Transforming growth factor‐β canceled the hepatocyte proliferation caused by transforming growth factor‐α when the two substances were mixed and administered through a disconnected central portal vein branch after creation of an Eck fistula. In contrast, transforming growth factor‐β had no antidotal action on the stimulatory effects of insulin or full test doses of insulinlike factor‐2, hepatocyte growth factor, epidermal growth factor or triiodothymanine. A minor antidotal effect on hepatic stimulatory substance activity could be detected, but only with hepatic stimulatory substance was given in doses smaller than those known to cause maximum stimulatory response. These results suggest a highly specific pharmacological and physiological interaction between transforming growth factor‐α and transforming growth factor‐α in the modulation of liver growth control. (HEPATOLOGY 1992;16:1267–1270.) Copyright © 1992 American Association for the Study of Liver Disease

Alessandro Azzarone

Antonio Francavilla

Braun

Carlo Scotti-Fogueni

Carr

Fausto

Francavilla

Goustin

Gruppuso

Guiseppe Carrieri

Houck

Kendrick Porter

Mazzaferro

Mead

Michalopoulos

Qia-Hua Zeng

Richman

Roberts

Short

Starzl

Thomas E. Starzl

Umberto Cillo

English

PubMed

Francavilla, A

Azzarone, A

Carrieri, G

Scotti-Foglieni, C

Zeng, QH

Cillo, U

Porter, K

Starzl, TE

Name not available

Effect on the canine eck fistula liver of intraportal TGF-B alone or with hepatic growth factors

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Effect on the canine Eck fistula liver of intraportal TGF-β alone or with hepatic growth factors

