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| Research update from PRC laboratory: | See Structural Details... |
Presence of matrix degrading enzymes (MMPs) and their inhibitors in amniochorion:
MMPs are involved in tissue remodeling in most of the body's organs. As the pregnancy grows membrane remodeling is necessary to maintain membrane integrity. As the MMPs are regulated by cytokines in other systems we propose that pathologic over stimulation of the MMP system may result in PROM. For this reason we chose to investigate whether AC is a source of MMPs and TIMPs (tissue inhibitor of MMP).
Expression of TIMP1, TIMP2 and gelatinase A and B (MMP2 and 9) in human fetal membranes:
We tested for the presence of TIMP 1 and 2 mRNA expression in the AC using RT-PCR. Term AC and AC in culture demonstrated the presence of both of these TIMPs. Expression of this mRNA was constitutive throughout the culture period (ten days). We have also noted the expression of MMP2 and MMP-9 using RT-PCR. In situ hybridization demonstrated chorion, the cells in the reticular layer next to chorion and scattered amnion cells as sources of these mRNAs.. Immunocytochemistry using antibodies to TIMP1 and TIMP2 localized the protein in the reticular layer, amnion and chorion cells close to the decidual region. Production of gelatinases by the AC was documented using media samples run on substrate gel zymography. Tissue homogenates of term membranes not placed in culture indicated only 72 kD gelatinase. It seems that the culture process induces 92 kD gelatinase in the membrane. The same media samples when run with gelatin and EDTA yielded no matrix degradation, indicating that the enzyme observed was truly an MMP.
Induction of MMP9 during infection and PROM:
RT-PCR was performed using primers specific for MMP2 and MMP9 to study their expression pattern in human amniochorion. MMP2 was constitutively expressed throughout gestation and MMP9 was induced during culture conditions and labor. MMP9 was also documented in membranes from women with documented IAI and PROM. This finding was supported by our in vitro findings on cultured membranes. LPS and PGPS stimulation after a 48 h incubation induced AC MMP9 expression whereas controls were negative. We localized the site of production of these MMP mRNAs by in situ hybridization. Both MMP2 and 9 were produced by amnion, chorionic laeve, reticular and spongy layers of the connective tissue. MMP9 expression was scattered in amnion whereas MMP2 was more abundant.
Expression of stromelysin 1,2 and 3 in human fetal membranes and elevation of Stromelysins 1 AF levels in PROM::
Stromelysins are a group of MMPs which can degrade a wide variety of matrix components, including collagen Type IV, seen in the basement membrane of amnion and chorion. Our ongoing screening of amniochorion for the presence of other classes of MMPs revealed the expression of all three stromelysins (Stromelysins 1 [MMP3], Stromelysin 2 [MMP10], and stromelysin 3 [MMP11]) in amnion and chorion using RT-PCR and in situ hybridization. We were also able to localize the stromelysin1 protein to amnion and chorion. Tissues collected at the time of active labor and non labor (from C-section) showed mRNAs for all three stromelysins. We collected a total of 55 AF samples from women at term with no pregnancy complications and from women with PROM irrespective of labor and infectious status. ELISA performed to analyze the bio and immuno active stromelysin1 showed an increase in the levels of stromelysin1 in the AF of women with PROM compared to term. We also analyzed the levels of stromelysin1 in culture media after stimulation of AC with LPS. A dose dependent increase in stromelysin 1 was seen in the media. (For more details go to Our publications)
Absence of matrilysin in human fetal membranes:
Matrilysin (MMP7) belongs to the stromelysin class of MMPs.
We studied its expression in fetal membrane during active labor and during non laboring conditions using RT-PCR.
Two sets of primers were employed to study the expression of this MMP. None of our samples produced visible bands
for matrilysin. The specificity of the primers was later confirmed by amplifying matrilysin mRNA from breast carcinoma
cell lines known to express matrilysin.
Expression of MT1-MMP in human fetal membranes: MT1-MMP belongs to the novel class of MMPs which are membrane bound.
MT1-MMP is required for the activation of pro-MMP2. MT1-MMP forms a tri-molecular arrangement with TIMP2 to produce
active MMP2. After documenting the presence of MMP2 and TIMP2 in human fetal membranes, we have localized MT1-MMP
mRNA and peptide to the same areas of the fetal membranes that produce MMP2 and TIMP2. This experiment documented
the existence of the enzymatic machinery necessary to activate MMP2 in human fetal membranes.
