Effect of Lumican on the Migration of Human Mesenchymal Stem Cells and Endothelial Progenitor Cells: Involvement of Matrix Metalloproteinase-14

<div><h3>Background</h3><p>Increasing number of evidence shows that soluble factors and extracellular matrix (ECM) components provide an optimal microenvironment controlling human bone marrow mesenchymal stem cell (MSC) functions. Successful <em>in vivo</em> administration of stem cells lies in their ability to migrate through ECM barriers and to differentiate along tissue-specific lineages, including endothelium. Lumican, a protein of the small leucine-rich proteoglycan (SLRP) family, was shown to impede cell migration and angiogenesis. The aim of the present study was to analyze the role of lumican in the control of MSC migration and transition to functional endothelial progenitor cell (EPC).</p> <h3>Methodology/Principal Findings</h3><p>Lumican inhibited tube-like structures formation on Matrigel® by MSC, but not EPC. Since matrix metalloproteinases (MMPs), in particular MMP-14, play an important role in remodelling of ECM and enhancing cell migration, their expression and activity were investigated in the cells grown on different ECM substrata. Lumican down-regulated the MMP-14 expression and activity in MSC, but not in EPC. Lumican inhibited MSC, but not EPC migration and invasion. The inhibition of MSC migration and invasion by lumican was reversed by MMP-14 overexpression.</p> <h3>Conclusion/Significance</h3><p>Altogether, our results suggest that lumican inhibits MSC tube-like structure formation and migration <em>via</em> mechanisms that involve a decrease of MMP-14 expression and activity.</p> </div

Lumican inhibits MMP-14 expression and activity in MSC.

<p>(A): Lumican effect on MMP-14 expression in MSC and EPC analyzed by Western blotting using a mouse anti-MMP-14 monoclonal antibody directed against the catalytic domain of MMP-14 and probing with anti-β-actin. Semi-confluent MSC and EPC were seeded on plastic or in presence of lumican coating as described in Materials and Methods section. Inactive and active MMP-14 forms were detected in positive control cell lysates of fibrosarcoma cell line (HT-1080). Active MMP-14 was not detected in negative control cell lysates of HeLa cells. (B): MMP activity was measured using a SensoLyte® 520 MMP-14 Assay Kit as described in the Materials and Methods section. The activity of MMP-14 of cell lysates of MSC and EPC seeded on plastic or lumican coating (30 µg/cm²) was measured from four different donors in two independent experiments. Data presented as mean ± S.D. **<i>p</i><0.01. (C): MMP-2 and MMP-9 expression and activity in MSC and EPC. MMP-2 and MMP-9 expression and activity in cell lysates (left panel) and conditioned cell media (right panel) were analyzed by zymography.</p

Lumican inhibits MSC migration and invasion.

<p>MSC (A) or EPC (B) were plated on 12-well plates at 15 000 cells per chamber of culture-insert. Cells were incubated at 37°C and 5% CO₂ for 24 h. After withdrawal of the culture-insert, lumican (100 nM) was added to the serum-free cell culture medium. The migration speed of MSC and EPC was determined by means of computer-assisted phase contrast videomicroscopy during 48 hours as described in Materials and Methods. Representative images of cell positions after 48 h of migration are displayed on the right panels. The data are representative of three independent analyses. *<i>p</i><0.05. (C) MSC and EPC (D) invasion in presence of lumican. Cells were seeded on inserts as described in the Materials and Methods section. Medium with 10% or 2% FBS was used as a chemotactic agent for MSC or EPC, respectively, and the cells were cultured for a further 48 h. Negative control medium contained 2% BSA. Medium containing 0.5% BSA and 100 nM lumican was added to the upper chamber at the time of seeding. Invading cell nuclei were stained with Hoechst 33342 (5 µg/mL) and counted using fluorescence microscopy. Representative fluorescence images of invading cells are displayed on the upper panel. Results are expressed as the percentage of control (cells invading toward chemotactic medium) (mean ± S.D.) from at least three independent experiments with triplicate inserts. Scale bar: 100 µm. **<i>p</i><0.01; (E, upper panel): Lysates from transiently transfected MSC with GFP (mock) or MMP-14-GFP constructs were analyzed for MMP-14 overexpression by Western blotting with an antibody raised against MMP-14 after 48 h of transfection. Non-transfected MSC lysate is also included as control. (E, bottom panel): Overexpression of MMP-14 in MSC induces MMP-14 activity as shown by the enhancement of active MMP-2 band by zymography. (F): Migration of GFP- (mock) or MMP-14-GFP-transfected MSC was determined 48 h post-transfection using cell culture-insert assay. The migration speed of GFP-positive MSC, was determined by means of computer-assisted phase contrast and fluorescence videomicroscopy during 24 hours as described in supplemental methods section. The data are representative of two independent analyses. **<i>p</i><0.01. (G): GFP- (mock) or MMP-14-GFP-transfected MSC invasion in presence of lumican. Cells were cultured and inserts seeded as described in the supplemental methods section. Medium with 10% FBS was used as a chemotactic agent for MSC, and the cells were cultured for a further 48 h. Negative control medium contained 2% BSA. Medium containing 0.5% BSA and 100 nM lumican was added to the upper chamber at the time of seeding. Invading cell nuclei were stained with Hoechst 33342 (5 µg/mL) and counted using fluorescence microscopy. Representative fluorescence images of invading cells are displayed on the upper panel. Results are expressed as the percentage of control (mock-transfected cells invading toward chemotactic medium) (mean ± S.D.) from two independent experiments with triplicate inserts. Scale bar: 100 µm. *<i>p</i><0.05. ***<i>p</i><0.001.</p

Lumican inhibits MSC tube-like formation.

<p>(A): Lumican inhibits <i>in vitro</i> tube-like formation by MSC but not EPC. Tube formation on Matrigel® (a-d) in control (a, c) or 100 nM lumican-supplemented medium (b, d) was observed twenty four hours after MSC (a, b) and EPC (c, d) seeding. Representative photographs are presented on the left panel. The semi-quantitative evaluation of the tube network from ten randomly selected fields was performed using ImageJ software and NeuronJ plugin (upper right diagram for MSC, lower right diagram for EPC). Experiments were performed in triplicate on three different donors. Results represent the mean ± S.D. Scale bar: 180 µm *<i>p</i><0.05. (B, C): MSC and EPC tube-like structure formation in presence of TIMPs. Tube formation of MSC (B) or EPC (C) on Matrigel®, in control condition (a), with 200 ng/ml of TIMP-1 (b), TIMP-2 (c), TIMP-3 (d) in cell culture medium 24 h after seeding. Representative photographs are presented on the upper panels of the figure. The semi-quantitative evaluation of the branch network (lower diagrams) was performed as described above. Results represent the mean ± S.D. Scale bar: 180 µm *<i>p</i><0.05.</p