatinases [gelatinase A (=MMP-2), gelatinase B (=MMP-9)], stromelysins [stromelysin-1 (=MMP-3)] and the membrane-type matrixmetalloproteinases (MT-MMP). The extracellular matrix boundary for invasion, the basement membrane, mainly consists of type IV collagen [4,8,9]. Type IV collagen is also present next to the perivascular cells *
To whom correspondence should be addressed. Tel.: +31-503613020; Fax: +31-50-3611806; E-mail: [email protected] 0143-4004/$–see front matter
[10] and is a substrate for MMP-2 and -9. It has been demonstrated that human trophoblast invasiveness in vitro depends on the production of these MMPs and that both MMP-2 and MMP-9 are secreted by human trophoblasts isolated from first trimester placenta. Previous studies indicate that there is production of MMP-2 protein and expression of its mRNA in both cytotrophoblasts from cell columns of anchoring villi and from decidual cells of first trimester human pregnancies [11–13]. Immunohistochemistry of MMP-9 showed labelling of both syncytiotrophoblast and of extravillous cytotrophoblast [14–16]. Trophoblast from third trimester placentae secretes primarily MMP-9 and minimal amounts of MMP-2 [17]. Presence of the collagenase MMP-1 as well as the stromelysin MMP-3 was earlier described in trophoblast, but not in decidua [13]. MMP-8 and MMP-13 have not been described in trophoblast, but have a role in tumour growth and invasion [6]. MMP-13 preferentially cleaves type II collagen, and may act like a gelatinase [18]. It is not known whether placental-bed MMP-activity is compromised in the first trimester of pregnancies which develop pathological conditions like pre-eclampsia or 2003 Elsevier Ltd. All rights reserved.
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intra-uterine growth restriction in later trimesters. Therefore, our current study is designed to detect MMP-activity in total as well as in latent form in first trimester placental bed biopsies of pregnancies with a known outcome. Activity of MMP-1, -2, -3, -8, -9 and -13 was examined in pregnancies further complicated by hypertensive disorders or intra-uterine growth restriction and uncomplicated control pregnancies, using immunohistochemistry, zymography and immunocapture assays.
20 µm sections, subsequently adding of buffer and homogenization [buffer (pH 7.6): 50 m Tris, 5 m CaCl2, 1 µ ZnCl2, 1 per cent Triton X-100 (100 µl buffer/mg sample)]. Next, the homogenate was centrifuged and 50 µl aliquots of the supernatant were stored. All steps were performed on ice, and slides and supernatants were subsequently kept at 20(C. Protein concentrations were determined using a BCA Protein Assay Kit (23225, Pierce, IL, USA) according to the manufacturers recommendations.
MATERIALS AND METHODS
Zymography
Chorionic villus sampling (CVS) was performed vaginally between 10 and 12 weeks of gestation, largely for maternal age or serumscreening related risk for Down syndrome. Length of gestation was calculated according to the last menstrual period and subsequently confirmed by ultrasound (crown–rump length). Tissue was obtained using a biopsycatheter (Cook, K-CMA-5000). Follow-up of these pregnancies was available by a questionnaire returned by the patient postpartum. During CVS, surplus material was obtained containing decidua with little trophoblast. Under the microscope, this decidua was mechanically separated from the villi using needles and stored immediately at 20(C until further processing. From 1998 onwards, 700 samples with known outcome were collected. Out of these samples, a selection was made of 13 pregnancies complicated by hypertensive disorders like pre-eclampsia (PE) and HELLP-syndrome or intra-uterine growth restriction (IUGR). Pre-eclampsia was defined as a diastolic blood-pressure (BP) of 90 mmHg or a raise of 15 mm Hg compared with first trimester BP, measured twice, combined with proteinuria of at least 300 mg/day. HELLP syndrome was defined by laboratory