Evidence of angiogenesis in primary biliary cirrhosis: animmunohistochemical descriptive study

Jesus Medina1,†, Paloma Sanz-Cameno1,†, Luisa Garcıa-Buey1, Samuel Martın-Vılchez1,Manuel Lopez-Cabrera2, Ricardo Moreno-Otero1,*

1Unidad de Hepatologıa (planta 3), Hospital Universitario de la Princesa, Universidad Autonoma de Madrid, Diego de Leon 62, E-28006 Madrid, Spain2Unidad de Biologıa Molecular, Hospital Universitario de la Princesa, Universidad Autonoma de Madrid, Diego de Leon 62, E-28006 Madrid, Spain

0168-8278/$30.00 q 2004 European Association for the

doi:10.1016/j.jhep.2004.09.024

Received 30 July 2004; received in revised form 8

accepted 17 September 2004; available online 26 Octob

* Corresponding author. Tel.: C34 913093911; fax: CE-mail address: rmoreno.hlpr@salud.madrid.org (R.

† Both authors contributed equally to this work.

See Editorial, pages 7–11

Background/Aims: The intrahepatic inflammatory process occurring during primary biliary cirrhosis contributes to

bile duct destruction, but the cellular and molecular pathways involved are largely unknown. Furthermore, additional

pathogenetic mechanisms may exist. We aimed at evaluating the cellular infiltrate phenotype; the expression of

lymphocyte activation, antigen recognition and cell-adhesion molecules; the occurrence of hepatic angiogenesis and themolecules involved.

Methods: Immunohistochemical investigations were performed in frozen liver biopsy sections from primary biliary

cirrhosis patients.

Results: CD8C and CD69C T cells were predominant in inflammatory infiltrates around damaged cholangiocytes;

b2-microglobulin conformational epitope and intercellular adhesion molecule-1 expression were enhanced in bile ducts

and hepatocytes. Inflamed portal areas showed vascular cell adhesion molecule-1 up-regulation; formation of tubule-

like structures (neovessels) by endothelial cells expressing vascular endothelial-cadherin and CD-31; vascular

endothelial growth factor expression in surrounding sinusoidal endothelial cells; and enhanced expression ofangiopoietins 1 and 2, their receptor Tie-2 and endoglin, suggesting their involvement in new vascular structure

formation.

Conclusions: The inflammatory infiltrate in primary biliary cirrhosis shows an increased reactivity for lymphocyte

activation, antigen recognition and cell- and vascular-adhesion molecules. Additionally, intrahepatic angiogenesis

occurs, involving vascular endothelial growth factor, angiopoietins 1 and 2, Tie-2 and endoglin in neovessel formation.

q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Keywords: Primary biliary cirrhosis; Lymphocyte activation; Adhesion molecules; Angiogenesis; Vascular endothelial

growth factor; Angiopoietins

1. Introduction

Primary biliary cirrhosis (PBC) is a chronic inflamma-

tory liver disease of multifactorial etiopathogenesis, charac-

terized by the presence of an intrahepatic mononuclear

cell infiltrate, as well as circulating autoantibodies [1,2].

Study of the Liver. Pub

September 2004;

er 2004

34 914022299.

Moreno-Otero).

The frequent association of PBC with other autoimmune

conditions and its similarity with graft-versus-host disease

[3] emphasize the likelihood that host immune mechanisms

are decisive.

It has been suggested that immunological mechanisms

involving T-lymphocyte-mediated lysis are important in the

characteristic bile-duct and hepatocellular damage occur-

ring in PBC [4–7]. The induction of T-cell mediated

immune responses is triggered by antigen-specific recog-

nition and cell adhesion molecules, which are crucial for

inflammatory reactions. A noteworthy previous step is

extravasation of inflammatory cells through the vascular

Journal of Hepatology 42 (2005) 124–131

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Table 1

Baseline characteristics of patients with primary biliary cirrhosis

Sex (M/F) 1/18

Age (years) [range] 51G10 [35–63]

ALT (IU/l) 103G72

AP (IU/l) 893G593

Bilirrubin (mg/dl) 0.94G0.20

Histological stage

I 5

II 8

III 6

ALT, alanine aminotransferase; AP, alkaline phosphatase.

