Purification of a factor from human placenta that stimulates capillary endothelial cell protease production, DNA synthesis, and migration.

Abstract

A protein that stimulates the production of plasminogen activator and latent collagenase in cultured bovine capillary endothelial cells has been purified 106-fold from term human placenta by using a combination of heparin affinity chromatography, ion-exchange chromatography, and gel chromatography. The purified molecule has a molecular weight of 18,700 as determined by NaDodSO4/PAGE under both reducing and nonreducing conditions. The purified molecule stimulates the production of plasminogen activator and latent collagenase in a dose-dependent manner between 0.1 and 10 ng of protein/ml. The purified protein also stimulates DNA synthesis and chemotaxis in capillary endothelial cells in the same concentration range. Thus, this molecule has all of the properties predicted for an angiogenic factor.During neovascularization of tumors, new capillaries arise as sprouts from existing microvessels in response to local release of angiogenic factors by the tumor cells (1). It has been proposed that the response of microvascular endothelial cells to angiogenic factors has three major components: an increase in endothelial cell protease production that allows the endothelial cells to penetrate the surrounding tissues, a stimulation of endothelial cell migration toward the source of the angiogenic factor, and an increase in the rate of endothelial cell proliferation (2). Possible correlates of each of these components have been identified in the response of cultured microvascular endothelial cells to angiogenic preparations-increased collagenase and plasminogen activator (PA) production (2), increased endothelial cell motility and chemotaxis (3, 4), and increased rate of multiplication (5, 6). However, it has not been demonstrated whether the stimulation of these different processes is due to a single molecule in the preparations or to several different molecules. Several groups have used heparin-Sepharose affinity chromatography to purify endothelial cell mitogens from various sources (7-12). We have investigated whether heparin-Sepharose affinity chromatography could also be used to purify the PA and collagenase-inducing activity from a preparation with known angiogenic activity and whether the purified proteaseinducing molecule was also able to induce the other activities associated with angiogenesis-increased endothelial cell mitosis and motility. As a source of the protease-inducing activity, we have chosen term human placenta, which has been shown to contain an angiogenic activity (13) and to stimulate PA and collagenase production in cultured capillary endothelial cells (21). and the substance to be tested. After incubation at 370C for 24 hr, the medium was collected from the cultures and was assayed for collagenase as described (16). All collagenase was in a latent form and was activated with trypsin to detect activity. The cell layers from these same cultures were washed twice with cold phosphate-buffered saline, pH 7.5, and were extracted with 0.5% Triton X-100 in 0.1 M sodium phosphate, pH 8.1, and the cell...

Phosphorylation of STAT-3 in Response to Basic Fibroblast Growth Factor Occurs through a Mechanism Involving Platelet-activating Factor, JAK-2, and Src in Human Umbilical Vein Endothelial Cells

“…DISCUSSION PAF is now considered as a major lipid second messenger in the regulation of a number of biological responses including tumor expansion and angiogenesis (25,26). PAF induces endothelial cell proliferation (27) and migration through the basement membrane by inducing the production of plasminogen activator protein and DNA synthesis (28,29). There are data to suggest that the production of PAF may result through the binding of many different pro-angiogenic protein growth factors to their tyrosine receptors.…”

Inhibition of Cell Migration and Angiogenesis by the Amino-terminal Fragment of 24kD Basic Fibroblast Growth Factor

“…1 is a mitogen produced and secreted by endothelial cells, which promotes cell proliferation, chemotaxis, protease production, and integrin expression (1)(2)(3)(4). As a consequence, it is an integral component of the angiogenic process.…”

Purification from a human hepatoma cell line of a basic fibroblast growth factor-like molecule that stimulates capillary endothelial cell plasminogen activator production, DNA synthesis, and migration.

“…Since the FGF-like molecule purified from placenta stimulated both PA and collagenase production (15), the effect of the purified hepatoma factor on collagenase production was investigated. The conditioned medium and cell extracts used above were immunoprecipitated with antibodies to interstitial collagenase, and the immunoprecipitated material was run on SDS-PAGE.…”

“…The purified molecule, an 18,700-dalton protein, stimulates PA and collagenase production, cell replication, and chemotactic movement in cultured capillary endothelial cells, indicating that a single molecule is able to induce the complete "angiogenic phenotype" in the endothelial cell (15). Moreover, this molecule induces angiogenesis in vivo in the chicken chorioallantoic membrane (15). This factor has been identified as human basic fibroblast growth factor (FGF) on the basis of its affinity for heparin-Sepharose, cross-reactivity with antibodies to basic FGF, and amino acid sequence data.…”