Azzarone, A

Carrieri, G

Cillo, U

Fagiuoli, S

Francavilla, A

Gasbarrini, A

Scotti-Foglieni, C

Starzl, TE

Zeng, QH

PubMed

Azzarone A

Francavilla A

Gasbarrini A

Scotti Foglieni C

Fagiuoli S

Cillo U

Zeng QH

Starzl TE

CARRIERI, GIUSEPPE

Archivio Istituzionale della Ricerca- Università degli Studi di Foggia

Effects on in vivo and in vitro hepatocyte proliferation of methylprednisolone, azathioprine, mycophenolic acid, mizoribine, and prostaglandin E1.

D-Scholarship@Pitt

\4S9 Effects on In Vivo and In Vitro Hepatocyte Proliferation of Methylprednisolone, Azathioprine, Mycophenolic Acid, Mizoribine, and Prostaglandin E1 A. Azzarone, A. Francavilla, G. Carrieri, A. Gasbarrini, C. Scotti-Foglieni, S. Fagiuoli, U. Cillo, a.H. Zeng, and T.E. Starzl I N both rats and dogs, the immunosuppressive drugs cyclosporine (CyA) and FK 506 augment liver regen-eration and possess other hepatotrophic qualities. J-4 In contrast, rapamycin (RPM), a powerful immunosuppres-sant that is chemically related to FK 506 but targeted to a different stage of T-cell activation,5,6 was recently shown to have antiproliferative properties, including inhibition of regeneration of the livers, as well as of the intestine and kidney.7,8 However, there is a paucity of information about the influence of other immunosuppressive drugs on growth and regeneration, Using in vivo (partial hepatec-tomy and portacaval shunt) and in vitro (tissue culture) experimental models described elsewhere,2-4,8-10 we have investigated the effect on hepatocyte proliferation of meth-ylprednisolone (MP), mycophenolic acid (MPA), mizorib-ine (MZ), azathioprine (AZA), and prostaglandin EJ (POE J ), MATERIALS AND METHODS In Vitro Study Hepatocytes in Primary Culture. The livers were removed from 7-week-old male rats (Fischer 344 purchased from Hilltop Lab Animals Inc, Scottdale, Pal weighing 180 to 200 g. Hepato-cytes were isolated by a modification of the in situ two-step collagenase perfusion technique of Seglen and Jirtle et al. 11.12 The hepatocytes were dispersed and washed twice with cold Ca2+ free perfusion buffer and resuspended in basal medium (MEM) sup-plemented with pyruvate (1 mmollL), proline (0,26 mmollL), insulin (10-7 mollLJ and 5% fetal calf serum. Viability was determined by Trypan blue exclusion, and only preparations having >90% viability at the outset were used. Cell number was determined with a hemocytometer. The cells were plated at a cell density of 6,5 x 104 per well in Coming 35 mm tissue culture dishes (Coming, NY) containing 1,5 mL medium and maintained at 37°C in a 5% CO2 atmosphere. After a 3-hour attachment period, the medium was aspirated and 1.5 mL MEM containing epidermal growth factor (EOF) and insulin at concentrations of 10 ng/mL and 10-7 mollL, respectively, were added. The substances were dissolved in DMSO (MPA), saline (MZ), or ethanol (POE I , MP, AZA) and added in the appropriate concentrations. The amount of alcohol or DMSO added to the medium was never more than 2 JLL/mL, which does not affect hepatocyte proliferation. In Vitro f H] Thymidine Incorporation.