Department of Medicine, Royal Free and University College Medical School,
The Rayne Institute, London, WC1E 6JJ, UK
* Present address: University Department of Surgery, University of Western
Australia, Royal Perth Hospital, Perth, Western Australia, 6000,
Australia
Author for correspondence (e-mail: a.foley-comer{at}ucl.ac.uk )
Accepted 8 January 2002
![]() |
Summary |
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Key words: Mesothelium, Wound healing, Tight junction, Fluorescent dyes, Confocal microscopy
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Introduction |
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The mechanisms involved in mesothelial regeneration following injury are
controversial. Hertzler (Hertzler,
1919) was the first to observe that small and large peritoneal
injuries healed at the same rate and concluded that the mesothelium could not
regenerate solely by centripetal migration of cells at the wound edge as
occurs for healing of squamous epithelia. Subsequently, several hypotheses
have been proposed for the origin of the cells in regenerating mesothelium;
these cells include subserosal mesenchymal precursors
(Ellis et al., 1965
;
Raftery, 1973
;
Bolen et al., 1986
), bone
marrow-derived precursors (Wagner et al.,
1982
), free-floating macrophages
(Eskeland and Kjærheim,
1966
; Ryan et al.,
1973
) and free-floating mesothelial cells
(Cameron et al., 1957
;
Watters and Buck, 1973
;
Whitaker and Papdimitriou, 1985). To date, the most accepted proposal is that
repopulating mesothelial cells originate from a pool of pluripotent subserosal
fibroblast-like cells, which migrate to the serosal surface, divide and
differentiate into mesothelial cells (Ellis
et al., 1965
; Raftery,
1973
). However, irradiation studies have demonstrated impaired
local mesothelial regeneration, which was recoverable by addition of
peritoneal lavage cells (Whitaker and
Papadimitriou, 1985
), suggesting that subserosal fibroblasts are
not the source of regenerating mesothelial cells. In addition, studies of the
kinetics of serosal repair demonstrated that subserosal cells were not
essential for mesothelial healing and that the regenerating cells were likely
to originate from the surrounding uninjured serosal surface
(Mutsaers et al., 2000
).
In 1957, Cameron and colleagues proposed that mesothelial healing involved
attachment of free-floating mesothelial cells to the injured surface.
Peritoneal lavage fluid recovered from experimental animals following injury
to the mesothelium was found to contain a significantly higher number of
viable free-floating mesothelial cells two days post injury than the controls
(Whitaker and Papadimitriou,
1985). The increased free-floating cell population was thought to
be caused by the proliferation of mesothelial cells adjacent to
(Johnson and Whitting, 1962
;
Mutsaers et al., 2000
) and
opposing (Watters and Buck,
1973
; Fotev et al.,
1987
) the serosal injury.
In this study, we have conclusively demonstrated that serosal healing
involves incorporation of free-floating mesothelial cells into the
regenerating mesothelium. Fluorescently labelled cell tracking confirmed
implantation of cultured and peritoneal lavage-derived mesothelial cells onto
the denuded wound surface in a well characterised rodent model of normal
serosal repair (Fotev et al.,
1987; Mutsaers et al.,
1997
). Furthermore, proliferation and incorporation of
fluorescently labelled mesothelial cells was demonstrated by
immunolocalisation of proliferating cell nuclear antigen (PCNA) and the tight
junction-associated protein, zonula occludens-1 (ZO-1), respectively.
![]() |
Materials and Methods |
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Primary fibroblasts, used as a control in cell tracking studies, were isolated from the lungs of male Lewis rats. Peritoneal fibroblasts were not used owing to possible contamination with surface mesothelial cells. Diced lung parenchyma (carefully avoiding the serosal surface) was incubated in 1 mg/ml of type II collagenase (Worthington Biochemical Corp, Lakewood, New Jersey, USA) in DMEM for 2 hours at 37°C. The cell suspension was centrifuged at 1000 rpm for 5 minutes, resuspended and cells maintained in DMEM supplemented with 10% FCS, 4mM L-glutamine and antibiotics (penicillin, 100,000 units/l and streptomycin, 50 mg/l). All cells were grown in a humidified atmosphere of 10% CO2 in air at 37°C.
