Mesothelial cells in tissue repair and fibrosis

1Mesothelial cells form a monolayer, known as the mesothelium, that line the pleural, peritoneal, and pericardial cavities, with visceral and parietal surfaces covering the internal organs and body wall, respectively. They attach to a thin basement membrane supported by sub-serosal connective tissue, and are bathed in a small volume of serosal fluid that resembles an ultrafiltrate of plasma containing blood proteins, sugars, resident inflammatory cells, and various enzymes.

2Mesothelial cells synthesize and secrete lubricants including glycosaminoglycans and surfactant to prevent friction and adhesions forming between adjacent parietal and visceral surfaces. They play critical roles in the maintenance of serosal homeostasis in response to injury, inflammation, and immunoregulation (reviewed in ). Mesothelial cells are also central cells in serosal repair, secreting inflammatory mediators, chemokines, growth factors, and extracellular matrix (ECM) components.

3Mesothelial cells display different phenotypes which, depending on their location and state of activation, are likely to reflect functional differences. Although morphologically they resemble epithelial cells and possess many epithelial characteristics; surface microvilli, apical/basal polarity, cytokeratins, and junctional complexes, embryologically they derive from the mesoderm and express mesenchymal features including vimentin and desmin. Upon stimulation they can also undergo morphological and functional changes consistent with an epithelial-to-mesenchymal transition (EMT;;; ) which has recently been termed mesothelial-to-mesenchymal transition (MMT; ).

4The ability of mesothelial cells to undergo MMT suggests that the mesothelium is a likely source of fibrogenic cells during serosal inflammation and tissue repair and therefore play important roles in pleural and peritoneal fibrosis and adhesion formation. In addition, it has been hypothesized that mesothelial cells may be a source of (myo)fibroblasts in interstitial lung fibrosis.

5This review will focus on aspects of the mesothelium that contribute to fibrosis including coagulation and fibrinolysis, inflammation, ECM production, and EMT/MMT, and discuss some common fibrotic conditions attributed to changes in mesothelial cell structure and function (Figure 1).

6Mesothelial cells are important regulators of fibrin levels in the serosal cavities following injury. Fibrin deposition is an early step in normal wound repair but persistence of fibrin can lead to fibrosis and post-operative adhesion formation. For example in serosal cavities, the denudation of the mesothelium can cause impairment in the regulation of fibrinolytic activity by mesothelial cells and an accumulation of fibrin. If this fibrin is not removed it is replaced by granulation tissue that will be substituted by dense fibrous tissue.

7The regulation of fibrin deposition by mesothelial cells is mediated by the secretion of both procoagulant and fibrinolytic enzymes. Procoagulant activity is due to production and regulation of tissue factor (TF), the main cellular initiator of the extrinsic coagulation cascade. TF is produced by mesothelial cells and complexes with other coagulation cascade proteins to activate thrombin which in turn cleaves serum fibrinogen to form fibrin. This is regulated by TF pathway inhibitor (TFPI), also produced by mesothelial cells. It has been shown in pleural injury that a relative excess of TF activity is expressed so that the inhibitory capacity of TFPI and other endogenous inhibitors are exceeded and local coagulation is thereby promoted.

8Fibrinolytic activity is mediated through secretion of tissue plasminogen activator (tPA), urokinase PA (uPA), and uPA receptor (uPAR), and their inhibitors plasminogen activator inhibitors (PAI)-1 and PAI-2. The clearance of fibrin is based on the balance of the expression of the components of the fibrinolytic system and their net influence on local fibrinolytic activity.

9Mesothelial cells express tPA, uPA, uPAR, and PAI-1. In the pleura, all these components, together with plasminogen, the substrate for uPA and tPA, can be detected in the pleural fluid. The fibrinolytic pathway can be activated directly by tPA or via expression of uPAR on the surface of pleural mesothelial cells, lung fibroblasts, and macrophages. Because uPA binds to uPAR with high affinity, the bound form retains PA activity even in the presence of protease inhibitors. Apart from its fibrinolytic properties, uPA can also initiate signaling through uPAR which also contributes to the pathogenesis of serosal inflammation and repair. uPA can upregulate uPAR in mesothelial cells and also contributes to chemotactic and mitogenic responses induced by pleural mesothelial cells and lung fibroblasts.