· To determine in vitro DNA synthesis, 3 jLCWHlthymidine (Dupont New England Nu-clear Research Products, Boston, Mass) was added to each well and maintained for 24-48 hours of the culture period. When the cells were harvested, DNA content was determined by the mi-crofluorometric method of Setara and Morley,13 and DNA syn-thesis was measured by the method of Michelopoulos et al. 14 Table 1, Regimens Dose Used Group Drugs (mglkg/dose) Route Vehicle 1 (n = 5) 1M Saline 2 (n = 10) MP 1M Saline 3 (n = 5) 1M Saline 4 (n = 10) MZ 20 1M Saline 5 (n = 5) PO 0.5% CMC; 0.4% Tween 0.9 alcohol in saline 6(n = 10) MPA 15 PO 0.5% CMC; 0.4% Tween 0.9% alcohol in saline 7 (n = 5) 1M 1 % CMC in saline 8(n = 10) AZA 6 1M 1 % CMC in saline 9 (n = 5) 1M 10% ethanol in saline 10 (n = 10) PGE, 0.2 1M 10% ethanol in saline MP and MZ were dissolved in saline; MPA was dissolved in 0.5% carbossil-methil-cellulose (CMC), 0.4% Tween, and 0.9% alcohol in saline; AZA was mixed wnh olive oil or dissolved w~h 1% CMC in saline; PGE, was dissolved in alcohol and diluted 10 times with saline. In Vivo Study 70% Partial Hepatectomy: Rat Model. Rats similar to those used in the in vitro experiments were assigned to groups and treated for 4 days as controls or with drugs (Table I). On the 4th day, between 0900 and 1030 hours, the rats received a standard 70% hepatectomy under light ether anesthesia. Food and drink were allowed immediately. Parenteral fluid and electrolyte sup-port were not required. Twenty-four hours after the hepatectomies, 185 x 10-4 Bq [3Hl thymidine was administered to all rats by intraperitoneal injection. The rats were killed 2 hours later by guillotine. Extraction and purification of hepatic DNA were accomplished by the method of Ove et al l5 and DNA content was measured with calf thymus DNA (Sigma, St Louis, Mo) as the standard. 16 Specimens from each liver were prepared for histological examination with hema-toxylin-eosin and the proportion of labeled hepatocytes was counted. From the Department of Gastroenterology, University of Bari, Bari, Italy (A.A., A.F.), and the Department of Surgery, University Health Center of Pittsburgh, Pittsburgh, Pennsylvania (A.A., A.F., G.C., A.G., C.S-F., S.F., U.C., Q.H.Z., T.E.S.). Supported by Research grants from the Veterans Administration and Project Grants OK 29961 and CA 35373 from the National Institutes of Health, Bethesda, Maryland. Address reprint requests to A. Francavilla, MD, 3601 Fifth Ave, 5C Falk Clinic, Pittsburgh, PA 15213. © 1992 by Appleton & Lange 0041-1345/92/$3.001 + 0 2868 Transplantation Proceedings, Vol 24, No 6 (December), 1992: pp 2868-2871 HEPATOCYTE PROLIFERATION c( z o CI ! b )( a. E CJ III 1\1 i 40 30 20 10 Methylprednisolone Ilg/ml . . • 11-1.--11 -. .J 11----- .. ---...... ,/iNo Insulin III III ! l~--D---~--r_-~r_D~-I-illD--q--~ a. )( UJ c .Q ~ o e-o CJ c CD 40 30 .S 20 'C E >. .r: ~ 10 J: ~ o 0.6 1.2 2.4 5 10 . . . II--kll--l- : ff~ o ....L..--,---T---r_---, __ II----,--II--"T----u-.., o 3 6 12 25 50 100 Mizoribine ng/ml 2869 Prostaglandin E1 Ilg/ml + EGF 10ng/ml L.._.........t ..... ---t-------....... -II---II---... ,/iII --1--11 ----Ko~ ·EGF ~---I-----~----r_---r_--ft-~r_-fft--rf---KKKKKI o 1.25 2.5 5 10 15 u-i : ~--K---~--~--~:~I~==~:~ o 0.6 1.2 2.4 5 10 Mycophenolic Acid Ilg/ml Fig 1. Effect of different doses of MP, PGE" MZ, and MPA on DNA synthesis in hepatocy1es cultured in the presence of EGF (10 ng/mL). In some experiments, MP was tested in the absence of insulin, and PGE, in the absence of EGF. N = 9 for all data points, which represent three experiments with triplicate determinations. Data expressed as mean ± SO. *P < .05; **P < .01. Portacaval Shunt: Dog Model. Conditioned female beagle dogs underwent a functional end-to-side portacaval shunt (PCS) as previously described. 