Mesothelial cells, which are embryologically derived from the mesoderm,
share characteristics of both epithelial and mesenchymal cells
(Whitaker et al., 1982) and so
were distinguished from fibroblasts using monoclonal antibodies directed
against human pancytokeratin (dilution 1:20) and human vimentin (dilution
1:400; Dako Ltd, Ely, UK).
Fluorescence labelling of cultured cells
To examine the role of free-floating cell populations in vivo, cultured
mesothelial cells and fibroblasts were fluorescently labelled with the
celltracking probe DiI
(1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbacyanine
perchlorate; Molecular Probes Inc., Eugene, Oregon, USA). Briefly,
subconfluent cultures, up to passage three, were incubated in serum-free DMEM
containing 10 µM DiI for 20 minutes under standard conditions, washed with
phosphate buffered saline (PBS; pH 7.3) and then incubated in standard
supplemented medium for a further 30 minutes according to the manufacturers
instructions. In order to perform studies examining cell proliferation, the
chloromethylbenzamido derivative of DiI, CM-DiI, was used for cell labelling
because of its ability to withstand histological tissue processing
(Andrade et al., 1996).
Previous studies have demonstrated negligible transfer of DiI between
adjacent membranes (Honig and Hume,
1986), an important property for cell tracking. To confirm this
finding, six replicate suspensions containing equal numbers
(2.5x105) of unlabelled and DiI-labelled mesothelial cells
were plated and incubated under standard conditions for 3 days. The proportion
of cells labelled with DiI was then determined by direct cell counting using a
Zeiss MC80 DX fluorescent microscope.
Fluorescence labelling of peritoneal lavage cells
To collect peritoneal lavage cells, male Lewis rats received a widespread
abrasion injury to the anterior peritoneal wall with a sterile gauze swab.
Animals were sacrificed 2 days post injury, when the free-floating mesothelial
cell population was maximal (Whitaker and
Papadimitriou, 1985), and the peritoneal cavity was lavaged with
20 ml serum-free DMEM. The lavage fluid was centrifuged at 1000 rpm for 5
minutes, the cell pellet re-suspended in serum-free DMEM containing 10 µM
DiI and incubated for 30 minutes at 37°C, before washing with PBS and
resuspending the labelled cells in serum-free DMEM.
Peritoneal free-floating macrophages have been suggested as a potential
source of regenerating mesothelial cells
(Eskeland and Kjærheim,
1966; Ryan et al.,
1973
). Therefore, peritoneal free-floating cells were labelled
using the red fluorescent dye, PKH26-PCL (Sigma Aldrich, Poole, UK), which is
specifically taken up by phagocytic cells and remains within the cells for
more than 21 days in vivo (Melnicoff et
al., 1989
). Rats were injected intraperitoneally (i.p.) with 0.5
µM PKH26-PCL 2 days post injury, killed 2 hours later and the peritoneal
cavity lavaged with 20 ml serum-free DMEM to retrieve labelled lavage
cells.
Mesothelial healing model
7 to 9 week old male Lewis rats (Harlan, Bicester, UK) weighing 160-170 g
were used throughout this study (n=3 for each experimental
treatment). Animals were housed in groups of five and fed on a commercial diet
and water ad libitum. A testicular thermal injury model
(Fotev et al., 1987;
Mutsaers et al., 1997
) was
used to examine normal serosal healing. Briefly, a metal probe, consisting of
a mica-coated brass rod with a 1 cm diameter tip heated to 60°C, was
applied to a standard site on both testicular serosal surfaces for 3 seconds.
The tunica vaginalis and scrotal skin were closed using 4-0 silk sutures.
In vivo cell tracking
An equal number of labelled and unlabelled cells, suspended in serum-free
DMEM at a concentration of 1x106 cells/ml were used for all
in vivo cell tracking studies. The inclusion of unlabelled cells allowed clear
distinction of DiI-labelled cells. Aliquots (1 ml) containing labelled and
unlabelled, cultured or lavage-derived cells were injected i.p. immediately
following serosal injury. To assess whether mesothelial cell implantation was
restricted to the wound site, an additional set of uninjured animals was
injected i.p. with DiI-labelled cultured mesothelial cells.
