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The hepatic stimulatory substance (HSS) extracted from weanling rat livers was purified 381,000-fold using chromatographic techniques including nondissociating polyacrylamide gel electrophoresis (nondenaturing PAGE). The activity of this highly purified HSS, named Acr-F4, was assessed in two in vivo models. In 40% hepatectomized rats, it produced a fivefold increase in the proliferative rate normally seen following this partial hepatectomy. In Eck fistula dogs, the level of base increase in hepatocyte renewal was amplified threefold by an infusion of Acr-F4 (50 ng/kg/day). Acr-F4 had no influence on the regenerative response of the kidney following a unilateral nephrectomy or of the bowel following a 40% resection of the small bowel. On the basis of these findings, it can be concluded that HSS (Acr-F4) has a high biological activity and is organ specific.
That liver regeneration could be augmented by extracts prepared from livers in an active proliferative state has been demonstrated by several investigators (1–4). However, all attempts focused on purification and characterization of the putative growth factor in these extracts have produced few and generally inconclusive results. A partially purified factor, hepatic stimulatory substance (HSS), capable of stimulating in vivo hepatocyte proliferation (5, 6) has been isolated from weanling rat liver homogenates.
In this paper, data concerning further purification and characterization of HSS is reported, made possible by the use of new laboratory techniques and the introduction of a very sensitive and reliable animal model for assessing HSS activity in vivo. Specifically, nondissociating polyacrylamide gel electrophoresis allowed us to obtain a 381,000-fold purification of HSS, eliminating almost all contaminating proteins in the preparation. In addition, the dog with a portacaval shunt coupled with the availability of specific antibodies against HSS made it possible to achieve this level of purification.
Type-V neuraminidase, trypsin, aprotinin, and proteins used as molecular weight markers were purchased from Sigma Chemical Company, St. Louis. Missouri, [methyl-3H]Thymidine (50–80 Ci/mmol) was obtained from New England Nuclear, Boston, Massachusetts. l-l-Tosylamido-2-phenylethyl chlorome-thyltrypsin ketone was purchased from Worthington Biochemical Corporation, Boston, Massachusetts. Amicon ultrafiltration membrane filters were purchased from Amicon Corporation, Danvers, Massachusetts. The chemicals required for electrophoresis were purchased from Bio-Rad Laboratories, Richmond, California.
Adult male Fischer (F344) rats (180–200 g), weanling male rats (60–90 g), and male mongrel dogs (15–20 kg) were purchased from Hilltop Lab Animals, Scottsdale, Pennsylvania. They were maintained in temperature- and light- (6 am to 6 pm) controlled rooms until used. They were given food and water ad libitum.
Adult rats underwent either a 40% hepatectomy or a sham operation consisting of a laparotomy and manual manipulation of the liver between 7:30 and 9:30 am using the method of Higgins and Anderson (7). Unilateral nephrectomy and 40% resections of the small bowel were performed as described previously (8).
In dogs, large side-to-side portacaval shunts were constructed with an excision of an ellipse of tissue from both the portal vein and the inferior vena cava and anastomosing the two vessels side to side (Figure 1). The shunts were made completely diverting by individually ligating the main right and left portal trunks distal to the anastomosis of the portal vein to the IVC (9). The tip of a small infusion catheter was placed into the ligated left portal branch within the liver and led through the body wall and via a long subcutaneous tunnel to a small calibrated finger pump that was placed into a dog jacket (Figure 1).
The steps for HSS preparation and purification are summarized in Table 1. These methods (6) yielded an active fraction that has been identified as F150 because it elutes from the column with a 150 mM NaCl gradient.
An aliquot of 0.6 mg lyophilized fraction F150 resuspended in Tris buffer 0.025 M, pH 8.3, underwent electrophoresis using nondissociating PAGE (10, 11) on 8% acrylamide. With this technique, F150 generates several distinct bands, and the gel can be divided in four zones from which its proteins can be eluted. The eluates, acrylamide fractions 1–4 (Acr F1–F4) are dialyzed against 150 mM ammonium acetate, lyophilized, and stored at −70° C until being tested further.
In rats, 6 hr after a 40% partial hepatectomy, control rats were given intraperitoneal injections of 2 ml of 5 mM phosphate buffer, pH 7.4, whereas HSS-treated rats received either F150 or an acrylamide fraction dissolved in 2 ml phosphate buffer, 5 mM, pH 7.4, at the protein concentrations indicated in the tables. Seventeen hours later, 50 µCi [3H]thymidine were injected intraperitoneally, and the animals were sacrificed 1 hr later. Six hours after surgery, the rats that had received a unilateral nephrectomy or 40% resection of the small bowel were treated as described above for the 40% hepatectomized rats.
[3H]Thymidine incorporation and mitotic index determinations were made as described previously (9, 12). An augmentation of all parameters, beyond the modest response that is present after sham surgery, was considered to be indicative of biologic activity of the liver extracts.
