Rat liver perisinusoidal lipocytes (PL) cultured on uncoated plastic transform spontaneously within 6-10 d to myofibroblastlike cells (MFBIC). Parallel to the transformation the TGFa-and TGF#1-mRNA expression increased and was highest in MFBIC. Competitive radioligand binding assays demonstrated that in contrast to untransformed PL the MFBIC synthesize and secrete transforming growth factor (TGF)-a (15 fmol/cell per 24 h) and predominantly the latent form of TGFWI (0.2 fmol/cell per 24 h). Medium conditioned by MFBIC (MFBcM) significantly stimulated PL proliferation with little effect on PL proteoglycan synthesis. By transient acidification of the MFBcM, known to activate the latent form of TGFj@1, the stimulatory effect on PL proteoglycan synthesis was enhanced and furthermore PL transformation (measured by expression of iso-a smooth muscle actin and loss of retinylpalmitate) was accelerated. Preincubation of this medium with neutralizing antibodies to TGF,6 resulted in (a) the growth inhibitory effect was converted to a growth stimulation and (b) the stimulatory effect on proteoglycan synthesis was abolished.In summary our data indicate that progressive activation of PL on plastic (transformation to MFBIC) leads to an enhanced expression of the TGFa-and TGFjdl-mRNAs and secretion of the corresponding proteins. Medium conditioned by MFBIC stimulates proliferation, transformation, and PG synthesis of untransformed PL. These mechanisms are suggested to be relevant in self perpetuation of liver fibrogenesis. (J. Clin. Invest.

A central problem in the study of the pathogenesis of liver fibrosis (fibrogenesis) is the identification of the cellular sources of the extracellular matrix and the dissection of the molecular mediators stimulating connective tissue synthesis in certain hepatic target cells. In the present study the role of platelets and of some platelet-derived polypeptide growth factors in the proliferation and proteoglycan synthesis of rat liver fat storing cells in culture (the principle connective tissue-producing cell type in liver) was determined. Fat storing cell proliferation was determined by measurement of the DNA-content, and [ 3 H]thymidine-and bromodeoxyuridine-incorporation. Glycosaminoglycan synthesis was determined by the measurement of [ 35 S]sulphate incorporation. Human platelet lysate (0.3 to 2.6 g protein per litre medium) stimulated, in a dose-dependent manner, both the proliferation and glycosaminoglycan synthesis of rat liver fat storing cells kept äs a primary culture in Dulbecco's modificätion of Eagle's medium in the absence of foetal calf serum. More than 70% of the newly synthesized glycosaminoglycans were found in the medium. Among the various thrombocyte-derived polypeptides tested äs candidate mediators of the platelet-derived fibrogenic activity, platelet derived growth factor was not effective in enhancing glycosaminoglycan synthesis, and it stimulated the proliferation of fat storing .cells only about 2 fold. On the other hand, epidermal growth factor proved to be a Stimulus of both processes. Transforming growth factor (> 10 pmol/1) inhibited foetal calf serum (Dulbecco's modificätion of Eagle's medium with a fraction of foetal calf serum of 0.1) and epidermal growth factor stimulated proliferation but enhanced the synthesis of sulphated glycosaminoglycans about 2-fold. These results suggest the possible role of transforming growth factor äs a negative modulator for fat storing cells proliferation but a positive modulator for fat storing cell transformation and extracellular glycosaminoglycan matrix synthesis. Furthermore, our results indicate a cpoperation between different hepatic and extrahepatic cell types by paracrine Stimulation of fat storing cells. Transfonning growth factor in combination with epidermal growth factor appear to be candidate mediators of the platelet-derived fibrogenic activity, which stimulates fat storing cells in culture, and might also be effective in vivo during hepatic repair processes following liver injury.

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The synthesis of total sulfated glycosaminoglycans (GAG) was stimulated by transforming growth factors (TGFcl l.Cfold at 5 ng/ml, and TGFDI 2.05fold at 2.5 ng/ml) in primary cultures of rat liver fat storing cells (FSC). The combination of both TGFs resulted in an additively stimulated synthesis of total sulfated GAG (more than 3-fold), chondroitin sulfate (more than &fold) and hyaluronate (3.8fold), respectively, whereas the formation of dermatan sulfate was unchanged and that of heparan sulfate was slightly reduced. In summary, TGFs were identified as important mediators of stimulated GAG synthesis in those cells of the liver (FSC), which are the primary site of matrix glyc~onjugate production.

Because proteases free in the body are damaging to the tissues, animals have evolved various agents for their inactivation and clearance. Mammals, for instance, have a diverse array of active site protease inhibitors in the plasma. In addition, mammals have alpha 2-macroglobulin (alpha 2M), which binds active proteases, and the alpha 2M-protease complex is then cleared from the plasma by a receptor-mediated endocytotic process. alpha 2M is also present in the plasma of many invertebrates, and in the American horseshoe crab, Limulus polyphemus, it is the only protease inhibitor in the plasma. To search for a clearance process for proteases in Limulus, fluorescein isothiocyanate (FITC)-labeled proteins were injected into the blood, and the fluorescence in the plasma and associated with the blood cells was determined. FITC-labeled trypsin was cleared with an initial mixing period (0-10 min) and a rapid clearance period (10-30 min), followed by the reappearance of FITC in the plasma (45-90 min). Before and during the clearance process, the labeled trypsin was associated with a complex having a molecular mass identical to that of Limulus alpha 2M, and that was precipitated by antibodies directed against Limulus alpha 2M. The fluoresceinated material that reappeared in the plasma after 45 min was of low molecular mass (< 10 kDa) and thus appears to have experienced degradation. The clearance of trypsin requires its protease activity, since phenylmethylsulfonyl fluoride-inactivated, FITC-labeled trypsin was cleared only very slowly if at all (t1/2 > 180 min). FITC-labeled, trypsin-reacted Limulus alpha 2M was cleared rapidly from the plasma of Limulus, whereas FITC-labeled, native Limulus alpha 2M persisted undiminished in excess of 400 min. The blood cells of Limulus bound FITC-labeled trypsin-reacted Limulus alpha 2M, and the peak of recovery from the blood cells coincided with the minimum concentration of FITC-labeled protein in the plasma, suggesting that the blood cells participate in the clearance of alpha 2M-protease complex from the plasma. Thus, we have demonstrated the existence of a clearance pathway in Limulus that operates selectively on enzymatically active proteases and have shown that Limulus alpha 2M is the probable agent for protease clearance. This is the first documentation of a protease clearance pathway in invertebrates and represents the first identified physicological function for alpha 2M in invertebrates.