Targeting carcinoma-associated mesothelial cells with antibody-drug conjugates in ovarian carcinomatosis.

Ovarian carcinomatosis is characterized by the accumulation of carcinoma-associated mesothelial cells (CAMs) in the peritoneal stroma and mainly originates through a mesothelial-to-mesenchymal transition (MMT) process. MMT has been proposed as a therapeutic target for peritoneal metastasis. Most ovarian cancer (OC) patients present at diagnosis with peritoneal seeding, which makes tumor progression control difficult by MMT modulation. An alternative approach is to use antibody-drug conjugates (ADCs) targeted directly to attack CAMs. This strategy could represent the cornerstone of precision-based medicine for peritoneal carcinomatosis. Here, we performed complete transcriptome analyses of ascitic fluid-isolated CAMs in advanced OC patients with primary-, high-, and low-grade, serous subtypes and following neoadjuvant chemotherapy. Our findings suggest that both cancer biological aggressiveness and chemotherapy-induced tumor mass reduction reflect the MMT-associated changes that take place in the tumor surrounding microenvironment. Accordingly, MMT-related genes, including fibroblast activation protein (FAP), mannose receptor C type 2 (MRC2), interleukin-11 receptor alpha (IL11RA), myristoylated alanine-rich C-kinase substrate (MARCKS), and sulfatase-1 (SULF1), were identified as specific actionable targets in CAMs of OC patients, which is a crucial step in the de novo design of ADCs. These cell surface target receptors were also validated in peritoneal CAMs of colorectal cancer peritoneal implants, indicating that ADC-based treatment could extend to other abdominal tumors that show peritoneal colonization. As proof of concept, a FAP-targeted ADC reduced tumor growth in an OC xenograft mouse model with peritoneal metastasis-associated fibroblasts. In summary, we propose MMT as a potential source of ADC-based therapeutic targets for peritoneal carcinomatosis. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Novel Aspects of the Immune Response Involved in the Peritoneal Damage in Chronic Kidney Disease Patients under Dialysis.

Chronic kidney disease (CKD) incidence is growing worldwide, with a significant percentage of CKD patients reaching end-stage renal disease (ESRD) and requiring kidney replacement therapies (KRT). Peritoneal dialysis (PD) is a convenient KRT presenting benefices as home therapy. In PD patients, the peritoneum is chronically exposed to PD fluids containing supraphysiologic concentrations of glucose or other osmotic agents, leading to the activation of cellular and molecular processes of damage, including inflammation and fibrosis. Importantly, peritonitis episodes enhance peritoneum inflammation status and accelerate peritoneal injury. Here, we review the role of immune cells in the damage of the peritoneal membrane (PM) by repeated exposure to PD fluids during KRT as well as by bacterial or viral infections. We also discuss the anti-inflammatory properties of current clinical treatments of CKD patients in KRT and their potential effect on preserving PM integrity. Finally, given the current importance of coronavirus disease 2019 (COVID-19) disease, we also analyze here the implications of this disease in CKD and KRT.

Mesothelial-to-Mesenchymal Transition and Exosomes in Peritoneal Metastasis of Ovarian Cancer.

Most patients with ovarian cancer (OvCA) present peritoneal disseminated disease at the time of diagnosis. During peritoneal metastasis, cancer cells detach from the primary tumor and disseminate through the intraperitoneal fluid. The peritoneal mesothelial cell (PMC) monolayer that lines the abdominal cavity is the first barrier encountered by OvCA cells. Subsequent progression of tumors through the peritoneum leads to the accumulation into the peritoneal stroma of a sizeable population of carcinoma-associated fibroblasts (CAFs), which is mainly originated from a mesothelial-to-mesenchymal transition (MMT) process. A common characteristic of OvCA patients is the intraperitoneal accumulation of ascitic fluid, which is composed of cytokines, chemokines, growth factors, miRNAs, and proteins contained in exosomes, as well as tumor and mesothelial suspended cells, among other components that vary in proportion between patients. Exosomes are small extracellular vesicles that have been shown to mediate peritoneal metastasis by educating a pre-metastatic niche, promoting the accumulation of CAFs via MMT, and inducing tumor growth and chemoresistance. This review summarizes and discusses the pivotal role of exosomes and MMT as mediators of OvCA peritoneal colonization and as emerging diagnostic and therapeutic targets.

