Hepatic stellate cell/myofibroblast subpopulations in fibrotic human and rat livers

Hepatic stellate cell/myofibroblast subpopulations in fibrotichuman and rat livers

David Cassiman1,*, Louis Libbrecht2, Valeer Desmet2, Carl Denef1, Tania Roskams2

1Laboratory of Cell Pharmacology, Herestraat 49, B-3000 Leuven, Belgium2Laboratory of Morphology and Molecular Pathology, University of Leuven (K.U. Leuven), Leuven, Belgium

Background/Aims: Hepatic stellate cells (HSC) are commonly considered the precursor population of septal myofi-broblasts (MF) in cirrhosis. We studied the distribution and expression profile of mesenchymal (myo)fibroblast-likepopulations in fibrotic and cirrhotic liver, in an attempt to elucidate their possible interrelationships.

Methods: Fibrotic/cirrhotic livers (from 22 human explants and from two rat models: carbon tetrachloride intoxica-tion, bile duct-ligation) were studied by means of immunohistochemistry (single and double immunostaining) withantibodies raised against desmin, alpha-smooth muscle actin (aSMA), glial fibrillary acidic protein (GFAP), neural-celladhesion molecule (N-CAM), synaptophysin, neurotrophins, neurotrophin receptors and alpha B-crystallin(ABCRYS).

Results: Septal MF showed the same expression profile as portal MF, in human and rat, being aSMA/ABCRYS/brain-derived nerve growth factor/GFAP-expression, with additional N-CAM- and desmin-expression in rat portal/septal MF. Perisinusoidally located HSC stained with all tested markers, MF at the septal/parenchymal interfaceshowed an expression profile, intermediate between the profiles of HSC and portal/septal MF.

Conclusions: In advanced fibrosis and in cirrhosis, regardless of cause or species, three distinct mesenchymal(myo)fibroblast-like liver cell subpopulations can be discerned: portal/septal MF, interface MF and perisinusoidallylocated HSC. The fact that septal MF share more characteristics with portal MF than with HSC might suggest descent.q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.

Keywords: Hepatic stellate cell; Myofibroblast; Alpha-smooth muscle actin; Neural-cell adhesion molecule; Glial fibril-lary acidic protein; Neural; Neuroendocrine; Cirrhosis; Bile duct-ligation; Carbon tetrachloride

1. Introduction

Liver extracellular matrix constituents are synthesized by

hepatocytes, bile duct epithelial cells and endothelial cells,

but the largest contribution to extracellular matrix and fibro-

genesis probably comes from mesenchymal (myo)fibro-

blast-like subpopulations of the liver [1–4]. In the last

three decades, several of these subpopulations have been

described in the liver. They all share ultrastructural charac-

teristics (an electron-microscopical ‘mesenchymal’ pheno-

type) and immunohistochemical features (vimentin and/or

desmin and/or alpha-smooth muscle actin (aSMA) expres-

sion). In the space of Disse, typical hepatic stellate cells

(HSC), transitional cells (intermediate between HSC and

myofibroblasts (MF)) and MF have been described. Based

on sequential ultrastructural and immunohistochemical

studies, HSC are thought to transdifferentiate to transitional

cells and then on to MF-like cells, following repeated

damage to the liver [5–7]. Perisinusoidal and pericellular

fibrosis is thought to result from an imbalance between

extracellular matrix production and degradation by HSC,

transitional cells and perisinusoidal MF.

Another mesenchymal (myo)fibroblast-like cell type in

the liver is the portal (myo)fibroblast, which in normal

conditions resides in the portal mesenchyme. Periductal

(myo)fibroblasts appear to constitute a distinct subpopula-

tion of mesenchymal cells in the portal tract, as well as

vascular smooth muscle cells residing in the walls of portal

vein branches and portal arteries. Periductal (myo)fibro-

blasts have been suggested to proliferate and transdifferenti-

Journal of Hepatology 36 (2002) 200–209

www.elsevier.com/locate/jhep

0168-8278/02/$20.00 q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.

PII: S0168-8278(01)00260-4

Received 17 July 2001; received in revised form 19 September 2001;

accepted 25 October 2001

* Corresponding author. Tel.: 132 16 34 58 01; fax: 132 16 34 56 99.