4•9 . 'o POE, was dissolved daily in vehicle solution (5 mmollL ammonium acetate. 5 mg/L bovine serum albumin in saline) and infused for 4 days into the left branch of the portal vein. At day 4, 0.2 mCilkg of intravenous (IV) [3Hl thymidine was given with a specific activity of 80-90 Cilmmol. Two hours later, while the dogs were under sodium pentobarbital anesthesia, specimens were taken from left and right lobes of the liver and fixed in 10% normal buffered formalin. The dogs were killed with an IV bolus of potassium chloride. Hepatocyte size and organelle structure were quantitated, and proliferation was estimated by nuclear thymidine incorporation (classical autoradiography). These parameters were compared in the left (treated) vs the right (untreated) lobes. Statistical Analysis. Data were reported as mean ± SD. Stu-dent's one-tailed t test was used to determine the significance of differences. A P value <.05 was considered significant. RESULTS In Vitro Hepatocy1e Viability MP A and MZ that were added after a 3-hour attachment period did not alter the DNA or AL T concentration in the medium after 48 hours of incubation (data not shown). In contrast, AZA killed the hepatocytes, making continuation of the experiment impossible. Figure 1 shows the effect in vitro on hepatocyte prolif-eration maintained for 48 hours in the presence of MP, MPA, MZ, and POE •. MZ and MPA inhibited DNA synthesis, whereas MP caused a near doubling of thymine incorporation. POE. feebly stimulated hepatocyte prolif-eration at the higher concentration. This effect was over-lapped by EOF stimulation. In Vivo Results Table 2 reports the results obtained in the experiment in vivo using the 70% partial hepatectomy rat model. After partial hepatectomy, the different vehicle solu-tions showed considerable variability in liver regeneration. It suggested the need for concurrent controls for each test group. The only drug that significantly augmented regeneration relative to the controls was MP. The only drug that significantly inhibited regeneration was MZ (Table 2). The MZ inhibition was profound, reducing DNA synthesis and 2870 AZZARONE, FRANCAVILLA, CARRIERI, ET AL Table 2. Effects of MP, MZ, MPA, AZA, and PGE, on Liver Regeneration in 70% Partial Hepatectomy Rat Model Group 1 (n = 5) 2 (n = 10) 3 (n = 5) 4 (n = 10) 5 (n = 5) 6 (n = 10) 7 (n = 5) 8 (n = 10) 9 (n = 5) 10 (n = 10) Substances Vehicle MP Vehicle MZ Vehicle MPA Vehicle AZA Vehicle PGE, • P < .05 vs their own control group. Dose Used (mglkg/dose) 20 15 6 0.2 the labeled nuclei rate. MPA, AZA, and PGE I had no effect on regeneration in the whole animal (Table 2). In contrast, PGE I was found profoundly active when infused continuously for 4 days into the left branch of the portal vein in the pes dog model (Table 3). CONSIDERATIONS AZA, MPA, and MZ are cytotoxic agents. They act by selectively inhibiting the synthesis of purine nucleotides (adenine for AZA and guanine for MPA and MZ), thereby reducing DNA synthesis of a variety of immunologic and other specialized cells, including hepatocytes. In accord with this, our in vitro results show both an inhibitory effect of MP A and MZ and a toxic one of AZA. In in vivo experiments, only MZ confirmed the inhibition found in vitro, whereas AZA and MPA at the doses