Scotti‐Fogueni, C

Zeng, Q‐

D-Scholarship@Pitt

l I Reprinted from HEPATOLOGY, St. Louis Vol. 16, No.5, PP. 1267-1270, November, 1992 (printed in the U.S.A.) (Copyright 0 1992 by The American Association for the Study of Liver Diseasel) Effect on the Canine Eck Fistula Liver of Intraportal qdc-~ Alone or with Hepatic Growth Factors ANTONIO FRANCAVlLLA,l,2,3 ALESSANDRO AzzARONE,3 GurSEPPE CARRIERI,3 CARLO SCOTTI-FOGLIENI,1,2 QIA-HUA ZENG, 1,2 UMBERTO CILLO, 1,2 KENDRICK PORTERl , 2 AND THOMAS E. STARZL1,2 ITransplantation Institute and ZDepartment of Surgery, University Health Center of Pittsburgh, Pittsburgh, Pennsylvania 15213; and 3Department of Gastroenterology, University of Ban, Ban, Italy Transforming growth factor-p canceled the hepa-tocyte proliferation caused. by transforming growth factor-Of when the two substances were mixed and administered through a disconnected central portal vein branch after creation of an Eck fistula. In con-trast. transforming growth factor-p had no antidotal action on the stimulatory effects of insulin or full test doses of insulinlike factor-2, hepatocyte growth factor, epidermal growth factor or triiodothymanine. A minor antidotal effect on hepatic stimulatory substance ac-tivity could be detected., but only with hepatic stimu-latory substance was given in doses smaller than those known to cause maximum stimulatory response. These results suggest a highly specific pharmacological and physiological interaction between transforming growth factor-a and transforming growth factor-p in the modulation of liver growth control. (HEPATOLOGY • q92; 16: 1267-1270.) The Eck fistula (portacaval shunt) in dogs is a useful tool in the identification and study of hepatic growth-modulating substances, of which some of the most potent have little or no effect on hepatocytes in culture (1). This operation is followed by tripling of hepatocyte proliferation, atrophy of hepatocytes within 4 days to about half their original volume and establishment of a new stable state after 4 days (2). Growth stimulating substances, when infused into the tied-off central portal vein after this operation, augment further the heightened proliferation of the Eck fistula liver and prevent liver atrophy after creation ofthe fistula (1, 3-7). These effects have been termed hepatotrophic. In con-trast, transforming growth factor-(3 (TGF-(3), a known inhibitor of hepatocyte proliferation in vitro (8, 9), has the opposite effect in that it suppresses hepatocyte proliferation after creation of the Eck fistula and further reduces the size of the atrophied hepatocytes (1). The striking TGF-(3 effect is easily overridden with in-sulin (1). Received May 4. 1992; accepted July 9. 1992. This work was aided by grant no. DK 29961 from the National Institutes of Health. Address reprint requesta to: Thomas E. Stan!. M.D .. Ph.D .• Department of Surgery, 3601 Fifth Avenue. 5C Falk Clinic. UniversIty of Pittsburgh. Pitts· burgh. PA 15213. 31/1/40940 In this study, we examined further the interaction of TGF -(3 with the five substances that were the most potently hepatotrophic in the Eck fistula model (1): insulin, insulinlike growth factor-2 (IGF -2), TGF -a, hepatocyte growth factor (HGF) and hepatic stimulatory substance (HSS). In addition, the interaction of TGF-(3 was tested with other molecules: epidermal growth factor (EGF), which is highly stimulatory in vitro (10) but not in vivo; and triiodothymanine (T 3)' which causes feeble stimulation only in the in vivo system (1,11). The results revealed a strong, although not absolute, speci-ficity of the TGF-a interactions with TGF-(3. MATERIALS AND METHODS The Experimental Model. Details of the experimental model have been described (1, 3, 4). In experiments testing a single drug, the left portal vein was used for infusion (Fig. lA) and the right portal vein was ligated. For double infusions, catheters were placed in both main portal branches and connected to separate infusion pumps (Fig. IB). The dogs were active after surgery and appeared clinically well. They were fed sugar water ad libitum on the day of surgery and a regular diet thereafter. The pathological endpoints were the same as those previ-ously described (1-7). Four days after creation of the portacaval shunt, 0.2 mCiJkg of intravenous [3Hlthymidine was given (specific activity = 80 to 90 Cilmmol; Du Pont-New England Nuclear, Boston. MA). Two hours later, while dogs were under sodium phenobarbital anesthesia, specimens were taken from the left and right lobes of the liver and fixed in 10% normal buffered formalin. At autopsy, after each dog was