E-mail address: [email protected] (D. Cassiman).

ate in response to bile duct-ligation, causing periductal/peri-

ductular and periportal ‘biliary type’ of fibrosis [8,9]. In

schistosomiasis, vascular smooth muscle cells residing in

the portal vein wall were thought to proliferate and trans-

differentiate to matrix-producing MF, causing periportal

fibrosis [10].

Yet another mesenchymal (myo)fibroblast-like cell type

of the liver are the MF located around the centrolobular vein

(so-called second-layer cells). They were suggested to

proliferate in the livers of alcohol-fed baboons, causing

typical ‘alcoholic type’ of pericentral fibrosis [11]. Finally,

(myo)fibroblasts residing in Glisson’s capsule form a poten-

tial source of extracellular matrix in the liver and show

ultrastructural resemblances with HSC/MF [1].

Liver MF and/or perisinusoidal HSC were shown to

express neural/neuroendocrine features such as silver stain-

ing [12], neural-cell adhesion molecule (N-CAM) [13–15],

glial fibrillary acidic protein (GFAP) [16–19] and nestin

expression [20], in addition to classical mesenchymal

HSC markers as desmin for rat HSC [21–25], aSMA for

activated human and rat HSC [26–28] and vimentin for

human and rat HSC [29]. We recently studied several new

in vivo markers for human and rat HSC/MF: synaptophysin

(SYN), neurotrophins (nerve growth factor (NGF), brain

derived nerve growth factor (BDNF), neurotrophin (NT) 3

and NT-4) and NT receptors (tyrosine kinases (Trk) A, B

and C, low-affinity nerve growth factor receptor p75) [31]

and alpha B-crystallin (ABCRYS) [32]. p75 [33] and

ABCRYS [34] were also described as HSC markers by

others.

We performed an immunohistochemical study on the

distribution of functional HSC markers in human and rat

fibrotic liver specimens, to elucidate possible interrelation-

ships between mesenchymal (myo)fibroblast-like popula-

tions of the liver. We studied four frequent fibrotic

disorders of the human liver: biliary type of fibrosis in

primary biliary cirrhosis (PBC) and primary sclerosing

cholangitis (PSC) specimens, post-hepatitic cirrhosis due

to chronic hepatitis C virus (HCV) infection and post-alco-

holic cirrhosis. All specimens showed cirrhosis, since they

were all obtained from explant livers at the end-stage of

disease. In rats, two paradigmatic models of fibrogenesis

were studied: carbon-tetrachloride-induced cirrhosis

(CCl4) and bile duct-ligation-induced fibrosis (BDL).

D. Cassiman et al. / Journal of Hepatology 36 (2002) 200–209 201

Table 1

Primary antibodiesa

Primary antibody to Type Source Working dilution

Alpha B-crystallin Rb poly Novocastra 1/500

NGF Rb poly Santa-Cruz 1/100

BDNF Rb poly Santa-Cruz 1/100

NT-3 Rb poly Santa-Cruz 1/100

NT-4/5 Rb poly Santa-Cruz 1/50

Trk-B Rb poly Santa-Cruz 1/100

Trk-C Rb poly Santa-Cruz 1/50

P75 Rb poly Santa-Cruz 1/50

Synaptophysin Rb poly Dako 1/50

N-CAM Rb poly Chemicon 1/100

N-CAM Mo mono Sigma 1/25

GFAP Rb poly Dako 1/300

GFAP Mo mono Biogenex 1/40

Desmin Mo mono Roche 1/20

aSMA Mo mono Dako 1/40

a Mo mono: mouse monoclonal antibody; Rb poly: rabbit polyclonal

antibody. NGF: nerve growth factor, BDNF: brain-derived nerve growth

factor, NT: neurotrophin, Trk: tyrosine kinase, p75: low-affinity nerve

growth factor receptor p75, N-CAM: neural-cell adhesion molecule,

GFAP: glial fibrillary acidic protein, aSMA: alpha-smooth muscle actin.

Novocastra: Newcastle-upon-Tyne, UK. Santa-Cruz Biotechnology: Santa-

Cruz, California, USA. Dako: Copenhagen, Denmark. Chemicon: Teme-

cula, California, USA. Sigma: Steinheim, Germany. Biogenex: San Ramon,

California, USA. Roche Diagnostics: Brussels, Belgium.