results indicating haemolysis, a raise in alanin-aminotransferase (ALAT), aspartateaminotransferase (ASAT) and lactatedehydrogenase (LDH), and a platelet count lower than 100109/l. For IUGR, criteria according to Kloosterman [19] were used. Birthweight was corrected for gestational age, and percentiles were computed. IUGR was defined as a corrected birthweight below the 5th percentile. Each case was matched with two controls, matching for maternal age, parity and gestational age at time of sampling. Patients with concurrent morbidity (e.g. diabetes), smoking or on medication were excluded. Fetal karyotyping showed no chromosomal abnormalities. Patients were informed that surplus material could be used for research, according to the ‘Guideline Good Use’ by the FMWV-committee (Federation of Medical Scientific Associations). The placental bed biopsies were divided into parts of which from the center of the sample cryostat sections were cut for histology (H&E, Cytokeratin 7, MMP-2 and MMP-9). The remaining sample was homogenized for further assessment of MMP-activity and protein concentrations. Homogenization was performed, after weighing, by cutting the samples into
Presence of MMPs was analysed by zymography, using a 10 per cent polyacrylamide gel (108 cm in SE 245 Mighty Small device of Hoefer, San Fransisco, USA), containing 0.1 per cent gelatin (type A: from procine skin, Sigma) as a substrate. The gel was loaded with 15 µl samples of a 1 : 1 mixture of supernatant and non-reducing loading buffer (4 per cent SDS, 20 per cent Glycerol, 0.005 per cent phenolblue and 0.125 Tris pH 6.8), incubated for 20 min. Electrophoresis was performed in a cold chamber (4(C) with 20 mA per gel, followed by incubation for 30 min with renaturing fluid (2.5 per cent Triton X-100) and a wash with developing buffer (50 m Tris pH 7.6; 5 m CaCl2; 0.2 NaCl; 0.02 per cent Triton X-100). The gels were incubated overnight in fresh developing buffer at 37(C. After staining with Coomassie Blue (10 per cent glacial acetic acid; 30 per cent methanol; 1.25 g Coomassie Brilliant Blue) for 30 min, destaining (10 per cent glacial acetic acid; 30 per cent methanol) took 1 h. Proteolytic activities were visualized by clear zones against a dark blue background, indicating lysis of gelatin. No proteolytic activity was seen when after electrophoresis the gel was incubated in EDTA containing buffers (10 m EDTA) confirming true MMP activity. Moleclular weights (MW) were determined running a standard panel of seven proteins with known MWs ranging from 6.5–200.0 kD (Kaleidoscope Prestained Standards, Bio-Rad, Hercules, CA, USA) together with the samples.
Immunohistochemistry (cytokeratin 7, double-staining MMP-2 and -9/cytokeratin 7) Cryostat sections (4 µm) were dried and fixed in acetone for 10 min. All steps were performed at room temperature and followed by rinsing with Phosphate Buffered Saline, pH 7.4 (PBS) for 5 min. Next, sections were incubated with a monoclonal antibody against cytokeratin 7 (RKC 105, Eurodiagnostics, Apeldoorn, the Netherlands) in PBS/ 1 per cent BSA 1 : 50, for 60 min. Subsequently, endogenous peroxidase was blocked by incubation in 0.075 per cent H2O2 in PBS for 30 min. The secondary antibody, peroxidase conjugated Rabbit anti-Mouse antibody (DAKO, Glostrup, Denmark), was used in a 1 : 50 dilution in PBS/1 per cent BSA, containing 1 per cent AB serum and incubated for