10Both pro- and anti-fibrinolytic mediators are regulated by inflammatory factors including lipopolysaccharide, tumor necrosis factor alpha (TNF-α), and interleukin (IL)-1 and fibrogenic mediators such as transforming growth factor beta (TGF-β) and thrombin. If the fibrinolytic capacity is insufficient and fibrin accumulation is not resolved, fibrous adhesions/plaques form between opposing serosal surfaces.

11Control of fibrin deposition and lysis is particularly important in the pleura. Cytokines implicated in the pathogenesis of pleural injury, including TNF-α, can upregulate uPAR expression at the surface of cell types involved in pleural injury and thereby influence local remodeling of transitional fibrin. Exposure of mesothelial cells to asbestos can also influence uPAR expression. The fibrinolytic system can also be controlled transcriptionally and post-transcriptionally through changes in uPAR mRNA stability and translational control. The regulatory mechanism involves the interaction and destabilization of uPAR mRNA through formation of cis–trans complexes between uPAR mRNA binding proteins and specific sequences of uPAR mRNA.

12Mesothelial cells play a critical role in the modulation of serosal inflammation through their ability to synthesize cytokines/chemokines, growth factors, ECM proteins, and intracellular adhesion molecules as well as their ability to present antigen. When the serosa is challenged by infection or agents such as dialysis fluid or asbestos, there is a massive influx of leukocytes from the vasculature into the serosal space. Mediators released from activated macrophages such as TNF-α, IL-1β, and interferon gamma (IFN-γ) stimulate mesothelial cells to produce cytokines such as monocyte chemotactic protein-1 (MCP-1) also known a chemokine (C–C motif) ligand 2 (CCL2), RANTES also known as CCL5 and IL-8 also known as chemokine (C–X–C motif) ligand 8 (CXCL8) and adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), E-cadherin, N-cadherin, CD49a, CD49b, and CD29 to further recruit more leukocytes to the site of injury and facilitate leukocyte adherence and migration across the mesothelium.

13Mesothelial cells also mediate inflammation through the local synthesis of hyaluronan, which is able to sequester free radicals and initiate tissue repair responses. Synthesis of hyaluronan fragments are increased by exposure to IL-1β, IL - 6, TNF-α, TGF-β1, and platelet-derived growth factor (PDGF; ) and can activate the inflammatory cascade in mesothelial cells by inducing IL-8 and MCP-1 production via activation of the NF-κB signaling pathway. In the peritoneum, induction of these inflammatory cytokines by long-term exposure to peritoneal dialysis (PD) fluid may promote the development of chronic peritoneal inflammation, leading to long-term peritoneal damage and exacerbation of the fibrotic pathway.

14Mesothelial cells also contribute to controlling inflammation both in normal and inflamed tissue by producing cyclooxygenase and metabolizing arachidonic acid to release prostaglandins and prostacyclin.

15Mesothelial cells secrete a variety of ECM molecules, which physiologically are important for cell function and repair of serosal membranes. Mesothelial cells synthesize ECM molecules including collagen types I, III, and IV, elastin, fibronectin, laminin, and proteoglycans and they can also regulate ECM turnover by secreting matrix metalloproteinases and tissue inhibitors of metalloproteinases. In culture, mesothelial cells can be further stimulated to produce ECM when exposed to peritoneal effluent from patients with acute peritonitis or various cytokines and growth factors such as IL-1β, TNF-α, epidermal growth factor (EGF), PDGF, and TGF-β.

16The renin–angiotensin system also stimulates ECM production. During PD and peritonitis, angiotensin II levels are increased. This promotes mesothelial cell production of fibronectin via the induction of the ERK1/2 and MAPK pathways thereby contributing to peritoneal injury and inflammation. The increased production of fibronectin by mesothelial cells can also be induced by the presence of advanced glycation end products (AGEs; ).

