The placental membrane protects the fetus from mechanical and physical factors during gestation. Rupture of the membranes is seen during labor and sometimes before the onset of labor (premature rupture of membranes [PROM]). Increased matrix metalloproteinase activity has been documented in human placental membrane cells near term, at the time of labor, and when PROM occurs.1 The matrix metalloproteinases are enzymes belonging to a family of zinc- and calcium-dependent endopeptidases that have the combined ability to degrade the components of the connective tissue of the membranes.2,3 It is speculated that controlled matrix metalloproteinase activity maintains the mechanical and structural integrity of the fetal membrane by a regulated collagenolytic and remodeling process throughout pregnancy.4 Vadillo-Ortega et al5 documented increased activity of the matrix metalloproteinases and reduced activity of their inhibitors in the amniotic fluid of women with PROM. It is theorized that the collagen-rich extracellular matrix of placental membranes can be degraded by increased matrix metalloproteinase activity or by reduced activity of the tissue inhibitors of metalloproteinases.4–6
Although the causes of PROM are multifactorial, several exogenous and endogenous factors have been linked to it.6 The main exogenous factor is infection, with consequent host inflammatory response, present in over 60% of cases of PROM.6–8 We speculate that infection could cause an imbalance in the expression pattern of matrix metalloproteinases and their natural inhibitors, which might result in increased activity of matrix metalloproteinases leading to PROM.
In this study we examined the pattern of expression of gelatinase A and B (matrix metalloproteinase 2 and 9) because these proteases are essential for degradation of amniochorion basement membrane in response to infectious stimuli. We also examined changes in the expression of tissue inhibitor of metalloproteinase 2, which can inhibit both gelatinases. This inhibitor is also involved in the activation process of gelatinase A.
Materials and Methods
This study was approved by the institutional review board at The Women's Hospital at Centennial Medical Center, Nashville, Tennessee.
Amniochorionic membranes (n = 6) were collected, placed in an organ explant system, and stimulated with lipopolysaccharide (1000 ng/mL) as described in our previous studies.9,10 Membranes were collected at random from women who had cesareans at term and were not in labor. The selection of this group was random, and average gestational age was 38.4 weeks. Women with any complications of pregnancy, twin gestations, or signs of infection or who were taking antibiotic therapy were excluded from the study. The lipopolysaccharide (endotoxin) used in this study (a component of gram-negative bacterial cell wall outer membrane) was obtained from Escherichia coli strain O55:B5 (Sigma Chemicals, St. Louis, MO). During the course of gram-negative infection this toxin is released into the tissue environment when bacterial cells die. A 24-hour stimulation of membranes was done after a 48-hour preincubation period. At the end of stimulation, tissue and media samples were collected and stored at −70C until assayed.
Total RNA extracted from the cultured tissues was reverse transcribed.9–11 A constant amount of the resultant complementary DNA (cDNA) was subjected to quantitative competitive polymerase chain reaction (PCR). In this competitive PCR assay, one set of primers was used to amplify both the target gene cDNA and a neutral DNA fragment. The neutral DNA fragment competes with the target cDNA fragment for the same primers (gelatinase and tissue inhibitor of metalloproteinase 2 specific) and acts as an internal standard by providing a PCR product of a different size. This protocol used a nonhomologous DNA fragment (Clontech, Palo Alto, CA) engineered to contain the desired gene template primers and thus was recognized by the pair of gene-specific primers. Serial dilutions of the internal standards were added to the PCR reaction, which contained constant amounts of experimental cDNA samples. This cDNA was then coamplified with known concentrations of the internal standards for 30 cycles (the concentration of internal standard used was between 6 × 106 and 0.6 molecule/μL). After PCR, the products were resolved by 1.5% ethidium bromide–stained agarose gel electrophoresis. When the intensity of the target gene product band equaled that of the internal standard band, the molar concentrations of both were approximately equal. Quantitation was achieved by gel band spot densitometry using the Alpha Ease software program (AlphaInnotech Corp., San Leandro, CA). Integrated density values for the two matching bands (internal standard and target genes) were obtained, and the densitometric ratio was calculated. This value was multiplied by the known concentration of the internal standard to obtain the approximate number of transcripts per 0.5 μg of total RNA. Because the molar concentrations of the internal standards were known, the relative levels of expression of target messenger RNA could be estimated (number fold increase or decrease). Glyceraldehyde 3 phosphate dehydrogenase (a housekeeping gene) was used as a PCR control to document the quality of RNA and reaction conditions.
