These studies suggest that IGF-1 is a mitogen for hRPE cells and also stimulates production of the angiogenic factor, VEGF. Additionally, PD98059 inhibits the production of VEGF, suggesting that the MAP kinase pathway is involved in IGF-1-mediated angiogenesis.

Impaired liver regeneration in cirrhosis complicates the surgical treatment of liver tumors which arise in this setting. We developed a rat model to investigate the regenerative response of cirrhotic liver after hepatectomy and studied the effect of exogenous transforming growth factor-alpha (TGF-alpha), a potent liver mitogen. Micronodular cirrhosis was established by the simultaneous administration of CCl4 and phenobarbital. Hepatic DNA synthesis ([3H]thymidine incorporation into DNA) 24 hr after partial hepatectomy in cirrhotic rats was 15.6 +/- 3.4 cpm/micrograms DNA (means +/- SEM), which was significantly lower than in normal rats (37.3 +/- 3.4 cpm/micrograms DNA, P less than 0.05). Exogenous TGF-alpha (30 nmol/kg, sc every 12 hr) significantly improved [3H]thymidine incorporation (35.6 +/- 8.2 cpm/micrograms DNA, P less than 0.05). An autoradiographic nuclear labeling index also confirmed increased DNA synthesis (6.7% vs 13.4%). TGF-alpha had no effect on normal regenerating liver (42.5 +/- 8.8 cpm/micrograms DNA, NS). Although the significance of TGF-alpha-enhanced liver regeneration in cirrhosis has yet to be assessed, this model may be useful for the study of mechanisms which control hepatic proliferation.

Somatostatin (SS-14) is known as an antigrowth factor for a variety of cell types, including gastrointestinal mucosa, exocrine pancreas, lymphocytes, and some tumors. We have recently identified and biochemically characterized SS-14-binding protein on rat liver plasma membranes (S. E. Raper, P. C. Kothary, and J. DelValle, Gastroenterology 96: A408, 1989; P. C. Kothary et al., Digestion 46 (Suppl 1): 58, 1990). We hypothesized that SS-14 may affect liver growth as well and investigated cellular mechanisms of this phenomenon focusing on the second messenger cAMP. Freshly isolated rat hepatocytes were plated on tissue culture dishes coated with Matrigel (laminin, heparan sulfate, and type IV collagen). The medium was not supplemented with serum or hormones. Either dibutyryl-cAMP (1 mM) or isobutylmethylxanthine (IBMX, 0.1 mM) was added in the presence or absence of SS-14 (10 nM). DNA synthesis was estimated by the rate of [3H]thymidine incorporation into DNA and by the labeling index (an autoradiographic measurement of the number of labeled nuclei). SS-14 significantly inhibited both [3H]thymidine incorporation and labeling index of rat hepatocytes stimulated by dibutyryl-cAMP or IBMX. SS-14 also inhibited intracellular cAMP accumulation stimulated by IBMX. We conclude that SS-14 exerts at least part of its antiproliferative effects via the adenylate cyclase system. Further study using other signal transduction systems may yield more information about mechanisms of hepatocyte growth.

(i) In the rat, SS-14 effectively blocks insulin-stimulated [3H]thy incorporation into DNA, possibly by blocking intracellular cAMP accumulation. (ii) Pertussis toxin blocks the growth inhibitory effects of SS-14, suggesting that inhibitory G proteins are involved in the mechanism of SS-14 action. Somatostatin may be useful in studying the role of second messengers in cell growth.

The antiproliferative effects of somatostatin on hepatocytes stimulated by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) were investigated using primary cultures of adult rat hepatocytes. Somatostatin inhibits HGF-induced (at a dose of 10 ng/mL) or EGF-induced (at a dose of 100 ng/mL) 3H-thymidine incorporation into hepatocytes in a dose-dependent manner (10(-10) to 10(-8) M). This inhibition was confirmed by autoradiography. The effect of somatostatin was nontoxic as judged by preserved albumin synthesis, a marker for differentiated hepatocyte function. In the presence or absence of somatostatin, neither HGF nor EGF significantly altered intracellular cyclic adenosine monophosphate (cAMP). We conclude that somatostatin is a potent inhibitor of HGF- or EGF-induced deoxyribonucleic acid synthesis in adult rat hepatocytes. The mechanism of this inhibition appears to be independent of cAMP. The significance of somatostatin in liver regeneration has yet to be assessed.

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Liver regeneration following partial hepatectomy is significantly impaired in rats with hereditary vasopressin (AVP) deficiency. This suggested that AVP might have a direct effect on cultured rat hepatocytes. Hepatocytes from male Sprague-Dawley rats were isolated using a two-step collagenase perfusion technique and plated at a density of 10(5)/16-mm Primaria plate. After a suitable attachment period, hepatocytes were incubated with minimal essential media, AVP, AVP plus a specific AVP antagonist, or oxytocin. Hepatocyte proliferation was measured by [3H]thymidine incorporation ([3H]Thy) into hepatocyte DNA. AVP (10 nM) increased [3H]Thy significantly (and this effect was blocked by an AVP-specific antagonist (50 nM). Oxytocin had no effect on hepatocyte DNA synthesis. To further investigate the influence of AVP on hepatocyte proliferation, the effect of AVP on transforming growth factor-alpha (TGF-alpha)-stimulated hepatocyte proliferation was also studied. This combination was chosen based on the ability of AVP to inhibit the biologic effects of EGF (a TGF-alpha analog). There was significant attenuation of TGF-alpha (50 nM)-stimulated [3H]Thy in the presence of AVP (10 nM). In summary: (1) AVP stimulates proliferation of cultured rat hepatocytes. (2) The effect of AVP can be significantly abolished by a specific AVP antagonist. (3) The proliferative response of AVP is specific. (4) AVP significantly attenuates TGF-alpha-stimulated hepatocyte hepatic DNA synthesis. Further studies should elucidate the mechanisms for the effects of AVP on hepatic proliferation alone or in combination with other factors.

Prevention of postoperative hepatic failure is important after hepatic resection. In patients with cirrhosis, impaired liver function and regenerative capacity after major hepatic resection are associated with increased morbidity and mortality. In this study, a combination of epidermal growth factor (EGF) and insulin were used as hepatotrophic factors in an attempt to stimulate DNA synthesis after 70% hepatectomy (HTX). Regenerative capacity was evaluated in normal and cirrhotic rat liver by measuring DNA synthesis in vivo. Micronodular liver cirrhosis was established by the simultaneous oral administration of CCl4 and phenobarbital. Epidermal growth factor plus insulin was injected subcutaneously immediately after and 12 h after HTX or sham operation was performed. Rats were killed 24 h after the operation and liver regeneration was estimated by [3H]-thymidine incorporation into DNA as well as an autoradiographic nuclear labelling index. Hepatectomy increased [3H]-thymidine incorporation significantly in both normal and cirrhotic rats. In cirrhotic rats, [3H]-thymidine incorporation after HTX was significantly lower than in normal rats and administration of a combination of EGF and insulin after HTX enhanced [3H]-thymidine incorporation. In conclusion, DNA synthesis 24 h after HTX is decreased in cirrhotic rats compared with normal rats and EGF supplementation with insulin accelerates DNA synthesis in hepatectomized cirrhotic rats. The data suggest that administration of combinations of exogenous hepatotrophic factors may play a useful role in the treatment of cirrhotic patients undergoing major hepatic resection.