Table 2

Secondary and tertiary antibodiesa

Application Antibody Source Dilution

Po-labelled single immunostaining Rb poly primary Ab, in human and rat GtaRb-EnVision (Po) Dako undiluted

Mo mono primary Ab, in human 2nd RbaM-Po Dako 1/200

3rd SwaRb-Po Dako 1/200

Mo mono primary Ab, in rat 2nd GtaM-Po (RAg) Sigma 1/100

3rd RbaGt-Po (RLP) Jackson 1/100

Fluorescence-labelled double immunostaining Rb poly primary Ab SwaRb-TRITC Dako 1/200

N-CAM and ABCRYS Rb poly primary Ab 2nd GtaRb (RAg) Jackson 1/200

3rd BoaGt-FITC Santa-Cruz 1/200

Mo mono primary Ab DoaM-FITC/TRITC (RAg) Jackson 1/200

aSMA primary Ab 2nd GtaM-FITC (RAg) Sigma 1/200

3rd BoaGt-FITC Santa-Cruz 1/200

a Po: peroxidase-labelled, Rb poly: rabbit polyclonal, Ab: antibody, Mo mono: mouse monoclonal. 2nd: secondary, 3rd: tertiary, GtaRb: goat anti-rabbit,

RbaM: rabbit anti-mouse, SwaRb; swine anti-rabbit, GtaM: goat anti-mouse, RbaGt: rabbit anti-goat, BoaGt: bovine anti-goat, DoaM: donkey anti-mouse,

TRITC: tetramethylrhodamine isothiocyanate, FITC: fluoresceine isothiocyanate, (RAg): pre-adsorbed with rat serum antigens by the manufacturer, (RLP):

pre-adsorbed with rat liver powder (from Sigma), at 48C overnight. Dako: Copenhagen, Denmark. Sigma: Steinheim, Germany. Jackson: Jackson Laboratories,

West Grove, Pennsylvania, USA. Santa-Cruz: Santa-Cruz Biotechnology, Santa-Cruz, California, USA.

2. Material and methods

2.1. Human liver specimens

A series of 22 cirrhotic human liver specimens, taken from explant livers

for diagnostic purposes, was used: post-HCV ðn ¼ 8Þ; post-alcoholic

ðn ¼ 6Þ; PBC ðn ¼ 6Þ; PSC ðn ¼ 2Þ. Diagnoses were based on histopatho-

logical examination of routinely processed tissue and on clinical and

laboratory data. Part of each fresh biopsy was snap frozen in liquid nitro-

gen-cooled isopentane and stored at 2708C until use.

2.2. Rat models

Male Wistar rats, 250–300 g body weight, were used in the CCl4 and

BDL models. CCl4 cirrhosis and BDL were induced as described [31,32].

Three rats that had received 10 weeks of CCl4 (full cirrhosis) and three rats

D. Cassiman et al. / Journal of Hepatology 36 (2002) 200–209202

at 4 weeks after BDL were killed. Liver specimens taken from these rats

were snap frozen in liquid nitrogen-cooled isopentane and stored at 2708C

until use. All animals were fed ad libitum and received humane care in

accordance with the guidelines established by the University of Leuven

Ethical Committee.

2.3. Immunohistochemistry

Single immunostaining on serial cryosections of human and rat liver and

double immunostaining on cryosections of rat liver were performed as

described [31,32]. Primary antibodies: see Table 1. Secondary and tertiary

antibodies: see Table 2. Double staining experiments on BDL-fibrotic and

CCl4-cirrhotic rat livers for desmin in combination with GFAP, N-CAM

and ABCRYS were performed, as well as double staining for aSMA in

combination with GFAP and N-CAM. Double staining was detected using a

Zeiss Axioplan 2 microscope (Zeiss, Weesp, Netherlands), coupled to a

Spot Advanced camera, version 2.2.2. Images were reproduced digitally,

using Carl Zeiss Vision Imaging Systems KS 300 (release 3.0) software.

