J. Anat. (1997) 191, pp. 291–299, with 4 figures Printed in Great Britain 291

Increase in immunoreactivity for endothelin-1 in blood vessels

of rat liver metastases : experimental sarcoma and carcinoma

A. LOESCH1, M. TURMAINE1, M. LOIZIDOU2, R. CROWE1, S. ASHRAF1, I. TAYLOR2

AND G. BURNSTOCK1

"Department of Anatomy and Developmental Biology and Centre for Neuroscience, and #Department of Surgery, University

College London, UK

(Accepted 20 May 1997)

Using electron immunocytochemistry, blood vessels in the normal rat liver and in 2 different animal models

of liver metastases : (1) Hooded Lister rat with MC28 tumour, a sarcoma, and (2) nude rat with HT29

tumour, a carcinoma, were investigated for the presence of endothelin-1. In the normal livers, small

subpopulations of vascular endothelial cells displayed discrete immunoreactivity for endothelin-1. In the

livers with malignant tumours, there was a substantial increase in endothelin-1-immunoreactive endothelial

cells in vessels located at the tumour periphery. In the controls, antibody to endothelin-1 also labelled

sporadically some fibroblast}fibroblast-like cells associated with the blood vessels. In contrast, intense

immunoreactivity for endothelin-1 was frequently associated with the tumour cells and}or fibroblast cells in

both types of tumour examined.

Key words : Vasculature ; endothelium; fibroblasts ; liver metastases.

Hepatic metastases from colorectal cancer are a

frequent clinical problem. Despite the availability of a

large number of treatment options, cure remains

elusive. Hepatic artery infusion chemotherapy with 5-

fluorouracil and systemic leucovorin is probably the

best available form of adjuvant therapy (Taylor,

1992). Vascular manipulation of hepatic artery in-

fusion chemotherapy by vasoconstrictor agents

appears to improve drug delivery to the tumour as

well as tumour response rates (Goldberg et al. 1990).

A better understanding of vascular control mech-

anisms in the normal liver and colorectal liver

metastases may help to improve the vascular ma-

nipulation of hepatic artery infusion chemotherapy by

vasoconstrictor agents.

In mammalian species the neuronal control of the

normal liver vasculature utilises noradrenaline, tyro-

sine hydroxylase and various peptides, including

substance P, vasoactive intestinal polypeptide, calci-

tonin gene-related peptide, somatostatin, and neuro-

Correspondence to Professor G. Burnstock, Department of Anatomy and Developmental Biology and Centre for Neuroscience, University

College London, Gower Street, London WC1E 6BT, UK. Tel. -44171 387 7050; Fax: -44171 380 7349.

peptide Y (Gulbenkian et al. 1985; Goehler et al.

1988; Burt et al. 1989; Inoue et al. 1989; Ding et al.

1991; Ueno et al. 1991; Fehe! r et al. 1992). The blood

vessels of the experimental rat liver tumour(s) do not

have smooth muscle layer in their walls and are

devoid of autonomic perivascular innervation (Ashraf

et al. 1997). It is therefore likely that endothelial cells

in the tumour blood vessels play a major part in the

regulation of local blood flow. Standard electron

microscopy of human and rat liver tumours has

already disclosed variations in the ultrastructural

organisation of the blood vessels, including variations

in the diameter of the vessel lumen and the com-

position of the perivascular}adventitial region (Ashraf

et al. 1996, 1997). All these vessels, however, contained

endothelial cells displaying ultrastructural features

similar to those observed in the uninvolved healthy

regions of the liver.

As far as we are aware, to date the ultrastructural

localisation of endothelial vasoactive agents in tumour

vasculature have not been investigated in any animal

model of colorectal liver metastases. In the present

292 A. Loesch and others

study we have examined 2 different types of animal

model : the syngeneic MC28 fibrosarcoma, a con-

nective tissue tumour which was originally raised in

Hooded Lister rats (Murphy et al. 1986; Loizidou et

al. 1991), and the xenogeneic HT29 human colorectal

cancer cell line in nude rats. The latter rat model was

developed in a small series of experiments in our

laboratory (Department of Surgery, University Col-

lege London, London, UK). In the present paper, the

immunoreactivity to endothelin-1 (ET-1), a potent

vasoconstrictor agent and mitogenic agent

(Yanagisawa et al. 1988; Hirata et al. 1989), was

examined at the ultrastructural level in the rat liver

tumours of epithelial origin (HT29) and fibroblast

origin (MC28).