The release of immunoreactive gelatinases A and B and tissue inhibitor of metalloproteinase 2 into the culture medium was quantitated using enzyme-linked immunosorbent assay (ELISA). The assay procedure involved a multiple-site, two-step sandwich immuno-assay using monoclonal antibody directed against the protein of interest (Amersham Life Sciences, Buckinghamshire, UK). Manufacturer's instructions were followed for ELISA. Standard curves were developed using duplicate samples of known quantities of recombinant human gelatinases A or B or tissue inhibitor of metalloproteinase 2, as appropriate. Concentrations were determined by relating the absorbance obtained by spectrophotometry to the standard curve by linear regression analysis. Controls consisted of assay buffer, plain media, and lipopolysaccharide-containing media incubated in wells without tissue. Colorimetric absorption was read at 405 nm using an IncStar Automatic microplate reader (IncStar Corp., Still Water, MN).
Statistical significance of the data from quantitative competitive PCR and ELISA on culture media samples was analyzed by unpaired nonparametric, two-sample Mann-Whitney U test. A two-tailed P ≤ .05 was considered significant.
Messenger RNA for gelatinase A (matrix metalloproteinase 2) and gelatinase B (matrix metalloproteinase 9) increased in response to lipopolysaccharide stimulation. Quantitative competitive PCR performed on amniochorionic membrane stimulated with 1000 ng/mL of lipopolysaccharide produced approximately 3.6 × 106 transcripts (median 3.3 × 106, range 5.5 × 105–7.6 × 106) of gelatinase A messenger RNA/0.5 μg of total RNA compared with only 5.9 × 104 (median 6 × 104, range 5.7 × 104–6.0 × 104) transcripts in control tissues (P = .009) (Figure 1).
Similarly, gelatinase B (matrix metalloproteinase 9) messenger RNA expression also increased by 100-fold after lipopolysaccharide stimulation. Control tissues produced approximately three molecules (median 3, range 0–6), whereas lipopolysaccharide stimulation resulted in approximately 366 transcripts (median 570, range 55–619) of gelatinase B per 0.5 μg of total RNA tested (P = .006) (Figure 2).
Transcripts for tissue inhibitor of matrix metalloproteinase 2 showed no significant change after lipopolysaccharide stimulation. An approximate number of 4.1 × 105 transcripts per 0.5 μg of total RNA (median 5.6 × 105, range 5.5 × 104–6.1 × 105) was found after lipopolysaccharide stimulation compared with 5.7 × 105 transcripts (median 5.7 × 105, range 5.2 × 104–6.4 × 105) in controls (P = .69) (Figure 3).
Immunoreactive levels of gelatinases and tissue inhibitor of matrix metalloproteinase 2 in culture media were quantitated by ELISA. Gelatinase A (matrix metalloproteinase 2) levels were 210 ng/mL (median 200 ng/mL, range 150–330 ng/mL) after 1000 ng/mL of lipopolysaccharide stimulation compared with 120 ng/mL (median 100 ng/mL, range 100–160 ng/mL) in controls (P = .01).
Gelatinase B (matrix metalloproteinase 9) protein level increased from 4.6 ng/mL (median 5 ng/mL, range 1–7 ng/mL) in controls to 7 ng/mL (median 7.5 ng/mL, range 1–13 ng/mL) after lipopolysaccharide stimulation. This increase was not statistically significant (P = .3).
Tissue inhibitor of matrix metalloproteinase 2 levels decreased after lipopolysaccharide stimulation. The levels were 114 ng/mL (median 104 ng/mL, range 84–143 ng/mL) in control culture media compared with 68 ng/mL (median 76 ng/mL, range 48–84 ng/mL) after lipopolysaccharide stimulation (P = .007).