killed with an overdose of anesthesia, the patency of the anastomosis and the correct position of the catheter tip were confirmed. The liver tissue was processed and stained with hematoxylin and eosin. Autoradiography was carried out with Kodak NTB2 liquid emulsion (Eastman Kodak Co., Rochester, NY) with an exposure time of at least 30 days. The number of replicating hepatocytes as an index of hepatocyte regeneration was determined by counting the number of [3Hlthymidine-Iabeled nuclei per 1,000 hepatocytes. The sizes of individual hepato-cytes (index of hypertrophy or atrophy) were determined by tracing the outlines of at least 500 midzonal liver cells pro-jected onto standard-thickness paper, cutting out the indi-vidual silhouettes and weighing them. This method has been shown to be accurate in determining hepatocyte size and has been validated by planimetry and studies of unicellular or-ganisms, the sizes of which have been determined directly (12). 1267 1268 FRANCAVILLA ET AL. HEPATOLOGY A Pump B , ~ , \ BOdy wall Pump Subltance Tested '" J ~ Pump Insulin + Inhibitor FIG. 1. Eck fistula model for postoperative infusion into the (A) left portal vein and (B) the left and right portal vein. In control experiments (data not shown), we established that the vehicle used for the test substances is inert. Each animal served as its own control because the cell replication and cell sizes in the directly infused liver lobes could be compared with these parameters in the contralateral lobes that were not infused or infused with different solutions. In addition, we used as fresh controls animals not subjected to surgery and animals with Eck fistula only. Animals from the group of Eck fistula controls were killed on days 1 to 4 after surgery, enabling us to note the pace of change and the difference in changes of different liver lobes (Table 1). Growth Stimulatonl Tested. The hormones we used to stimulate growth were semisynthetic human insulin (E.R. Squibb & Sons, Inc., Princeton, NJ) and T 3 (Sigma Chemical Co., St. Louis, MO). Growth stimulating factors used were HSS produced in our laboratory (13); recombinant HGF (also called hematopoietin [14]), generously donated by Dr. George Michalopoulos of the University of Pittsburgh; IGF-2 and EGF <Collaborative Research, Inc., Bedford, MA); and TGF-a, (Peninsula Laboratories, Inc., Belmont, CAl. Growth Factor Inhibitonl Tested. TGF-13 <Collaborative Research, Inc., Bedford, MA) was infused alone (Fig. lA) or mixed with selected growth stimulating factors (Fig. 18) and infused into one of the branches of the portal vein. Statistical Analysis. Data are reported as mean ::!:: S.D. Student's one-tailed t test was used to determine the signifi-cance of differences. A p value less than 0.05 was considered significant. RESULTS Controls. The day-to-day changes in the liver after creation of Eck fistula were the same as those reported previously (1-4) with rapid atrophy that was equal in the right and left lobes. We saw a nearly threefold increase in cell mitosis (Table 1). TGF·f3lnfusion. At a TGF-13 dose of 5 ng/kglday, th normal proliferative response of 1.5 to 4.5 labelet hepatocytes/1,000 hepatocytes was cut to about 2.: labeled hepatocytes/1,000 hepatocytes in the TGF -13-infused left liver lobes; in the uninfused right lobes WE found a full proliferative effect. In addition, the hepa tocytes were significantly more atrophied in the lef1 lobes than in the uninfused right lobes (Table 1). TGF· 13 Plus Growth Stimulator.. As previously reo ported (1), a strong hepatotrophic response was evident in the lobes infused only with insulin, IGF-2, HSS, HGF or TGF-a. Modest responses to T3 and EGF were seen (Table 2). TGF·13 at a dose 10 times greater than that shown to be antihepatotrophic in the control experi-ments documented in Table 1 caused no reduction in these responses except for those induced by TGF-o. (Table 2). TGF-13 completely eliminated the TGF-a actvity. In addition, it suppressed the normally occurring prolifer-ative response characteristic of Eck fistula and aggra-vated hepatocyte atrophy (Table 2). Do8f!