| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||
Cells and Matrix |
Department of Medicine, University of Hong Kong, Hong Kong SAR, PR China
Correspondence to: S. Yung, Department of Medicine, Room 302, New Clinical Building, Queen Mary Hospital, Pokfulam, Hong Kong SAR, PR China. ssyyung{at}hkucc.hku.hk
 |
ABSTRACT |
|---|
 TOP ABSTRACT DISCUSSIONCONCLUSIONSREFERENCES |
|---|
Background: The introduction of peritoneal dialysis
(PD) as a modality of renal replacement therapy has provoked much interest in
the biology of the peritoneal mesothelial cell. Mesothelial cells isolated
from omental tissue have immunohistochemical markers that are identical to
those of mesothelial stem cells, and omental mesothelial cells can be
cultivated in vitro to study changes to their biologic functions in
the setting of PD. Method: The present article describes the structure and
function of mesothelial cells in the normal peritoneum and details the
morphologic changes that occur after the introduction of PD. Furthermore, this
article reviews the literature of mesothelial cell culture and the limitations
of in vitro studies. Results: The mesothelium is now considered to be a
dynamic membrane that plays a pivotal role in the homeostasis of the
peritoneal cavity, contributing to the control of fluid and solute transport,
inflammation, and wound healing. These functional properties of the
mesothelium are compromised in the setting of PD. Cultures of peritoneal
mesothelial cells from omental tissue provide a relevant in vitro
model that allows researchers to assess specific molecular pathways of disease
in a distinct population of cells. Structural and functional attributes of
mesothelial cells are discussed in relation to long-term culture,
proliferation potential, age of tissue donor, use of human or animal in
vitro models, and how the foregoing factors may influence in
vitro data. Conclusions: The ability to propagate mesothelial cells
in culture has resulted, over the past two decades, in an explosion of
mesothelial cell research pertaining to PD and peritoneal disorders.
Independent researchers have highlighted the potential use of mesothelial
cells as targets for gene therapy or transplantation in the search to provide
therapeutic strategies for the preservation of the mesothelium during chemical
or bacterial injury.
KEY WORDS: Mesothelial cells; omentum; peritoneum; senescence; proliferation.
Mesothelial cells are specialized epithelial cells that line the internal organs and body wall in the peritoneal, pleural, and pericardial cavities. Irrespective of species (human, rodent, rabbit, or horse) or anatomic origin, mesothelial cells constitute a homogeneous population that adopts a predominantly elongated, flattened, and squamous morphology (1,2). Mesothelial cells of cuboidal morphology have also been identified in close proximity to parenchymal organs such as the spleen, liver, and diaphragm, and to milky spots of the omentum, and also in areas where the mesothelium is injured (2–4). Ultrastructure studies have highlighted distinct differences between squamous and cuboidal mesothelial cells (Table 1).
|
Abundant microvilli and cilia are found on the luminal surface of the mesothelial cell; they protect the mesothelial surface from frictional injury by entrapment of water and serous exudates (microvilli) and by regulation of surfactant secretion (cilia) (5,6). Mesothelial cells contribute to the normal homeostasis and physiology of serosal cavities. They are the first line of defense during chemical, bacterial, and surgical insult. The present brief review focuses on the structural and functional properties of peritoneal mesothelial cells in vivo and in vitro, and on the potential applications of these properties in peritoneal dialysis (PD) studies.
|
DISCUSSION |
|---|
|
TOPABSTRACTDISCUSSIONCONCLUSIONSREFERENCES |
|---|
|
The mesothelium is actively involved in the transport and movement of cells and fluid across the serosal cavities through pinocytic vesicles, intracellular junctions, and stomata (2,7). Transport is primarily by convection and diffusion, because the mesothelium offers limited resistance as a biologic barrier. The presence of anionic sites (perlecan, for example) on mesothelial cells endows the peritoneal membrane with a net negative charge that acts as a selective permeability barrier to the passage of plasma proteins (13–15).