At 3, 5 and 8 days post injury, animals were sacrificed and both testes
excised. In order to assess cell implantation and expression of the
tight-junction-associated protein, ZO-1, and the mesothelial cell surface
marker HBME-1, the testes were washed with PBS, the serosal surface dried with
compressed air and mesothelial monolayer imprints obtained on 5% gelatin
coated microscope slides (Mutsaers et al.,
1997). This technique removed almost all cells from the
regenerating surface, although occasional cells failed to adhere to the
gelatin. Whole testes from animals injected with CM-DiI labelled cultured
mesothelial cells were removed 4 days post injury, when there is maximal
mitotic activity on the wound surface
(Watters and Buck, 1973
;
Mutsaers et al., 2000
), and
processed for histology and PCNA immunohistochemistry.
Cell proliferation
Paraffin-wax-embedded sections (3 µm) of whole testes were microwaved in
10 mM citrate buffer, pH 6.4, for 10 minutes to allow nuclear antigen
retrieval. Endogenous peroxidase activity and non-specific binding sites were
blocked by incubating sections in 1% hydrogen peroxide and 1.5% normal rabbit
serum, respectively. Sections were incubated with a monoclonal antibody
directed against PCNA (dilution 1:75; Dako Ltd, Ely, UK) for 2 hours in a
humidified chamber at room temperature. Negative controls were treated with
isotype-specific mouse IgG2a antibody (PharMingen, San Diego,
California, USA). Sections were then incubated with biotinylated rabbit
anti-mouse antisera (dilution 1:100; Dako Ltd) for 1 hour followed by
streptavidin-HRP (dilution 1:200; Dako Ltd) for 30 minutes with subsequent
detection using the chromogenic substrate 3,3'-diaminobenzidine (DAB;
Sigma Aldrich, Poole, UK). Sections were mounted with DPX (BDH, Poole, UK) and
consecutive sections were examined by confocal laser scanning microscopy for
the presence of CM-DiI labelled cells.
HBME-1 and ZO-1 localisation
Immunolocalisation of the mesothelial cell surface marker HBME-1 and the
tight-junction-associated protein ZO-1 on mesothelial imprints was performed
to demonstrate incorporation of labelled mesothelial cells into the
reconstituted serosal surface. Imprints were fixed with 4% (w/v)
paraformaldehyde, pH 7.4, for 5 minutes and permeabilised for 5 minutes in
PBS, pH 7.0, containing 20 mM HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid), 300 mM sucrose,
50 mM sodium chloride, 3 mM magnesium chloride and 0.5% Triton X-100.
Non-specific staining was blocked with 5% newborn calf serum before incubating
the imprints with monoclonal antibodies directed against ZO-1 (dilution 1:25;
Zymed Laboratories Inc, San Francisco, California, USA) or HBME-1 (dilution
1:100; Dako Ltd, Ely, UK) for 1 hour at room temperature in a humidified
chamber. Negative controls were treated with isotype-specific antisera (Dako
Ltd). Imprints were then incubated with rabbit anti-mouse fluorescein
isothiocyanate (FITC)-conjugated antisera (dilution 1:40; Dako Ltd) for 1 hour
at room temperature in a humidified chamber before being washed, mounted in
Immu-mount (Shandon, Runcorn, UK) and examined using confocal laser scanning
microscopy.
Microscopy and imaging
Gelatin imprints and tissue sections of regenerating mesothelium were
examined by confocal laser scanning microscopy using the Leica TCS NT system.
Photomultiplier tube voltage thresholds for confocal microscopy were set to
gate out background fluorescence produced by isotype-specific negative
controls. Fluorescent images were sequentially collected through regenerating
mesothelial imprints and tissue sections for FITC and TRITC (tetrarhodamine
isothiocyanate) fluorochromes at 488 and 568 nm emission wavelengths,
respectively. Tissue sections stained for PCNA were examined using an Olympus
BX40 light microscope, and images were captured using KS300 image analysis
software.
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Results |
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Transfer of DiI between cultured cells
Equal numbers of unlabelled and DiI-labelled mesothelial cells were
cultured for 3 days, and the proportion of DiI-labelled cells were determined.
Cell counts demonstrated no significant change in the proportion of
DiI-labelled mesothelial cells between the time of plating (50%) and 3 days
(45.8±2.7%; Student's two-tailed t-test: p > 0.05).