-respome Relatiomhip of HSS v •• that of TGF· 13. Although 50 ng/kglday HSS still had its full hepa-totrophic effect after mixing with 50 ng/kglday TGF-p, smaller HSS doses of 25 and 10 ng were partly or completely inactivated after mixing with the fixed large dose of TGF-13 (Table 3). DISCUSSION TGF -13, a 25,000-Da homodimeric peptide, is known to be a powerful in vitro inhibitor of various cell types (15, 16), including hepatocytes stimulated with EGF and TGF·a (9). During the liver regeneration that reaches its peak 24 hr after partial hepatectomy in rats, the tissue TGF.f3 level increases significantly, but not until after this peak of regeneration is reached. Thereafter the TGF -13 concentration rises four to eight times by 72 hr after hepatectomy and then recedes to the control level by 96 hr. Such findings have prompted speculation that TGF -13 modulates liver regeneration by counteracting the growth stimulation of TGF-a, EGF and other regeneration-promoting factors (17). In contrast to the classic "burst" ofliver regeneration after partial hepatectomy, the heightened cell renewal after creation of the Eck fistula builds to a new steady state within 4 days after surgery and remains constant thereafter. In this investigation it was demonstrated that 5 ng/kg/day TGF -13 prevented the increase in hepatocyte proliferation invariably seen in the Eck fistula liver. It also worsened the characteristic hepa-tocyte atrophy. In subsequent experiments, 50 ng/kglday TGF-13 was infused to simulate the high concentrations of endog-enous TGF-p seen during regeneration after hepa-Vol. 16, No.5, 1992 No. of nays after Group dollS PCS 1 2 No surgery 2 3 1 3 3 2 4 3 3 5 4 4 6 3 4 7 2 4 PCS = portacaval shunt. ECK FISTULA AND TGF-13 TABLE 1. Hepatocyte size and autoradiographic labeling ~qdc-fi dOlle (ng/kgldayl 5 50 No. of labeled hepatocytes per 1,000 hepatocytell" Left lobe 1.55 ± O.lS 1.43 ± 0.43 6.33 ± MKU~ 5.03 ± 0.27b 4.4 ± 0.3b 2.4 ± 0.5c• d 2.1 ± 0.05e• d Right lobe 1.55 ± 0.08 1.36 ± 0.4 6.26 ± 0.64b 4.96 ± 0.3b 4.2 ± 0.3b 4.6 ± O.S 4.9 ± 0.7 1269 Cell size unite" Left lobe 0.161 ± 0.002 0.192 ± 0.02 0.134 ± 0.008b 0.128 ± O.oogb 0.095 ± O.Olb 0.064 ± 0.02'" d 0.071 ± 0.006e• d Right lobe 0.160 ± 0.003 0.192 ± 0.023 0.134 ± O.oogb 0.130 ± O.OOSb 0.096 ± 0.015b 0.103 ± 0.01 0.106 ± 0.004 Group 1: dogs that did not undergo surgery; groups 2 to 5: dogs 1 to 4 days after PCS; groups 6 and 7: dogs with PCS and 4-day continuous infusion of TGF·13 into the left branch of the portal vein. aData expressed as mean ± S.D. bp < 0.001 vs. groups 1 and 2. cp < 0.01 VB. own right lobe. d p < 0.05 vs. left lobe of group 5. TABLE 2. Hepatocyte size and autoradiographic labeling after continuous infusion of growth stimulating factor into the right portal vein branch and infusion of growth stimulating factor plus TGF-ji (50 nglkg/day) into the left portal infusion of branch No. of labeled hepatocytes per 1,000 hepatocytell" Cell size units'" No. of Stimulating DOlle Right lobe Right lobe Group subjects substances (ng/kgldayl Left lobe (no inhibitor) Left lobe (no inhibitor) 1 3 Insulin 0.16 15.0 ± O.S 14.9 ± 0.4 0.lS7 ± 0.018 0.165 ± 0.01 2 1 IGF-2 50 11.0 10.3 0.148 0.1477 3 2 HSS 50 13.8 ± 0.2 13.3 ± 0.15 0.142 ± 0.006 0.143 ± 0.004 4 1 HGF 50 11.3 11.2 0.164 0.163 5 2 Ts 100 7.4 ± 0.65 7.3 ± O.S 0.126 ± 0.016 0.127 ± 0.013 6 2 EGF 150 5.15 ± 0.2 5.05 ± 0.05 0.119 ± 0.001 0.120 ± 0.001 7 2 TGF-a 50 3.4 ± 0.25b 12.6 ± 0.5 0.OS2 ± 0.006b 0.159 ± 0.002 aData expressed as mean ± S.D. bp < 0.001. TABLE 3. Hepatocyte size and autoradiographic labeling after continuous infusion of HSS into the right portal vein branch and HSS plus TGF·ji (50 ng/kg/day) into the left portal vein branch No. of labeled hepatocytell per 1,000 hepatocytell" Cell size units No. of KSS Group subjectll (ng/kglday) Left lobe 1 10 2.3 2 1 25 5.8 3 2 50 13.0 ± 0.2 aData expressed as mean ± S.D. tecto my . These high concentrations are considered the result of the development of resistance to qdc-~ (18). In the 4 days after creation of the Eck fistula, the larger dose ofqdc-~ (50 ng/kglday) abrogated the stimulation usually caused by TGF-o.. Because the reactivity to TGF-a alone was fully retained in the contralateral lobes, it appeared that qdc-~ was inactivated almost completely with one pass through the liver. Under the same conditions of testing, all of the other potent hepatotrophic factors that were examined (insulin, Right lobe (no inhibitor) 7.5 9.2 13.3 ± 0.15 Left lobe 0.093 0.104 0.142 ± 0.006 Right lobe (no inhibitor) 0.141 0.153 0.143 ± 0.004 IGF-2, HGF, HSS, T 3 and EGF) were unaffected by mixing and coadministration with qdc-~K These data indicate that growth-stimulating factors of hepatocytes can be divided into two groups: those resistant to modulation by TGF -~ and those whose proliferation-inducing qualities are dramatically re-duced or even canceled by qdc-~K However, it should also be emphasized that the foregoing classification of hepatic stimulatory factors according to their reaction to coadministration with qdc-~ depends in part on dose 1270 FRANCAVILLA ET AL. HEPATOLOGY