2.4. Analysis of immunohistochemical stainings

All stainings were evaluated by three independent, skilled liver pathol-

ogists (L.L., V.D. and T.R.). Perisinusoidally located, stellate-shaped cells,

some of them containing lipid droplets, residing in the parenchymal lobules

or nodules were named ‘HSC’. Stellate- or spindle-shaped cells at the inter-

face between parenchyma and portal tract or between parenchyma and

septa, i.e. at the borders of fibrotic septa, were named ‘interface MF’.

Stellate- or spindle-shaped cells residing in the portal tracts and in fibrotic

septa were named ‘portal MF’ and ‘septal MF’, respectively. The propor-

tion of HSC immunoreactive to one marker was given a relative quotation

(1¼ minority, 11 ¼ large subpopulation, 111¼ reference marker),

proportionally to the marker(s) that stained the largest quantity of HSC in

our hands. A similar procedure was applied to assess the number of immu-

noreactive interface MF, portal and septal MF, smooth muscle cells in

portal vessel walls and centrolobular veins (2¼ no staining, ^¼ less

than reference marker, 1¼ reference marker). In case of disagreement

on the attributed scores, consensus between the pathologists was reached

at a multi-headed microscope.

3. Results

3.1. HSC

3.1.1. Human specimens

The highest number of HSC in human cirrhotic livers was

found immunoreactive to aSMA and N-CAM (Fig. 1A,B).

All tested markers, with the exception of desmin, stained a

varying proportion of HSC, diffusely distributed throughout

the parenchymal nodules (Fig. 1C,D and Table 3).

3.1.2. Rat specimens

The largest proportion of HSC in the parenchymal

lobules or nodules of CCl4-cirrhotic (Table 4) and BDL

rat livers (Table 5) was immunoreactive to GFAP or

desmin (Fig. 1E–H). In CCl4-cirrhotic livers, all tested

markers stained a varying proportion of diffusely distribu-

ted HSC. GFAP showed co-localization with desmin in

many nodular HSC. Overall, less cells showed GFAP posi-

tivity, in comparison with desmin positivity. No clear

predominance in nodular localization could be distin-

guished for either desmin- or GFAP-immunoreactive

cells (Fig. 2A–C). Also in CCl4-cirrhosis, no co-localiza-

tion for GFAP and aSMA could be detected in HSC. In

BDL rat livers, desmin immunoreactivity was most abun-

dant in the periportal area, while pericentral HSC showed


Fig. 1. Single immunostaining. (A): Cirrhotic human liver specimen (chronic hepatitis C virus infection), stained with alpha-smooth muscle actin

antibody. Hepatic stellate cells (HSC) (arrowheads) are immunoreactive, as well as myofibroblasts (MF) at the interface between parenchyma and

septum (S) and in septum and portal tract (upper right corner: portal artery). (B): Cirrhotic human liver (primary biliary cirrhosis), stained with

neural-cell adhesion molecule antibody. HSC are immunoreactive (arrowheads), as well as MF located at the interface between parenchyma and

septum (S). Reactive bile ductules (arrows) are also immunoreactive. (C): Human liver specimen, alcoholic cirrhosis, stained with neurotrophin-3

antibody. HSC lining the sinusoids are immunoreactive (arrowheads). (D): Cirrhotic human liver (primary biliary cirrhosis), stained with alpha B-

crystallin antibody. HSC (arrowheads) are immunoreactive, as well as interface MF and septal MF (S: fibrotic septum). (E): Carbon-tetrachloride-

induced cirrhosis of the rat liver, stained with glial fibrillary acidic protein (GFAP) antibody. HSC are immunoreactive (arrowheads), as well as a

subpopulation of interface MF and septal MF (S). Upper left corner: portal tract. (F): Carbon-tetrachloride-induced cirrhosis of the rat liver, stained

with desmin antibody. HSC are immunoreactive (arrowheads), as well as a subpopulation of interface MF and septal MF (S). (G): Bile duct-ligated

fibrotic rat liver, stained with GFAP antibody. HSC (arrowheads) are immunoreactive, as well as a subpopulation of MF at the interface, co-

proliferating with reactive bile ductules (arrows) and a subpopulation of MF in the expanding portal tract (upper left corner). (clv: centrolobular

vein). (H): Bile duct-ligated fibrotic rat liver, stained with desmin antibody. HSC (arrowheads) are immunoreactive, as well as a subpopulation of MF

at the interface, co-proliferating with reactive bile ductules (arrows) and a subpopulation of MF in the expanding portal tract (lower right corner).