Cellular Integrin α5ß1 and Exosomal ADAM17 Mediate the Binding and Uptake of Exosomes Produced by Colorectal Carcinoma Cells.

Approximately 25% of colorectal cancer (CRC) patients develop peritoneal metastasis, a condition associated with a bleak prognosis. The CRC peritoneal dissemination cascade involves the shedding of cancer cells from the primary tumor, their transport through the peritoneal cavity, their adhesion to the peritoneal mesothelial cells (PMCs) that line all peritoneal organs, and invasion of cancer cells through this mesothelial cell barrier and underlying stroma to establish new metastatic foci. Exosomes produced by cancer cells have been shown to influence many processes related to cancer progression and metastasis. In epithelial ovarian cancer these extracellular vesicles (EVs) have been shown to favor different steps of the peritoneal dissemination cascade by changing the functional phenotype of cancer cells and PMCs. Little is currently known, however, about the roles played by exosomes in the pathogenesis and peritoneal metastasis cascade of CRC and especially about the molecules that mediate their interaction and uptake by target PMCs and tumor cells. We isolated exosomes by size-exclusion chromatography from CRC cells and performed cell-adhesion assays to immobilized exosomes in the presence of blocking antibodies against surface proteins and measured the uptake of fluorescently-labelled exosomes. We report here that the interaction between integrin α5ß1 on CRC cells (and PMCs) and its ligand ADAM17 on exosomes mediated the binding and uptake of CRC-derived exosomes. Furthermore, this process was negatively regulated by the expression of tetraspanin CD9 on exosomes.

Increased miR-7641 Levels in Peritoneal Hyalinizing Vasculopathy in Long-Term Peritoneal Dialysis Patients.

Peritoneal hyalinizing vasculopathy (PHV) represents the cornerstone of long-term peritoneal dialysis (PD), and especially characterizes patients associated with encapsulating peritoneal sclerosis. However, the mechanisms of PHV development remain unknown. A cross sectional study was performed in 100 non-selected peritoneal biopsies of PD patients. Clinical data were collected and lesions were evaluated by immunohistochemistry. In selected biopsies a microRNA (miRNA)-sequencing analysis was performed. Only fifteen patients (15%) showed PHV at different degrees. PHV prevalence was significantly lower among patients using PD fluids containing low glucose degradation products (GDP) (5.9% vs. 24.5%), angiotensin converting enzyme inhibitors (ACEIs) (7.5% vs. 23.4%), statins (6.5% vs. 22.6%) or presenting residual renal function, suggesting the existence of several PHV protective factors. Peritoneal biopsies from PHV samples showed loss of endothelial markers and induction of mesenchymal proteins, associated with collagen IV accumulation and wide reduplication of the basement membrane. Moreover, co-expression of endothelial and mesenchymal markers, as well as TGF-ß1/Smad3 signaling activation were found in PHV biopsies. These findings suggest that an endothelial-to-mesenchymal transition (EndMT) process was taking place. Additionally, significantly higher levels of miR-7641 were observed in severe PHV compared to non-PHV peritoneal biopsies. Peritoneal damage by GDPs induce miRNA deregulation and an EndMT process in submesothelial vessels, which could contribute to collagen IV accumulation and PHV.

Targeting Toll-like receptors with soluble Toll-like receptor 2 prevents peritoneal dialysis solution-induced fibrosis.

