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Qian Sun, Ilene Sugino, Marco Zarbin; Mmps In Bovine Corneal Endothelial Cell-conditioned Media (BCE-CM). Invest. Ophthalmol. Vis. Sci. 2011;52(14):1779.
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Mass spectroscopy and 2D gel studies show that BCE-CM is a complex mixture of secreted and intracellular proteins with a number of proteins that could contribute to the ability of BCE-CM to enhance cell survival on aged and AMD Bruch's membrane (e.g., laminin, collagens, fibronectin, proteoglycans, matrix metalloproteases (MMPs), growth factors). 2D gels indicated prominent spots, identified as MMP2. Since MMP2, a type IV collagenase, could contribute to the BCE-CM effect by modification of Bruch's membrane and modulation of cell activity, we determined whether MMP2 inhibition impaired the ability of BCE-CM to enhance cell survival on aged and AMD Bruch's membrane.
Zymography was performed on 4 different batches of BCE-CM to confirm the presence of MMP-2 and possibly other MMPs able to digest gelatin (e.g., MMP-8 or MMP-9). GM6001(Millipore/Chemicon), an inhibitor of collagenases (MMP-1, -2, -8, -9), was added to BCE-CM at a range of concentrations (0.1, 1, 10µM ) to determine an effective concentration for Bruch's membrane explant assays. Fetal RPE were seeded onto paired submacular Bruch's membrane explants (N=6; mean donor age ±SEM, 73.75±1.78 yrs.) for 21 day culture in BCE-CM+MMP inhibitor or BCE-CM+negative control (provided by inhibitor manufacturer). Explants were examined by scanning electron microscopy to determine the extent of surface coverage and surface morphology of cells.
Zymographyidentified several bands of BCE-CM proteins able to digest gelatin. The two strongest staining bands indicated MMP-2, active and latent forms. Zymography of BCE-CM showed MMP2 activity was completely inhibited by GM6001 concentrations of 1 and 10 µM. 10 µM GM6001 was used to inhibit MMP2 in BCE-CM used as culture medium for fetal RPE seeded onto Bruch's membrane. Explant pairs (cultured with MMP inhibitor or negative control for inhibitor) were resurfaced to a similar degree, regardless of presence or absence of MMP inhibition. However, in 3 of the 6 explants, cells cultured without MMP inhibition appeared to be more differentiated (more uniform cell size, smaller, more apical processes).
These data indicate that while MMP2 does not appear to be a major contributor to enhanced cell resurfacing and survival on aged and AMD submacular explants following culture in BCE-CM, MMP2 may play a role in cell differentiation.
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