(clv: centrolobular vein) (Fig. 3A is a parallel section). (Original magnification £ 200).

Table 3

Immunohistochemical expression profile of subpopulations of

mesenchymal (myo)fibroblast-like cells in fibrotic/cirrhotic human

livera

HSC Interface

MF

Septal

MF

Portal

MF

SMC

Desmin 2 2 2 2 ^

aSMA 111 1 1 1 1

N-CAM 111 ^ 2 2 2

GFAP 11 1 ^ ^ 2

SYN 1 /11 2 2 2 2

NGF 1 /11 ^ 2 2 2

BDNF 11 ^ ^ ^ Arteries 1

NT-3 11 2 2 2 2

NT-4 1 ^ 2 2 Arteries ^

Trk-B 1 2 2 2 2

Trk-C 1 2 2 2 2

P75 1 2 2 2 2

ABCRYS 1 1 ^ ^ 1

a HSC: hepatic stellate cells; MF: myofibroblasts; SMC: smooth muscle

cells in portal vessel walls.

less abundant or undetectable desmin immunoreactivity.

GFAP-staining showed highest abundance in the pericen-

tral region of BDL rat livers, while intensity of staining

diminished toward the periportal region (Fig. 1G,H).

Double staining with GFAP and desmin created a spectrum

ranging from red (GFAP single staining) in the pericentral

area, via orange and yellow (co-localization of GFAP and

desmin staining) in periportal lobular HSC and in interface

MF, to green (desmin single staining) in interface MF co-

proliferating with reactive bile ductules and in portal MF.

Occasional interface MF and portal MF showed single

staining for GFAP as well (Fig. 2D).

3.2. Interface MF


Interface MF were highly immunoreactive to aSMA

(Fig. 1A), GFAP (Fig. 2E) and ABCRYS (Fig. 1D).

GFAP-staining created a rim of immunoreactivity

surrounding the nodular parenchyma and lining the fibrotic

septa (Fig. 2E). Immunoreactivity to N-CAM (Fig. 1B),

BDNF and NT-4 was also present, but only in a subset

of interface cells. NGF, finally, was detected in occasional

cell clusters at the interface. Desmin, SYN, NT-3 (Fig. 1C),

Trk-B, Trk-C and p75 antibodies did not stain interface

cells (Table 3).


Interface MF in CCl4-cirrhosis and in BDL rat livers

were immunoreactive to desmin (Fig. 1E,G), GFAP (Fig.

1F,H), aSMA (Fig. 2F,H) and N-CAM (Figs. 2G and 3A)

antibodies, a subset was ABCRYS-positive. Double stain-

ing for desmin and ABCRYS in interface MF was present,

as well as for aSMA and N-CAM. Occasional desmin and

GFAP double staining could be detected at the interface,

although desmin 1 /GFAP-cells and desmin-/GFAP 1


Table 4


mesenchymal (myo)fibroblast-like cells in CCl4-cirrhotic rat liversa

HSC Interface MF SeptalMF Portal MF SMC

Desmin 111 1 ^ ^ 1

aSMA 1 1 ^ ^ 1

N-CAM 1 1 1 1 2

GFAP 111 1 ^ ^ 1

SYN 1 2 2 2 2

NGF 1 2 2 2 2

BDNF 1 2 2 2 ^

NT-3 1 2 2 2 2

NT-4 11 2 2 2 ^

Trk-B 1 2 2 2 2

Trk-C 1 2 2 2 2

P75 1 2 2 2 2

ABCRYS 11 ^ ^ ^ 1


cells in portal vessel walls.

Table 5


mesenchymal (myo)fibroblast-like cells in BDL rat liversa

HSC Interface MF Septal MF Portal MF SMC CV

Desmin 111 1 ^ ^ 1 ^

aSMA 11 ^ ^ ^ 1 1

N-CAM 11 1 1 1 2 ^

GFAP 111 ^ ^ ^ 1 1

SYN 1 2 2 2 2 2

NGF 11 ^ ^ ^ 2 2

BDNF 1 1 ^ ^ ^ 2

NT-3 1 2 2 2 2 2

NT-4 11 2 2 2 ^ 2

Trk-B 1 2 2 2 2 2

Trk-C 1 ^ 2 2 2 2

P75 1 2 2 2 2 2

ABCRYS 11 1 1 1 1 1


cells in portal vessel walls. CV: centrolobular vein.

