Zamir Halpern

Inhibition of experimentally-induced liver cirrhosis in rats by a nonpeptidic mimetic of the extracellualr matrix-associated Arg-Gly-Asp epitope

AIMS/METHODSnIn the present study, we examined whether a nonpeptidic mimetic of Arg-Gly-Asp (RGD), which specifically inhibits RGD-dependent adhesion of CD4+ T lymphocytes or fibroblasts to fibronectin, can prevent thioacetamide-induced liver cirrhosis in rats. The nonpeptidic RGD mimetic, rather than the RGD peptide was utilized, since the peptide is rapidly degraded, and therefore, relatively ineffective for in vivo use.nnnRESULTSnWe now report that rats treated with thioacetamide and the RGD analogue SF-6,5 for 12 weeks had lower histopathologic scores than those treated with thioacetamide alone. Further improvement in liver histology was observed after another 8 weeks of treatment with the analogue SF-6,5. Quantitative microscopic analysis by computerized imaging morphometry of liver biopsies from the three groups and controls confirmed the semi-quantitative histopathologic scores (p < 0.001). After 3 months of treatment, the spleen weights in the SF-6,5-treated rats were 30% less than those of rats that received only thioacetamide, which indicated that the analogue-treated rats were less portal hypertensive.nnnCONCLUSIONSnThe observed inhibition of the progression of cirrhosis in rats by the nonpeptidic RGD analogue suggests that RGD mimetics may be useful therapeutically in inhibiting pathological processes that involve RGD recognition.

Amphiregulin and hepatocyte-derived extracellular matrix regulate proliferation and autocrine growth factor expression in colon cancer cell lines of varying liver-colonizing capability.

We studied the effect of two members of the epidermal growth factor (EGF) family—amphiregulin and heparin‐binding EGF‐like growth factor (HB‐EGF)—on cell proliferation, growth factor and growth factor receptor expression, and cell differentiation in two human colon cell lines of varying liver‐colonizing potential. The effect of amphiregulin and HB‐EGF was assessed both in cells grown on plastic, as well as on cells grown on hepatocyte‐derived extracellular matrix (ECM). We found that both colon cell lines were sensitive to HB‐EGF stimulation of cell proliferation. Amphiregulin inhibited cell proliferation in KM12 cells and stimulated the strongly metastatic cell line KM12SM to a slight extent. When the cells were cultured on hepatocyte‐derived ECM, amphiregulin inhibited the weakly metastatic KM12 and stimulated the growth of KM12SM. HB‐EGF synergistically acted with hepatocyte‐derived ECM to enhance cell proliferation in both colon cell lines. Expression of ligands of the EGF family, such as transforming growth factor‐α (TGF‐α) and amphiregulin, was decreased in both cell lines when cultured on ECM. Hepatocyte‐derived ECM decreased expression of cripto in KM12 and increased it in KM12SM cells. Neither cripto nor TGF‐α mRNA levels was affected by growing the cells in the presence of amphiregulin. However, amphiregulin increased expression of its own mRNA in the weakly metastatic KM12 and decreased it in the strongly metastatic KM12SM when the cells were cultured on plastic. Amphiregulin and HB‐EGF stimulated expression of erb‐B2 in both cell lines cultured on plastic. Surprisingly, when the cells were grown on hepatocyte‐derived ECM, amphiregulin inhibited erb‐B2 expression in both cell lines. We observed no effect of amphiregulin on cell differentiation as assessed by alkaline phosphatase expression. Our studies demonstrate one mechanism that could play a role in site‐specific metastasis. We found an inhibitory response to an autocrine growth factor in the context of hepatocyte‐derived ECM in a weakly metastatic cell and a stimulatory effect of the same growth factor when strongly metastatic cells were cultured on the same ECM. J. Cell. Biochem. 76:332–340, 1999.

Isolated Colonic Loop in the Rabbit: An in vivo System for Studying Intestinal Mucus

A method for preparing an isolated colonic loop (Thiry-Vella) in a living rabbit is described. The loop, with its intact neurovascular supply, continues to secrete clear colonic mucus for more than 2 months. The chemical composition of the mucus, collected daily for 2 months, was analyzed and shown to be a high molecular weight glycoprotein composed of approximately equimolar amounts of protein and carbohydrate. The main sugars found were N-acetylgalactosamine, N-acetylglucosamine, galactose, and sialic acid. The most prominent amino acids were threonine, aspartic acid, glycine and serine. A considerable flattening and atrophy of the glandular structure of the isolated colonic loop was observed during the 2 months. This fact did not markedly affect the amount and chemical composition of the mucus that was collected daily from this loop. This model can be used in vivo to investigate colonic mucus in normal and diseased animals, or even following the administration of various drugs.

963 SORTILIN-DEFICIENT HEPATOCYTES ARE LESS SUSCEPTIBLE TO BILE ACIDS-INDUCED INFLAMMATION AND APOPTOSIS

Background and Aims: Sortilin is a trafficking molecule directing newly synthesized molecules from the trans-Golgi network to secretory pathways or cell surface. One of the molecules trafficked by sortilin is acidic sphingomyelinase (ASMase), whose product ceramide participates in apoptosis signaling. We hypothesized due to less production of ceramide during liver injury, hepatocytes from sortilin−/− mice would be less sensitive to apoptosis. We have investigated liver injury, apoptosis and necrosis, inflammation and fibrosis in a bile duct ligation model in wild type (WT) and sortilin−/− mice. Methods: We have assessed liver damage and fibrosis 7 days after BDL using WT and sortilin−/− mice. The effect of bile acids on hepatocyte apoptosis and expression of inflammatory cytokines was determined in vitro in primary hepatocytes from WT and sortilin−/− mice. Results: Sortilin−/− mice displayed reduced liver damage compared to WT mice, as determined by lower serum AST, ALT, alkaline phosphatase and bilirubin. Sortilin−/− mice also had fewer areas of hepatocyte necrosis that result from bile infarcts, reduced hepatocyte apoptosis compared to WT mice, as well as reduced mRNA levels of collagen I. We determined the in vitro effect of bile acids on hepatocyte apoptosis and expression of proinflammatory cytokines and observed strong reduction of apoptosis and inflammatory cytokines in sortilin-deficient hepatocytes. Conclusion: Sortilin−/− mice have attenuated fibrosis and liver damage after BDL, due in part to reduced hepatocyte apoptosis and reduced inflammatory cytokines.