Journal: Molecular Biology of the Cell
Article Title: Cells activated for wound repair have the potential to direct collective invasion of an epithelium
Figure Lengend Snippet: Mesenchymal leader cells in the 2D ex vivo injury culture model are enriched for molecules involved in invasion, CD44, MMP-2, and MMP-9. (A) Model of the different regions of the ex vivo mock cataract surgery explant that are separated by microdissection for this study from both top and side views. Cells found in the points of the star-shaped culture that contain the region of the lens that is occupied by epithelial cells in vivo are referred to as the original attachment zone (OAZ, yellow). In response to injury, cells move onto the denuded basement membrane (BM) into the central migration zone (CMZ, purple). (B–D) To examine whether there are migration-specific changes in expression of molecules associated with invasion after wounding, ex vivo injury explants were microdissected into OAZ and CMZ regions and extracted on days 1–3 for Western blot analysis for CD44, MMP-9, MMP-2, and GAPDH (loading control). Graphs depicting Western blot results from four independent studies show the relative expression of CD44, MMP-9, and MMP-2 to GAPDH. CD44, MMP-9, and MMP-2 were predominately expressed in the CMZ region. These molecules decreased in expression as wound healing progressed on day 2 and on day 3, when wound healing is typically completed. MMP-2 and MMP-9 were detected predominantly in their active forms (bottom arrows) compared with their inactive proenzymatic form (top arrows). For both MMP-2 and MMP-9, the active form (lower band) was quantified and is represented on the graphs as a ratio to GAPDH. Immunoblots were quantified using Kodak 1D software, and data were normalized to results at day 1 in the CMZ zone. Values for each graph and relative densities are plotted ±SEM. Representative Western blots are shown below each graph. (E–M) Ex vivo–wounded cultures were fixed and immunostained for vimentin (F, I, L; green) and colabeled for CD44 (E, G; red), MMP-9 (H, J; red), or MMP-2 (K, M; red). Vimentin-rich mesenchymal cells at the leading edge were enriched for MMP-2, MMP-9, and CD44. CD44 localized at cell–cell interfaces and to the tips of these cells at the leading edge (E–G, arrow). MMP-9 strongly localized to vimentin-rich cells, sometimes colocalized with vimentin (H–J, arrow) and also to regions in the area to be repaired just beyond the cells (H, J, arrowhead). Bar, 10 μm. (N) The role of MMP-2/9 function on wound healing in the 2D ex vivo injury cultures was evaluated using inhibitors to MMP-2, MMP-9, or MMP-2/9. Phase images were taken on days 0–3, from which the open wound area was calculated in at least six capsules and quantified using NIS Elements analysis software. Open wound area was plotted on the graphs ±SEM. Inhibition of MMP-2 and/or MMP-9 function resulted in only a slight delay in wound closure.
Article Snippet: Antibodies used for Western blotting included CD44 (monoclonal antibody [mAb]; Developmental Studies Hybridoma Bank, Iowa City, IA), MMP-9 (polyclonal; Millipore), MMP-2 (monoclonal; Millipore), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA).
Techniques: Ex Vivo, Laser Capture Microdissection, In Vivo, Migration, Expressing, Western Blot, Software, Inhibition