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30 min. Peroxidase activity was visualized using 3-Amino-9Ethyl-Carbazol (AEC). After rinsing with demiwater for 5 min, counterstaining was performed using haematoxylin. Sections were subsequently mounted in Kaiser’s glycerol gelatin. The slides were examined with a 40 objective microscope (Olympus BHC, Japan) and scored 0/+/++ for cytokeratin 7 as determination of the amount of trophoblast in the placental bed biopsy. A score of 0 was given when no trophoblast cells were present, + in case of few and ++ when trophoblast was abundant. For the double-staining with cytokeratin 7 and MMP-2 or MMP-9, first, endogenous peroxidase was blocked with 125 µl H2O2 in 50 ml of PBS for 30 min. Next, slides were incubated with a monoclonal antibody against MMP-2 (Ab-3, Oncogene/Calbiochem, Cambridge, MA, USA) in PBS/1 per cent BSA 1 : 50 or MMP-9 (#56-2A4, Oncogene/ Calbiochem, Cambridge, MA, USA) in PBS/1 per cent BSA 1 : 200 for 60 min. Subsequently the sections were incubated consecutively with peroxidase conjugated Rabbit anti-Mouse, Goat anti-Rabbit and Rabbit anti-Goat antibody (all DAKO, Glostrup, Denmark) with a 1 : 50 and 1 : 100 dilution in PBS/1 per cent BSA with 1 per cent AB serum during 30 min. For visualization of the peroxidase-activity, 3-Amino-9-EthylCarbazol (AEC) was used. To avoid aspecific staining of the second primary antibody 0.1 Glycin, pH=2 for 45 min was applicated. The slides were incubated with the second primary antibody, cytokeratin 7 (RCK 105, Eurodiagnostics, Apeldoorn, the Netherlands) 1 : 50 in PBS/1 per cent BSA for 1 h. As a secondary antibody Alkaline Phosphatase-Conjugated Goat anti-Mouse antibody (DAKO, Glostrup, Denmark) 1 : 50 in PBS/1 per cent BSA containing 1 per cent AB serum was used, and incubated for 30 min. For visualization of the second primary antibody, Fast Blue was used (10 mg Naphtol AS-MX in 50 ml 0.1 Tris/HCl pH=8.2 with 10 per cent MgSO4, 50 mg of Fast Blue BB and 12 mg Levamisol) for 30 min. After rinsing the slides were mounted with Kaisers glycerol gelatin. Slides stained for only cytokeratin 7 and slides that were double-stained were compared to confirm blocking after the first primary antibody.
Immunocapture-assay MMP Although there is a clear link between zymography and the MMP-assay [20], zymography is semi-quantitative and does not measure true activity of the MMPs. TIMP-MMP complexes dissociate with zymography and seem to increase the amount of active MMP, this in contrast to an immunocaptureassay. After dilution of all the samples to a protein concentration of 80 µg/ml, a specific immunocapture-assay (Amersham Biosciences, Buckinghamshire, UK) was used to determine the activity of the different MMPs [20–23]. The assay is an established method, and is based on a modified pro-urokinase, where the activation sequence, normally recognized by plasmin (Pro–Arg–Phe–Lys [Ile–Ile–Gly–Gly), is replaced by a sequence that is specifically recognized by
Figure 1. Immunohistochemistry with cytokeratin 7 (RCK 105) and Mayers haematoxylin. There is clear staining of cytotrophoblast in red with nuclei staining in blue. Patients scoring 0 (A), ‘+’ (B) or ‘++’ (C), corresponding with the amount of trophoblast cells in the samples. ‘0’ no trophoblast cells, ‘+’ few, and ‘++’ abundant. Scale bar=50 µm.
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3UR003 3UR003
Figure 2. Zymography of one of the homogenates. Two bands appear of 72 and 90 kD, corresponding with proMMP-2 and proMMP-9 respectively. The other bands have higher molecular weights and are believed to be complexes of MMP-9 with other proteins. Lanes 1, 2 and 3 are loaded with 15, 12 and 9 µg protein respectively. Lanes 4–9: serial dilutions of homogenate starting with 6 µg protein loaded in lane 4. Note that MMP-2 and MMP-9 are still visible in lane 7, loaded with less than 1 µg of protein.
MMPs (Arg–Pro–Leu–Gly ?Ile–Ile–Gly–Gly). A chromogenic peptide substrate for urokinase is then added to measure the active urokinase, generated through MMP activation of the modified urokinase. Specific MMP-1, -2, -8, -9 and -13 antibody-coated microtiter plates are used to confirm MMPspecificity. Immobilized latent MMP is then activated using p-aminophenylmercuric acetate (APMA) to measure total MMP. Differential activation with APMA enables the analysis of the activity of both already active and latent forms of MMPs. The activity is expressed as (DeltaA/ to2*1000)/10 µl.
