Purification and Properties of a New Endotherium Growth Suppressing Factor from Macrophages

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A carboxymethylated derivertives of a linear (1→3)-β-D-glucan (CMCD) acted directly on mouse peritoneal macrophages and mouse lymphoma P388D1 cells, and induced a growth suppressing activity for bovine artery endothelial cells (BAE) from themselves at a concentration of 100μg/mL. An endothelial cell growth-suppressing factor (EGSF) was purified from the conditioned medium of a mouse lymphoma P388D1 cell culture in the presence of 100μg/mL CMCD. The purification steps included, in order, ammonium sulfate fractionation and six stages of column chromatography. The purified EGSF showed two bands with silver staining on SDS-PAGE under reducing conditions and their molecular masses were estimated as approximately 55 and 63 kDa, while the molecular mass of the purified EGSF was estimated as about 65 kDa by gel filtration using Superdex 200HR. This factor strongly suppressed the proliferation of endothelial cells from bovine artery, human umbilical vein, and human dermal vas capillare and this … More suppression was observed to be reversible. The intravenous administration of the purified EGSF to sarcoma 180-bearing mouse caused a rapid decrease in the number of viable tumor cells in tumor lumps within 16h. Immunohistochemical study demonstrated that hemorrhagic disorder all over the tissue in the tumor lamp occurred under this circumstance. Thus, the purified factor exhibited not only growth suppressing activity for endothelial cells but also tumor regressing activity. The purified factor significantly inhibited in vitro tubulogenesis of bovine artery, human umbilical vein, and adult human darmal microvascular endothelial cells on collagen gel. After the tube formation of endothelial cells was completed on a collagen gel, the purified factor disrupted the tubes at a concentration of about 5 ng/ml within 48h. These findings demonstrate that EGSF is a potent inhibitor of angiogenesis as well as the growth of endothelial cells, and may bring about the regression of a solid tumor by inhibiting angiogenesis. Less