Low concentration of S100A8/9 promotes angiogenesis-related activity of vascular endothelial cells: bridges among inflammation, angiogenesis, and tumorigenesis?

Abstract

Previous studies showed that several members of the S100A family are involved in neovascularization and tumor development. This study checked whether low concentrations of S100A8 or S100A9 has any effect on the behaviour of vascular endothelial cells. A human umbilical vascular endothelial cell (HUVEC) line was used to measure vascular endothelial cell bioactivity related to angiogenesis, such as cell proliferation, migration, and vessel formation. In the low concentration range up to 10 μg/mL, either each alone or in combination, S100A8 and S100A9 proteins promoted proliferation of HUVEC cells in a dose-dependent manner. The presence of both proteins in culture showed additive effects over each single protein. Both proteins enhanced HUVEC cells to migrate across the transwell membrane and to form tube-like structures on the Matrigel surface. When mixed in Matrigel and injected subcutaneously in Balb/c mice, both proteins increased vessel development in the gel plugs. Microarray assay of HUVEC cells treated with 10 μg/mL S100A8 revealed that ribosome pathway, pathogenic Escherichia coli infection pathway, apoptosis, and stress response genes were modulated by S100A8 treatment. We propose that S100A8 and S100A9 proteins from either infiltrating inflammatory cells or tumor cells play an important role in the interplay among inflammation, angiogenesis, and tumorigenesis.

Figure 1

Stimulation of HUVEC proliferation following S100A8 or S100A9 protein treatment at different concentrations. Shown are representatives of three experiments with similar results.

Figure 2

Photographs of HUVEC cells migrated through transwell chambers. The numbers above each panel are the cell counts per microscope field (mean ± standard deviation, n = 6). *P < 0.05 versus medium control by the two-tailed, paired Student's t-test.

Figure 3

Tube-like structure formation of HUVEC seeded on Matrigel with or without S100A8 or S100A9 protein in vitro (both at 10 μg/mL). FGF plus heparin was used as a positive control.

Figure 4

Photographs (pictures, upper left panel), haemoglobin contents (solid columns, upper left panel), and histology of Matrigel plugs indicating increased neovascularization following S100A8 or S100A9 protein treatment. Matrigel mixed with PBS was the negative control group, and FGF plus heparin was the positive control group. Haemoglobin content of Matrigel plugs (mean ± standard deviation, n = 4) was expressed as the amount of haemoglobin per gram of Matrigel plug. Microscopic examination of the same Matrigel plugs showed inflammatory cells and vascular structures (arrowheads). Shown are representations of two experiments with similar results.

Figure 5

Roles of S100A8/A9 in tumorigenesis. In this hypothesis, S100A8/A9 are produced by newly formed tumor cells or infiltrating leukocytes. When they reach adequately low concentrations, they can stimulate (1) tumor cells proliferation, (2) vascular endothelial cell proliferation, migration, and formation of vascular structures, and (3) neutrophils to migrate into tumor mass or adhere to endothelium. By producing S100A8/A9, the newly entering neutrophils enhance local inflammation and neovascularization in turn. Other S100A family members like S100A4, S100A7, and S100A13 may join S100A8 and/or S100A9 in this episode as well.