RESULTS Microscopic examination of the tissues (H&E) showed the presence of decidual cells. Staining for cytokeratin 7 revealed a positive reaction in 18 out of 39 samples. Fifteen scored ‘+’, 3 scored ‘++’ for the amount of trophoblast in the tissue (Figure 1). The cytokeratin-positive samples were cases as well as controls, and no significant differences could be seen between the group of hypertensive disorders, IUGR or controls. Using zymography of four random samples, two bands of 72 kD and 90 kD appeared, corresponding with respectively proMMP-2 and proMMP-9. These bands could still be detected with a minimum of 25 µg of substrate, corresponding with 1 µg of protein (Figure 2). The assay for the activity of the different MMPs was first performed on a subset of samples of both groups (complicated (n=4) as well as uncomplicated pregnancies (n=4)), and no activity of MMP-1, -3, -8 or -13 could be measured, nor in the active form nor in the total complex. In case of MMP-2, total MMP-2 was detected in a range of 0.4 to 12.0 U/µl ((DeltaA/to2*1000)/10 µl). However, no significant differences were found between complicated pregnancies and their matched controls. Activity of total MMP-9 was detected in six out of the 39 samples, ranging from 1.4 to 26.7 U/µl ((DeltaA/to2*1000)/10 µl). Comparing the results of the scores for cytokeratin 7 with the activity of total MMP-2 or total MMP-9 for the cases and their controls, no relation between cases and their controls could be found. Immunohistochemistry for MMP-2 and MMP-9 of 2 selected samples showed clear staining for MMP-2 in case of high MMP-2 activity in the assay. Staining of the decidual
cells, as well as of the trophoblasts was seen (Figure 3A and B). MMP-9 activity was low in both samples, but in the sample where trophoblast was abundant, immunostaining for MMP-9 was profound (Figure 3D). Less staining for MMP-2 was related to low activity (Figure 3C). Comparing the complicated pregnancies (hypertensive and IUGR) with their controls, no significant differences could be found. Tables 1a and 1b shows the differences in the means of activity of total MMP-2. No significant differences could be found between cases and controls. For MMP-9, no difference between the medians of MMP-activity in first trimester placental bed biopsies in further complicated or uncomplicated pregnancies could be demonstrated.
DISCUSSION An essential feature of human placentation is the interaction of invading trophoblast with the uterine vasculature [24]. Interaction of trophoblast with the extracellular matrix is one of the main factors in providing a substrate for attachment and growth or migration. In pre-eclamptic patients this trophoblast invasion is impaired, mainly in the spiral arteries. Although many observations regarding impaired early placentation in pregnancies further complicated by pre-eclampsia or IUGR are known [25], the exact regulation is still to be elucidated. The decidualized endometrium has influence on trophoblast function by for example altering the expression of the metalloproteinases. Stromal cell differentiation is associated with loss of collagen VI, which facilitates invasion through the interstitial spaces of the decidua [26]. Regarding the profound changes in early pregnancy mentioned above, we hypothesized that differences in MMP activity exists in first trimester decidua of pregnancies that later develop complications like pre-eclampsia or IUGR. Storage of decidual samples, obtained by cvs, till after the outcome of the pregnancy was known, enabled us to investigate a case-control series. From the results of our current study we may conclude that in week 10 to 12 of pregnancy, MMP-2 and -9 can already be detected in small decidual samples. As shown earlier by Huppertz et al. [13], we found MMP-2 and MMP-9 expression in extravillous trophoblast as well as in decidual cells.
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Figure 3. Immunohistochemical double-staining for cytokeratin 7/MMP-2 or cytokeratin 7/MMP-9. Cytokeratin is stained blue, with the different MMPs stained brownish. Scale bar=50 µm. (A) Patient scoring ‘+’ for cytokeratin 7 with high total MMP-2 activity and low total MMP-9 activity in the assay. Double-staining for cytokeratin 7 and MMP-2. Strong staining for MMP-2 can be seen, of trophoblast cells as well as of decidual cells. (B) Patient scoring ‘+’ for cytokeratin 7 with high total MMP-2 activity and low total MMP-9 activity in the assay. Double-staining for cytokeratin 7 and MMP-9. The sample shows weak staining of MMP-9, corresponding with the low activity in the assay. (C) Patient scoring ‘++’ for cytokeratin 7 with low total MMP-2 and low total MMP-9 activity. Double-staining for cytokeratin 7 and MMP-2. Weak staining of the trophoblast and decidua with MMP-2. (D) Patient scoring ‘++’ for cytokeratin 7 with low total MMP-2 and low total MMP-9 activity. Double-staining for cytokeratin 7 and MMP-9. Strong staining of MMP-9, of the decidua as well as of the trophoblast.