Implantation of DiI- and PKH26 PCL-labelled cells onto a denuded
serosal surface
DiI-labelled cultured mesothelial cells, which demonstrated red
fluorescence localised to both the plasma membrane and vesicle-like structures
within the cell cytoplasm (Fig.
1A; inset), were injected i.p. into injured rats to determine
whether these cells were capable of implanting onto a denuded serosal surface.
5 days post injury, imprints comprised islands of predominantly rounded cells,
but after 8 days the wound surface was completely covered with cells, which
had assumed a more polygonal configuration. Imprints of regenerating
mesothelium at 5 and 8 days post injury demonstrated the presence of
DiI-labelled cells (Fig. 1A),
which were most numerous at the wound centre and least in number at the
periphery. However, labelled cells were absent on imprints following
transplantation of DiI-labelled fibroblasts at all time points examined
(Fig. 1B). In addition,
imprints of uninjured mesothelium, taken from animals injected with
DiI-labelled cultured mesothelial cells, did not demonstrate any incorporation
of DiI-labelled cells (data not shown).
|
When animals were injected with DiI-labelled peritoneal lavage cells, labelled cells were found on imprints of regenerating mesothelium at both 5 and 8 days post injury (Fig. 1C) and displayed a similar distribution to that found using cultured mesothelial cells. However, although PKH26-PCL-labelled peritoneal lavage phagocytes (Fig. 1D inset) were present on imprints 3 and 5 days post injury (Fig. 1D, E), they were completely absent by 8 days (Fig. 1F).
Proliferation of implanted DiI-labelled cultured mesothelial cells on
the regenerating serosal surface
Four days post injury, transverse sections of healing serosa, immunostained
for the proliferation marker PCNA, demonstrated positive cells within the
seminiferous tubules (Fig. 2A)
and the regenerating mesothelium (Fig.
2B). Isotype-specific negative controls did not reveal any
non-specific staining (data not shown). Adjacent sections revealed multiple
layers of CM-DiI-labelled cultured mesothelial cells at the wound site
(Fig. 2C), which corresponds to
the position of PCNA-positive cells.
|
Incorporation of fluorescence-labelled cells into the regenerating
mesothelium
To confirm the identity of implanted DiI-labelled peritoneal lavage cells,
imprints were examined for expression of the mesothelial cell surface marker
HBME-1. Normal mesothelium showed a plasma membrane distribution of HBME-1
expression (Fig. 3A), and this
was not present on isotype-specific-treated negative controls (data not
shown). Dual colocalisation of DiI and HBME-1 demonstrated that implanted
DiI-labelled lavage cells expressed the mesothelial cell surface marker at
both 5 and 8 days post injury (Fig.
3B).
|
The incorporation of DiI-labelled cultured mesothelial and peritoneal lavage cells into regenerating mesothelium was examined by immunolocalisation of the tight-junction-associated protein ZO-1 on mesothelial imprints. In normal mesothelium, ZO-1 immunoreactivity was detected at the plasma membrane with weak staining observed within the cell cytoplasm (Fig. 4A). In regenerating mesothelium, cells on the wound surface 5 days post injury demonstrated ZO-1 staining localised towards the plasma membrane and displayed a punctate distribution at sites of cell-to-cell contact (Fig. 4B), which was increased at day 8 (Fig. 4C). Confocal overlay images revealed cell membrane localised expression of ZO-1 in implanted DiI-labelled cultured mesothelial cells (Fig. 4B) and lavage-derived cells (Fig. 4C) at both 5 and 8 days post injury. Weak cytoplasmic expression of ZO-1 was also observed in regenerating mesothelium at all time points examined in both DiI-labelled and unlabelled cells. Negative controls treated with specific isotypes demonstrated negligible FITC staining (data not shown).
|
![]() |
Discussion |
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It has previously been shown that there is an increase in the number of
free-floating mesothelial cells in peritoneal fluid following serosal injury
(Whitaker and Papadimitriou,
1985). Peritoneal lavage fluid also contains inflammatory exudate
cells, predominantly macrophages, and it has been proposed that macrophages
can transform into mesothelial cells to reconstitute the mesothelium
(Eskeland and Kjærheim,
1966
; Ryan et al.,
1973
). To assess whether macrophages are a source of mesothelial
cells, free-floating phagocytic cells were labelled with PKH26-PCL and
transplanted into the peritoneal cavity following injury. Labelled cells were
present on the wound surface at 3 and 5 days but were absent at 8 days,
demonstrating that macrophage transformation into mesothelial cells does not
occur. This supports previous studies in which peritoneal macrophages labelled
with trypan blue (Ellis et al.,
1965
) or loaded with polystyrene spheres
(Raftery, 1973
) were not
identified within healed mesothelium.
