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Mesenchymal Transition in Human Mesothelial Cells In Vitro and Ex Vivo [TGF-β1 and IL-1β treatment]

ABSTRACT: Peritoneal dialysis (PD) is an effective form of renal replacement therapy. A significant proportion of patients who initiate PD suffer from PD-related clinical complications, including peritoneal membrane damage, which may limit the duration of treatment. Mesothelial-to-mesenchymal transition (MMT) significantly contributes to the peritoneal dysfunction related to PD. Hence, we analyzed the genetic reprograming of the MMT-process with the aim to identify new biomarkers that may be tested in PD-patients. Microarray analysis revealed a partial overlapping of MMT induced in vitro and MMT of effluent-derived mesothelial cells (ex vivo), and that MMT, both in vitro and ex vivo, is mainly a repression process being higher the number of genes that are down-regulated than those that are induced. According to cellular morphology and the number of altered genes and pathways, the MMT ex vivo could be subdivided into two stages: early/epitheliod and advanced/non-epitheliod. We could demonstrate by RT-PCR array analysis that a number of genes differentially expressed in effluent-derived non-epitheliod cells also showed significant differential expression when comparing standard versus low-GDP PD fluids. Among the secreted proteins that are up-regulated along the MMT process thrombospondin-1 (TSP1), collagen-13 (COL13), vascular endothelial growth factor A (VEGFA), and gremlin-1 (GREM1) were selected to be measured in PD effluents. TSP1, COL13 and VEGFA, but not GREM1, showed significant differences between early and advanced stages of MMT, and their expression were associated with high peritoneal transport status. The results establish a proof of concept about the feasibility of MMT-associated secreted proteins as biomarkers in PD.

ORGANISM(S): Homo sapiens

PROVIDER: GSE92454 | GEO | 2017/03/22

SECONDARY ACCESSION(S): PRJNA357607

REPOSITORIES: GEO

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Project description:Peritoneal dialysis (PD) is the worldwide recognized preferred dialysis treatment for children affected by end-stage kidney disease (ESKD). However, due to the unphysiological composition of PD fluids, the peritoneal membrane (PM) of these patients may undergo structural and functional alterations, which may cause fibrosis. Several factors may accelerate this process and primary kidney disease may have a causative role. In particular, patients affected by corticoresistant primary focal segmental glomerulosclerosis), a rare glomerular disease leading to nephrotic syndrome and ESKD, seem more prone to develop peritoneal fibrosis. The mechanism causing this predisposition is still unrecognized. To better define this condition, we carried out, for the first time, a new comprehensive comparative proteomic mass spectrometry analysis of mesothelial exosomes from peritoneal dialysis effluent (PDE) of 6 pediatric patients with focal segmental glomerular sclerosis (FSGS) versus 6 patients affected by other primary renal diseases (No FSGS). Our omic study demonstrated that, despite the high overlap in the protein milieu between the two study groups, machine learning allowed a complete distinction of the whole proteomic exosome mesothelial content of FSGS versus No FSGS (with 100% accuracy). Out of the 2490 identified proteins, 40% (995) were involved in epithelial-mesenchymal transition (EMT)/fibrosis and in the transforming growth factor-β pathway. Additionally, the Weight Gene Co-expression Network Analysis algorithm identified that some of the discriminative proteins (TIMP1, CTHRC1, SPARC, CHMP4B, COL5A2, ANXA13, FNC2 and CENP-E) were also highly correlated to PD vintage, fibrosis, EMT and non-neoplastic PM disease. All together our data demonstrated that mesothelial cells of FSGS patients are more prone to activate a pro-fibrotic machinery with exosomes having a primary role in this process. Moreover, they indicated that identified FSGS-associated elements in mesothelial exosome protein could be employed as potential new biomarkers of mesothelial integrity. Finally, our results highlighted that in FSGS patients particular attention should be paid to use more biocompatible dialysis solution, reduce the length of time on PD and personalize PD regimens to minimize the risk of rapid loss of PM function or development of encapsulating peritoneal sclerosis.