J. Medina et al. / Journal of Hepatology 42 (2005) 124–131 125

endothelium to accumulate in areas of inflammation and

damage. The mechanisms responsible for leukocyte

migration [8,9] are based on specific interactions between

proteins from leukocyte membranes and endothelial sur-

faces, known as vascular adhesion molecules [10–13].

Investigation of the expression pattern of these factors may

lead to a better understanding of the pathogenetic

mechanisms of PBC.

The excessive accumulation of inflammatory infiltrates,

together with the accumulation of extracellular matrix and

development of fibrosis in the livers of PBC patients, may

result in an increased resistance of the tissue to blood flow

and to the delivery of oxygen, which thereby becomes

hypoxic. Under these circumstances, it is conceivable that

an angiogenic switch occurs, leading to the upregulation of

pro-angiogenic factors, and to the formation of neovessels,

as described for other chronic inflammatory liver diseases

such as chronic viral hepatitis [14,15]. Vascular Endothelial

Growth Factor (VEGF) is the most thoroughly described

pro-angiogenic factor [16]. VEGF induces endothelial cell

(EC) proliferation by binding to two tyrosine kinase

receptors: kinase insert domain receptor (KDR) and fms-

like tyrosine kinase receptor (Flt-1). Its promoter contains

hypoxia-inducible factors—responsive elements. VEGF

plays a crucial role in virtually all pathological situations

in which angiogenesis occurs [16], including liver diseases

such as hepatocellular carcinoma [17] and chronic hepatitis

C [15,18], making it a suitable candidate target for

therapeutic blocking of angiogenesis [19–21]. Besides

induction of EC proliferation, effective angiogenesis also

requires stabilization of the nascent vessels, establishment

of interendothelial junctions and formation of a lumen [22].

Angiopoietin 1 (Ang-1) stabilizes neovessels by binding the

Tie-2 receptor, thereby affecting junctional molecules [23]

and facilitating communication between ECs and mural

cells [24]. However, an excess of Ang-1 makes vessels too

tight and inhibits sprouting [25]. Ang-2 may exert opposing

effects: in the absence of VEGF, Ang-2 acts as an antagonist

of Ang-1, destabilizes vessels and causes EC death, leading

to vessel regression [26], but it facilitates sprouting in the

presence of VEGF [25]. Finally, endoglin (CD105), a

vascular-specific Transforming Growth Factor-b (TGF-b)

coreceptor, is involved in a number of processes, including

inflammation, vascular adhesion, matrix remodelling and

angiogenesis: in this respect, endoglin promotes vessel

maturation, stimulates extracellular matrix generation,

induces differentiation of mesenchymal cells to

pericytes and participates in definition of arterio-venous

boundaries [27].

Our aims were: (1) to evaluate the phenotype of cellular

infiltrates, and the expression of lymphocyte activation,

antigen recognition and cell-adhesion molecules in the

livers of PBC patients; and (2) to investigate and

characterize at the molecular level the patterns of reactivity

of proangiogenic factors involved in the formation of

neovessels in PBC liver samples.

2. Material and methods

2.1. Patients and controls

The study protocol was approved by the Ethical Committee of theHospital, and signed informed consent was obtained from each patient.Nineteen PBC patients were studied (Table 1). All tested negative for

hepatitis B surface antigen (HBsAg) by enzyme-linked immunoassay(Abbott Laboratories, North Chicago, IL, USA) and for antibodies tohepatitis C (anti-HCV) by enzyme-linked immunoassay (Orto Diagnostic

Systems, Raritan, NJ, USA). Diagnosis was based on the presence of typicalclinical, serum biochemical, serological and liver histological findings. Allpatients were positive for antimitochondrial antibodies. At the time of liverbiopsy none of the patients was receiving ursodeoxycholic acid,

corticosteroids or other immunosuppressive therapy. Hepatic histologywas staged according to Ludwig et al. [28]. Briefly, stage I was defined asportal inflammation confined to the portal triads; stage II was characterized

by portal and periportal inflammation without septal fibrosis or bridgingnecrosis; in stage III, lobular fibrosis and/or bridging necrosis were presentand stage IV corresponded to cirrhosis. Liver biopsy samples from seven

patients with minimal reactive changes (normal liver) or with histologicalchanges of nonimmune-mediated cholestasis (mild portal inflammationand/or ductular hyperplasia) were also studied. Biopsies were obtained

during abdominal surgery for noncomplicated cholelithiasis and all thesepatients were negative for HBsAg, anti-HCV and autoantibodies.