Peritoneal membrane failure due to fibrosis limits the use of peritoneal dialysis (PD). Peritoneal fibrosis may potentially be induced by sterile inflammation caused by ongoing cellular stress due to prolonged exposure to PD solutions (PDS). Effective therapies to prevent this process remain to be developed. Toll-like receptors (TLRs) mediate sterile inflammation by recognizing damage-associated molecular patterns (DAMPs) released by cellular stress. We evaluated the involvement of TLRs and DAMPs in PDS-induced fibrosis models and the therapeutic potential of TLR-DAMP targeting for preventing fibrosis. A range of PDS elicited pro-inflammatory and fibrotic responses from PD patient peritoneal leukocytes, mesothelial cells and mouse peritoneal leukocytes. TLR2/4 blockade of human peritoneal cells or TLR2/4 knockouts inhibited these effects. PDS did not induce rapid ERK phosphorylation or IκB-α degradation, suggesting that they do not contain components capable of direct TLR activation. However, PDS increased the release of Hsp70 and hyaluronan, both TLR2/4 DAMP ligands, by human and mouse peritoneal cells, and their blockade decreased PDS-driven inflammation. Soluble TLR2, a TLR inhibitor, reduced PDS-induced pro-inflammatory and fibrotic cytokine release ex vivo. Daily catheter infusion of PDS in mice caused peritoneal fibrosis, but co-administration of soluble TLR2 prevented fibrosis, suppressed pro-fibrotic gene expression and pro-inflammatory cytokine production, reduced leukocyte/neutrophil recruitment, recovered Treg cell levels and increased the Treg:Th17 ratio. Thus, TLR2/4, Hsp70 and hyaluronan showed major roles in PDS-induced peritoneal inflammation and fibrosis. The study demonstrates the therapeutic potential of a TLR-DAMP targeting strategy to prevent PDS-induced fibrosis.

miR-21 Promotes Fibrogenesis in Peritoneal Dialysis.

Peritoneal dialysis (PD) is a life-saving form of renal replacement therapy for those with end-stage kidney disease. Mesothelial cells (MCs) line the peritoneal cavity and help define peritoneal response to treatment-associated injury, a major reason for treatment failure. miRNAs are important regulators, but their roles in peritoneal fibrosis are largely unknown. In this study, miR-21 was one of the most abundant miRNAs in primary MCs, and was up-regulated by the profibrotic cytokine transforming growth factor-ß1 and in PD effluent-derived MCs exhibiting mesenchymal phenotypic change. Increased miR-21 was found in peritoneal membrane biopsy specimens from PD patients compared to healthy controls (PD biocompatible, 5.86×, P = 0.0001; PD conventional, 7.09×, P < 0.0001, n = 11 per group). In PD effluent from a cohort of 230 patients, miR-21 was higher in those receiving the therapy long-term compared to new starters (n = 230, miR-21 3.26×, P = 0.001) and associated with icodextrin use (R = 0.52; 95% CI, 0.20-0.84), peritonitis count (R = 0.16; 95% CI, 0.03-0.29), and dialysate cytokines. miR-21 down-regulated programmed cell death 4 and programmed cell death 4 protein was decreased in peritoneal membrane biopsy specimens from PD patients compared to healthy controls. New miR-21 targets were identified that may be important during PD fibrogenesis. These data identify miR-21 as an important effector of fibrosis in the peritoneal membrane, and a promising biomarker in the dialysis effluent for membrane change in patients receiving PD.

Mesothelial-to-mesenchymal transition as a possible therapeutic target in peritoneal metastasis of ovarian cancer.

Peritoneal dissemination is the primary metastatic route of ovarian cancer (OvCa), and is often accompanied by the accumulation of ascitic fluid. The peritoneal cavity is lined by mesothelial cells (MCs), which can be converted into carcinoma-associated fibroblasts (CAFs) through mesothelial-to-mesenchymal transition (MMT). Here, we demonstrate that MCs isolated from ascitic fluid (AFMCs) of OvCa patients with peritoneal implants also undergo MMT and promote subcutaneous tumour growth in mice. RNA sequencing of AFMCs revealed that MMT-related pathways - including transforming growth factor (TGF)-ß signalling - are differentially regulated, and a gene signature was verified in peritoneal implants from OvCa patients. In a mouse model, pre-induction of MMT resulted in increased peritoneal tumour growth, whereas interfering with the TGF-ß receptor reduced metastasis. MC-derived CAFs showed activation of Smad-dependent TGF-ß signalling, which was disrupted in OvCa cells, despite their elevated TGF-ß production. Accordingly, targeting Smad-dependent signalling in the peritoneal pre-metastatic niche in mice reduced tumour colonization, suggesting that Smad-dependent MMT could be crucial in peritoneal carcinomatosis. Together, these results indicate that bidirectional communication between OvCa cells and MC-derived CAFs, via TGF-ß-mediated MMT, seems to be crucial to form a suitable metastatic niche. We suggest MMT as a possible target for therapeutic intervention and a potential source of biomarkers for improving OvCa diagnosis and/or prognosis. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

miR-9-5p suppresses pro-fibrogenic transformation of fibroblasts and prevents organ fibrosis by targeting NOX4 and TGFBR2.

