Ann. rheum. Dis. (1977), 36, Supplement p. 29

Collagen deposition in liver disease

J. O'D. McGEEFrom the Department ofPathology, Radcliffe Infirmary, Oxford

Collagen forms a small component of the totalprotein of normal liver, but in diseases which leadto cirrhosis and in cirrhosis itself it is deposited inlarge amounts throughout the organ. This leads tostructural disruption of normal liver architecture(Fig. 1). The effects of this are the creation ofportosystemic shunts within the liver (Hase, 1968)and perhaps the formation of a connective tissuediffusion barrier in the space of Disse betweenhepatocytes and their vascular supply.

This paper presents evidence that collagendeposition in liver disease is due, at least in part, toincreased collagen synthesis. Those liver cellswhich are responsible for collagen synthesis andthe regulation of the synthesis of this protein in thediseased liver are also briefly discussed.

Collagen synthesisCollagen synthesis may readily be studied in mouseliver after one dose of carbon tetrachloride. Afterone dose the hepatocytes in the centre of theliver lobule undergo necrosis, the necrotic cells arethen removed, and by the fifth day the centre of thelobule has become repopulated with new hepato-cytes derived from the periportal area. During thehealing of this lesion an increased amount ofcollagen can usually be visualized in the centre ofthe lobule three to five days after poisoning. Thesefibres disappear very rapidly and the liver is morpho-logically normal on the sixth to seventh day of theexperiment.

In this model the rate of collagen synthesis isincreased 24 hours after injury, reaches a maximum

FIG. 1 Percutaneous needle biopsy of liver showing alcoholic cirrhosis with pronounced connective tissue depositionthroughout the liver parenchyma. Reticulin stain.

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Suppl. p. 30 Annals of the Rheumatic Diseases

on the third to fourth day, and returns to normal bythe sixth day (Fig. 2). The level of prolyl hydroxylaseshows a similar response after injury (Fig. 3). Thisenzyme, which synthesizes the hydroxyproline ofcollagen, is considered to be almost unique tocollagen synthesizing cells. These data indicate,therefore, that those fibres deposited in the centreof the lobule may have their genesis in increasedcollagen synthesis. In human liver disease directevidence of increased collagen synthesis is difficultto obtain because the usual isotopic techniques formeasuring collagen synthesis cannot effectively be

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FIG. 2 Collagen synthesis indose of carbon tetrachloride (Cabout 20 g were given by oesphasolution Of CC14 in liquid para,liver was measured as describeiEach point is average of 2 deilivers. Rate of collagen syntAexpressed as a ratio of that foui

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Table I Prolyl hydroxylase in normal liver and incirrhosis. Enzyme estimations performed as describedby McGee et al. (1974)

Diagnosis Prolylhydroxylase(cpm/mgprotein)

Normal 1158 (726, 893, 1459, 1553)Gilbert's disease* 1310 (946, 1674)Cirrhosis 3585-9588 (3585, 6000, 9144, 9366

9588)

*Gilbert's disease is characterized by a defect in the liver's ability tohandle bilirubin. It is not associated with collagen deposition.

applied to the small amounts of tissue (about10-12 mg wet weight) obtained by percutaneousneedling of the liver. However, it can be shown inhuman cirrhosis (Table I) and in diseases whichlead to cirrhosis (unpublished data) that the hepaticlevel of prolyl hydroxylase is raised, suggesting thatthe rate of collagen synthesis is also increased(McGee et al., 1974).The site at which connective tissue synthesis

occurs in liver has been investigated by autoradio-graphy using 35S-sulphate, which is a precursor ofthe sulphated glycosaminoglycans of ground sub-stance. In a form of non-progressive hepatic fibrosis(congenital hepatic fibrosis) only occasional sinuso-idal cells and a few cells in fibrous bands were active

2 3 in the uptake of 35S04. In other diseases such asalcoholic hepatitis and non-suppurative destructive

mouse liver after single cholangitis (primary biliary cirrhosis), which may'Cl4). Male mice weighing progress to cirrhosis, there is marked uptake ofzgeal tube 0 2 ml ofa 40% isotope at the sites of connective tissue depositionffin. Collagen synthesis in and in portal tracts (Figs. 4, 5). Cells related tod by McGee et al. (1973). hepatic sinusoids rather surprisingly also showterminations in 2 separate marked uptake of isotope (Fig. 6). It can be con-iesis in poisoned animals cluded from these results that sulphated glycos-nd in control normal livers aminoglycan synthesis is increased in aggressive

liver disease and that this occurs not only whereconnective tissue is obviously being laid down butalso in hepatic sinusoids.