Animals

Inbred male Hooded Lister rats weighing 250–300 g

and nude (RH-rnu) rats (Harlan-Olac UK Ltd,

Bicester, UK) weighing 200–250 g were used. The

animals were housed in standard cages and main-

tained on a routine rat pellet and water diet ad

libitum. The animals were divided into 3 groups:

experimental (n¯ 6, injected with MC28 or HT29

cells, in Hooded Lister and nude rats respectively),

control (n¯ 6, injected with PBS) and normal (n¯ 2,

no surgical intervention).

Inoculation of cancer cells

Animals were anaesthetised with a mixture of halo-

thane and oxygen and a midline laparotomy was

performed. The caecum and terminal coils of the

ileum were delivered via the wound and the ileocolic

vein was identified in the mesentery. 2¬10' MC28

cells or 1¬10( HT29 cells (0±5 ml of cell suspension)

were injected into the ileocolic vein (of Hooded Lister

and nude rats, respectively) using a fine needle (27G).

To avoid backflow of blood, pressure was applied at

the puncture site on withdrawal of the needle. The

dose of cells used for the HT29 inoculation (1¬10()

was determined as optimal after a small dose response

study. Animals (n¯ 6 per group) were inoculated

with different doses (0±5, 0±75, 1, or 2¬10() as

described above. After 3 wk, the animals given

0±5¬10( tumour cells did not develop liver tumours,

while 2}6 of those which received 0±75¬10( cells and

5}6 from the 1¬10( cell group developed liver

tumours. Higher tumour doses were impractical to

administer because the high cell density resulted in cell

clumping and lower tumour take (3}6 animals).

Tissue specimens

Terminal anaesthesia by an overdose of sodium

pentobarbitone (Sagatal, RMB, Animal Health,

Dagenham, UK) was administered to the animals

2 wk after surgery in Hooded Lister rats and 3 wk

after surgery in nude rats for perfusion fixation

through the heart with 4% paraformaldehyde and

0±1% glutaraldehyde in 0±1 phosphate buffer

(pH 7±4). In the experimental group, tumour, along

with adjacent normal liver, was excised after

perfusion-fixation and divided into appropriate tissue

blocks. In the control group, the tissue was taken

from 2 different regions of the right lobe of the liver ;

one at the hilum and the other some distance away

from it. In the normal group, the tissue was taken

from the same 2 regions of each of the major lobes

(right, left and median). The specimens were then

placed in the same fixative for about 5 h at 4 °C,

washed in phosphate buffer and stored overnight at

4 °C in 0±05 Tris-buffered saline (TBS, Dako,

Carpinteria, CA, USA). The following day, thick

sections (100 µm) were cut on a vibratome and

processed for ET-1 by the pre-embedding peroxidase-

antiperoxidase (PAP) electron immunocytochemistry.

Specimens were exposed to 0±3% hydrogen peroxide

in 50% methanol for 30 min (for blocking of

endogenous peroxidases), washed in TBS, and then

exposed to normal goat serum (Nordic Immunology,

Tilberg, the Netherlands) diluted 1:9 in TBS con-

taining 0±1% sodium azide (this buffer was used for

the dilution of antibodies) for 1±5 h. After rinsing in

TBS the specimens were incubated for 48 h at 4 °Cwith rabbit polyclonal antibody to ET-1 at a dilution

of 1:1000. After washing in TBS, the specimens were

then exposed to goat-antirabbit immunoglobulin G

serum (Biogenesis, Bournemouth, UK) diluted 1:40.

After washing in TBS the specimens were incubated

for 3 h with a rabbit PAP complex (DAKO, Glostrup,

Denmark) diluted 1:75, and next treated with 3,3«-diaminobenzidine (Sigma, Poole, UK) and 0±01%

hydrogen peroxide. After washing in TBS and 0±1

cacodylate buffer (pH, 7±4), the specimens were trans-

ferred to 1% osmium tetroxide (in cacodylate buffer)

for 1 h, washed in cacodylate buffer, dehydrated in

graded series of ethanol and propylene oxide and flat

embedded in Araldite. Ultrathin sections were stained

with uranyl acetate and lead citrate and subsequently

examined with a JEM-1010 electron microscope.