Uncontrolled extracellular matrix (ECM) production by fibroblasts in response to injury contributes to fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Reactive oxygen species (ROS) generation is involved in the pathogenesis of IPF. Transforming growth factor-ß1 (TGF-ß1) stimulates the production of NADPH oxidase 4 (NOX4)-dependent ROS, promoting lung fibrosis (LF). Dysregulation of microRNAs (miRNAs) has been shown to contribute to LF. To identify miRNAs involved in redox regulation relevant for IPF, we performed arrays in human lung fibroblasts exposed to ROS. miR-9-5p was selected as the best candidate and we demonstrate its inhibitory effect on TGF-ß receptor type II (TGFBR2) and NOX4 expression. Increased expression of miR-9-5p abrogates TGF-ß1-dependent myofibroblast phenotypic transformation. In the mouse model of bleomycin-induced LF, miR-9-5p dramatically reduces fibrogenesis and inhibition of miR-9-5p and prevents its anti-fibrotic effect both in vitro and in vivo. In lung specimens from patients with IPF, high levels of miR-9-5p are found. In omentum-derived mesothelial cells (MCs) from patients subjected to peritoneal dialysis (PD), miR-9-5p also inhibits mesothelial to myofibroblast transformation. We propose that TGF-ß1 induces miR-9-5p expression as a self-limiting homeostatic response.

Mesothelial-to-mesenchymal transition in the pathogenesis of post-surgical peritoneal adhesions.

Peritoneal adhesions (PAs) are fibrotic bands formed between bowel loops, solid organs, and the parietal peritoneum, which may appear following surgery, infection or endometriosis. They represent an important health problem with no effective treatment. Mesothelial cells (MCs) line the peritoneal cavity and undergo a mesothelial-to-mesenchymal transition (MMT) under pathological conditions, transforming into myofibroblasts, which are abundant in peritoneal fibrotic tissue. The aim of this study was to investigate if peritoneal MCs undergo a MMT contributing to the formation of post-surgical adhesions. Biopsies from patients with PAs were analysed by immunohistochemistry, immunofluorescence, and quantitative RT-PCR. A mouse model of PAs based on ischaemic buttons was used to modulate MMT by blocking the transforming growth factor-beta (TGF-ß) pathway. The severity of adhesions and MMT-related marker expression were studied. We observed myofibroblasts derived from the conversion of MCs in submesothelial areas of patients with PAs. In addition, MMT-related markers were dysregulated in adhesion zones when compared to distant normal peritoneal tissue of the same patient. In animal experiments, blockage of TGF-ß resulted in molecular reprogramming of markers related to the mesenchymal conversion of MCs and in a significant decrease in the severity of the adhesions. These data indicate for the first time that MMT is involved in PA pathogenesis. This finding opens new therapeutic strategies to interfere with adhesion formation by modulating MMT with a wide range of pharmacological agents.

Immune-Regulatory Molecule CD69 Controls Peritoneal Fibrosis.

Patients with ESRD undergoing peritoneal dialysis develop progressive peritoneal fibrosis, which may lead to technique failure. Recent data point to Th17-mediated inflammation as a key contributor in peritoneal damage. The leukocyte antigen CD69 modulates the setting and progression of autoimmune and inflammatory diseases by controlling the balance between Th17 and regulatory T cells (Tregs). However, the relevance of CD69 in tissue fibrosis remains largely unknown. Thus, we explored the role of CD69 in fibroproliferative responses using a mouse model of peritoneal fibrosis induced by dialysis fluid exposure under either normal or uremic status. We found that cd69-/- mice compared with wild-type (WT) mice showed enhanced fibrosis, mesothelial to mesenchymal transition, IL-17 production, and Th17 cell infiltration in response to dialysis fluid treatment. Uremia contributed partially to peritoneal inflammatory and fibrotic responses. Additionally, antibody-mediated CD69 blockade in WT mice mimicked the fibrotic response of cd69-/- mice. Finally, IL-17 blockade in cd69-/- mice decreased peritoneal fibrosis to the WT levels, and mixed bone marrow from cd69-/- and Rag2-/-γc-/- mice transplanted into WT mice reproduced the severity of the response to dialysis fluid observed in cd69-/- mice, showing that CD69 exerts its regulatory function within the lymphocyte compartment. Overall, our results indicate that CD69 controls tissue fibrosis by regulating Th17-mediated inflammation.