Absence of MMP1 (Interstitial collagenase-1) in AC:
Recently we tested AC in culture for the presence of MMP1 by RT-PCR. AC in culture was shown to be negative for mRNA for MMP1, however, the same primers amplified MMP1 mRNA from decidual cells. In situ hybridization and immunohistochemistry tests are underway.
LPS stimulation results in an imbalance in fetal membrane MMP2/TIMP2 system:
Amniochorion membranes in culture stimulated with LPS produced increased MMP2 expression with no change in TIMP2 mRNA expression compared to control tissues. Media samples from these experiments were subjected to ELISA to document protein levels. MMP2 levels were increased whereas TIMP2 levels were decreased in a dose dependent manner giving an imbalance in the ratio between the enzyme and its inhibitor. This decreased level of TIMP2 may be enough in concert with MT1-MMP to activate MMP2, but its decreased molar concentration is less than adequate to maintain a 1:1 stoichiometric inhibition. MMP9 expression was induced in response to LPS, however, increases in MMP9 protein levels were not statistically significant. TIMP1 mRNA and protein levels were unaltered after LPS stimulation.
Table C.7.7.1.
|
Imbalance in MMP/TIMP ratio |
MMP2 |
TIMP2 |
MMP2/TIMP2 ratio |
|
|
# of molecules of mRNA |
Control tissues |
59,425 |
284000 |
0.3 |
|
LPS stimulated tissues |
3,688,666 |
493500 |
9.91 |
|
|
Protein levels |
Control tissues |
120 ng/ml |
114ng/ml |
1 |
|
50 ng/ml LPS |
165 ng/ml |
77.6 ng/ml |
2.1 |
|
|
100 ng/ml LPS |
196 ng/ml |
75.2 ng/ml |
2.6 |
|
|
1000 ng/ml LPS |
210 ng/ml |
67.6 ng/ml |
3.1 |
|
MMP2 and TIMP2 in amniotic fluid during PROM:
Our ELISA analysis of AF collected from women with PROM showed a significant drop in the levels of immunoreactive TIMP2 and an increase in the levels of immunoreactive (IR) MMP2 compared to the AF levels of these proteins present in women at term. A similar increase was seen in the levels of IR-MMP9 and TIMP1.
Amniotic fluid levels of MMPs and TIMPs
|
TERM |
PTL |
PROM |
p-Term vs PTL |
p-Term vs PROM |
p-PTL vs PROM | |
|
MMP2 |
1505 ± 650 |
1892 ±714 |
2106 ±438 |
ns |
<0.05 |
ns |
|
MMP9 |
0.2±0.8 |
3.75±10.09 |
15.03±14.54 |
ns |
<0.05 |
ns |
|
TIMP1 |
1891 ±1012 |
2406 ±1461 |
3163 ±1485 |
ns |
<0.05 |
ns |
|
TIMP2 |
176 ±68 |
231 ±130 |
104 ±57 |
ns |
<0.01 |
<0.001 |
|
ActiveMMP2(pg/ml) |
170.6±81 |
230±89 |
293±73 |
ns |
<0.01 |
ns |
|
Active MMP9(pg/ml) |
0 |
0 |
612 ±1.2 |
ns |
<0.001 |
<0.001 |
Bioactive MMP9 was seen only during PROM:
Induction of MMP9 mRNA and protein during PROM, infection and during bacterial toxin stimulation lead us to believe that MMP9 may have a significant role in matrix degradation in spite of lower concentrations than MMP2. Since ELISA detects IR-MMP9 both the active and inactive forms, we have assayed all our AF samples for the presence of bioactive MMP9. Bioactive levels of MMP9 were seen only in the amniotic fluid of women with PROM. No bioactive MMP9 was detected in the AF of women at term or with PTL although these samples showed the presence of IR-MMP9 (Table). Since the bioactive form of MMP9 is free of TIMP inhibition, its presence even at low concentration may contribute significantly to the degradation of the ECM.
Expression of TIMP3 and TIMP4 in amniochorion:
Our laboratory has recently documented the production of mRNA for TIMP3 and 4. They were shown to be produced by amnion and chorion cells along with some cells in the interstitium. mRNAs for these TIMPs were also seen in membranes at the time of labor as well as non laboring conditions. Upon completion of these experiments, our laboratory has documented the presence of all four TIMPs in human fetal membranes.