Matrilin-1 Is an Inhibitor of Neovascularization
In the course of conducting a series of studies whose goal was to discover novel endogenous angiogenesis inhibitors, we have purified Matrilin-1 (MATN-1) and have demonstrated, for the first time, that it inhibits neovascularization both in vitro and in vivo. Proteins were extracted from cartilage...
| Main Authors: | , , , , , , , |
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
| Published: |
American Society for Biochemistry and Molecular Biology (ASBMB)
2018
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| Online Access: | http://hdl.handle.net/1721.1/119675 https://orcid.org/0000-0002-0505-1435 https://orcid.org/0000-0003-4255-0492 |
| Summary: | In the course of conducting a series of studies whose goal was to discover novel endogenous angiogenesis inhibitors, we have purified Matrilin-1 (MATN-1) and have demonstrated, for the first time, that it inhibits neovascularization both in vitro and in vivo. Proteins were extracted from cartilage using a 2M NaCl, 0.01M HEPES buffer at 4°C, followed by concentration of the extract. The concentrate was fractionated by size exclusion chromatography and fractions were then screened for their ability to inhibit capillary EC proliferation in vitro. Fractions containing endothelial cell (EC) inhibitory activity were pooled and further purified via cation exchange chromatography. The resulting fractions from this step were then screened to isolate the anti-angiogenic activity in vitro. This activity was identified via tandem mass spectrometry (MS/MS) as being MATN-1. Human MATN-1 was cloned and expressed in Pichia pastoris and purified to homogeneity. Purified recombinant MATN-1, along with purified native protein, was shown to inhibit angiogenesis in vivo using the chick chorioallantoic membrane assay via the inhibition of capillary EC proliferation and migration. Finally, using a MATN-1-deficient mouse, we showed that angiogenesis during fracture healing was significantly higher in MATN-1-/- mice in comparison to the wild type mice as demonstrated by in vivo imaging and by elevated expression of angiogenesis markers including PECAM1, VEGFR, and VE-cadherin. |
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