The dipeptide alanyl-glutamine ameliorates peritoneal fibrosis and attenuates IL-17 dependent pathways during peritoneal dialysis.

Peritoneal dialysis (PD) can result in chronic inflammation and progressive peritoneal membrane damage. Alanyl-Glutamine (Ala-Gln), a dipeptide with immunomodulatory effects, improved resistance of mesothelial cells to PD fluids. Recently, interleukin-17 (IL-17) was found to be associated with PD-induced peritoneal damage. Here we studied the capacity of intraperitoneal Ala-Gln administration to protect against peritoneal damage by modulating IL-17 expression in uremic rat and mouse PD exposure models. Supplementation of PD fluid with Ala-Gln resulted in reduced peritoneal thickness, αSMA expression and angiogenesis. Addition of Ala-Gln also attenuated the IL-17 pathway expression induced by PD, reflected by substantial reduction or normalization of peritoneal levels of IL-17, transforming growth factor ß, IL-6, and the transcription factor retinoic acid receptor-related orphan receptor gamma T. Moreover, increased levels of IL-17 were associated with PD-induced peritoneal thickening. Conversely, Ala-Gln treatment prevented peritoneal extracellular matrix deposition, an effect seen with IL-17 blockade. Thus, intraperitoneal administration of Ala-Gln, a stable dipeptide commonly used in parenteral nutrition, ameliorates PD-induced peritoneal damage in animal models, in part by modulating IL-17 expression. Hence, Ala-Gln supplementation of dialysate may be a potential strategy to ameliorate peritoneal deterioration during PD.

Clathrin mediates infectious hepatitis C virus particle egress.

UNLABELLED: Although it is well established that hepatitis C virus (HCV) entry into hepatocytes depends on clathrin-mediated endocytosis, the possible roles of clathrin in other steps of the viral cycle remain unexplored. Thus, we studied whether cell culture-derived HCV (HCVcc) exocytosis was altered after clathrin interference. Knockdown of clathrin or the clathrin adaptor AP-1 in HCVcc-infected human hepatoma cell cultures impaired viral secretion without altering intracellular HCVcc levels or apolipoprotein B (apoB) and apoE exocytosis. Similar reductions in HCVcc secretion were observed after treatment with specific clathrin and dynamin inhibitors. Furthermore, detergent-free immunoprecipitation assays, neutralization experiments, and immunofluorescence analyses suggested that whereas apoE associated with infectious intracellular HCV precursors in endoplasmic reticulum (ER)-related structures, AP-1 participated in HCVcc egress in a post-ER compartment. Finally, we observed that clathrin and AP-1 knockdown altered the endosomal distribution of HCV core, reducing and increasing its colocalization with early endosome and lysosome markers, respectively. Our data support a model in which nascent HCV particles associate with apoE in the ER and exit cells following a clathrin-dependent transendosomal secretory route. IMPORTANCE: HCV entry into hepatocytes depends on clathrin-mediated endocytosis. Here we demonstrate for the first time that clathrin also participates in HCV exit from infected cells. Our data uncover important features of HCV egress, which may lead to the development of new therapeutic interventions. Interestingly, we show that secretion of the very-low-density lipoprotein (VLDL) components apoB and apoE is not impaired after clathrin interference. This is a significant finding, since, to date, it has been proposed that HCV and VLDL follow similar exocytic routes. Given that lipid metabolism recently emerged as a potential target for therapies against HCV infection, our data may help in the design of new strategies to interfere specifically with HCV exocytosis without perturbing cellular lipid homeostasis, with the aim of achieving more efficient, selective, and safe antivirals.

A pathogenetic role for endothelin-1 in peritoneal dialysis-associated fibrosis.