Although new mesothelial cells do not originate from inflammatory cells,
previous studies have suggested that macrophages secrete mitogens, which
stimulate mesothelial proliferation and initiate healing
(Fotev et al., 1987;
Rodgers and diZerega, 1992
).
Mesothelial cells surrounding a serosal lesion proliferate between 24 and 48
hours after injury, when collections of macrophages are present on the wounded
area. Maximal cell proliferation at the centre of the wound occurs 4 days post
injury following attachment of free-floating mesothelial cells and migration
of cells at the edge of the lesion towards the wound centre
(Whitaker and Papadimitriou,
1985
; Watters and Buck,
1973
; Mutsaers et al.,
2000
). Fotev and coworkers
(Fotev et al., 1987
)
demonstrated mitogenic activity for mesothelial cells in wound lavages from
injured serosal tissue and conditioned medium from macrophage cultures.
Subsequently, Mutsaers et al. (Mutsaers et
al., 1997
) identified fibroblast growth factor-2, tumour necrosis
factor-
and platelet-derived growth factor as cytokines with
significant mesothelial cell mitogenic potency in vivo. Our studies
demonstrated numerous CM-DiI-labelled mesothelial cells and corresponding
PCNA-positive nuclei on the healing serosal surface 4 days post-injury,
confirming proliferation of free-floating mesothelial cells once implanted
onto the wound. It is likely that these cells proliferate in response to
mediators secreted by macrophages present on the wound surface early in the
process of regeneration.
To show that implanted mesothelial cells become incorporated into the
reconstituted mesothelium, we examined the formation of apical junctional
complexes between mesothelial cells in the healing monolayer. Mesothelial
cells form a number of junctional complexes including tight junctions
(Baradi and Hope, 1964;
Kluge and Hovig, 1967
). ZO-1,
a plaque protein associated with apical junctions, links the cadherin-catenin
complex with the actin-based cytoskeleton
(Itoh et al., 1997
). In
uninjured mesothelium, ZO-1 expression localised towards the plasma membrane
at sites of cell-to-cell contact. During mesothelial regeneration, ZO-1
expression was predominantly cytoplasmic, owing to the loss of intercellular
communicating junctions. By 5 days, however, ZO-1 was detected towards sites
of cell-to-cell contact, implying the reformation of apical junctional
complexes. Mobilisation of ZO-1 from the cytoplasm has previously been
demonstrated in Madin-Darby canine kidney cells in which low calcium levels
prevented apical junction formation, whereas switching to normal calcium
levels resulted in a re-distribution of ZO-1 to the cell surface
(Rajasekaran et al., 1996
).
The extent and intensity of ZO-1 staining at the cell membrane appeared to
have increased by 8 days after injury, which coincided with the
reestablishment of an intact mesothelial monolayer. Whether the intensity of
staining was due to an upregulation of ZO-1 was not determined.
To show that mesothelial cell implantation and incorporation occurs on other serosal surfaces in different models of injury, we repeated these studies using an abrasion injury to the peritoneal wall. All results were consistent with the testicular injury model (data not shown), suggesting that free-floating mesothelial cells are a source of new mesothelium on all serosal surfaces.