Fig. 2. Single and double immunostaining. (A–C): Carbon-tetrachloride-induced cirrhosis of the rat liver, stained with desmin (green, A and B) and

glial fibrillary acidic protein (GFAP) (red, B and C) antibodies. Desmin (green, A and B) stains hepatic stellate cells (HSC) (arrowheads), myofi-

broblasts (MF) at the interface between septa and nodular parenchyma and portal/septal MF. GFAP (red, B and C) mainly stains HSC (arrowheads)

and nerve endings or smooth muscle cells in the wall of a portal artery branch. Co-localization of desmin and GFAP (yellow, B) is only seen in HSC

(arrowheads). (PV: portal vein branch; original magnification £ 400). (D): Bile duct-ligated fibrotic rat liver, stained with desmin (green) and GFAP

(red) antibodies. Upper left corner shows the expanding portal tract (PT), with multiple reactive bile ductules, lower right corner is the pericentral

area. Desmin (green) stains MF in the portal tract, MF at the interface between portal tract and parenchyma – co-proliferating with reactive bile

ductules – and HSC. GFAP (red) stains the same cell types, but co-localization (yellow) is only seen in interface MF and in occasional HSC. In the

portal tract, cells are either desmin-positive (green) or GFAP-positive (red) and no co-localization is detected. In the lobule, the intensity of GFAP

expression increases with the distance from the portal tract, while desmin decreases (original magnification £ 400). (E): Cirrhotic human liver

specimen (chronic hepatitis C virus infection), stained with GFAP antibody. HSC are immunoreactive (arrowheads), as well as MF at the interface

between parenchyma and septum (S). Occasional septal MF are immunoreactive to GFAP (original magnification £ 200). (F): Carbon-tetrachloride-

induced cirrhosis of the rat liver, stained with a-smooth muscle actin (aSMA) antibody. HSC (arrowhead) are immunoreactive, as well as a

subpopulation of interface and septal (S) MF (original magnification £ 200). (G): Carbon-tetrachloride-induced cirrhosis of the rat liver, stained

with neural-cell adhesion molecule antibody. HSC (arrowhead) are immunoreactive, as well as MF at the interface, in fibrotic septa (S) and in the

portal tract (upper left corner and middle of the right side) (original magnification £ 200). H: Bile duct-ligated fibrotic rat liver, stained with aSMA

antibody. HSC (arrowhead) are immunoreactive, as well as MF at the portal/parenchymal interface, co-proliferating with reactive bile ductules

(arrows) and cells in the expanding portal tract (right half of the micrograph). (clv: centrolobular vein) (Fig. 3F is a parallel section) (original

magnification £ 200).

cells were also present (Fig. 2A–D). No co-localization of

desmin and N-CAM, nor of GFAP and aSMA could be

detected at the interface (Fig. 3B–E). Interface MF did not

stain with SYN, NT-3, NT-4, Trk-B or p75 antibodies. In

BDL, additional weak expression of NGF, BDNF and Trk-

C could be detected at the interface (Fig. 3F–H and Tables

4 and 5).

3.3. Portal/septal MF


Portal MF were uniformly immunoreactive to aSMA

(Fig. 1A), a subset showed GFAP (Fig. 2E), BDNF or

ABCRYS (Fig. 1D) expression. (Myo)fibroblastic cells

forming the core of the fibrotic septa, defined as the area


bilaterally lined by the GFAP-positive interface, showed

exactly the same expression profile as portal tract MF.

Comparison of serial sections showed that the number of

portal/septal MF staining with aSMA clearly exceeded the

number of cells stained with any other marker (not shown).

No desmin, N-CAM, SYN, NGF, NT-3, NT-4, Trk-B, Trk-

C or p75 immunoreactivity could be detected in either

septal or portal cells (Fig. 1B,C and Table 3).


CCl4-cirrhotic and BDL-fibrotic rat livers showed portal

and septal MF immunoreactive to N-CAM (Figs. 2G and

3A) and aSMA (Fig. 2F,H) antibodies. A subset of portal/

septal cells was desmin-, GFAP- or ABCRYS-positive (Fig.