In patients undergoing peritoneal dialysis (PD), chronic exposure to nonphysiologic PD fluids elicits low-grade peritoneal inflammation, leading to fibrosis and angiogenesis. Phenotype conversion of mesothelial cells into myofibroblasts, the so-called mesothelial-to-mesenchymal transition (MMT), significantly contributes to the peritoneal dysfunction related to PD. A number of factors have been described to induce MMT in vitro and in vivo, of which TGF-ß1 is probably the most important. The vasoconstrictor peptide endothelin-1 (ET-1) is a transcriptional target of TGF-ß1 and mediates excessive scarring and fibrosis in several tissues. This work studied the contribution of ET-1 to the development of peritoneal damage and failure in a mouse model of PD. ET-1 and its receptors were expressed in the peritoneal membrane and upregulated on PD fluid exposure. Administration of an ET receptor antagonist, either bosentan or macitentan, markedly attenuated PD-induced MMT, fibrosis, angiogenesis, and peritoneal functional decline. Adenovirus-mediated overexpression of ET-1 induced MMT in human mesothelial cells in vitro and promoted the early cellular events associated with peritoneal dysfunction in vivo. Notably, TGF-ß1-blocking peptides prevented these actions of ET-1. Furthermore, a positive reciprocal relationship was observed between ET-1 expression and TGF-ß1 expression in human mesothelial cells. These results strongly support a role for an ET-1/TGF-ß1 axis as an inducer of MMT and subsequent peritoneal damage and fibrosis, and they highlight ET-1 as a potential therapeutic target in the treatment of PD-associated dysfunction.

IL-17A is a novel player in dialysis-induced peritoneal damage.

The classical view of the immune system has changed by the discovery of novel T-helper (Th) subsets, including Th17 (IL-17A-producing cells). IL-17A participates in immune-mediated glomerulonephritis and more recently in inflammatory pathologies, including experimental renal injury. Peritoneal dialysis patients present chronic inflammation and Th1/Th2 imbalance, but the role of the Th17 response in peritoneal membrane damage has not been investigated. In peritoneal biopsies from dialyzed patients, IL-17A immunostaining was found mainly in inflammatory areas and was absent in the healthy peritoneum. IL-17A-expressing cells included lymphocytes (CD4+ and γδ), neutrophils, and mast cells. Elevated IL-17A effluent concentrations were found in long-term peritoneal dialysis patients. Studies in mice showed that repeated exposure to recombinant IL-17A caused peritoneal inflammation and fibrosis. Moreover, chronic exposure to dialysis fluids resulted in a peritoneal Th17 response, including elevated IL-17A gene and protein production, submesothelial cell infiltration of IL-17A-expressing cells, and upregulation of Th17 differentiation factors and cytokines. IL-17A neutralization diminished experimental peritoneal inflammation and fibrosis caused by chronic exposure to dialysis fluids in mice. Thus, IL-17A is a key player of peritoneum damage and it may be a good candidate for therapeutic intervention in peritoneal dialysis patients.

Carcinoma-associated fibroblasts derive from mesothelial cells via mesothelial-to-mesenchymal transition in peritoneal metastasis.

Peritoneal dissemination is a frequent metastatic route for cancers of the ovary and gastrointestinal tract. Tumour cells metastasize by attaching to and invading through the mesothelial cell (MC) monolayer that lines the peritoneal cavity. Metastases are influenced by carcinoma-associated fibroblasts (CAFs), a cell population that derives from different sources. Hence, we investigated whether MCs, through mesothelial-mesenchymal transition (MMT), were a source of CAFs during peritoneal carcinomatosis and whether MMT affected the adhesion and invasion of tumour cells. Biopsies from patients with peritoneal dissemination revealed the presence of myofibroblasts expressing mesothelial markers in the proximity of carcinoma implants. Prominent new vessel formation was observed in the peritoneal areas harbouring tumour cells when compared with tumour-free regions. The use of a mouse model of peritoneal dissemination confirmed the myofibroblast conversion of MCs and the increase in angiogenesis at places of tumour implants. Treatment of omentum MCs with conditioned media from carcinoma cell cultures resulted in phenotype changes reminiscent of MMT. Adhesion experiments demonstrated that MMT enhanced the binding of cancer cells to MCs in a ß1-integrin-dependent manner. Scanning electron microscopy imaging showed that the enhanced adhesion was mostly due to increased cell-cell interaction and not to a mere matrix exposure. Invasion assays suggested a reciprocal stimulation of the invasive capacity of tumour cells and MCs. Our results demonstrate that CAFs can derive from mesothelial cells during peritoneal metastasis. We suggest that MMT renders the peritoneum more receptive for tumour cell attachment/invasion and contributes to secondary tumour growth by promoting its vascularization.