Localisation of endothelin in experimental liver metastases 293

Controls

The rabbit polyclonal ET-1 antibody to synthetic

human ET-1 (Cambridge Research Biochemicals

(CRB), Cambridge, UK) has been used in PAP

electron immunocytochemistry of endothelial cells

(Loesch et al. 1991; Loesch & Burnstock, 1995, 1996;

Gorelova et al. 1996) as well as immunoassay to detect

ET levels in the perfusate of freshly harvested

endothelial cells (Milner et al. 1990). Preabsorption of

ET-1 antibody with its respective antigen (synthetic

human ET-1, CRB) at a concentration of 10−%

eliminated positive labelling in human and animal

vascular endothelial cells (Loesch et al. 1991; Loesch

& Burnstock, 1995). An inhibition enzyme-linked

immunoabsorbent assay (ELISA) of ET-1 antibody

showed that this antibody cross-reacted with pro-

endothelin 39 (7%), ET-2 (15%) and ET-3 (100%)

(Bodin et al. 1992). Preincubation of ET-1 antibody

with 10 nmol of human ET-1, ET-2 or ET-3 sub-

stances per ml of optimally diluted antibody was

sufficient to abolish immunostaining (CRB). The

dilution of anti-ET-1 antibody used (1:1000) was in

concert with the previous immunocytochemical

studies showing that at this concentration the anti-

body produces a good quality ET-1-immunosignal

(and very low background, if any) in endothelial cells

of various vascular beds (Loesch et al. 1991; Loesch &

Burnstock 1995; Gorelova et al. 1996). In the present

PAP study, the specificity of the immunolabelling was

investigated routinely by omission of the primary

antibody and IgG steps, independently, as well as by

replacement of primary antibody by nonimmune

normal rabbit serum (Nordic Immunology). No

labelling was observed in these control preparations.

Semiquantitative assessment

Limited quantitation has been made on endothelial

cells. In order to establish the percentage of en-

dothelial cells positive and negative for ET-1, cells

were counted by direct examination with the electron

microscope of ultrathin sections taken from (1)

hepatic parenchyma with sinusoidal and portal tract

vessels of the 2 specimens of normal liver from 2 rats

and (2) from the 2 specimens of HT29 liver tumour (2

rats) and 2 specimens of MC28 liver tumour (2 rats).

This semiquantitative data was based on analysis of

relatively small fragments of normal and tumour

livers (approximately 5–6 mm# of each specimen) and

therefore should only be treated as a guide to the

proportion of immunopositive and immunonegative

endothelial cells observed.

Normal liver

In the normal}healthy livers of Hooded Lister and

nude rats, only weak immunoreactivity for ET-1 was

displayed in endothelial cells (Fig. 1a–c) and stronger

in fibroblast}fibroblast-like cells (Fig. 1a). Immuno-

reactivity for ET-1 was associated with the cytoplasm

and membranes of intracellular organelles}structures

in vascular endothelial cells and fibroblast-like cells.

In both types of rats, only about 2% of sinusoidal and

portal tract vessel endothelial cells displayed ET-1

immunoreactivity (8 out of total 420 endothelial cells

examined); no immunoreactivity to ET-1 was

observed in portal artery and central vein. Blood

vessels showing weak endothelial immunoreactivity

for ET-1 and}or lack of ET-1-immunoreactivity were,

in fact, similar to those observed in procedural control

preparations for immunocytochemistry (data not

shown).

Liver metastases

A postmortem examination of the liver, performed on

d 15 of laparotomy in Hooded Lister rats and on d 21

of laparotomy in nude rats, revealed 1 to 5 foci of

metastatic tumour. Their size ranged from 0±5 to

5 mm in diameter.

HT29 tumour

This tumour showed moderate to poor differentiation.

Cells were generally arranged in irregular clusters

sometimes forming alveoli. At the junction of the

tumour with the normal liver, large blood vessels

resembling those of the portal tracts were sometimes

observed. Tumour blood vessels consisted of a layer

of endothelial cells resting on a basement membrane

and surrounded by a layer of fibroblast-like cells.

Vascular smooth muscle and perivascular nerve

fibres}varicosities were absent in these vessels. Some

endothelial cells showed fenestrations.

In this tumour, immunoreactivity for ET-1 was

observed. Immunoreactivity for ET-1 was more

prominent in the tumours of larger size. The en-

dothelial cells of various size blood vessels were ET-1-

positive (Fig. 2a–c). The blood vessels with the ET-1-

positive endothelium, however, were located in the

tumour periphery near the border (junction) with the

normal-looking liver tissue. Cross-sections of the

tumour showed the presence of approximately 50

‘peripheral ’ vessels (per 5–6 mm# of tumour tissue

examined in each specimen). In the central part of

294 A. Loesch and others

Fig. 1. Blood vessels of the portal tract region of normal rat liver labelled for ET-1. (a) Note discrete ET-1-labelling—black precipitate

(arrows) in endothelial cells (En) of small portal vein; stronger labelling is seen in a fibroblast (Fib). Ductal endothelial cells (Ep) can also

be seen. N, nucleus ; lu, lumen of small portain vein; ibd, interlobular bile duct (¬21400). (b) A capillary displaying endothelial labelling

for ET-1 (¬10700). (c) A magnified fragment of capillary illustrated in (b) showing ET-1-immunoprecipitate in endothelial cell (¬20000).

tumour there was scarcity of the vessels and lack of

ET-1-positive endothelium. Not all endothelial cells in

tumour ‘peripheral ’ vessels were ET-1 positive: about

10% of endothelial cells displayed ET-1-immuno-

reactivity (23 out of 227 endothelial cells examined).