2 3

FIG. 3 Collagen prolyl hydroxylase in normal liver aftercarbon tetrachloride injury. Mice were poisoned as out-lined in legend to Fig. 2. Prolyl hydroxylase was measuredas detailed by McGee et al. (1973). Results expressed as

in Fig. 2

Liver cells responsible for collagen formationThe nature of the cell type responsible for collagenformation in the liver has been investigated by avariety of methods (Popper and Udenfriend, 1970).Electron microscopic investigations have shownthat connective tissue deposition in portal tracts isassociated with typical fibroblastic activity. Untilrelatively recently, however, cells with the ultra-structural characteristics of fibroblasts had not beenidentified within the liver lobule itself. It has nowbeen shown that perisinusoidal cells (also known asIto or fat storage cells), which are found in thespace of Disse in the normal lobule, contain all ofthe subcellular machinery needed for the synthesisof proteins for export-for example, a well developedrough endoplasmic reticulin and Golgi apparatus

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II. Fibrosis in disease Suppl. p. 31

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FIG. 4 Histological appearance of liver in non-suppurative destructive cholangitis (prinmary biliary cirrhosis).Inflammatory cell infiltration and increased connective tissue formation in a portal tract (upper half of figure)

FIG. S Autoradiograph of 35S04 uptake in same liver as in Fig. 4. Shows marked uptake of isotope in portaltracts where connective tissue is being laid down (McGee et al., 1974)

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Suppl p. 32 Annals of the Rheumatic Diseases i'S : t s'WA.:~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~A4~~~~~~~~~~~~~~~~~~k.

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FIG. 6 Autoradiograph of 35SO4 uptake in same liver as in Fig. S. Higher power magnification shows that isotopeis being utilized by cells related to hepatic sinusoids

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FIG. 7 Electron micrograph ofnormal human hepatic sinusoid. Perisinusoidal cell with well developed rough endoplasmicreticulum (RER) lies in space of Disse and is closely associated with collagen fibrils (C). It is separated from sinusoidallumen (L) by sinusoidal cells. H=hepatocyte. D= lipid droplet

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II. Fibrosis in disease Suppl. p. 33

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FIG. 8 Electron micro-graph of perisinusoidalcell in normalhuman liver.Perisinusoidal cell isclosely associated withcollagen fibrils (C) andhas prominent Golgiapparatus (G)

FIG. 9 Electron micro-graph of healing zone inmouse liver 4 days aftersingle dose of40% CC] 4in liquid paraffin. Numer-ous cells throughout thearea contain a well devel-oped rough endoplasmicreticulum (RER) and areassociated with numerouscollagenfibrils (C). Manycells contain lipid droplets(D)

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Suppi. p. 34 Annals of the Rheumatic Diseases

(Figs. 7 and 8). In the normal liver these cells maybe intimately associated with collagen fibrils (Fig.8). In addition these cells increase in number andsize in zones where collagen is deposited in ex-perimental liver injury (Fig. 9). This, then, is primafacie evidence that perisinusoidal cells may be theintralobular liver fibroblasts. The presence ofprominent lipid droplets within these cells (Fig. 7)has been construed as evidence for a fat storagefunction. It should be noted, however, that collagen-producing cells in many situations contain intra-cytoplasmic lipid droplets (McGee and Patrick,1972).Claims have been made that hepatocytes have a

collagen productive function (Rojkind and Martinez-Palomo, 1976). This is based mainly on the findingthat isolated hepatocytes contain prolyl hydroxylase.The isolated cells used in these experiments, however,were only 95% pure and perhaps a contaminatingcell was the source of the enzyme measured, becauseisolated hepatic mesenchymal cells entirely free ofhepatocytes also contain prolyl hydroxylase (Shabaet al., 1973). More recently it has been shown thatisolated mesenchymal cells (a heterogeneous cell