In view of earlier described differences in the third trimester, and the presumed role of MMPs in early pregnancy, it is remarkable that we did not find any differences between cases and controls. However, several factors can be taken into account. First, it may be that the most important area for MMP action is at the cell surface and activities in the tissue homogenates are not informative. Also possible is that, though proMMP-2 and -9 are present and total activity can be measured, they are not discriminating between further complicated and normal pregnancies. This is in contrast to studies in third trimester pregnancies, where differences were found. It may be too early in pregnancy to detect a difference in MMP-activity in decidua.
A third possibility is that the activity of the MMPs in decidual cells is not compromised in complicated pregnancies, but the activity in trophoblast cells is, as shown in placentae at term [27]. However, there is no difference in the relation between the presence of trophoblast and MMP-activity in our cases compared to controls, although we do see profound expression of MMP-9 immunohistochemistry in case of abundant trophoblast. Finally, MMP activity is a balance between proMMP with their inhibitors (TIMPs) and activators. Though measurement of total MMP-activity may not differ, it is still possible that a shift in balance has taken place in compromised pregnancies from activity in trophoblastic to decidual cells or vise versa.
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Table 1a. Means of total MMP-2 activity for cases and their matched controls grouped for pre-eclampsia or HELLP syndrome and for IUGR (n.s.: not significant. Student’s t test) Complication
IUGR PE/HELLP
Total MMP-2 activity (DeltaA/to2*1000/10 µl)
Difference
Mean of cases (SD)
Mean of matched controls (SD)
5.54 (2.7) (n=9) 5.02 (0.6) (n=4)
5.32 (2.6) (n=18) 5.72 (2.6) (n=8)
n.s. n.s.
Table 1b. Medians of total MMP-9 activity for cases and their matched controls (n.s.: not significant. Wilcoxon test) Complication
IUGR PE/HELLP
Total MMP-9 activity (DeltaA/to2*1000/10 µl)
Difference
Median of cases (ranges)
Median of matched controls (ranges)
0.0 (0.0–26.1) (n=9) 0.0 (0.0–0.0) (n=4)
0.0 (0.0–7.4) (n=18) 0.0 (0.0–1.4) (n=8)
The necessary degradation of the extracellular matrixcomponents for the invasion of trophoblast in the maternal decidua seems a complex interaction. MMPs might act from both sides: Trophoblasts producing MMPs to enable invasion, and decidual cells allowing invasion. However, in conclusion, our study does not support the currently held concept of deranged MMP-activity in early pregnancy in further complicated pregnancies.
[14] [15] [16] [17]
REFERENCES [1] Fisher SJ, Leitch MS, Kantor MS, Basbaum CB, Kramer RH. Degradation of extracellular matrix by the trophoblastic cells of first-trimester human placentas. J Cell Biochem 1985;27:31–41. [2] Woessner J-FJ. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 1991;5:2145–54. [3] Jiang W, Bond JS. Families of metalloendopeptidases and their relationships. FEBS Lett 1992;312:110–4. [4] Nagase H, Woessner J-FJ. Matrix metalloproteinases. J Biol Chem 1999; 274:21491–4. [5] Stetler-Stevenson WG, Liotta LA, Kleiner D-EJ. Extracellular matrix 6: role of matrix metalloproteinases in tumor invasion and metastasis. FASEB J 1993;7:1434–41. [6] Mignatti P, Rifkin DB. Biology and biochemistry of proteinases in tumor invasion. Physiol Rev 1993;73:161–95. [7] Bischof P, Meisser A, Campana A. Paracrine and autocrine regulators of trophoblast invasion—a review. Placenta 2000;21:S55–60. [8] Docherty AJ, Murphy G. The tissue metalloproteinase family and the inhibitor TIMP: a study using cDNAs and recombinant proteins. Ann Rheum Dis 1990;49:469–79. [9] Cross JC, Werb Z, Fisher SJ. Implantation and the placenta: key pieces of the development puzzle. Science 1994;266:1508–18. [10] Autio-Harmainen H, Sandberg M, Pihlajaniemi T, Vuorio E. Synthesis of laminin and type IV collagen by trophoblastic cells and fibroblastic stromal cells in the early human placenta. Lab Invest 1991;64:483–91. [11] Fernandez PL, Merino MJ, Nogales FF, Charonis AS, Stetler-Stevenson W, Liotta L. Immunohistochemical profile of basement membrane proteins and 72 kilodalton type IV collagenase in the implantation placental site. An integrated view. Lab Invest 1992;66:572–9. [12] Polette M, Gilbert N, Stas I, Nawrocki B, Noel A, Remacle A et al. Gelatinase A expression and localization in human breast cancers. An in situ hybridization study and immunohistochemical detection using confocal microscopy. Virchows Arch 1994;424:641–5. [13] Huppertz B, Kertschanska S, Demir AY, Frank HG, Kaufmann P. Immunohistochemistry of matrix metalloproteinases (MMP), their
[18] [19] [20]
[21]
[22] [23]
[24] [25] [26] [27]
n.s. n.s.