MORPHOLOGY OF HUMAN PERITONEAL MESOTHELIAL CELLS IN VIVO
The introduction of PD more than three decades ago aroused much interest in
the physiology and biology of peritoneal mesothelial cells. Ultrastructure
studies of peritoneal samples from PD patients have underscored the
detrimental effects of unphysiologic PD solution on the structural integrity
of mesothelial cells. Those effects include cell enlargement, transformation
from squamous to cuboidal or columnar morphology, increased
nucleus-to-cytoplasm ratio, vacuolation, activation, and loss of microvilli
(16,17).
Prolonged exposure of mesothelial cells to PD solutions also results in the
collapse of cell–matrix interactions and abrogation of cell–cell
contact inhibition, with subsequent shedding of mesothelial cells into the
peritoneal cavity. The loss of mesothelial cells from the peritoneal membrane
correlates with peritoneal vasculopathy and increased thickness of the
submesothelial compact zone
(17).
Structural and morphologic changes to the peritoneal mesothelium during PD therefore have serious consequences with regard to the functional integrity of the peritoneum. An intriguing question relates to the fate of these cells after shedding into the peritoneal cavity: Do they represent degenerative cells that are destined for apoptosis and removal from the peritoneal cavity by phagocytosis? Or are they still viable in suspension, capable of maintaining function within the peritoneum?
The fact that mesothelial cells from PD effluent can be cultured suggests that detached cells are viable—at least in part. However, these cells exhibit varied phenotypes and can consist of cells with a cobblestone epithelial morphology; large, senescent cells containing multiple nuclei and multiple vacuoles; or cells with an elongated, fibroblastic appearance. Independent researchers have hypothesized that restoration of the mesothelium after insult involves the detachment of mesothelial cells from adjacent areas free of damage, with subsequent migration to areas of injury where the cells reattach and proliferate to remesothelialize the injured monolayer (18). It is therefore possible that a proportion of the cells shed into PD effluent are destined to assist in peritoneal remesothelialization. Further studies to confirm this hypothesis are warranted.
To delineate the mechanisms by which PD fluid constituents, alone or in combination, modulate the structural and functional integrity of the mesothelium, a reproducible model that can mimic to the greatest extent possible the in vivo environment must be established. Using a model of this sort, researchers can perform experiments in a controlled manner. A cell culture—a three-dimensional cell culture in particular—provides such a model.
MESOTHELIAL CELL CULTURES
The human peritoneal mesothelial cells (HPMCs) isolated from omental
specimens possess biochemical and morphologic characteristics identical to
those found in peritoneal mesothelial stem cells. Therefore cultured
mesothelial cells provide an excellent tool for studying structural and
functional properties under baseline and stimulated conditions.
The culture of HPMCs from omental tissue is well established and reproducible (19,20). Care is required to prevent contamination of mesothelial cell cultures with peritoneal fibroblasts and, to a lesser extent, endothelial cells. Peritoneal fibroblasts can be distinguished by their elongated appearance and negative staining for cytokeratin (20). Endothelial cells can be identified by their ability to form capillary-like tubules in vitro.
The von Willebrand factor was once considered the classical marker for endothelial cells, but studies by Chung–Welch et al. showed that mesothelial cells can also express von Willebrand factor, albeit with less intensity and distinct localization (21). The use of a comprehensive panel of cell markers and phase-contrast microscopy are the best methods for distinguishing HPMCs from endothelial cells (20).