population composed of sinusoidal lining cells andpresumably perisinusoidal cells) contain moreprolyl hydroxylase per cell than isolated hepatocytes.Furthermore, when mesenchymal cells and hepato-cytes are isolated from regenerating rat liver inwhich collagen synthesis is increased isolatedmesenchymal cells contain at least eight times moreprolyl hydroxylase than isolated hepatocytes (Martinand Patrick, unpublished). Since it is difficult toisolate hepatocytes and mesenchymal cells in anentirely pure form the interpretation of experimentssuch as these is at present not clear.Although the evidence available is against a

significant collagen biosynthetic role for hepatocytesit has been shown recently that malignant hepato-cytes may assume this function. In human hepa-tomas and in the same tumour induced by an azodye carcinogen (unpublished) it has been shown byimmunohistochemical methods that malignant hepa-tocytes contain both prolyl hydroxylase (Fig. 10)and collagen antigen (Fig. 11), suggesting that the'transformed' hepatocyte may assume a collagen-productive function. Neither enzyme nor collagencan be detected in normal liver cells by this method.

FIG. 10 Prolyl hydroxylase in human hepatoma cells identified by the immunoperoxidase procedure using aspecific antibody to prolyl hydroxylase. All tumour cells contain brown reaction product indicating presence ofprolyl hydroxylase. The nuclei (appearing as white holes) do not contain enzyme. For details of technique see Al-Adnani et al. (1975)

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II. Fibrosis in disease Suppl. p. 35

FIG. 11 Collagen antigen in human hepatoma cells identified as in Fig. 10 except that an antibody to neutralsalt soluble collagen was used. Collagen antigen present in the cytoplasm ofmalignant hepatocytes but not in the nuclei.Collagen fibres are also stained (arrows)

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FIG. 12 Isolation of collagen stimu-lating factors by Sephadex G25 chro-matography from mouse liver 3 daysafter single dose of carbon tetrachloride.Factors labelled F1-F4 (U iimliE) wereisolated from a 15 000 x g supernatantderived from livers homogenized in aphosphate buffer (McGee et al., 1973).The assay for collagen-stimulatingfactors based on their ability to stimu-late prolyl hydroxylase activity inculturedfibroblasts is describedby McGeeet al. (1973) with one modification-namely, the fibroblasts used in this assaywere grown in medium containing 10%fetal calf serum and other supplements.*6 * = OD280

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Suppl. p. 36 Annals of the Rheumatic Diseases

Regulation of cola syntesisThe regulation of collagen synthesis in collagen-productive diseases such as cirrhosis has only begunto be investigated (McGee et al., 1973). A series offactors can be isolated from experimental liverdisease in which collagen synthesis is increased(Table II) which will cause an increase in prolyl

Table II Prolyl hydroxylase and collagen synthesisin mouse liver 3 days after carbon tetrachloride

Treatment Prolyl hydroxylase Collagen synthesis(cpm x 10-5/g) (cpmlg)

Nil (control) 745 510CCI 4 2448 1731

*Two mice were given 0 - 2 ml of 40% CCl4 in olive oil by eosphagealtube. Three days later two normal and the two poisoned mice received5 ,UCi of 14C-proline (SA 285 mCi/mMol) intraperitoneally twohours before killing by cerivcal dislocation. All animals were starvedovernight before kiUing. Prolyl hydroxylase and collagen synthesiswere measured in the same livers by methods described elsewhere byMcGee et al. (1973). Each value is the mean enzyme level and collagensynthetic rate of the two animals in each group.

hydroxylase in fibroblast cultures (Fig. 12). Similarfactors have also been isolated from human cirrhotictissue (Smith and McGee, unpublished data). Morerecently it has been shown that the effluents fromthese columns contain four factors which alsostimulate collagen chain synthesis in the same tissueculture system (Fallon et al., unpublished data).Since these collagen stimulating factors are notpresent in normal liver it has been postulated thatthey may play a role in the regulation ofthe increasedcollagen biosynthetic response observed in liverinjury (McGee et al., 1973).In conclusion, there is evidence that in experi-

mental and human liver disease collagen depositionis due in part to increased collagen synthesis. Itseems likely that this protein is synthesized byfibroblasts in portal tracts and by perisinusoidalcells in non-neoplastic liver disease but that hepato-cytes may assume this function when they becomeneoplastic. Finally, collagen biosynthesis in pre-cirrhotic and cirrhotic liver may be controlled bycollagen stimulating factors.

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