2.2. Monoclonal antibodies (mAb)

The mAbs used in this study were HP2/6 anti-CD4 (helper T

lymphocytes) [29], B 9.4.2 anti-CD8 (cytotoxic T lymphocytes) [30], TP1/55 anti-CD 69 [31], HP-1H8 anti-b2-MG [32], RR 1/1 anti-CD54 (anti-ICAM-1) [33], 4B9 anti-Vascular Cell Adhesion Molecule-1 (anti-

VCAM-1) (cytokine-activated ECs) [34], TEA 1/5 anti-endoglin (macro-phages, ECs) (F. Sanchez-Madrid, unpublished), TP1/15 anti-CD31 (ECs)[35] and TEA 1/3 anti-Vascular Endothelial cadherin (anti-VE-cadherin)(ECs) [36]. The RR 1/1 mAb was kindly provided by Dr T.A. Springer. All

the mentioned mAbs were hybridoma culture supernatants and were used ata 1/1 dilution in TBS buffer. The P3X63 mouse myeloma supernatant wasused as a negative control in all immunostaining studies.

2.3. Liver tissue studies

All liver biopsies were divided into two parts. One was fixed informaldehyde and embedded in paraffin for routine histological examin-

ation, and the other was snap-frozen and stored at K80 8C until used forimmunohistochemistry. Liver biopsy specimens were evaluated by twopathologists, establishing the histological diagnosis of PBC or cholestaticliver disease from other etiology. Additionally, immunohistochemical

analysis using an indirect immunoperoxidase staining technique wasperformed, as previously described [37,38].

J. Medina et al. / Journal of Hepatology 42 (2005) 124–131126

3. Results

3.1. Histological findings

Staging of fibrosis showed that 5 PBC patients had stage

I, 8 stage II and 6 stage III (Table 1). None of the studied

patients presented an established cirrhosis.

3.2. Phenotype of intrahepatic lymphocytes and expression

of activation molecules

As expected [39], most liver-infiltrating lymphocytes in

PBC were CD4C T cells (data not shown). In areas of bile

duct damage and hepatocellular necrosis, activated CD8C T

lymphocytes and CD69C T lymphocytes were predominant

(Fig. 1(A) and (B)).

3.3. Expression of antigen recognition molecules

An enhanced expression of a b2-microglobulin confor-

mational epitope on bile duct cells as well as on lobular

hepatocytes from patients with PBC was observed (Fig. 1(C)).

Fig. 1. Immunohistochemical characterization of the inflammatory

infiltrate in liver biopsies from patients with primary biliary cirrhosis

(PBC). (A) CD8C T cells were predominant in the inflammatory

infiltrate surrounding damaged bile ducts and zones of hepatocellular

necrosis. (B) These T lymphocytes presented an activated phenotype, as

shown by their CD69 positivity. (C) Both bile duct cells and lobular and

periportal hepatocytes of PBC patients showed a marked staining after

the use of a mAb against an conformational epitope of b2-

microglobulin. (D) An up-regulated ICAM-1 expression was observed

in bile ducts and periportal hepatocytes of PBC patients. (E) Both

sinusoidal lining cells and interstitial cells in portal tracts (here

adopting a dendritic cell-like pattern) presented a clear expression of

VCAM-1. (F) Endoglin was expressed in sinusoidal endothelial cells,

particularly in periportal areas, in association with the inflammatory

infiltrates. Original magnification, !250.

This reactivity to mAb HP-1H8 correlated with inflammatory

activity in a similar pattern to that observed in patients with

chronic viral hepatitis C before antiviral treatment [32].