HDAC1/2 control mesothelium/ovarian cancer adhesive interactions impacting on Talin-1-α5ß1-integrin-mediated actin cytoskeleton and extracellular matrix protein remodeling.

BACKGROUND: Peritoneal metastasis, which accounts for 85% of all epithelial ovarian carcinoma (EOC) metastases, is a multistep process that requires the establishment of adhesive interactions between cancer cells and the peritoneal membrane. Interrelations between EOC and the mesothelial stroma are critical to facilitate the metastatic process. No data is available so far on the impact of histone acetylation/deacetylation, a potentially relevant mechanism governing EOC metastasis, on mesothelial cells (MCs)-mediated adhesion. METHODS: Static adhesion and peritoneal clearance experiments were performed pretreating mesenchymal-like MCs and platinum-sensitive/resistant EOC cell lines with MS-275-a Histone deacetylase (HDAC)1-3 pharmacological inhibitor currently used in combination trials. Results were acquired by confocal microscopy and were analyzed with an automated Opera software. The role of HDAC1/2 was validated by genetic silencing. The role of α4-, α5-α1 Integrins and Fibronectin-1 was validated using specific monoclonal antibodies. Quantitative proteomic analysis was performed on primary MCs pretreated with MS-275. Decellularized matrices were generated from either MS-275-exposed or untreated cells to study Fibronectin-1 extracellular secretion. The effect of MS-275 on ß1 integrin activity was assessed using specific monoclonal antibodies. The role of Talin-1 in MCs/EOC adhesion was analyzed by genetic silencing. Talin-1 ectopic expression was validated as a rescue tool from MS-275-induced phenotype. The in vivo effect of MS-275-induced MC remodeling was validated in a mouse model of peritoneal EOC dissemination. RESULTS: Treatment of MCs with non-cytotoxic concentrations of MS-275 caused a consistent reduction of EOC adhesion. Proteomic analysis revealed several pathways altered upon MC treatment with MS-275, including ECM deposition/remodeling, adhesion receptors and actin cytoskeleton regulators. HDAC1/2 inhibition hampered actin cytoskeleton polymerization by downregulating actin regulators including Talin-1, impairing ß1 integrin activation, and leading to abnormal extracellular secretion and distribution of Fibronectin-1. Talin-1 ectopic expression rescued EOC adhesion to MS-275-treated MCs. In an experimental mouse model of metastatic EOC, MS-275 limited tumor invasion, Fibronectin-1 secretion and the sub-mesothelial accumulation of MC-derived carcinoma-associated fibroblasts. CONCLUSION: Our study unveils a direct impact of HDAC-1/2 in the regulation of MC/EOC adhesion and highlights the regulation of MC plasticity by epigenetic inhibition as a potential target for therapeutic intervention in EOC peritoneal metastasis.

Calprotectin blockade inhibits long-term vascular pathology following peritoneal dialysis-associated bacterial infection.