In summary, the following model is proposed. Mesothelial regeneration
requires recruitment of inflammatory cells to the wound surface and release of
mitogenic cytokines to activate and stimulate mesothelial cell proliferation
surrounding the wound (Fotev et al.,
1987; Mutsaers et al.,
1997
). Activated mesothelial cells break their cell-to-cell
contacts and migrate onto the wound surface
(Whitaker and Papadimitriou,
1985
). Recent evidence suggests that this may be induced by
hepatocyte growth factor, which is secreted by mesothelial cells
(Warn et al., 2001
) and
surrounding fibroblasts (Yashiro et al.,
1996
). Additional mesothelial cells detach and become
free-floating (Whitaker and Papadimitriou,
1985
), accounting for a 12-fold increase in peritoneal lavage
mesothelial cell counts 2.5 days after serosal injury
(Fotev et al., 1987
). However
the mechanisms by which these cells become detached from the basement membrane
and remain viable in the serosal fluid is not known. Free-floating mesothelial
cells move down chemotactic gradients, attach to ECM components exposed
beneath the mesothelium or are deposited from the serosal fluid, then
proliferate and reconstitute an intact mesothelial monolayer.
Our findings complement the studies of Bertram et al.
(Bertram et al., 1999) who
demonstrated a significant reduction in adhesion formation following the
intraperitoneal transplantation of autologous mesothelial cells, suggesting
that the implanted cells may enhance serosal healing. This may be of
therapeutic significance to certain subgroups of patients at high risk of
peritoneal adhesion formation, for example those receiving continuous
ambulatory peritoneal dialysis as they have ready access to peritoneal lavage
fluid. In conclusion, we have shown that cultured and lavage-derived
mesothelial cells, but not cultured lung fibroblasts or macrophages, implant
onto areas of serosal injury, proliferate and become incorporated into the
reconstituted mesothelium, conclusively demonstrating that free-floating
mesothelial cells are an origin of the regenerating mesothelium.
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Acknowledgments |
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References |
---|
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---|
Andrade, W., Seabrook, T. J., Johnston, M. G. and Hay, J. B. (1996). The use of the lipophilic fluorochrome CM-DiI for tracking the migration of lymphocytes. J. Immunol. Methods 194,181 -189.[Medline]
Baradi, A. F. and Hope, J. (1964). Observations on ultrastructure of rabbit mesothelium. Exp. Cell Res. 34,33 -44.[Medline]
Bertram, P., Tietze, L., Hoopmann, M., Treutner, K.-H., Mittermayer, C. and Schumpelick, V. (1999). Intraperitoneal transplantation of isologous mesothelial cells for prevention of adhesions. Eur. J. Surg. 165,705 -709.[Medline]
Bolen, J. W., Hammar, S. P. and McNutt, M. A. (1986). Reactive and neoplastic serosal tissue. A light microscopic, ultrastructural and immunocytochemical study. Am. J. Surg. Pathol. 10,34 -47.[Medline]
Cameron, G. R., Hassan, S. M. and De, S. N. (1957). Repair of Glisson's capsule after tangential wounds of the liver. J. Path. Bact. 73, 1-10.
Ellis, H., Harrison, W. and Hugh, T. B. (1965). The healing of peritoneum under normal and pathological conditions. Br. J. Surg. 52,471 -476.[Medline]
Eskeland, G. and Kjærheim, Å. (1966). Regeneration of parietal peritoneum in rats. An electron microscopical study. Acta. Pathol. Microbiol. Scand. 68,379 -395.[Medline]
Fotev, Z., Whitaker, D. and Papadimitriou, J. M. (1987). Role of macrophages in mesothelial healing. J. Path. 151,209 -219.[Medline]
Hertzler, A. E. (1919). In The peritoneum vol. 1, (ed. A.E. Hertzler), pp. 264-265. St Louis, CV Mosby Company.
Honig, M. G. and Hume, R. I. (1986). Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures. J. Cell Biol. 103,171 -187.[Abstract]
Itoh, M., Nagafuchi, A., Moroi, S. and Tsukita, S.
(1997). Involvement of ZO-1 in cadherin-based cell adhesion
through its direct binding to catenin and actin filaments.
J. Cell Biol. 138,181
-192.