17Mesothelial cells undergo MMT, a similar process to EMT in epithelial cells. EMT is a well characterized process, involving a number of overlapping and sequential events that require the appropriate spatiotemporal expression, interaction, and modification of a number of intra- and extra-cellular factors to cause a change in cell phenotype. The process is controlled primarily by three main families of transcription factors: zinc finger Snail (SNAI1, SNAI2) basic helix–loop–helix (Twisted1), and ZEB (ZEB1, ZEB2; ). Epithelial cells initially lose cell–cell junctions by down-regulating E-cadherin and other junctional proteins, reduce attachment to the basal lamina and subsequently lose apical–basal cell polarity. With cell migration and invasion of the basement membrane and a change in cytoskeletal components, a full change to a mesenchymal phenotype occurs. Expression of a multitude of mesenchymal markers, including alpha smooth muscle actin (α - SMA), EDA-fibronectin, vimentin, and fibroblast specific protein - 1 (FSP-1), is proposed as an unequivocal indicator of EMT. The fibrogenic mediator TGFβ is the most well described inducer of EMT whereas bone morphogenic protein-7 has been identified as a repressor in certain tissues. MicroRNAs have emerged as important regulators of EMT as they are able to target multiple signaling pathways.

18Mesothelium-specific genetic lineage tracing studies in mice have clearly demonstrated that during development, mesothelial cells contribute to smooth muscle in the developing vasculature of the gut, heart, liver, and lungs through EMT, which will subsequently be referred to as MMT. The transcription factor Wilms tumor-1 (WT-1), expressed by mesothelium regulates its functional properties during development. During lung development, WT-1 expressing mesothelial cells migrate into the lung parenchyma and undergo a transition to form subpopulations of bronchial smooth muscle cells, vascular smooth muscle cells, and fibroblasts, through the action of sonic hedgehog signaling. This process has also been shown to occur in the adult. For example, Lachaud and colleagues isolated murine uterine-derived mesothelial cells and stimulated them to undergo MMT and become functional vascular smooth muscle-like cells expressing smoothelin-B typical of contractile cells.

19In vitro, numerous groups have shown upregulation of mesenchymal markers and downregulation of junctional components by human mesothelial cells following exposure to various injurious agents. TGF-β1 induced MMT in human mesothelial cell cultures isolated from the pleura, omentum, or mesenteric tissue, with evidence of downregulation of junction components (E-cadherin, ZO-1), upregulation of mesenchymal markers (α-SMA), and deposition of ECM. A number of studies have shown an upregulation of transcription factors in mesothelial cells associated with MMT (SNAI1/SNAI2, ZEB1/2, Twist1) following exposure to TGF-β1 as well as other cytokines including hepatocyte growth factor (HGF), PDGF, and IL-Iβ. Lipopolysaccharide, a derivative of the bacterial cell wall, has also been found to induce MMT and is proposed to be a mechanism whereby peritonitis is linked to peritoneal fibrosis.

20In vivo, a number of studies have reported the importance of mesothelial cells in the development of fibrosis following injury. In a rat peritoneal scrape injury model, DiI-labeled rat mesothelial cells injected into the peritoneal cavity were found to incorporate into the mesothelial layer, eventually appearing in the subserosa. Furthermore, adenovirus-mediated overexpression of TGF-β1 in the lung and peritoneum induced fibrosis in mice that was associated with MMT; reduced E-cadherin and increased COL1, α-SMA, MMP-2, and 9. These changes are likely to be mediated by both Smad3-dependent and independent signaling pathways. Such findings confirm the ability of mesothelial cells to undergo MMT following damage. The possibility that there may be a genetic basis to this process was demonstrated by a study investigating mouse strain differences in susceptibility to TGF-β1-induced peritoneal fibrosis. Interestingly, an increase in markers of MMT was associated with enhanced peritoneal fibrosis in the susceptible mouse strain (C57/Bl6) whereas the resistant strain (SJL) showed minimal response.

