Inhibition of LDL degradation in cultured human fibroblasts induced by endothelial cell-conditioned medium.
Cultured porcine aortic endothelial cells (EC) secrete a potent mitogen into their serum-free culture medium which stimulates DNA synthesis in quiescent human fibroblasts. Previous work has shown that endothelial cell-conditioned medium (ECCM) also stimulates parallel, concentration-dependent increases in low density lipoprotein (LDL) cell surface binding and internalization. LDL degradation, however, was inhibited in ECCM-treated cells. Studies were conducted to determine whether the EC-derived growth factor (EDGF) and the endothelial-derived inhibitor of LDL degradation (EDID) are separate and distinct components, and to further delineate some of the characteristics of EDID. Time-course studies showed that the mitogenic activity released by EC into ECCM increased steadily during the first 24 hours and came to a plateau at about 48 hours of conditioning, whereas inhibitory activity increased linearly in ECCM for at least 6 days. Mitogenic activity in ECCM was stable under the same conditions. Ultrafiltration experiments showed that EDGF activity has an apparent molecular weight in excess of 30,000; in contrast EDID activity was not retained by filters with a 2000 dalton retention limit. EDID activity also failed to bind to a Blue Sepharose affinity column, while EDGF was bound and could be eluted with 1M NaCl in the presence of ethylene glycol. The ammonium ion concentration in ECCM was measured to determine the possible contribution of this ion to EDID activity. Confluent cultures of porcine aortic EC in serum-free culture medium produced substantial amounts of NH4+. After 96 hours of conditioning, the level of NH4+ in ECCM rose from near zero to 2-4 mM. When equivalent amounts of NH4Cl were added to fresh nonconditioned medium, a level of inhibitory (EDID) activity similar to that found with ECCM was observed. These studies indicate that EDGF and EDID activities are distinct and separate components of ECCM. Since NH4+ ion is present in ECCM in concentrations that are sufficient to account for all the observed EDID activity, it is likely that NH4+ is responsible for this activity.
D000641:Ammonia / Q000032:analysis; D000818:Animals; D001011:Aorta / Q000166:cytology*; D002478:Cells, Cultured; D003470:Culture Media; D004727:Endothelium / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006133:Growth Substances / Q000032:analysis / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000032:analysis / Q000276:immunology* / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D013552:Swine
D003470:Culture Media; D006133:Growth Substances; D008077:Lipoproteins, LDL; C019455:low density lipoprotein inhibitor; D000641:Ammonia