Johnson, F. R. and Whitting, H. W. (1962). Repair of parietal peritoneum. Br. J. Surg. 49,653 -660.[Medline]
Kluge, T. and Hovig, T. (1967). The ultrastructure of human and rat pericardium. 1. Parietal and visceral mesothelium. Acta. Path. Microbiol. Scand. 71,529 -546.[Medline]
Kuffler, D. P. (1990). Long-term survival and sprouting in culture by motoneurons isolated from the spinal cord of adult frogs. J. Comp. Neurol. 302,729 -738.[Medline]
Leavesley, D. I., Stanley, J. M. and Faull, R. J. (1999). Epidermal growth factor modifies the expression and function of extracellular matrix adhesion receptors expressed by peritoneal mesothelial cells from patients on CAPD. Nephrol. Dial. Transplant. 14,1208 -1216.[Abstract]
Melnicoff, M. J., Horan, P. K. and Morahan, P. S. (1989). Kinetics of changes in peritoneal cell populations following acute inflammation. Cell Immunol. 118,178 -191.[Medline]
Menzies, D. and Ellis, H. (1990). Intestinal obstruction from adhesions: how big is the problem? Ann. R. Coll. Surg. Engl. 72,60 -63.[Medline]
Mutsaers, S. E., McAnulty, R. J., Laurent, G. J., Versnel, M. A., Whitaker, D. and Papadimitriou, J. M. (1997). Cytokine regulation of mesothelial cell proliferation in vitro and in vivo. Eur. J. Cell Biol. 72,24 -29.[Medline]
Mutsaers, S. E., Whitaker, D. and Papadimitriou, J. M. (2000). Mesothelial regeneration is not dependent on subserosal cells. J. Path. 190,86 -92.[Medline]
Raftery, A. T. (1973). Regeneration of parietal and visceral peritoneum. A light microscopical study. Br. J. Surg. 60,293 -299.[Medline]
Rajasekaran, A. K., Hojo, M., Huima, T. and Rodriguez-Boulan, E. (1996). Catenins and zonula occludens-1 form a complex during early stages in the assembly of tight junctions. J. Cell Biol. 132,451 -463.[Abstract]
Rodgers, K. E. and diZerega, G. S. (1992). Modulation of peritoneal reepithelialization by postsurgical macrophages. J. Surg. Res. 53,542 -548.[Medline]
Ryan, G. B., Grobéty, J. and Majno, G. (1973). Mesothelial injury and recovery. Am. J. Path. 71,93 -112.[Medline]
Stylianou, E., Jenner, L. A., Davies, M., Coles, G. A. and Williams, J. D. (1990). Isolation, culture and characterization of human peritoneal mesothelial cells. Kidney Int. 37,1563 -1570.[Medline]
Tietze, L., Bornträeger, J., Klosterhalfen, B., Amo-Takyi, B., Handt, S., Günther, K. and Merkelbach-Bruse, S. (1999). Expression and function of ß1 and ß3 integrins of human mesothelial cells in vitro. Exp. Mol. Path. 66,131 -139.[Medline]
Wagner, J. C., Johnson, N. F., Brown, D. G. and Wagner, M. F. (1982). Histology and ultrastructure of serially transplanted rat mesotheliomas. Br. J. Cancer 46,294 -299.[Medline]
Wang, N.-S. (1974). The regional difference of pleural mesothelial cells in rabbits. Am. Rev. Respir. Dis. 110,623 -633.[Medline]
Warn, R., Harvey, P., Warn, A., Foley-Comer, A., Heldin, P., Versnel, M., Arakaki, N., Daikuhara, Y., Laurent, G. J., Herrick, S. E. and Mutsaers, S. E. (2001). HGF/SF induces mesothelial cell migration and proliferation by autocrine and paracrine pathways. Exp. Cell Res. 267,258 -266.[Medline]
Watters, W. B. and Buck, R. C. (1973). Mitotic activity of peritoneum in contact with a regenerating area of peritoneum. Virchows Arch. (Cell Pathol.) 13, 48-54.
Whitaker, D., Papadimitriou, J. M. and Walters, N.-I. (1982). The mesothelium and its reactions: a review. CRC Crit. Rev. Toxicol. 10, 81-144.
Whitaker, D. and Papadimitriou, J. M. (1985). Mesothelial healing. Morphological and kinetic investigations. J. Path. 145,159 -175.[Medline]
Yashiro, M., Chung, Y. S., Inoue, T., Nishimura, S., Matsuoka, T., Fujihara, T. and Sowa, M. (1996). Hepatocyte growth factor (HGF) produced by peritoneal fibroblasts may affect mesothelial cell morphology and promote peritoneal dissemination. Int. J. Cancer 67,289 -293.[Medline]