Some sections of the vessels, however, displayed

whole endothelium positive for ET-1, as for example

observed in sinusoidal capillaries at the normal-

tumour junction (Fig. 2c). In ET-1-immunoreactive

endothelial cells the immunoprecipitate was localised

in the cytoplasm and in association with the mem-

branes of intracellular organelles and structures

including the endoplasmic reticulum, mitochondria,

nucleolemma and cytoplasmic}subplasmalemmal

vesicles.

In addition to ET-1-positive endothelium, the

stroma cells and}or fibroblast}fibroblast-like cells,

but not the tumour cells displayed immunoreactivity

for ET-1 (Fig. 3a, b). These ET-1-positive cells

contained a number of mitochondria. Cisternae of

Localisation of endothelin in experimental liver metastases 295

Fig. 2. Blood vessels with different diameters in HT29 liver metastases labelled for ET-1. (a) Small vessel in the tumour periphery showing

one ET-1-positive and two ET-1-negative (black asterisks) endothelial cells. Note that the ET-1-positive cell contains an inclusion (star)

resembling a large sized multivesicular body; ET-1-negative cells contain numerous mitochondria (m) and multivesicular bodies (mvb). er,

endoplasmic reticulum; lu, lumen (¬13300). (b) A larger vessel in the tumour periphery displays endothelial cells positive for ET-1; ET-

1-negative cells can also be seen (black asterisk). In the perivascular region note richness of collagen fibres (col). Fib, fibroblast (¬16500).

(c) Whole sinusoidal endothelium lining capillary from the border region displays immunoreactivity for ET-1. N, nucleus ; Go, Golgi complex;

H, hepatocyte (¬10900).

296 A. Loesch and others

Fig. 3. HT29 liver metastases labelled for ET-1. (a) Two fibroblast}stroma-like cells display intense cytoplasmic immunoreactivity for ET-

1. N, nucleus ; m, mitochondria ; col, collagen fibres (¬16400). (b) ET-1-positive fibroblast-like cell containing electron-lucent}empty

vacuoles (va). Unlabelled cells can also be seen (black stars) (¬9400).

endoplasmic reticulum were frequently enlarged}swollen. Some ET-1-positive cells displayed large

electron-lucent vacuoles (Fig. 3b). Similar to en-

dothelial cells, the ET-1-immunoprecipitate was

localised in the cytoplasm and in association with the

membranes of intracellular organelles and structures.

MC28 tumour

This tumour was poorly differentiated. It consisted of

masses of fibroblast-like cells with very little stroma;

the latter contained thin walled blood vessels. The

tumour blood vessels ranged in size from 8 to 60 µm.

They were more frequently observed in the periphery

of the tumour and were absent in the centre of the

tumour. The wall of these blood vessels consisted of a

single layer of endothelial cells resting on a continuous

basement membrane. Vascular smooth muscles and

perivascular nerve fibres were not observed. As in the

HT29 tumour, the blood vessels resembling those of

the portal tract were observed at the junction of the

tumour tissue with normal hepatic parenchyma.

Localisation of endothelin in experimental liver metastases 297

Fig. 4. MC28 liver metastases labelled for ET-1. (a) A blood vessel from the peripheral region of the tumour displays immunolabelling for

ET-1 in the vascular endothelium (En) ; an unlabelled endothelial cell is also seen (black asterisk). N, nucleus ; lu, lumen (¬8250). (b) The

endothelium in the sinusoidal capillary is ET-1-negative, whilst a pericyte-like cell (pe) is ET-1-positive. er, endoplasmic reticulum (¬15600).

(c) Note two tumour cells displaying cytoplasmic reactivity for ET-1; ET-1-negative tumour cells can also be seen (black stars). Go, Golgi

complex; m, mitochondria (¬10300).

This tumour displayed immunoreactivity for ET-1.