The proliferative potential of HPMCs is limited in culture. Cells can be maintained in culture without significant loss in cell morphology up to the second or third passage. Thereafter, cells become enlarged and flattened, with numerous nuclei and vacuoles. Cells beyond the sixth passage fail to attach to their substratum. Senescence that occurs after a predetermined number of cell divisions is called replicative senescence, a result of critical shortening or uncapping of telomeres (22). Alternatively, senescence can occur in a telomere-independent manner in response to non-optimum culture conditions (23). In this respect, Ksiazek et al. recently reported that the onset of senescence in cultured omentum-derived mesothelial cells is associated with oxidative stress–induced upregulation of the cyclic dependent kinase inhibitor p16INK4a (24). The age of the omentum donor may also contribute to the rate of mesothelial cell senescence (25).
In the absence of an external stimulus, the aging process of HPMCs is associated with an increase in the inflammatory phenotype. Therefore, to prevent interexperimental discrepancies, the age of the omentum donor should be taken into account when analyzing data. The use of mesothelial cells from rodents has an advantage over use of HPMCs, because neonatal and fetal tissue may provide a population of rapidly dividing cells, often resulting in the establishment of immortal cell lines without terminal differentiation or senescence (26).
The number of mesothelial cells lining the peritoneum depends on the fine balance between cell proliferation and cell death, the latter occurring through apoptosis, necrosis, or senescence. Under normal peritoneal homeostasis, mesothelial cells exhibit limited cell proliferation, with only 0.1%–0.5% of cells in the mesothelium undergoing mitosis at any one time. However, injury to mesothelial cells significantly induces cell proliferation and cell death, thereby altering the structural integrity of the peritoneal membrane. The measurement of HPMC proliferation is a good indicator of a biologic response to any given stimulus. However, merely observing a difference in cell number or a secondary endpoint related to cell proliferation such as [3H]-thymidine incorporation or dye uptake gives limited information.
To analyze the growth characteristics of cultured HPMCs under various stimuli pertaining to PD (for example, elevated glucose concentration, glucose degradation products, advanced glycation end-products, and lactate or bicarbonate buffers), the establishment of growth curves can provide essential information relating to the lag phase (the time taken by HPMCs to recover from subculture and to attach and spread), log phase (exponential increase in cell number), and plateau phase (period when cultures become confluent and the growth rate slows or stops). Data regarding subtle changes to mesothelial cell proliferation, such as the effect of the stimulus on cell attachment, shortening or lengthening of the lag phase, progression through the cell cycle, and cell death can also be obtained from growth curves. The correlation of cell proliferation with changes in cell shape and structure, increased numbers of nuclei or vacuoles, and altered synthesis of mRNA and protein under control and experimental conditions can provide essential information on the impact of any given stimulus on mesothelial cell structure and function (26).
Human peritoneal mesothelial cells have also been isolated from PD effluent (27). It must be emphasized that cells isolated in this way cannot be termed "normal," because they have been exposed to systemic influences pertaining to end-stage renal failure and also to constituents of PD fluids. The significance of these cells with regard to peritoneal change is currently under debate. Yanez–Mo and colleagues have suggested that mesothelial cells shed into PD effluent are representative of the cells lining the mesothelium and therefore reflect the structural and functional status of the peritoneum during PD; however, others have argued that effluent-derived mesothelial cells are instead emblematic of cells committed to peritoneal detachment and cell death (17,27).
Effluent-derived mesothelial cells in culture possess morphologic characteristics identical to those observed in mesothelial cells found in peritoneal biopsies. Such cells therefore provide essential information relating to structural and functional changes in vivo. The limiting factor in the study of effluent-derived mesothelial cells in the poor cell yield (mean: 25,569 ± 2971 cells per overnight bag)—a yield that depends on dwell time, dialysate glucose concentration, posture of the patient during the dwell, and frequency of peritonitis (27,28). The prevalence of non-polygonal HPMCs in PD effluent correlates with the duration for which the patient has been maintained on PD and the frequency of peritonitis (27).