3.4. Expression of cellular and vascular adhesion molecules

ICAM-1 was only detected in some bile ducts, but its

expression was clearly enhanced in hepatocytes of PBC

patients (Fig. 1(D)) as compared with controls. A notable

finding in PBC, as compared with controls, was the

up-regulated expression of VCAM-1 both in sinusoidal

lining cells and in interstitial cells in portal tracts, here

adopting a dendritic cell-like pattern (Fig. 1(E)).

Endoglin expression was observed in sinusoidal ECs as

well as in vascular ECs in inflamed portal tracts (Fig. 1(F)).

3.5. Expression of endothelial markers

Endothelial immunostaining of liver sections with an

anti-CD31 mAb evidenced the formation of tubular-like

structures (microvessels) in inflamed portal tracts (Fig. 2(B)

and (C)) of PBC patients. This phenomenon was not

observed in the livers of controls, where only sinusoidal

cells showing a typical scattered distribution were stained

(Fig. 2(A)). The neovessels formed in the livers of PBC

patients also showed a marked staining with VE-cadherin, a

molecule present in adherens junctions, that provides

mechanical strength and tightness to the vessels (Fig. 2(E)

and (F)). VE-cadherin was, however, barely detectable in

control livers (Fig. 2(D)).

3.6. Expression of pro-angiogenic molecules

Sinusoidal ECs in PBC livers showed a marked

expression of VEGF, particularly in the periphery of

inflamed portal tracts (Fig. 3(B)). No immunostaining was

observed in control livers (Fig. 3(A)). Angiopoietin 1, a

molecule with a constitutive expression in healthy tissues

and organs (due to its role in homeostatic vessel

stabilization) [23,24], was uniformly expressed throughout

the parenchyma in the livers of both controls and PBC

patients (Fig. 3(C) and (D)). However, detailed visual

observation of the slides suggested an enhanced expression

of Ang-1 in periportal areas of PBC livers, surrounding

areas of inflammatory infiltration (Fig. 3(D)). Angiopoietin

2, a molecule that facilitates vessel sprouting in the presence

of VEGF [25], was not expressed in control livers, whereas

a pronounced expression was observed in the livers of PBC

patients (Fig. 3(E) and (F), respectively). Although a clear

hepatocellular staining was evident, sinusoidal ECs and the

newly tubule-like structures (neovessels) were particularly

positive (Fig. 3(F)). Finally, the expression of Tie-2, the

common receptor of Ang-1 and Ang-2, was investigated.

Interestingly, not only ECs were positive: a diffuse

expression throughout the parenchyma was observed

in the livers of controls (Fig. 3(G)) and in PBC patients

Fig. 2. Immunohistochemical assessment of angiogenesis in liver biopsies from patients with primary biliary cirrhosis (PBC) and controls. Staining of

PBC liver sections with a mAb against CD31 showed that endothelial cells in inflamed portal tracts and in periportal areas acquired a tubule-like

conformation, reflecting the formation of neovessels, i.e. the occurrence of an angiogenic process (panels B and C). Only sinusoidal endothelial cells

stained positive for CD31 in normal livers (panel A). Similarly, staining of endothelial cells with VE-cadherin unveiled the presence of vascular

structures in inflamed portal tracts (panels E and F), which was not observed in normal livers (panel D).

J. Medina et al. / Journal of Hepatology 42 (2005) 124–131 127

(Fig. 3(H)). In addition, an intense immunostaining with

the anti-Tie-2 mAb was detected in clusters of cells within

the inflamed portal areas of PBC patients, consistent with

the formation of new vascular structures (Fig. 3(H)).

4. Discussion

This immunohistochemical study of liver tissue from

patients with PBC sheds light on two phenomena that may

contribute to the pathogenesis of the disease: the elicitation

of a local immune reaction and the occurrence of

angiogenesis.

PBC is an autoimmune liver disease of unknown

etiology. One potential causative factor may be a defect of

lymphocyte function, responsible for an abnormal inhibition

of immune responses [40]. The defective suppressor

function found in patients with PBC may lead to several

humoral and cellular autoimmune phenomena [41–44]. We

previously demonstrated that in patients with PBC,

circulating CD4C, Leu-8C T cells were present in normal

numbers [45] but exhibited abnormal activation and

function [46]. The further demonstration that these abnorm-

alities can be corrected by exposing CD4C, Leu-8C T cells

to an inducer of protein kinase C [47] confirms the central

role that the abnormal function of T lymphocytes plays in

the pathogenesis of PBC. As a result, an immune reactivity

against self structures occurs: a sequential inflammatory and

often granulomatous destruction of small interlobular bile

ducts can be observed in the livers of PBC patients [1,2,48].