We performed a molar ratio analysis between gelatinases and tissue inhibitor of matrix metalloproteinase 2 based on the protein levels in culture media divided by their respective molecular weight. The ratio between gelatinases and tissue inhibitor of matrix metalloproteinase 2 was 1:1 in culture media from control tissues. However, this ratio increased to 3.1:1 in favor of the gelatinases after lipopolysaccharide stimulation.
Amniochorion in culture responds to infectious stimuli (lipopolysaccharide) with increased expression and release of gelatinases, especially gelatinase A (matrix metalloproteinase 2). Gelatinase B (matrix metalloproteinase 9) expression was induced during culture conditions,10 and its messenger RNA levels also increased significantly after lipopolysaccharide stimulation. This increase in messenger RNA expression did not translate into a significant increase in protein concentration. This discrepancy is indicative of an already well-documented complex posttranslational regulation of the matrix metalloproteinases.12
Tissue inhibitor of metalloproteinase 2 messenger RNA expression did not change after lipopolysaccharide stimulation. There was a significant decrease in the protein level in culture media, indicating posttranslational inhibition of tissue inhibitor of metalloproteinase 2. This also suggests independent pathways of regulation for gelatinases and tissue inhibitor of metalloproteinase 2 exerted by infection or inflammatory mediators.
Tissue inhibitor of metalloproteinase 2 can inhibit both gelatinases, although it is also required for gelatinase A zymogen activation. Tissue inhibitor of matrix metalloproteinase 2 anchors gelatinase A for presentation to membrane-type matrix metalloproteinase 1 on the cell membrane surface, which results in proteolytic cleavage and activation.13 After activation, tissue inhibitor of metalloproteinase 2 can inhibit active forms of gelatinases A and B if the inhibitor is present in excess molar concentration.13,14 It can also block the progelatinase binding site in membrane-type matrix metalloproteinase to abort the activation of the proenzyme.14 If the concentration of tissue inhibitor of metalloproteinase 2 is reduced, it can still cause the activation of gelatinase A because of its high-affinity binding to membrane-type metalloproteinase 1, which effectively anchors the progelatinase. However, this reduced concentration is not sufficient to inhibit active gelatinase A.14,15
In vitro resting human fetal membranes show a balanced gelatinolytic activity in culture conditions. This is indicated by a stoichiometric ratio of 1:1 between gelatinases and tissue inhibitor of metalloproteinase 2 in control cultures. After lipopolysaccharide stimulation, amniochorion shows a shift in the metalloproteinase-to-inhibitor ratio produced by an increased gelatinase concentration and a decreased inhibitor concentration. This imbalance in human fetal membranes during infection favors increased gelatinase activity. Although the assay used in this study documents only immunoreactive forms and not active forms of gelatinases, ongoing studies in our laboratory have found that a small percentage of these proenzymes are actually active and free of tissue inhibitor of metalloproteinase (unpublished data).
We have not yet studied the response of other tissue inhibitors of metalloproteinases (1 and 3) that can potentially inhibit gelatinases. The expression of these other tissue inhibitors of metalloproteinase has already been documented in fetal membranes by our laboratory.16 Recent studies in our laboratory suggest that there is a general trend in favor of protease activity during in vitro lipopolysaccharide stimulation.17
Gelatinases are enzymes that are capable of degrading the type IV collagen-rich basement membrane of human amniochorion. This structure bonds the two cell layers (amnion and chorion) to the extracellular matrix region, and type IV collagen also glues together the cells of the chorion cytotrophoblasts. Gelatinase A can also degrade laminin and fibronectin, which are two major noncollagenous components of the fetal membrane extracellular matrix. In normal fetal membranes, a balanced stoichiometric ratio between inhibitor and enzyme can promote remodeling of the tissue during placental growth throughout gestation. This study documented the increased presence of these enzymes and decreased concentration of inhibitors in fetal membranes in response to an infectious stimulus. This increase perturbs the natural balance and could lead to increased membrane degradation. Destruction of the basement membrane exposes the underlying collagen forms, which promotes upregulation and activation of other matrix metalloproteinases.18 In vivo intra-amniotic infection might cause similar effects that can result in PROM.
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