Abstract
A low-mol.-wt compound isolated from rat Walker 256 carcinoma and found to induce neovascularization in vivo was tested on cultures of cow brain-derived endothelial cells (CBEC) growing on plastic and collagen substrates. This factor had a mitogenic effect on CBEC cultured on native collagen gels and for this reason has been called "endothelial-cell-stimulating angiogenesis factor" (ESAF). CBEC growing on plastic culture dishes or denatured collagen films were not stimulated by ESAF. The mitogenic effect of ESAF was equally apparent when added to cells already attached to the native collagen substrate or when the collagen substrate was pre-incubated with ESAF before plating the cells. A floating collagen gel pre-incubated with ESAF in cultures of CBEC growing on plastic dishes did not stimulate cell growth. Our data indicate that the substrate influences cell behaviour and that CBEC only respond to ESAF when growing on a native collagen substrate.
Highlights
This factor had a mitogenic effect on cow brainderived endothelial cells (CBEC) cultured on native collagen gels and for this reason has been called "endothelial-cell-stimulating angiogenesis factor" (ESAF)
In a previous study we demonstrated structure of the LMW ESAF isolated in this that a crude tumour extract containing angiogenic activity in vivo was mitogenic for endothelial cells in vitro, provided that the cells were growing on a native collagen substrate in the presence of platelet-release factors
ESAF stimulated CBEC proliferation in growth medium containing either 8% or 16% foetal calf serum (FCS) when the cells were cultured on native collagen, but not on plastic or denatured collagen
Summary
The growth characteristics of the cowbrain endothelial cells (CBEC) on plastic tissue-culture dishes and native collagen gels has been described (Schor et al, 1979). As a result of the differences in the attachment and growth characteristics of the cells on the various substrates, twice as many CBEC were routinely plated on the collagen gels than on the plastic dishes in order to have comparable cell numbers attached 2-3 days later. The number of attached cells was determined (Day 0 in Fig. 1) and all cultures received either 100 ,ul of BSS alone or 100 pl of BSS containing 5 Hg of ESAF + filler. The presence of ESAF did not further increase the cell proliferation on plastic or gelatin films, but did produce a significant increase in cell growth on the collagen gels Under these conditions, ESAF did not shorten the lag period before growth began on collagen gels, nor did it have a significant effect on the cell attachment to the substrate, as estimated by the number of cells present in the supernatant of ESAF-treated and control cultures. The final cell density was not affected by ESAF (data not shown)
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