The immunological injury caused by this mononuclear

infiltration has been suggested to be responsible for ductal

damage. This has been further supported by evidence from

previous immunohistochemical studies showing that (1)

most of the liver infiltrating mononuclear cells were T

lymphocytes, and (2) B cells were found in the center of

portal tracts, and T lymphocytes around the damaged ducts

and in areas of interfase hepatitis [39,49–51].

Our results on phenotype analysis are consistent with

these data: CD8C T cells were predominant in the

inflammatory infiltrate around damaged cholangiocytes;

furthermore, these CD8C T cells were actively engaged in

a cytotoxic immune reaction, as shown by its marked

positivity for the activation inducer molecule CD69 [52,53];

in addition, the enhanced biliary and hepatocellular

expression of a b2-microglobulin conformational epitope

[32,54] and ICAM-1 [55] could play a critical role in

antigen recognition and adhesion of T lymphocytes to target

cells; finally, the up-regulated expression of VCAM-1 in

inflamed portal areas [14] suggests that these molecules may

be important for the recruitment and priming of T cells in

the liver of PBC patients.

The occurrence of hepatic angiogenesis in the livers of

PBC patients is a novel finding of this study. CD31 and

VE-cadherin positive ECs assemble to form new vascular

structures, mainly in portal and periportal areas, in

association with inflammatory infiltrates and fibrosis.

The observation of an enhanced expression of VEGF,

Ang-1, Ang-2, their receptor Tie-2 and endoglin, suggests

their involvement in EC proliferation and nascent vessel

stabilization.

The formation of neovessels in the liver has been

described both in healthy and pathological conditions [22].

In the first case (e.g. during early developmental phases of

the organism and during liver regeneration), a normal

hepatic vascular network architecture is formed, with

Fig. 3. Immunohistochemical investigation of pro-angiogenic factors in

liver biopsies from patients with primary biliary cirrhosis (PBC) and

controls. VEGF was not expressed in the livers of control individuals

(panel A), whereas a marked expression of this mitogenic factor was

observed in sinusoidal cells and neovessels of PBC livers (panel B). In

panel C, the diffuse hepatocellular expression of angiopoietin 1 can be

observed. This reactivity was more intense in samples from PBC

patients, particularly in those areas of marked inflammation (panel D).

As shown in panel E, angiopoietin 2 was not expressed in control livers,

whereas in PBC samples, hepatocytes and endothelial cells (both those

in the sinusoids and in the neovessels) presented a marked positivity for

this factor (panel F). The mAb against the Tie-2 receptor stained

endothelial cells and lobular hepatocytes of both controls (panel G) and

PBC patients (panel H). However, clusters of cells were also stained

within the inflamed portal areas of the latter (panel H).

J. Medina et al. / Journal of Hepatology 42 (2005) 124–131128

sinusoids representing the main vascular structure respon-

sible for parenchymal irrigation. In liver disease-associated

angiogenesis (e.g. liver tumor, HCV and fibrotic diseases,

and as reported here, PBC), vascular structures of capillary

type are formed (Fig. 4).

This study shows an increased number of vascular

structures in inflamed portal tracts of PBC patients in

comparison with controls. Some authors previously

reported a tendency to vasopenia and decreased peribili-

ary capillary plexus in the livers of PBC, primary

sclerosing cholangitis and autoimmune hepatitis patients

[56,57]. This was attributed to destruction of vascular

structures by autoimmune mechanisms, similarly to the

process undergone by bile ducts in PBC and primary

sclerosing cholangitis. This apparent controversy might

be related to the different dynamics of neoangiogenesis

and autoimmune destruction of vascular structures in

PBC. It seems reasonable to hypothesize that hepatic

angiogenesis is stimulated in PBC, at least in part, by

chronic inflammation and fibrosis, (pro-angiogenic

mediators are locally produced during inflammation;

hypoxia has been described in fibrotic tissue, leading to

stimulation of angiogenesis [22]). At later stages in PBC,

however, vessels (including newly formed capillaries)

might be destroyed during the scarring phases of the

disease. Both processes might coexist in the same

individual. On the other hand, autoimmune destruction

of tissue in PBC is mainly directed against bile ducts.

