.lournaloffHepatoiog)l, 1992; 14: 259-264 @ 1992 Elsevier Science Publishers B.V. All rights reserved. 0168-8278/92/%05.00

259

HEPAT 00940

ncrease

e ex

Giorgio

Annoni I, Francis

R. Weiner2

and

ark A. Zern3

‘istituto chica Medica 111. universitci di Miiano, Milano, Italy, ‘Liver Research Center, Albert Einstein College of Medecine, Bronx, NY and 3Depar:me?2!of Medicine, Roger Williams General Hospital and Brown iinivers !y, Providence, RI, United States of America (Received 13 December 1930)

We recently demonstrated that transforming growth factor-/31 stimulates collagen synthesis in hepatic cells in vitro, and that the synthesis of this cytokine is markedly increased in two rodent models of hepatic fibrosis. In. the present study, we investigated the association of transforming growth factor-p1 (TFG-01) gene expression in human liver disease. Sixteen patients with active liver disease had percutaneous liver biopsies performed for diagnostic purposes. Total RNA was extracted from an unused portion of each biopsy and then subjected to hybridization analysis with the following human cDNA clones: albumin, pro cwl(I) collagen, and TGF-/31. Surgical liver biopsy specimens from two patients without hepatic disease were used as controls. Whes compared to controls, the patients with active liver disease had a 19% decrease in albumin, a 97% increase in type I collagen, and a 120% increase in transforming growth factor-/31 mRNA levels. Moreover, steady-state levels of TGF-Bl and procollagen mRNAs were significantly correlated. Nuclear run-on assays showed that livers from two patients with fibrosis had TGF-Pl transcription rates that were more than 2-fold higher than rates in control livers. These findings indicate that transforming growth factor-01 gene expression is significantly enhanced in man during active liver disease.

The fibrotic liver is characterized by a disordered and increased deposition of several matrix proteins. However, the increase in collagen content is particularly significant in pathophysiology of this disorder (l), with type I collagen predomtnating in advanced cirrhosis (2). Recent studies have investigated the importance of soluble factors as initiators of the fibrogenic process. Some incompletely characterized fibrogenic factors have been shown to play a role in liver fibro::ls (3,4), and some authors have suggested that cytokines released by macrophages or lymphocytes may be important in the induction of hepatic fibrosis (5,6). Transforming growth factor-01 (TGF-01) is a cytokine that has been shown to augment fibrosis in a number of experimental models (7-14). While many properties have been ascribed to TGF-01, its major role in extracellular matrix protein synthesis, deposition, and

remodeling is particularly relevant to the study of liver fibrosis. For example, TGF-01 has L _n shown to induce collagen formation in rodent (7), chicken (8), and human fibroblasts (9), and in rat myofibroblasts (7). This increase in collagen synthesis has been shown to be associated with enhanced steady-state procollagen mRNA levels (7,9). A number of recent studies have demonstrated the significance of TGF$l in hepatic systems (10-14). We have observed that TGF-01 treatment of cultured hepatocytes (10) or Ito cells (11) leads to increased type I procollagen mRNA levels. Moreover, TGF-/31 mRNA levels were shown lo be increased in parallel with type I collagen gene expression in two animal model systems of bepatic fibrosis (10). Messenger RNA levels of this cytokine have been shown to be increased in whole liver (12) and in isolated hepatic cells (13) during acute injury with Ccl,, and in

Correspondence:Giorgio Annoni M.D., istituto Clinica Medica III, UniversiG di Milano. Via Pace 15, 20123 Milano, Italy.

260 situ hybridization techniques have localized an increase in TGF-01 transcripts to Ito cells during CC&-induced fibrosis in rats (14). These in vitro and in vivo findings strongly suggest a role for TGF-fil in models of liver fibrosis. However, no previous study has evaluated the association of TGF-01 with human liver disease. Therefore, in this study we have attempted to delineate the possible role of TGF-/31 in human hepptir: fiibrogenesis by examining its gene expression and its correlation with type I col!agen gene expression.