In dogs, the active electrophoretic fraction, Acr-F4 was infused in the left hepatic lobes through the left portal branch as described in Table 2. At the time of sacrifice, liver tissue was obtained from the left and right hepatic lobes and shunt patency and catheter position were verified. The labeling index was determined as described earlier (9).
DNA synthesis in kidney and small intestine was determined as described previously (8).
SDS-polyacrylamide gradient slab gel, using 7.5–20% gel with a 5% stacking gel, was prepared and developed according to the method of Laemmli (15). Both F150 and Acr-F4 undergo electrophoresis under these conditions. Protein bands were visualized using Coomassie blue R250 according to the method of Weber and Osborn (16).
Murine monoclonal antibodies against Acr-F4 were raised using PHC 43 and PHC 67 cells (17). The cells were cultured in serum-free medium and the monoclonal Abs were separated by protein-A chromatography. Activity was assessed by ELISA and found to be in the IgG fraction.
The unpaired Student's t test was used for the statistical analysis of all data.
In earlier investigations (6), the greatest degree of purification of HSS was obtained using fast protein liquid chromatography. By this technique, F150 (Figure 2), which was the most active fraction in 40% hepatectomized rats, was prepared. The use of nondissociating PAGE made it possible to further purify F150 (Figure 3).
The activity of F150 and its PAGE fractions were compared using 40% hepatectomized rats (Figure 4). The administration of 150 (3 µg/rat) produced results as previously reported (6). When fractions of Acr F1–F4 were tested, the only fraction with stimulatory activity similar to that of F150 was found in Acr-F4.
The results shown in Figure 5 demonstrate a dose–effect relation between the amount of Acr-F4 injected and the resultant increase in hepatocyte DNA synthesis and the number of mitoses enumerated. In addition, with the highest dose of Acr-F4 (0.6 µg/100 g body wt), the activity achieved was fivefold greater than the background response in control animals.
To evaluate the organ specificity of Acr-F4, rats with either a unilateral nephrectomy or a 40% resection of the small bowel were tested. No increase in DNA synthesis was found in the contralateral residual kidney or in the remaining small intestine (Figure 6).
The experiments performed in dogs demonstrate that when Acr-F4 was administered as a continuous infusion beginning 6 hr after portacaval shunt in the left portal vein, the mitotic rate tripled in the left liver lobe while no effect was seen in the right side of the liver. This effect was completely eliminated with the addition of anti-Acr-F4 monoclonal antibody to the infusion fluid (Table 3). The monoclonal antibody vehicle was inert when tested alone.
Table 4 summarizes the physicochemical characteristics of Acr-F4. It contains one major protein band with a molecular weight of about 14,000 (Figure 7). Experiments conducted in 40% hepatectomized rats demonstrated that Acr-F4 is heat-resistant and is not digested by neuroaminidase, whereas it is sensitive to proteolitic enzymes (data not shown).
The idea of a specific intrinsic liver growth factor was conceived almost 40 years ago when Teir and Ravanti (20) and Blomqvist (21) first reported a growth stimulatory activity in crude mesh extracts of weanling and regenerating rat liver but not in extracts from normal adult rat liver. Since then, a large number of studies (1–6) have suggested that regenerating liver is a source of a growth stimulator which is specific for the liver (Table 5).
Data reported previously (6) and the new data in this paper regarding Acr-F4 are summarized in Table 6. A 381,000-fold increase in activity over the original material was achieved using the 40% hepatectomized rat model as the test system. The activity present in this fraction (Acr-F4) is not species-specific, as demonstrated by the results obtained in dogs as well as rats and produced a dose–response that was specific for the liver (Figure 6).
The HSS found in weanling rat liver also has a powerful regenerating or growth effect on dog liver as assessed by the Eck fistula model. The degree of stimulation achieved with 50 ng/kg/day was as potent as gram quantities of crude cytosol (3, 4) and was as pronounced as the most potent well-recognized hepatotrophic substance currently available: insulin (9). In common with insulin (9) and crude cytosol (4), purified HSS affects only the directly infused liver tissue with little spillover to the uninfused liver. This suggests that it is largely degraded or consumed within a single pass through the liver, leaving little or none available to effect the contralateral hepatic lobes.
In an earlier report (6), it was shown that HSS prepared under these conditions loses its in vitro activity while retaining its in vivo activity. Thus it has not been possible to compare the HSS purified by LaBrecque et al (22) and Fleig and Hoss (23), which remained active in vitro with Acr-F4. The explanation for the disparities between in vivo and in vitro growth stimulation seen with Acr-F4 and these other fractions will not be resolvable until these substances are known.
In conclusion, the retention of in vivo activity of a highly purified HSS fraction, the ability to abolish the stimulatory activity of this fraction with specific monoclonal antibodies, and the organ specificity of Acr-F4 suggests that its complete identification should be close at hand.
Supported by research grants from the Veterans Administration and Project Grant DK 29961 from the National Institutes of Health, Bethesda, Maryland, and grant 87/01291-44 from Consiglio Nazionale delle Ricerche, Italy.
Presented at the Proceedings of the International Meeting on Normal and Neoplastic Growth in Hepatology, Bari, Italy, June 1989.