Shedding of the matrix metalloproteinases MMP-2, MMP-9, and MT1-MMP as membrane vesicle-associated components by endothelial cells

Abstract

Production of matrix-degrading proteases, particularly matrix metalloproteinases (MMPs), by endothelial cells is a critical event during angiogenesis, the process of vessel neoformation that occurs in normal and pathological conditions. MMPs are known to be highly regulated at the level of synthesis and activation, however, little is known about the regulation of MMP secretion by endothelial cells. We found that cultured human umbilical vein endothelial cells shed vesicles (300 to 600 nm) originating from localized areas of the cell plasma membrane, as revealed by ultrastructural analysis. Normal and reverse zymography, Western blot, and immunogold analyses of the vesicles showed two gelatinases, MMP-2 and MMP-9, in both the active and proenzyme forms, the MT1-MMP proenzyme located on the external side of the vesicle membrane and the two inhibitors TIMP-1 and TIMP-2. Serum and the angiogenic factors, fibroblast growth factor-2 and vascular endothelial growth factor, stimulated the shedding of MMPs as vesicle components. Shedding the vesicle was rapid, as it was already completed after 4 hours. Addition of shed vesicles to human umbilical vein endothelial cells resulted in autocrine stimulation of invasion through a layer of reconstituted basement membrane (Matrigel) and cord formation on Matrigel. We conclude that endothelial cells shed MMP-containing vesicles and this may be a mechanism for regulating focalized proteolytic activity vital to invasive and morphogenic events during angiogenesis.

Figure 1.

Ultrastructural analyses of the vesicle-shedding process. A and B: Scanning electron micrograph of HUVEC-releasing vesicles. The phenomenon occurs in two different areas of cell surface (arrows). C: Transmission electron microscopic picture of the surface of HUVEC, in cross-section, showing vesicle release from the plasma membrane. Scale bar, 0.5 μm. D: Morphological picture of isolated membrane vesicle (350 nm) with negative staining. Scale bar, 0.5 μm.

Figure 2.

Vesicle-associated proteolytic activity. A: Zymographic analysis of gelatinases. Bands corresponding to the proenzyme forms or the gelatinases (proMMP-2 and proMMP-9) were detected in the isolated vesicles (20 μg, lane 2). Lower molecular weight bands correspond to the activated enzymes. Supernatant of WM983A cells (lane 1) was used as a standard for proMMP-2 and proMMP-9. B: Western blot analysis of MMP-9. Vesicles (20 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with an anti-MMP-9 antibody as described in Materials and Methods. C: Reverse zymographic analysis of TIMPs in vesicles (40 μg). TIMP-1 and TIMP-2 appear as dark bands of, respectively, 28 and 21 kd.

Figure 3.

Analysis of MT1-MMP on shed vesicles. A: Western blot analysis of MT1-MMP. Vesicles (20 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with an anti-MT1-MMP antibody as described in Materials and Methods. B: Electron microscopy localization of MT1-MMP in the vesicles. The application of heavy metal salt solution on the sample reveals an electron-translucent membrane (arrow) on a dark background and the gold particles (10 nm) show the presence of MT1-MMP on the external site of the vesicle.

Figure 4.

Expression of β1 integrin on HUVEC-shed vesicles. Isolated vesicles were subjected to Western blot analysis as described in Materials and Methods.

Figure 5.

Modulation of vesicle-associated proteolytic activity by serum and angiogenesis regulatory factors. Zymographic analysis of vesicles isolated from supernatants of HUVECs exposed for 4 hours to medium containing the indicated concentrations of serum (A) or FGF-2 (10 ng/ml) or VEGF (10 ng/ml) (B).

Figure 6.

Effect of vesicles on endothelial cell invasion, assessed in the Boyden chamber, using a Matrigel-coated filter. HUVECs were added to the upper compartment of the chamber with or without the indicated amount of vesicles. Results are expressed as the number of migrated endothelial cells in 10 high-power fields (mean and SD of triplicates). The results are from one experiment representative of three.

Figure 7.

Effect of vesicles on the formation of capillary-like structures by endothelial cells. HUVECs were plated on Matrigel, in medium with 5% serum (A), complete medium (B), medium with 5% serum containing vesicles 0.01 μg/well (C), or 1 μg/ml (D). Pictures were taken after 24 hours. Original magnifications, ×100.