Materials and Methods

Patients Sixteen patients (ten males and six females, aged 31 to 60 years) admitted to hospital for a diagnostic liver biopsy were included in this study. None of the subjects were taking medication upon entering the study. Informed consent was obtained from all the patients and the study protocol was approved by the ethics committee of the University of Milano. The epidemiological and clinical characteristics of the patients are shown in Table 1. Liver biopsy and histology Liver biopsies were obtained from all patients, during laparoscopy, using a Tru-cut needle (Travenol). Half of each sample was processed for histological purposes including staining for hematoxylin-eosin and trichrome. *Coded biopsies were read blindly by one of us with no knowledge of the clinical histories of the patients. Patients were characterized according to histological diagnosis: alcoholic hepatitis (four patients), chronic active hepatitis (two patients), and active cirrhosis (ten patients). All the diagnoses fulfilled the internationally agreed-upon criteria (15,16). The remaining portion of each liver biopsy was immediately frozen in liquid nitrogen and stored at -80 “C until used in molecular studies. RNA extraction and Northern blot hybridization The liver specimens were used for RNA extraction employing a modification of the Chirgwin procedure (17) as described previously (18). The samples were homogenized in 3.5 ml of 4 M guanidine thiocyanate solution and after a low speed centrifugation to remove cellular debris, the RNA was pelleted through a cesium chloride gradient. Following a series of ethanol precipitations, total RNA content was calculated by A,,,, spectroscopy. Total RNA yields ranged from 15 to 30 pg per sample. Steady-state mRNA levels were determined by Northern or dot blot hybridization analysis. For Northern blots,

G. ANNONI et

al.

samples of 2.5 to 15 pg of total RNA from each human liver sample were denatured in 0.5 M glyoxal, 50% dimethyl sulfoxide, and 10 nM phosphate buffer, electrophoresed in a 1% agarose gel, transferred to a Gene Screen filter (New England Nuclear, Boston, MA) and baked for 2 h at 80 “C. The equality of RNA samples after transfer to a membrane was substantiated by ultraviolet illumination of ethidium bromide stained blots. After prehybridization, the filters were probed with the following human cDNA clones: albumin (19), proclll (I) collagen (20), and TGFPl (21). The cDNA probes were labeled with [32P]dCTP to a specific activity of 2-5 - 10s cpm/pg of DNA using a primer extension kit (Amersham, U.K.). Filters were hybridized overnight at 42 “C in 50% formamide, 5 x SSC, 1% Denhardt’s, and 1% SDS. The hybridization filters were washed under stringent conditions, exposed to X-ray film and the developed film scanned by densitometry. Surgical liver biopsy specimens from two pat&n5 without hepatic disease were used as controls, and as a reference standard for each liver sample. Fot dot blots, RNA was diluted serially in 3% formaldehyde and spotted on GeneScreen. Then in the same manner as with Northern blots, these filters were prehybridized, hybridized, developed, and autoradiography was performed. At least one Northern blot was done of each sample before dot blots were used for quantification, because Northern blots demonstrate that the sample is relatively undegraded and that the hybridization signal is in the correct location. Mostly dot blots were used for densitometry studies TGF-01 and type I collagen because less RNA is required for dot blots than for Northern blots. Since two controls were run on the same gel, the densitometry recording of each patient sample was compared to the mean of the two control samples. Transcriptional rate analysis Nuclear run-on assays were performed on four wedge liver biopsy samples of approx 2 g each using methods previously described (22). The two controls consisted of uninvolved normal liver tissue obtained at surgery for metastatic cancer. The two fibrotic livers were obtained at the time of liver transplantation. One control was compared to one fibrotic liver in each of two experiments. The liver cell nuclei were isolated by centrifugation through 2.1 M sucrose as described by Clayton and Darnell (23). Nuclei were labeled for 15 min with high specific activity [3’P]UTP (23), and the labeled RNA transcripts were isolated by lysis of the nuclei in a hypotonic solution followed by DNA digestion with RNase-free DNase, proteinase ir. digestion, phenol extraction, and ethanol precipitation. The labeled RNA was then hybridized with

TRANSFORMING

GROWTH FACTOR-/31 IN LIVER

261

at least a IO-fold excess of the cDNA probes which were bound to a nitrocellulose membrance (23). A complementary DNA clone of pBR322 was used as a negative control. After hybridization, the filters were washed extensively, digested with RNase A, exposed to X-ray film and densitometric scanning was performed.