Project description:Chronic exposure to biological incompatibility of peritoneal dialysis solution is one of the principal causes of ultrafiltration failure (UF). However, the changes of peritoneal cell composition and molecular mechanism of peritoneal microenvironment after long-term PD are unclear. Single-cell RNA sequencing was performed to profile cell composition and transcriptome characteristics in effluent obtained from patients undergoing PD with short vintage (SV), long vintage without ultrafiltration failure (LV_NOT_UF), and long vintage with ultrafiltration failure (LV_UF). A total of 6 SV patients, 6 LV_NOT_UF patients, and 4 LV_UF patients were enrolled. The median PD duration of the three groups were 5 months (range:1.0–6.0 months), 137 months (range: 92–189 months) and 132 months (range: 120–161 months), with no significant differences in age (38.3 ± 21.5 years, 45.7 ± 10.6 years and 41.0 ± 16.7 years, P>0.05) and gender (male%: 33.3%, 50.0% and 25.0%, P>0.05). scRNA-seq data annotated 8 main cell types including epithelial-like mesothelial cells (E-MCs), mesenchymal-like mesothelial cells (M-MCs), fibroblast, plasma B cells, conventional dendritic cells, plasmacytoid dendritic cells, natural killer T cells, and myeloid cells. LV_UF patients showed higher proportion of E-MCs, which exhibited activated autophagy and had properties of both anti-inflammatory and pro-inflammatory, and neutrophil- and autophagy-related DEGs of E-MCs were upregulated in the LV_UF group. In contrast, the M-MCs and the AQP1 expression in the peritoneal mesenchyme were reduced in the LV_UF group compared to the other groups. The neutrophils in PD effluent were elevated in the LV_UF group, while the M2-like MRC1-macrophages gradually decreased from the SV, LV_NOT_UF to LV_UF group. Compared with the LV_NOT_UF group, the upregulated DEGs of monocytes/macrophages in the LV_UF group not only involved in pro-inflammatory response, but also promoted EMT progress. Additionally, neutrophils and monocytes/macrophages both interacted with MCs. Our study uncovers peritoneal different individual cell populations and cellular functions in different stages of PD which indicates the dynamic changes of microenvironments of peritoneal membrane. These findings provide new insights into the novel roles of mesothelial cells and inflammatory cells in UF.

Project description:Despite their emerging relevance to fully understand disease pathogenesis, we have as yet a poor understanding as to how biomechanical signals are integrated with specific biochemical pathways to determine cell behaviour. Mesothelial-to-mesenchymal transition (MMT) markers colocalized with TGF-beta1-dependent signalling and yes-associated protein (YAP) activation across biopsies from different pathologies exhibiting peritoneal fibrosis, supporting mechanotransduction as a central driving component of these class of fibrotic lesions and its crosstalk with specific signaling pathways. Transcriptome and proteome profiling of the response of mesothelial cells (MCs) to linear cyclic stretch revealed molecular changes compatible with bona fide MMT, which (i) overlapped with established YAP target gene subsets, and (ii) were largely dependent on endogenous TGF-beta1 signaling. Importantly, TGF-beta1 blockade blunts the transcriptional upregulation of the se gene signatures, but not the mechanical activation and nuclear translocation of YAP per se. We studied the role therein of caveolin-1 (Cav1), a plasma membrane mechanotransducer. Exposure of Cav1-deficient MCs to cyclic stretch led to a robust upregulation of MMT-related gene programs, which was blunted upon TGF-beta1 inhibition. Conversely, Cav1 depletion enhanced both TGF-beta1 and TGFBRI expression. Cav1 genetic deficiency exacerbated MMT and PA fibrosis in an experimental model of peritoneal ischaemic buttons. Taken together, these results support that Cav1-YAP/TAZ fine-tune the fibrotic response through the modulation of MMT, onto which TGF-beta1-dependent signaling coordinately converges. Our findings reveal a cooperation between biomechanical and biochemical signals in the triggering of MMT, representing a novel potential opportunity to intervene mechanically-induced disorders coursing with peritoneal fibrosis, such as post-surgical adhesions. This SuperSeries is composed of the SubSeries listed below.

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Mesenchymal Transition in Human Mesothelial Cells In Vitro and Ex Vivo [TGF-β1 and IL-1β treatment]

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