used did not affect liver regeneration in rats after 70% partial hepatectomy. The inhibition of hepatocyte proliferation by cytotoxic drugs is consistent with previous reports about AZA.17 A seemingly obvious explanation could be that AZA, MP A, and MZ selectively inhibit synthesis of purine nucleotides, which are required for DNA synthesis. MP augmented liver regeneration in intact animals, and caused a striking increase in hepatocyte proliferation in culture. The absence of insulin in the medium drastically reduces MP stimulation to a level that is no longer signif-icant (Figure 1). MP is known to inhibit the synthesis and expression of DNA Percent of Route (cpm/mg x 10-3 ) Labeled Nuclei 1M 91 ± 8 18 ± 3 1M 153±11· 39 ± 4-1M 96 ± 9 18 ± 2 1M 17 ± 4· 6 ± 2-PO 66 ± 5 17 ± 3 PO 63 ± 6 16 ± 4 1M 59 ± 4 16 ± 3 1M 56 ± 5 15 ± 3 1M 88 ± 7 17 ± 2 1M 96 ± 10 18 ± 3 mUltiple cytokines, including IL-l, IL-2, and migration inhibitor factor. ls •l ,) Although IL-J and IL-2 are thought from reported in vitro experiments to be growth suppres-sors,20 this could not be demonstrated by our laboratory-sensitive in vivo test system, in which the recombinant cytokines in question were infused directly into the tied off portal vein of the Eck fistula liver. 21 Thus, the proliferative response to MP reported herein both in vivo and in vitro cannot be explained with what is currently known about steroid actions. PGE I has been successfully used in the therapy of posttransplant patients, as well as in the therapy of fulmi-nant hepatic failure (FHF).22-24 The administration of this drug after transplantation may reduce immunorejec-tion22 ,25 and drastically reverses primary graft nonfunction after orthotopic liver transplantation. 23 We demonstrated that PGE1 has hepatotrophic quali-ties, as is well known for other PGS.26-28 It stimulated hepatocyte proliferation in both in vitro and in vivo mod-els. However, a continuous and topic infusion of PGE1 seems necessary to stimulate liver regeneration. In fact, we were not able to obtain any proliferation in rats treated with only one daily if\iection for 4 days. Instead, when the drug was injected continuously in one lobe of the liver, we noted a stimulation just of the infused lobe. It could mean that after the PGE I passes through the liver it is much too diluted to stimulate the noninfused lobe or that it is promptly degradated as soon as it arrives at the lung.29 It is not possible to explain the growth effects of PGE1 by the well-known properties of this drug. Recently, a Table 3. Hepatocyte Size and Autoradiographic Labeling After Continuous Infusion of Different Doses of PGE, Into the Left Portal Vein Branch of Dogs With Eck's Fistula No. of Labeled Hepatocytes per 1,000 Dose Hepatocytes Cell Size (U) Group N (l'9/d) Left lobe Right lobe Left lobe Right lobe 1 2 4.8 12.5 ± 0.5· 5.1 ± 0.7 0.158 ± 0.01· 0.087 ± 0.007 2 2 0.48 10 ± 0.3- 3.9 ± 0.3 0.161 ± 0.005· 0.100 ± 0.005 3 2 0.24 6 ± 0.2· 3.8 ± 0.1 0.131 ± 0.004· 0.095 ± 0.009 4 2 0.048 5.1 ± 0.3 4.6 ± 0.4 0.104 ± 0.002 0.099 ± 0.004 • P < .05. left lobe vs right lobe. HEPATOCYTE PROLIFERATION linkage has been proposed between TGFa and PG.28 It seems that TGFa may induce hepatocyte proliferation in vitro by regUlating the metabolism of arachidonic acid and the formation of prostaglandins. However, it is unlikely that the beneficial effect of PGE1 in the therapy