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  • In vitro secretion and activity profiles of matrix metalloproteinases, MMP-9 and MMP-2, in human term extra-placental membranes after exposure to Escherichia coli.

In vitro secretion and activity profiles of matrix metalloproteinases, MMP-9 and MMP-2, in human term extra-placental membranes after exposure to Escherichia coli.

Reproductive biology and endocrinology : RB&E (2011-01-27)
Veronica Zaga-Clavellina, Guadalupe Garcia-Lopez, Arturo Flores-Pliego, Horacio Merchant-Larios, Felipe Vadillo-Ortega
ABSTRACT

Premature rupture of fetal membranes (PROM) complicated with intrauterine infection has been associated to alterations of the extracellular matrix (ECM) homeostasis. The aim of this work was to evaluate the integral/functional response of the amnion (AMN) and choriodecidua (CHD) to synthesis, secretion, and activity of MMP-2 and MMP-9 and of their inhibitors TIMP-1, -2, and -4, after stimulation with Escherichia coli. Full-thickness membranes were mounted on a Transwell device, constituting two independent chambers, Escherichia coli (1×10 (6) CFU/mL) were added to either the amniotic or the choriodecidual face or to both. Secretion profiles of MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-4 were quantified by ELISA and gelatinolytic activity by zymography. Immunoreactivity for MMP-2 and MMP-9 was revealed by immunohistochemistry and the collagen content was assessed by the hydroxyproline assay. Levels of MMP-9 in CHD and AMN increased 4- and 8-fold, respectively, after simultaneous infection. MMP-2 secreted to the medium by CHD increased a mean of 3 times after direct stimulation. Secretion profiles of TIMP-1, TIMP-2, and TIMP-4 remained without significant changes. Collagen content was significantly decreased (4-fold) in infected membranes, and was associated with loss of structural continuity and co-localization with immunoreactive forms of MMP-2 and MMP-9. Infection of chorioamniotic membranes with E. coli induces an increase in the secretion of inactive forms and an association to ECM of active forms of MMP-2 and MMP-9 without changes in TIMP-1, -2, and -4. These changes could explain the significant decrease of collagen content and loss of structural continuity.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Toluidine Blue O, Technical grade
Sigma-Aldrich
Brilliant Blue G, pure
Sigma-Aldrich
Brilliant Blue G, 250, for microscopy
Sigma-Aldrich
Toluidine Blue, for microscopy (Hist., Vit.)
Sigma-Aldrich
Toluidine Blue O, certified by the Biological Stain Commission
Sigma-Aldrich
Brilliant Blue R, pure
Sigma-Aldrich
Brilliant Blue R, Dye content ~50 %, Technical grade
Sigma-Aldrich
Brilliant Blue R, 250, for microscopy
Sigma-Aldrich
Anti-MMP-2 Antibody, a.a. 468-483 hMMP2, clone 42-5D11, clone 42-5D11, Chemicon®, from mouse