1E–H and Tables 4 and 5). Double staining for aSMA and

N-CAM was present in portal/septal MF, but co-localization

of neither desmin and GFAP (Fig. 2A–D), nor of desmin


and N-CAM (Figs. 1H and 3A are serial sections, double

staining in Fig. 3E), nor of GFAP and aSMA (Fig. 3B–D)

could be detected. No SYN, NGF (Fig. 3G), NT-3, NT-4,

Trk-B, Trk-C or p75 immunoreactivity could be detected in

either septal or portal cells (Tables 4 and 5).

As was pointed out by our group [8] and others [9]

previously, periductal MF appear to constitute a particular

subtype of portal MF. In CCl4-cirrhotic rat livers, periductal

MF formed a rim of MF, intensely immunoreactive to

aSMA, GFAP and ABCRYS. In BDL rat livers, periductal

MF were immunoreactive to aSMA and GFAP (results not

shown).

4. Discussion

The present immunohistochemical study shows that, in

the final stages of development of cirrhosis in human and

rat, three mesenchymal (myo)fibroblast-like subpopulations

can be distinguished in the liver, based on their localisation

and their expression profile, but regardless of the underlying

disorder.

The first subpopulation we discerned – HSC – stained

with all tested markers and showed most intense and most

widespread staining with aSMA (Fig. 1A) and N-CAM

(Fig. 1B) antibodies in humans, while in rat liver GFAP

(Fig. 1E,G) and desmin (Fig. 1F,H) antibodies stained the

largest proportion. The second subpopulation – interface

MF – showed no expression of SYN, Trk-B, Trk-C, p75

or NT-3, in contrast to HSC [30,31]. The interface MF did

express GFAP, N-CAM, aSMA, NGF, BDNF, NT-4 and

ABCRYS, however, in a varying proportion of cells. The

third subpopulation – portal/septal MF – only showed stain-

ing with aSMA, GFAP, BDNF, ABCRYS, N-CAM and

desmin (the latter two only in rat), in a varying proportion

of cells (Figs. 1–3).

Portal/septal MF in human serial sections showed aSMA

immunoreactivity in a proportion of cells that was clearly

larger than the proportion stained with any of the other

markers (Fig. 1A), while the same was true for N-CAM

expression in portal/septal MF in rat serial sections (Figs.

2G and 3A). Double immunostaining in rat sections showed

no overlap between N-CAM and desmin (Fig. 3E) or

between N-CAM and GFAP in these portal/septal MF,

while they did show co-expression of N-CAM and aSMA.

In normal rats, N-CAM expression is restricted to cells in

the portal tract ([13], own unpublished data). Desmin-posi-

tive cells in the septa of cirrhotic rats were already described

to be restricted to the interface, while the cells in the core of

septa were aSMA-positive, desmin-negative [25]. In

normal human liver, the portal tract contains some

aSMA-immunoreactive mesenchymal cells, while the

majority of lobules is devoid of aSMA-positive cells

([28], own unpublished data). The combination of these

findings could suggest the existence of a large subpopula-

tion of portal/septal MF in fibrotic/cirrhotic liver that

expresses N-CAM (and aSMA) in rats, aSMA in humans,

and that this subpopulation would be related, not to lobular

HSC but to the portal mesenchymal cells that are detected in

normal liver (in rat: N-CAM 1 , in human: aSMA 1 portal

cells). Recently, analogous findings led to the postulation of

at least two lineages (HSC and septal MF) in rat liver, char-

acterized by distinct behaviour and expression profile in

vivo and in vitro [35,36]. Transition from one lineage to

the other, for instance by (trans)differentiation, was not

excluded conclusively, however. Our study largely corrobo-

rates the results – not the conclusions – of Knittel et al. [35],

albeit through the application of a distinct panel of func-

tional markers, and extends their findings to human pathol-

ogy. In addition to the two mesenchymal (myo)fibroblast-

like cell populations they discriminated, we define a third –

the interface MF – that shows a phenotype, intermediate

between portal/septal MF and HSC.