selection. This was demonstrated when HSS (which is usually unaffected by qdc-~F was given in reduced quantities (lO or 25 ng/kg/day) that by themselves evoked minimum proliferative responses. Under these circumstances, the minor HSS-induced response could be eliminated with qdc-~K Further discussion of these findings does not seem warranted. However, we do emphasize the seemingly high degree of specificity of the qdc-~/qdc-a inter-action. Even EGF, which ostensibly has the same binding sites as TGF -a (19), was not affected by TGF -13. This pharmacological specificity is consonant with a critical role of the balance of these growth factors in the control of cell proliferation, as has been proposed by others (20, 21). Acknowledgment: We thank John Prelich for lending us his experience and technical assistance. REFERENCES 1. Francavilla A, Starzl TE, Porter K, Scotti-Foglieni C, Micha-lopoulos GK, Carrieri G, Trejo J, et al. Screening for candidate hepatic growth factors by selective portal infusion after canine Eck fistula. HEPATOLOGY 1991;14:665-670. 2. Starzl TE, Porter KA, Francavilla A. The Eck fistula in animals and humans. Curr Probl Surg 1983;20:688-767. 3. Starzl TE, Watanabe K, Porter KA, Putnam CWo Effects of insulin, glucagon, and insulin/glucagon infusions on liver mor-phology and cell division after complete portacaval shunt in dogs. Lancet 1976;1:821-825. 4. Starzl TE, Jones AF, Terblanche J, Usui S, Porter KA, Mazzoni G. Growth-stimulating factor in regenerating canine liver. Lancet 1979;1:127-130. 5. Mazzaferro V, Porter KA, Scotti-Foglieni CL, Venkataramanan R, Makowka L, Rossaro L, Francavilla A, et al. The hepatotrophic influence of cyclosporine. Surgery 1990;107:533-539. 6. Starzl TE, Porter KA, Mazzaferro V, Todo S, Fung J, Francavilla A. Hepatotrophic effects of FK 506 in dogs. Transplantation 1991;51:67-70. 7. Starzl TE, Schrieber SL, Albers MW, Porter KA, Foglieni CS, Francavilla A. Hepatotrophic properties in dogs of human FKBP, the binding protein for FK 506 and rapamycin. Transplantation 1991;52:751-753. 8. Braun L, Mead JE, Panzica M, Mikumo R, Bell 01, Fausto N. Transforming growth factor B mRNA increases during liver regeneration: a possible paracrine mechanism of growth regu-lation. Cell Bioi 1988;85:1539-1543. 9. Carr BI, Hayashi I, Branum EL, Moses HL. Inhibition of DNA synthesis in rat hepatocytes by platelet-derived type B trans-forming growth factor. Cancer Res 1986;46:2330-2334. 10. Richman RA, Claus TH, Pilkis SJ, Friedman DL. Hormonal stimulation of DNA synthesis in primary cultures of adult hepatocyte. Proc Nat! Acad Sci USA 1976;73:3589-3593. 11. Short JA, Brown RF, Husakova A, Gilbertson J, Zemel R, Lieberman I. Induction of deoxyribonucleic acid synthesis in the liver of the intact animal. J BioI Chern 1972;247: 1757-1766. 12. Starzl TE, Francavilla A, Halgrimson CG, Francavilla FR, Porter KA, Brown TH, Putnam CWo The origin, hormonal nature, and action of hepatotrophic substances in portal venous blood. Surg Gynecol Obstet 1973;137:179-199. 13. Francavilla A, Ove P, Polimeno L, Coetzee M, Makowka L, Rose J, Van Thiel DH, et ai. Extraction and partial purification of hepatic stimulatory substance in rats, mice and dogs. Cancer Res 1987;47:5600-5605. 14. Michaiopoulos B, Houck KA, Dolan ML, Lvetteke NC. Control of hepatocyte replication by two serum factors. Cancer Res 1984;44: 4414-4419. 15. Roberts AB, Sporn MH. Transforming growth factors. Cancer Surv 1985;4:683-705. 16. Goustin AS, Leof EB, Shipley GD, Moses HL. Growth factors and cancer. Cancer Res 1986;46:1015-1029. 17. Mead JE, Fausto N. Transforming growth factor alpha may be a physiological regulator of liver regeneration by means of an endocrine mechanism. Proc Natl Acad Sci USA 1989;86:1558-1562. 18. Houck KA, Michalopoulos GK. Altered responses of regenerating hepatocytes to norepinephrine and transforming growth factor type beta. J Cell PhysioI1989;141:503-509. 19. Gruppuso PA, Mead E, Fausto N. Transforming growth factor receptors in liver regeneration following partial hepatectomy in the rat. Cancer Res 1990;50:464-469. 20. Fausto N. Hepatology: a textbook of liver disease. 2nd ed. Philadelphia: W.B. Saunders Co., 1990:49-64. 21. Michalopoulos GK. Liver regeneration: molecular mechanisms of growth control. FASEB J 1990;4:176-187. 

Effect on the canine Eck fistula liver of intraportal TGF‐β alone or with hepatic growth factors

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Effect on the canine Eck fistula liver of intraportal TGF‐β alone or with hepatic growth factors

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