21Of note, it is apparent that MMT may not just be of relevance to peritoneal fibrosis and that a similar process occurs in other organs/tissues and possibly re-activating developmental programs in the adult. For instance,, using conditional cell lineage murine studies, demonstrated that hepatic stellate cells and myofibroblasts are derived from mesothelial cells expressing WT-1 during liver fibrogenesis. In addition, a study using similar techniques in mice found that WT-1 positive pleural mesothelial cells migrated into the lung parenchyma leading to lung fibrosis following TGF-β1 treatment. Lansley and colleagues also demonstrated that mesothelial cells undergo MMT during differentiation into osteoblast-like and adipocyte-like cells in culture, and suggested mesothelial cells may have progenitor/stem cell-like properties.

22Pleural fibrosis resembles fibrosis in other tissues and may be the consequence of an organized hemorrhagic effusion, tuberculous effusion, empyema, asbestos-related pleuracy and chronic inflammatory conditions such as systemic lupus erythematosus, rheumatoid arthritis, and scleroderma. In addition, certain medications have also been associated with the development of pleural fibrosis including procainamide, hydralazine, isoniazid, and targeted therapies such as tyrosine kinase inhibitors imatinib and dasatinib. Pleural fibrosis can manifest itself as discrete localized lesions (pleural plaques) or diffuse pleural thickening and fibrosis. The mesothelial cell plays an important role in the fibrotic process through interaction with inflammatory cells, profibrotic mediators and both the coagulation and fibrinolytic pathways.

23Fibrin is not normally present in the pleural space but rapidly accumulates in response to pleural injury. This was shown in an experimental rabbit model using intrapleural administration of tetracycline (TCN) to induce an acute pleural injury. Fibrin coated the pleural surfaces soon after injury and induced a peripheral pneumonitis with an exudative pleural effusion, leading to the formation of fibrinous adhesions within the exudative effusion. These fibrinous adhesions were rapidly remodeled with deposition of collagen within a few days. This model parallels the temporal course of loculation and fibrosis often observed in patients with complicated parapneumonic effusions.

24Fibrinolytic therapy, predominantly with streptokinase and urokinase, is often used for pleural loculations associated with parapneumonic effusions or hemothoraces. The rapid appearance of intrapleural fibrin resembles fibrin deposition within the lung which can lead to accelerated pulmonary fibrosis, for example in severe cases of acute respiratory distress syndrome (ARDS; ). TF is released locally by mesothelial cells and other resident and inflammatory cells into the pleural space together with various coagulation factors including TFPI.

25Although the primary target cell for pleural fibrosis is thought to be the subpleural fibroblast, studies have shown the importance of mesothelial cells in the pleural fibrotic response. A number of agents can induce fibrosis, including infection, radiation, and inorganic particles such as talc and asbestos. It is unclear how asbestos fibers induce subpleural fibroblasts and mesothelial cells to synthesize collagen but it is likely to be through the generation of cytokines, growth factors, and reactive oxygen species (ROS). ROS are cytotoxic and can stimulate fibroblasts to synthesize ECM components as well as induce expression of genes for profibrotic mediators such as TGF-β and TNF-α.

26TGF-β is considered the most potent pro-fibrotic cytokine with a central role in the pathogenesis of many fibrotic diseases including pleural fibrosis. TGF-β stimulates collagen synthesis by mesothelial cells, is present within pleural fluids in fibrosing forms of pleural injury and induces pleural fibrosis when administered intrapleurally. In addition, TGF-β lowers the ratio of matrix-degrading metalloproteinase-1 (MMP-1) to tissue inhibitors of metalloproteinases (TIMPs), promoting ECM accumulation. TGF-β has also been implicated in talc-induced pleurodesis, the most commonly used agent to induce pleurodesis. Patients with tuberculous pleurisy also have elevated pleural fluid levels of TGF-β which was shown to correlate with increased levels of pleural thickening, an index of pleural fibrosis.