The demonstration of ultrastructural parallels between

perisinusoidally located HSC, perisinusoidal transitional

cells, perisinusoidal MF and septal MF [1,5–7] is no

proof of a direct precursor–product relationship between

perisinusoidal HSC and septal MF. Moreover, MF-like


Fig. 3. Single and double immunostaining. (A): Bile duct-ligated fibrotic rat liver, stained with neural-cell adhesion molecule (N-CAM) antibody.

Hepatic stellate cells (HSC) (arrowheads) are immunoreactive, as well as myofibroblasts (MF) at the portal/parenchymal interface, co-proliferating

with reactive bile ductules (arrows) and MF in the expanding portal tract (lower right corner). (clv: centrolobular vein) (Fig. 1H is a parallel section)

(original magnification £ 200). (B–D): Bile duct-ligated fibrotic rat liver, stained with alpha-smooth muscle actin (aSMA, green: B, C) and glial

fibrillary acidic protein (GFAP) (red: C, D) antibodies. aSMA (green) stains MF in the portal tract (which lies around the portal vein (PV)), MF co-

proliferating with reactive bile ductules (arrow, granular staining pattern in B, C) and vessel walls (portal artery: upper right corner, B, C). GFAP

(red) stains HSC (lower right corner, C, D), MF in the portal tract, MF co-proliferating with reactive bile ductules (arrow) and vessel walls (upper

right corner, C, D). Except in the portal vessels (yellow, C), no co-localization of aSMA and GFAP could be detected (original magnification £ 200).

(E): Bile duct-ligated fibrotic rat liver, stained with desmin (green) and N-CAM (red) antibodies. Desmin (green) stains HSC, MF at the interface and

MF in the expanding portal tract (PT), while N-CAM (red) mainly stains MF at the interface and portal MF. No co-localization is seen in any of these

compartments (original magnification £ 400). (F): A section adjacent to the section shown in Fig. 2H, stained with neurotrophin-4 antibody. HSC

(arrowheads) are immunoreactive, as well as reactive bile ductules (arrows). (clv: centrolobular vein) (original magnification £ 200). (G): Carbon-

tetrachloride-induced cirrhosis of the rat liver, stained with nerve growth factor antibody. HSC are immunoreactive (arrowheads), while interface

and septum (S) are negative (original magnification £ 200). (H): Carbon-tetrachloride-induced cirrhosis of the rat liver, stained with brain-derived

nerve growth factor antibody. HSC (arrowhead) are immunoreactive. Reactive bile ductules (arrows) show immunoreactivity located at the luminal

membrane, smooth muscle cells in portal vessel walls are also positive. (Lower left corner: portal vein and artery in portal tract; S: septum) (original

magnification £ 200).

cells have been shown to reside in the perisinusoidal space

in normal baboon liver [11] and could be the precursors of

proliferating perisinusoidal MF in the diseased liver,

instead of HSC. The existence of functional in vivo and

in vitro parallels between HSC and septal MF, e.g. produc-

tion of extracellular matrix constituents or expression of

‘promiscuous’ differentiation-related markers, is no final

proof of direct descent either, nor is the demonstration of

in vitro transdifferentiation of HSC to MF-like cells by

culturing them on plastic [2,3]. In addition, our study and

similar discontinuous, sequential or ‘snapshot’ in vivo

studies – immunohistochemical or ultrastructural – do not

allow to follow the fate of individual cells or cell types

during the development of liver fibrogenesis. In conse-

quence, we have to conclude that, at the moment, we do

not know which mesenchymal (myo)fibroblast-like cell

type of the liver (see Section 1) is the precursor pool of

septal extracellular matrix-producing MF, in spite of poten-

tial clinical relevance.

In conclusion, our study shows that in cirrhosis, three

subpopulations of mesenchymal (myo)fibroblast-like cells

can be discriminated, largely independent of the cause of

cirrhosis or the species studied and that septal MF share

more characteristics with portal MF than with HSC. The

latter finding appears conflicting with the commonly postu-

lated precursor–product relation between HSC and septal

MF, but does not exclude it.

Acknowledgements

Paula Aertsen and Martine Verhoeven were indispensa-

ble for this study, due to their excellent technical skills. The

help of Anita Omasta in establishing the rat models was also

greatly appreciated. D.C. is supported by an ‘Aspirant

FWO’ grant from the F.W.O.-Vlaanderen.

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Hepatic stellate cell/myofibroblast subpopulations in fibrotic human and rat livers

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