of FHF is due to its growth qualities; in fact, as we described elsewhere30 it is not possible to reverse FHF by adminis-tration of liver growth factors. A more likely explanation for the therapeutic action of PGE1 could be the protection of the endothelial cells' integrity and an improvement of liver blood flOW?t.32 REFERENCES I. Makowka L, Svanas G, Esquivel CO, et al: Surg Forum 37:353, 1986 2. Francavilla A, Barone M, Todo S, et al: Lancet 2: 1248, 1989 3. Francavilla A, Starzl TE, Barone M, et al: Hepatology 14:140, 1991 4. Starzl TE, Porter KA, Mazzaferro V, et al: Transplantation 51:67, 1991 5. Dumont FJ, Staruch MK, Koprak SL, et al: J Immunol 144:251, 1990 6. Bierer BE, Mattila PS, Standaert RF, et al: Proc Natl Acad Sci USA 87:9231,1990 7. Francavilla A, Starzl TE, Scotti C, et al: Transplantation 53:496, 1992 8. Francavilla A, Carr B, Starzl TE, et al: Hepatology 15:871, 1992 9. Starzl TE, Porter KA, Putnam CW: Lancet 2: 1241, 1975 10. Starzl TE, Watanabe K, Porter KA, et al: Lancet 1:821, 1976 II. Seglen PO: Methods Cell Bioi 13:29, 1976 2871 12. Jirtle RL, Michalopoulos G, McLaine JR, et al: Cancer Res 41:3512, 1981 13. Setaro F, Morley CGD: Anal Biochem 71:313,1976 14. Michalopoulos G, Cianciulli HD, Novotny R, et al: Cancer Res 42:4673, 1982 15. Ove P, Francavilla A, Coetzee M, et al: Liver and Hor-mones. New York: Raven, 1987, p 265 16. Burton K: Methods Enzymol 12: 163, 1968 17. Gonzalez EM, Krejczy K, Malt R: Surgery 68:254, 1970 18. Kern JA, Lamb RJ, Reed JC, et al: J Clin Invest 81:237, 1988 19. Horst HJ, Flad HD: Exp ImmunoI68:156, 1987 20. Nakamura T, Arakaki R, Ichihara A: Exp Cell Res 179:488, 1988 21. Francavilla A, Starzl TE, Porter K, et al: Hepatology 14:665, 1991 22. Mozes MF, Moran M, Ketel B, et al: Kidney Int 35:520, 1989 23. Greig PD, Woolf GM, Abecassis M, et al: Transplant Proc 21:2385, 1989 24. Sinclair S, Levy G: Ital J Gastro 22:205, 1990 25. Pollak R, Dumble LJ, Wiederkehr JC, et al: Transplanta-tion 50:834, 1990 26. Miura Y, Fukui N: Cell Mol Bioi 25:179, 1979 27. Andreis PG, Whitfield JF, Armato U: Exp Cell Res 134: 265, 1981 28. Skouteris GG, McMenamin M: Biochem J 281:729, 1992 29. Anggard E, Samuels son B: J BioI Chern 239:4097, 1964 30. Francavilla A, Azzarone A, Carrieri G, et al: Hepatology (submitted for publication) 31. Levy GA, Abecassis M: Rev Infect Dis 11:712, 1989 32. Richardson PD, Withrington PG: Br J Pharmacol 48:337, 1978 

Effects on in vivo and in vitro hepatocyte proliferation of methylprednisolone, azathioprine, mycophenolic acid, mizoribine, and prostaglandin E<inf>1</inf>

Name not available

Effects on in vivo and in vitro hepatocyte proliferation of methylprednisolone, azathioprine, mycophenolic acid, mizoribine and prostaglandin E1

AZZARONE A

FRANCAVILLA A

CARRIERI G

GASBARRINI A

SCOTTI FOGLIENI C

FAGIUOLI S

ZENG QH

STARZL TE

CILLO, UMBERTO

Archivio istituzionale della ricerca - Università di Padova

Effects on in vivo and in vitro hepatocyte proliferation of methylprednisolone, azathioprine, mycophenolic acid, mizoribine, and prostaglandin E1

http://d-scholarship.pitt.edu/4845/1/31735062112523.pdf

Effects on in vivo and in vitro hepatocyte proliferation of methylprednisolone, azathioprine, mycophenolic acid, mizoribine, and prostaglandin E<inf>1</inf>

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Archivio istituzionale della ricerca - Università di Padova