27Peritoneal dialysis (PD) is an effective renal replacement therapy used for patients with end stage kidney disease. The major disadvantage associated with this therapy is that PD solutions are bio-incompatible and contribute to the development of peritoneal fibrosis in most patients within two years of PD commencing. During PD, the mesothelial cells that line the peritoneum are exposed to a hypertonic environment with high glucose levels. As a consequence, mesothelial cells undergo structural and functional alterations that contribute to the development of fibrotic lesions in the peritoneum.

28Peritoneal biopsies taken from PD patients show a reactive mesothelium with enlarged, weakly adhesive, degenerated mesothelial cells with a reduced number of microvilli and alterations in the number of endoplasmic reticulum and micropinocytotic vesicles. In many patients, there is denudation of the mesothelial layer which is associated with vasculopathy and submesothelial thickening. PD patients with subsequent peritonitis show even more pronounced mesothelial degeneration and a more prominent exfoliation of mesothelial cells. In these patients, there is also an acute infiltration of inflammatory cells into the submesothelium that contribute to the thickening of this layer.

29Alterations to the structure of the peritoneum may be attributed to changes in mesothelial cell proteoglycan production. Proteoglycans are anionic macromolecules and important components of ECM in the peritoneum. Mesothelial cells produce a number of small proteoglycans including perlecan, biglycan, and decorin. As PD progresses, there is an induction of versican while decorin and perlecan levels are reduced. These changes are associated with peritoneal ECM remodeling and expansion of the submesothelium. However, direct evidence for a role of these proteoglycans in serosal remodeling has yet to be demonstrated.

30The chronic exposure of peritoneal mesothelial cells to high levels of glucose and glucose degradation products contributes to loss of the mesothelial layer by decreasing mesothelial cell viability and altering normal mesothelial cell function through the induction of proinflammatory factors such as vascular endothelial growth factor (VEGF) and TGF-β1. VEGF is associated with neoangeogenesis and the down-regulation of the mesothelial cell intercellular tight junction proteins ZO-1, occludin, and claudin-1 while TGF-β1 is associated with lymphangiogenesis, the promotion of MMT, and the production of collagen type I, III, and IV.

31The fibroblast-like characteristics induced in mesothelial cells that have undergone MMT allow these cells to invade into the submesothelial stroma where they contribute to angiogenesis, fibrosis, and ultrafiltration failure. These cells are often observed in patients who have undergone PD for more than 12 months. MMT is associated with polymerization of the actin cytoskeleton and an increase in hyaluronan and is mediated by proinflammatory factors such as IL-1β, EGF, HGF, AGEs and their receptor RAGE. The prolonged expression of these factors during peritoneal inflammation delays the regression of mesothelial cells back to their epithelial phenotype thereby promoting fibrotic changes in the peritoneum. Other factors recently identified to be associated with MMT include MCP-1, ROS, and the small non-coding regulatory microRNAs miR-589, miR-30a, miR-30b, and miR-200c.

32Recently, JAK/STAT signaling was also identified as a mediator of PD-induced peritoneal membrane changes. Twice daily PD fluid infusions in rats for 10 days induced phospho-JAK, mesothelial cell hyperplasia, inflammation, fibrosis, and hypervascularity. These changes were attenuated following the administration of a JAK1/2 inhibitor. These findings are consistent with recent observations in a mouse model of lung fibrosis where blocking STAT3 attenuated the fibrotic response. Therefore, targeting the JAK/STAT signaling pathway may be a novel therapeutic strategy used to reduce PD related peritoneal changes that contribute to the development of peritoneal fibrosis in patients.