However, the surrounding peribiliary capillary plexus

might be selectively damaged during the process,

consistent with the mentioned reports [56,57]. The

occurrence of angiogenesis that we describe follows a

different pattern: neovessels are formed mainly in

periportal areas, in association with inflammatory infil-

trates. These spatial characteristics might also account for

the mentioned differences, through mechanisms that

remain unveiled. Therefore, the observation of one or

the other phenomenon (angiogenesis or vasopenia) could

strongly depend on the degree of disease progression at

the time of biopsy and on the area investigated.

We have previously reported that inducible nitric

oxide synthase is induced in the livers of PBC patients,

leading to the local formation of nitric oxide, which may

contribute to the vasodilatation required during the initial

phases of angiogenesis [58,59]. One of the factors that

lead to nitric oxide-mediated vasodilation is VEGF [16],

which is overexpressed in the livers of PBC patients.

Further evidence supporting an active angiogenic process

in PBC is the previously reported finding of positive

immunostaining for b1 integrins and a marked staining

for fibronectin and laminin on ECs of small periductal

vessels [60]; these molecules are directly involved in the

regulation of the angiogenic response [22]. Although

these findings have been primarily associated with the

recruitment of inflammatory cells and the pathogenesis of

bile duct destruction, they might also participate in the

angiogenic process that characterizes PBC.

The results of this study suggest that, in addition to the

already known factors that participate in liver damage in

PBC [1], angiogenesis may play a pathogenetic role.

However, clarification of the extent of this contribution

would require monitoring of markers of vascularization and

quantification of changes in larger numbers of patients. Our

preliminary data provide a qualitative characterization of

Fig. 4. Physiological and pathological hepatic angiogenesis. (A) During liver regeneration, hepatocytes divide and form avascular parenchymal

islands. Then, sinusoidal endothelial cells invade the clusters of hepatocytes in a complex process, which involves degradation of extracellular matrix,

proliferation in response to hepatocellular signals, and chemotactic stimuli-guided migration. Patent sinusoids with a normal, physiological structure

are formed. (B) During pathological liver angiogenesis (e.g. in fibrotic livers), the fenestration of sinusoids is lost, stellate cells become activated,

fibrillar extracellular matrix accumulates, and hepatocytes lose microvilli. The result is a distorted sinusoidal structure characterized by

capillarization and formation of neovessels.

J. Medina et al. / Journal of Hepatology 42 (2005) 124–131 129

the phenomenon that represents the necessary basis for

future studies.

Another issue undoubtedly worth investigating, although

beyond the scope of this study, is the modulation by

ursodeoxycholic acid, corticosteroids and/or other immu-

nosuppressants of the molecular mechanisms involved in

PBC-associated hepatic angiogenesis. A foreseeable diffi-

culty in this type of investigation is the need to biopsy

treated patients for follow-up purposes, which may not be

justifiable for ethical reasons. Finally, it will certainly be of

interest to assess the usefulness of anti-angiogenic therapies

in the treatment of PBC. Based on the results of this study,

those therapeutic agents targeting VEGF or its receptors

may be potential candidates [21].

Acknowledgements

This work has been supported in part by grants C03/02

from Instituto de Salud Carlos III, SAF 2001-1414 from

Ministerio de Ciencia y Tecnologıa (to R.M.O.) and

02/3015 from Fondo de Investigaciones Sanitarias (to

J.M.). The authors thank Dr A. Garcıa-Sanchez and Dr S.

Nieto for histological studies and valuable comments,

Dr T.A. Springer and Dr F. Sanchez-Madrid for

kindly providing monoclonal antibodies, Juan A. Martın

(Accion Medica) for help with illustrations and Brenda

Ashley for her assistance with English.

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