Immunoreactivity for ET-1 was localised in approxi-

mately 20% of endothelial cells (38 out of 201

endothelial cells examined) of blood vessels located in

the tumour periphery rather than in the central parts

(Fig. 4a). Sinusoidal capillaries, however, were nega-

298 A. Loesch and others

tive for ET-1. In general, in the blood vessels with

immunoreactive endothelium as well as in vessels

with immunonegative endothelium, perivascular}adventitial structures were scarce. Occasionally, how-

ever, cell profiles resembling pericytes were positive

for ET-1 (Fig. 4b). Some tumour cells were also

positive for ET-1 (Fig. 4c).

The present data show that both types of tumours

examined (HT29 carcinoma and MC28 fibrosarcoma)

display an increase in endothelial immunoreactivity

for ET-1. Immunoreactivity for ET-1 was also seen in

fibroblast-like cells in the control livers and more

frequently in both types of tumours; MC28 tumour

but not HT29 also contained some ET-1-positive

tumour cells.

The present data suggest an increased content of

vasoconstrictor ET-1 in endothelial cells from liver

metastases. However, a decreased sensitivity of tu-

mour blood vessels to ET-1 as well as to angiotensin

II and platelet-activating factor has been demon-

strated in sponge implants (Colon 26) in mice

(Andrade et al. 1992). Whether the increased pro-

duction (‘overproduction’) of ET-1 in the endo-

thelium of tumour vessels examined was part of a

compensatory mechanism to balance the depletion of

sensitivity of the vessels to ET-1 or whether ET-1 did

produce substantial vasoconstriction is not clear. It

has been shown that the neovasculature in tumour

does not respond to vasodilator agents (e.g. hy-

dralazine), suggesting that these vessels are in a state

of near-maximal vasodilation (for review see Peterson,

1991).

It is also possible that the increased production of

ET-1 in the endothelium of the tumour liver is, in fact,

linked with stimulation of the growth of the vessels

rather than with the vasomotor control of neo-

vasculature. It has already been shown that ET-1 may

stimulate the growth of endothelial cells and vascular

smooth muscle (Yanagisawa et al. 1988; Hirata et al.

1989). Interestingly, in patients with liver metastases a

significant increase in plasma levels of ET-1 is

observed (A. Shankar et al. unpublished results).

While the presence of ET-1 in vascular endothelial

cells can be related to vasoactive action of this agent

and endothelial control of the diameter of the normal

and}or tumour blood vessels examined in the present

study, the role of ET-1 in some stroma}tumour

and}or fibroblast}fibroblast-like cells (cells immuno-

reactive for ET-1) is enigmatic at this stage. Recent

studies suggest that fibroblasts associated with non-

malignant pathological conditions, such as fibrosis}cirrhosis are contractile in response to ET-1 (Bauer et

al. 1995; Pinzani et al. 1996) ; therefore fibroblast cells

surrounding tumour blood vessels may also be capable

of contraction. It seems rather unlikely that these cells

represent ‘neoendothelial cells ’ involved in angio-

genesis. However, most of the ET-1-positive cells

displayed features of cells with high metabolic state,

e.g. rich in mitochondria and endoplasmic reticulum,

vacuoles and large Golgi complex. Whether, there is

any physiological relationship between the ET-1-

positive cells in the stroma and the ET-1-positive

endothelium in the walls of blood vessels supplying

MC28 or HT29 tumour in the Hooded Lister rat or

nude rat livers remains unknown at this stage.

In this study, the MC28 tumour cells were immuno-

reactive for ET-1. A number of human tumour cell

lines have been shown to produce ET-1 in vitro and it

has been suggested that the cells use it in an

autocrine}paracrine fashion (Kuhusura et al. 1990;

Shichiri et al. 1991; Ishibashi et al. 1993). However, it

is interesting to note that HT29 when transplanted

and grown in vivo for these experiments did not

produce ET-1, unlike its behaviour in vitro where the

cells not only expressed ET-1 mRNA but also

produced a secreted ET-1 (Kuhusara et al. 1990).

In conclusion, the present data indicate that

endothelial cells of blood vessels in both types of liver

metastases (HT29 and MC28) examined displayed

increased immunoreactivity for ET-1. Further studies

on endothelial localisation of various vasoactive

agents (including nitric oxide synthase, the enzyme

synthesising the potent vasorelaxant nitric oxide;

Ignarro et al. 1987; Palmer et al. 1987) in liver

metastases are needed to understand the role of

endothelium in the control of flow in these blood

vessels and the significance of increased ET-1 in

several cell types. Identification of ET receptors would

also contribute to better understanding of vascular

involvement in pathology of liver tumours.

The authors thank Mr R. Jordan for editorial as-

sistance. Financial support of the British Heart

Foundation (A.L.), the Ministry for Science and

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