33The processes by which the peritoneum repairs following PD associated injury are yet to be fully defined. Viable mesothelial cells are exfoliated into the peritoneal cavity during PD and it is likely that these cells re-populate and restore the damaged mesothelium. Therefore it has been proposed that mesothelial cell transplantation could be used therapeutically to regenerate the PD injured mesothelium. Studies have shown that mesothelial cell transplantation is feasible in animals and humans and that genetically modified mesothelial cells can also be used to deliver proteins critical to the healing process. However, in one study the transplantation of mesothelial cells in rats was shown to activate the peritoneum and induce inflammation and recently, the morphology of the mesothelial cell was shown to be important for cell therapy used for peritoneal regeneration. Mesothelial cells harvested from the PD effluent of patients were separated based on morphology into epithelial-like and fibroblastic-like cells and transplanted into nude mice with an injured peritoneum. The mice transplanted with epithelial-like cells showed very few adhesions and exhibited no thickening of the peritoneum. However, transplantation of fibroblast-like cells did not inhibit peritoneal adhesion or thickening, highlighting the need for further optimization before this approach can be trialed in patients. Other cell sources that may be used for mesothelial repair include bone marrow derived cells, adipose-derived stem cells, and mesenchymal stem cells. Alternative therapeutic strategies being investigated to reduce mesothelial cell-mediated inflammation and prevent peritoneal fibrosis include targeting TGFβ1-mediated mechanisms, reducing mesothelial cell production of fibronectin developing a more bio compatible PD solution, altering PD daily dwelling time, and stimulating fibrinolytic agents.

34The formation of postoperative intra-abdominal and pelvic adhesions is a significant clinical and surgical problem. Adhesions are bands of fibrous tissue that form between apposing tissue and organs usually arising as a result of injury sustained during surgery. They are a leading cause of chronic pelvic pain, intestinal obstruction, and female infertility. The most severe consequence of adhesion formation is small bowel obstruction which can occur up to 20 years or more after the initial surgical procedure and is associated with mortality rates ranging between 3% and 30%. Postoperative adhesions have been reported to occur in up to 93% of patients undergoing abdominal surgery. A substantially increased risk of post-surgical complications is also likely where adhesions are present as a result of previous surgery.

35Adhesions are thought to occur when there is dysregulation of the normal serosal healing process. Many cell types including macrophages, lymphocytes, granulocytes, and fibroblasts play important roles in serosal repair, however the mesothelial cell is central to this process but may also play a critical role in the development of adhesions following injury. As discussed, mesothelial cells secrete a variety of coagulation and inflammatory mediators following serosal injury and it is these factors that are the essential inducers of adhesion development.

36Following serosal trauma (such as during surgery), the mesothelial layer is disrupted resulting in brief vasoconstriction followed by increased vascular permeability and chemotaxis of inflammatory cells to the site of injury. Mesothelial cells stimulate fibrin deposition through the production of TF and themselves become embedded in the developing fibrin scaffold. Under normal conditions the fibrin is degraded following release of fibrinolytic mediators from the mesothelial cells, such as tPA, but if there is a persistent fibrinolytic imbalance, there is subsequent deposition of ECM components by mesothelial cells, fibroblasts, and myofibroblasts. Ultimately this results in the formation of fibrin bands between tissues and organs which then become organized into fibrous adhesions.

37Detrimental effects of surgical techniques on peritoneal mesothelial cells have been reported which are thought to contribute to adhesion formation. For example, use of the common insufflation agent carbon dioxide gas (CO2) as well as the amount of insufflation pressure used during laparoscopy can result in morphological and biochemical changes to mesothelial cells and can cause hypoxia and dehydration. Therefore, several changes have been made to surgical techniques in order to prevent the mesothelial cell denudation and bleeding that also form the basis of peritoneal adhesion formation. These have included development of new microsurgical techniques (minimally invasive surgery), the use of specialized equipment and unpowdered gloves and humidifying and changing the temperature and composition of the gases used for laparoscopy.

38Currently, there are no definitive strategies to prevent the formation of adhesions during surgery. Many methods have been developed and tested using a variety of post-surgical adhesion animal models as well as in human clinical trials but with varying degrees of success. Addition of surgical barriers that provide anti-adhesive separation of denuded serosal tissues have proved beneficial but none completely prevent adhesion development in all patients.

39Strategies targeting the pathophysiological mechanisms involved in dysregulated serosal repair, such as the coagulation and inflammatory pathways, have also been trialed in an effort to prevent adhesion formation. Many anti-inflammatory and anti-coagulant substances have been used both systemically and locally including steroids, cyclo-oxygenase inhibitors, heparin, and tPA but to date, none of these agents have shown significant promise.