Results Sixteen patients with a variety of liver diseases served as subjects in the study. All had moderate liver injury with elevated aspartate aminotransferase (AST) levels between 39 and 258 W/l and none had albumin levels below 3.5 g/dl. All had histological evidence of active disease and increased collagen deposition by liver histology. The clinical characteristics of the patient population are provided in Table 1. The two normal controls had normal AST levels and a mean serum albumin level of 4.7 g/d], whereas the patients had a mean serum albumin level of 4.08 g/dl. The patients had a somewhat lower albumin mRNA content that control patients (81 f 9% S.D. of control) when the albumin mRNA blots were analyzed by densitometry. This difference in mRN4 content reflected the trend towards lower serum albumin levels in the patients with

subjected to Northern or dot blot analysis, there was an increase in the levels of TGF-PI and pro crl (I) collagen mRNA content in the patients’ livers when compared to controls. Northern blots which depict the changes that occur with fibrosis are shown in Fig. 1. The results of densitometry tracings of all the Northern and dot blots are shown in Table 2. Of note, the steady-state mRNA levels of TGF-PI and procollagen are significantly correlated (r= 0.50; pC.05). Unfortunately, assays of TGF/3activity were not possible given the Imitations posed by the percutaneous biopsy sizes. Previous studies in the murine schistosomiasis model (10) did show immunohistochemical correlation between TGF-01 mRNA content and protein levels. Moreover, no previous studies have shown any discordance between TGF-01 mRNA lev-

liver disease. When

the RNA isolated

from the patients’

livers was

TABLE 1 Characteristics

of the patient population

Patient

Sex

Etiologya

AST Albumin (U11) (g/d\)

Histological diagnosis

C.F. M.G. P.A. S.G.F. B.C.

M M F M M

ETOH ETOH ETOH ETOH HBV

47 39 131 145 108

4.1 4.0 4.1 3.5 4.8

M.S.

M

HBV

79

4.3

SC. M.N.

M F

HBV ETOH

87 58

4.4 3.8

P.N.

M

ETOH

165

4.3

Z.E.

M

ETOH

60

4.1

G.R. M.W. F.R. J.B. K.E. B.G.

M F M F F F

NANB PBC PBC HBV PBC (rlAT

92 123 99 258 241 190

3.6 3.9 3.5 4.1 4.2 3.9

alcoholic hepatitis alcoholic hepatitis alcoholic hepatitis alcoholic hepatitis chronic active hepatitis chronic active hepatitis active cirrhosis cirrhosis and alcoholic hepatitis cirrhosis and alcoholic hepatitis cirrhosis and alcoholic hepatitis active cirrhosis stage III-IV stage IV active cirrhosis stage IV active cirrhosis

a ET-OH, alcoholic liver disease; HBV, hepatitis B viral disease; NAN& non-A, non-B hepatitis disease; PBC, primary biliary cirrhosis; ol AT, (I, antitrypsin deficiency.

Fig. 1. Autoradiogram of a representative Northern blot hybridization study of total RNA isolated from the liver of a control patient (CON) compared to RNA from a patient with fibrotic liver disease (PT). RNA was isolated from surgical or percutaneous liverbiopsy

samples, electrophoresed, transferreti to a solid matrix, hybridized with a labeled cDNA probe, washed under stringent conditions, and exposed to X-ray film. The autoradiogram shows RNA hybridized with either a human albumin (ALB), pro al(l) collagen (COL I), or (TGF-61) cDNA probe. The ,markers represent ribosomal RNA sizes.

G. ANNONI

262

et al.

the two patients with liver disease than in controls. However, the signals were only slightly over background (data not shown).

in

TABLE 2 Densitometry scanning of total RNA extracted from patients with liver disease hybridized with TGF-01, human albumin and pro al(l) collagen cDNA ciones n

Probe

TGF-01 Albumin Pro nl(1) collagen

16 16 16

Mean f SD. as % of control

Range as Vo of control

220 + 66 81 + 9 197 f 22

(160-380) (66-98) (165-245)

TGF-01 and pro al(I) colla,

Increased transforming growth factor-beta 1 gene expression in human liver disease.

We recently demonstrated that transforming growth factor-beta 1 stimulates collagen synthesis in hepatic cells in vitro, and that the synthesis of thi...
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