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Published on: June 2, 2017
Microvesicles shed from fibroblasts act as metalloproteinase carriers in a 3-D collagen matrix
Valentina Laghezza Masci1, Anna Rita Taddei2, Gabriella Gambellini2
1Department of Innovation in Biological, Agrifood and Forestry Systems (DIBAF), Tuscia University, Viterbo, Italy.
Abstract:
This study shows that fibroblasts migrating into a collagen matrix release numerous microvesicles into the surrounding medium. By spreading in regions of the matrix far distant from cells of origin, microvesicles carry metalloproteinase 9 (MMP-9) to act upon the collagen fibrils. As a result, the collagen matrix is gradually transformed from a laminar to a fibrillar type of architecture. As shown by western blots and gelatin zymography, MMP-9 is secreted as a 92 kDa precursor and activated upon release of 82 kDa product into the culture medium. Activation is more efficient under three-dimensional than in two-dimensional culturing conditions. While MMP-9 labeling is associated with intraluminal vesicles clustered inside the microvesicles, the microvesicle's integrin β1 marker is bound to the outer membrane. The intraluminal vesicles are recruited from the cortical cytoplasm and eventually released following uploading inside the microvesicle. Here, we propose that fusion of the intraluminal vesicles with the outer microvesicle's membrane could work as a mechanism controlling the extent to which MMP-9 is first activated and then released extracellularly.
Insights
Fibroblasts release microvesicles carrying metalloproteinase 9 (MMP-9) to remodel collagen matrices. This process transforms matrix architecture and involves the activation and extracellular release of MMP-9 via intraluminal vesicles within microvesicles.
Area of Science:
- Cell Biology
- Biochemistry
- Extracellular Matrix Remodeling
Background:
- Fibroblast migration is crucial for tissue remodeling.
- The extracellular matrix (ECM) provides structural support and signaling cues.
- Matrix metalloproteinases (MMPs) are key enzymes involved in ECM degradation.
Purpose of the Study:
- To investigate the role of microvesicles in fibroblast-mediated collagen matrix remodeling.
- To elucidate the mechanism of metalloproteinase 9 (MMP-9) activation and release by fibroblasts.
- To understand how microvesicles contribute to changes in collagen architecture.
Main Methods:
- Cell culture (2D and 3D) of fibroblasts.
- Microvesicle isolation and characterization.
- Western blotting and gelatin zymography for MMP-9 analysis.
- Immunofluorescence microscopy for protein localization (MMP-9, integrin β1).
Main Results:
- Fibroblasts release microvesicles containing MMP-9 into the collagen matrix.
- Microvesicles facilitate MMP-9 activation and extracellular release, transforming collagen from laminar to fibrillar architecture.
- MMP-9 precursor (92 kDa) is activated to an 82 kDa product, with more efficient activation in 3D cultures.
- Integrin β1 is on the outer membrane, while MMP-9 is within intraluminal vesicles inside microvesicles.
Conclusions:
- Microvesicles act as carriers for active MMP-9, driving significant collagen matrix remodeling.
- A proposed mechanism involves the fusion of intraluminal vesicles with the microvesicle membrane for MMP-9 activation and release.
- This study reveals a novel pathway for extracellular enzyme regulation and matrix modification by fibroblasts.
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