40Studies have also examined the effect of mesothelial cell transplantation on preventing adhesion formation and this approach has shown some promise. However, how this approach can be used routinely in patients still needs to be determined. Clearly a better understanding of the mechanisms underlying adhesion formation is therefore critical to developing novel approaches to prevent their formation.

41Interstitial lung diseases (ILDs) represent a collection of heterogeneous parenchymal lung disorders characterized by inflammation and fibrosis that lead to impairment of gas-exchange in the lungs. Approximately 50% of ILDs have unknown etiology, of which idiopathic pulmonary fibrosis (IPF) is a well-defined subset.

42Histologically, the lungs in IPF demonstrate a pattern of usual interstitial pneumonia, which includes septal thickening, honeycombing, fibroblastic foci, and minimal interstitial inflammation. IPF occurs predominantly from middle age onwards affecting five million people worldwide. It is a debilitating and ultimately lethal disease, with a mortality rate worse than that seen with many cancers. It has a median survival of only 2–3 years from diagnosis, and there is currently no known cure. Recent phase III trial results showed that current drugs such as pirfenidone and nintedanib could only slow the progression of the disease. Pirfenidone works through downregulation of growth factor and procollagen I and II production and nintedanib is a small molecule tyrosine kinase inhibitor that blocks receptors for VEGF, PDGF, and FGF.

43The pathogenesis of IPF remains poorly understood although the mechanisms driving the fibrotic response are often considered to follow a similar pathway to other forms of tissue fibrosis where there is a chronic progression of the repair response resulting in excessive deposition of ECM without resolution.

44In IPF, the myofibroblast, characterized by α-SMA and vimentin expression, is recognized as the effector cell contributing to the deposition of ECM, mainly types I and III collagen. However, the cellular origin of the lung myofibroblast remains controversial and a combination of different cell types likely serves as precursors of myofibroblasts. A number of cellular sources of myofibroblast have been proposed, including existing peribronchial and perivascular adventitial fibroblasts, alveolar epithelial cells, bone marrow-derived cells, tissue-resident cells, and pericytes.

45As previously discussed, mesothelial cells can be induced to undergo MMT and transition into myofibroblasts. Decologne and colleagues used adenoviral gene transfer of TGF-β to the pleural mesothelium in rats and showed that as well as development of a progressive pleural fibrosis, the pleural fibrosis extended into the lung parenchyma supporting a possible role for mesothelial cells in pulmonary fibrosis. More recent mouse models of fibrogenic lung injury have also supported this observation by showing that mesothelial cells invade the lung parenchyma and adopt a myofibroblast phenotype after intratracheal TGF-β1 administration, leading to fibrosis. This was recently shown to be mediated through the TGF-β1-Smad2/3 signaling pathway. Blocking this pathway using novel TGF - β regulators, such as the nuclear receptor NR4A1, are likely to block MMT and attenuate tissue fibrosis.

46As a further validation of the in vitro and in vivo findings, immunohistochemical analysis of human IPF lung sections showed Wilms tumor-1 (WT-1)-positive mesothelial cells in the pleura and lung parenchyma, which corresponded with immunostaining of the mesothelial cell marker calretinin. In contrast, lung tissue sections from patients with chronic obstructive pulmonary disease, cystic fibrosis, and pulmonary arterial hypertension were all negative for WT-1. Collectively, these studies indicate potential contributions of pleural mesothelial cells as a source of myofibroblast in IPF and possibly a new avenue to identify therapeutic targets.

47Mesothelial cells clearly play an important role in serosal homeostasis and repair following injury, but following a breakdown in the normal regulatory mechanisms, mesothelial cells can also contribute to the development of tissue fibrosis. The mechanisms underlying this process are slowly being elucidated but more research is needed to investigate how mesothelial cells interact with their local environment and to identify ways to limit fibrosis and promote normal repair.

48The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.