substrates, and their inhibitors (TIMP) during trophoblast invasion in the human placenta. Cell Tissue Res 1998;291:133–48. Sawicki G, Radomski MW, Winkler-Lowen B, Krzymien A, Guilbert LJ. Polarized release of matrix metalloproteinase-2 and -9 from cultured human placental syncytiotrophoblasts. Biol Reprod 2000;63:1390–5. Isaka K, Usuda S, Ito H, Sagawa Y, Nakamura H, Nishi H et al. Expression and activity of matrix metalloproteinase 2 and 9 in human trophoblasts. Placenta 2003;24:53–64. de Jager CA, Linton EA, Spyropoulou I, Sargent IL, Redman CW. Matrix metalloprotease-9, placental syncytiotrophoblast and the endothelial dysfunction of pre-eclampsia. Placenta 2003;24:84–91. Shimonovitz S, Hurwitz A, Dushnik M, Anteby E, Geva-Eldar T, Yagel S. Developmental regulation of the expression of 72 and 92 kd type IV collagenases in human trophoblasts: a possible mechanism for control of trophoblast invasion. Am J Obstet Gynecol 1994;171:832–8. Pendas AM, Balbin M, Llano E, Jimenez MG, Lopez-Otin C. Structural analysis and promoter characterization of the human collagenase-3 gene (MMP13). Genomics 1997;40:222–33. Kloosterman GJ. Over intra-uteriene groei en de intra-uteriene groeicurve. [Intrauterine growth and intrauterine growth curves]. Ned Tijdschr Verloskd Gynaecol 1969;69:349–65. Sier CF, Casetta G, Verheijen JH, Tizzani A, Agape V, Kos J et al. Enhanced urinary gelatinase activities (matrix metalloproteinases 2 and 9) are associated with early-stage bladder carcinoma: a comparison with clinically used tumor markers. Clin Cancer Res 2000;6:2333–40. Hanemaaijer R, Visser H, Konttinen YT, Koolwijk P, Verheijen JH. A novel and simple immunocapture assay for determination of gelatinase-B (MMP-9) activities in biological fluids: saliva from patients with Sjogren’s syndrome contain increased latent and active gelatinase-B levels. Matrix Biol 1998;17:657–65. Capper SJ, Verheijen J, Smith L, Sully M, Visser H, Hanemaaijer R. Determination of gelatinase-A (MMP-2) activity using a novel immunocapture assay. Ann N Y Acad Sci 1999;878:487–90. Hanemaaijer R, Sier CF, Visser H, Scholte L, van-Lent N, Toet K et al. MMP-9 activity in urine from patients with various tumors, as measured by a novel MMP activity assay using modified urokinase as a substrate. Ann N Y Acad Sci 1999;878:141–9. Pijnenborg R, Vercruysse L, Verbist L, Van AF. Interaction of interstitial trophoblast with placental bed capillaries and venules of normotensive and pre-eclamptic pregnancies. Placenta 1998;19:569–75. Brosens JJ, Pijnenborg R, Brosens IA. The myometrial junctional zone spiral arteries in normal and abnormal pregnancies: a review of the literature. Am J Obstet Gynecol 2002;187:1416–23. Aplin JD, Charlton AK, Ayad S. An immunohistochemical study of human endometrial extracellular matrix during the menstrual cycle and first trimester of pregnancy. Cell Tissue Res 1988;253:231–40. Kolben M, Lopens A, Blaser J, Ulm K, Schmitt M, Schneider KT et al. Proteases and their inhibitors are indicative in gestational disease. Eur J Obstet Gynecol Reprod Biol 1996;68:59–65.