Bacterial infections and the concurrent inflammation have been associated with increased long-term cardiovascular (CV) risk. In patients receiving peritoneal dialysis (PD), bacterial peritonitis is a common occurrence, and each episode further increases late CV mortality risk. However, the underlying mechanism(s) remains to be elucidated before safe and efficient anti-inflammatory interventions can be developed. Damage-Associated Molecular Patterns (DAMPs) have been shown to contribute to the acute inflammatory response to infections, but a potential role for DAMPs in mediating long-term vascular inflammation and CV risk following infection resolution in PD, has not been investigated. We found that bacterial peritonitis in mice that resolved within 24h led to CV disease-promoting systemic and vascular immune-mediated inflammatory responses that were maintained up to 28 days. These included higher blood proportions of inflammatory leukocytes displaying increased adhesion molecule expression, higher plasma cytokines levels, and increased aortic inflammatory and atherosclerosis-associated gene expression. These effects were also observed in infected nephropathic mice and amplified in mice routinely exposed to PD fluids. A peritonitis episode resulted in elevated plasma levels of the DAMP Calprotectin, both in PD patients and mice, here the increase was maintained up to 28 days. In vitro, the ability of culture supernatants from infected cells to promote key inflammatory and atherosclerosis-associated cellular responses, such as monocyte chemotaxis, and foam cell formation, was Calprotectin-dependent. In vivo, Calprotectin blockade robustly inhibited the short and long-term peripheral and vascular consequences of peritonitis, thereby demonstrating that targeting of the DAMP Calprotectin is a promising therapeutic strategy to reduce the long-lasting vascular inflammatory aftermath of an infection, notably PD-associated peritonitis, ultimately lowering CV risk.

p38 maintains E-cadherin expression by modulating TAK1-NF-kappa B during epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells is a pathological process that occurs during peritoneal dialysis. EMT leads to peritoneal fibrosis, ultrafiltration failure and eventually to the discontinuation of therapy. Signaling pathways involved in mesothelial EMT are thus of great interest, but are mostly unknown. We used primary mesothelial cells from human omentum to analyze the role of the p38 MAPK signaling pathway in the induction of EMT. The use of specific inhibitors, a dominant-negative p38 mutant and lentiviral silencing of p38α demonstrated that p38 promotes E-cadherin expression both in untreated cells and in cells co-stimulated with the EMT-inducing stimuli transforming growth factor (TGF)-ß1 and interleukin (IL)-1ß. p38 inhibition also led to disorganization and downregulation of cytokeratin filaments and zonula occludens (ZO)-1, whereas expression of vimentin was increased. Analysis of transcription factors that repress E-cadherin expression showed that p38 blockade inhibited expression of Snail1 while increasing expression of Twist. Nuclear translocation and transcriptional activity of p65 NF-κB, an important inducer of EMT, was increased by p38 inhibition. Moreover, p38 inhibition increased the phosphorylation of TGF-ß-activated kinase 1 (TAK1), NF-κB and IκBα. The effect of p38 inhibition on E-cadherin expression was rescued by modulating the TAK1-NF-κB pathway. Our results demonstrate that p38 maintains E-cadherin expression by suppressing TAK1-NF-κB signaling, thus impeding the induction of EMT in human primary mesothelial cells. This represents a novel role of p38 as a brake or 'gatekeeper' of EMT induction by maintaining E-cadherin levels.

Blocking TGF-ß1 protects the peritoneal membrane from dialysate-induced damage.

During peritoneal dialysis (PD), mesothelial cells undergo mesothelial-to-mesenchymal transition (MMT), a process associated with peritoneal-membrane dysfunction. Because TGF-ß1 can induce MMT, we evaluated the efficacy of TGF-ß1-blocking peptides in modulating MMT and ameliorating peritoneal damage in a mouse model of PD. Exposure of the peritoneum to PD fluid induced fibrosis, angiogenesis, functional impairment, and the accumulation of fibroblasts. In addition to expressing fibroblast-specific protein-1 (FSP-1), some fibroblasts co-expressed cytokeratin, indicating their mesothelial origin. These intermediate-phenotype (Cyto(+)/FSP-1(+)) fibroblasts had features of myofibroblasts with fibrogenic capacity. PD fluid treatment triggered the appearance of CD31(+)/FSP-1(+) and CD45(+)/FSP-1(+) cells, suggesting that fibroblasts also originate from endothelial cells and from cells recruited from bone marrow. Administration of blocking peptides significantly ameliorated fibrosis and angiogenesis, improved peritoneal function, and reduced the number of FSP-1(+) cells, especially in the Cyto(+)/FSP-1(+) subpopulation. Conversely, overexpression of TGF-ß1 in the peritoneum by adenovirus-mediated gene transfer led to a marked accumulation of fibroblasts, most of which derived from the mesothelium. Taken together, these results demonstrate that TGF-ß1 drives the peritoneal deterioration induced by dialysis fluid and highlights a role of TGF-ß1-mediated MMT in the pathophysiology of peritoneal-membrane dysfunction.