The molecular and cellular mechanisms of activation of fat-storing cells (Ito cells or parasinusoidal lipocytes), a prerequisite of the fibrogenic response of injured liver, were studied by analysis in vitro of some aspects of the intercellular communication between parenchymal liver cells and fat-storing cells. Conditioned medium harvested from early serum-free monolayer cultures of hepatocytes isolated from normal rat liver stimulated strongly, reproducibly and dose-dependently the proliferation of nonconfluent fatstoring cells maintained under serum-reduced conditions. During exposure of fat-storing cells for 48 hr to the conditioned medium, the incorporation of [3H]thymidine into DNA was stimulated four to six times over control values, the DNA content per culture well was elevated by 40% above control values and the immunocytochemical detection of bromodeoxyuridine-labeled cell nuclei was increased from 13% stained nuclei in controls to 70% stained nuclei in treated fat-storing cells. The mitogenic effects of hepatocyte-conditioned medium were similar to or even higher than those of 10% fetal calf serum. No mitoinhibitory activity could be detected in the hepatocyte-conditioned medium when arginase, as a potential inhibitor, was excluded. Rat skin fibroblasts could not be stimulated under conditions where the proliferation activity of fatstoring cells was greatly enhanced. The occurrence of the mitogenic activity in the medium is not dependent on de novo synthesis or secretion because the media of hepatocytes cultured under anoxic conditions in the presence of cycloheximide, brefeldin A or ethylenediaminetetraacetate were highly active in promoting fat-storing cell proliferation, although hepatocyte viability was greatly reduced under some of these conditions. A significant positive correlation (r = 0.95, p < 0.01) was found between lactate dehydrogenase activity and the mitogenic potency of the conditioned medium. The proliferation factor for fat-storing cells could also be demonstrated in the lysate of freshly isolated hepatocytes from normal liver. The stimulatory activity in the medium was partially enriched by a combination of gel permeation and anion exchange fast protein liquid chromatography and characterized as a protein with an apparent molecular weight of about 60 kD that is heat and pH sensitive but insensitive to reducing agents. It does not bind to immobilized heparin; nor does soluble heparin or proteinase inhibitor affect the mitogenic activity of the factor. The hepatocyte-conditioned medium promotes neither transformation, as judged from immunocytochemical monitoring of the expression of desmin and smooth muscle α-actin filaments and cytochemical detection of lipid droplets, nor matrix synthesis-the rate and the profile of medium proteoglycan synthesis of fat-storing cells were not significantly altered. Furthermore, the immunofluorescence microscopic appearance of collagen types 1 and 3, fibronectin and laminin were significantly stronger in fat-storing cell cultures exposed to hepatocyte-conditioned medium because of the augmentation of cell number, but matrix expression per individual cell seemed to be unaffected.

The data led us to conclude that isolated hepatocytes contain a presumably cytosolic protein with potent mitogenic effects on fat-storing cells. The factor may be released from hepatocytes by membrane damage or other forms of parenchymal cell injury and provides during the onset of fibrogenesis the first mitogenic signal for fat-storing cells, which are located in the immediate proximity of hepatocytes. The proposed preinflammation step of paracrine activation of fat-storing cells is followed by paracrine stimuli elaborated from inflammatory cell types and platelets, respectively. (HEPATOLOGY 1992;16:1250–1266.)