EPITHELIAL-TO-MESENCHYMAL TRANSDIFFERENTIATION
Epithelial-to-mesenchymal transition (EMT) is a central feature of the
normal development of tissues and organs by which epithelial cells acquire a
mesenchymal, fibroblastic appearance, reduced intercellular adhesions, and
increased motility. In vitro, omentum-derived HPMCs adopt a
fibroblastic appearance before reaching confluence
(20). Mechanical injury of a
mesothelial monolayer can also induce EMT in a proportion of cells situated at
the periphery of the wound
(29). In HPMCs, EMT can be
induced by transforming growth factor β1 and is associated with increased
expression of the receptor for advanced glycosylation end-products
(30,31).
Effluent-derived HPMCs cultured ex vivo have also demonstrated EMT in
which decreased E-cadherin and cytokeratin expression is associated with an
induction of the transcription factor SNAIL. Such changes in
mesothelial morphology could be reversed by the addition of BMP-7
(32).
Whether EMT actually occurs in the peritoneum during PD is controversial. Yanez–Mo et al. demonstrated the presence of elongated mesothelial cells positive for cytokeratin and ICAM-1 embedded in the peritoneal submesothelium, but Williams et al. were unable to detect fibroblastic phenotype changes in mesothelial cells. This discrepancy may be attributable to the different methodologies used by the respective authors (17,27).
MESOTHELIAL CELLS AS TARGETS FOR GENE THERAPY AND TRANSPLANTATION IN PD
The omentum is a highly vascularized connective tissue that has been used
in reconstructive surgery for many years. Free omentum grafts have also been
used successfully in the treatment of various disorders, including Alzheimer
disease and leg and gastric ulcers
(33–35).
How the omentum facilitates the healing process in numerous disorders has not
been elucidated, although it has been suggested that mesothelial cells within
the omentum release growth factors into the injured tissue or are themselves
incorporated into the tissue.
To preserve the dialytic efficacy of the peritoneum during PD, cultured HPMCs might perhaps be incorporated into the peritoneum after significant and detrimental structural changes. Independent researchers have suggested that genetic engineering could offer a novel therapeutic strategy (36,37). Briefly, such a procedure would entail the removal of omental specimens from pre-dialysis patients at the time of catheter implantation, followed by isolation, expansion, and genetic modification of the retrieved HPMCs. These cells would then be stored in liquid nitrogen until required. Advantages that ex vivo gene therapy might confer to the peritoneal membrane include increased healing capability or the synthesis of a protein whose expression decreases during PD.
When mesothelial denudation is observed (for example, after peritonitis episodes), genetically modified HPMCs could be infused into the peritoneal cavity through the catheter and allowed to attach to the peritoneal membrane. Theoretically, such an intervention is a most exciting concept, but is this technique feasible in reality? Can a sufficient number of mesothelial cells be generated from uremic patients?
Williams et al. observed the absence of a mesothelium in 18.1% of peritoneal biopsies obtained from predialysis and hemodialysis patients, and in patients that possessed an intact mesothelium, the cells adopted a reactive state (17). Given that HPMCs in culture have a defined lifespan and that they enter senescence after the second passage, can sufficient cells be generated to maintain the cobblestone polygonal morphology without loss of proliferative potential?
In a recent study by Hekking et al., transplantation of mesothelial cells into the peritoneum in a rat model of peritonitis-induced submesothelial thickening prolonged activation and inflammation within the peritoneum (38). If (supposedly) sufficient cells can be generated and stored for future use, then before clinical trials can even be contemplated, it is essential that the mechanism of transplanted cell activation and methods of inhibiting such activation be determined.
The identification, isolation, and exploitation of mesothelial progenitor cells may offer an alternative opportunity to effectively preserve the structural and functional integrity of the peritoneal membrane during PD.
|
CONCLUSIONS |
|---|
|
TOPABSTRACTDISCUSSIONCONCLUSIONSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTDISCUSSIONCONCLUSIONSREFERENCES |
|---|
and IL-6 signaling
governs neutrophil trafficking and apoptosis during acute inflammation.
J Clin Invest 2003;112
: 598-607.[Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
