Proteomic study of mesothelial and endothelial cross-talk: key lessons.

INTRODUCTION: The peritoneum, pleura, and pericardium are yet understudied multicellular systems where mesothelial cells (MCs) and endothelial cells (ECs) are in close proximity. Crosstalk between these cell types likely plays role in molecular transport, immunological reactions, and metabolic processes in health, disease, and therapeutic intervention. AREAS COVERED: In this review, we discuss recent proteomic efforts to characterize the crosstalk between MC and EC. We describe the proteomic methods necessary for investigation of crosstalk between MC and EC, as well as the in-vitro models that can be employed. Potential experimental approaches range from conditioned medium, via co-culture on semi-permeable membranes, to 3D cell culture based organoid models. While the biological and clinical relevance of the models may increase with their ability to mimic close cell communication, the practicality of these complex experiments corresponds vice versa, making standardization more difficult and expensive. EXPERT OPINION: Currently, data and reports on mesothelial-to-endothelial crosstalk are still very scarce. In our opinion, the in-vitro model using semi-permeable cell culture inserts will allow to establish a basic understanding of cellular crosstalk that may occur between those cell types. Later-on, more sophisticated 3D cell cultures may be better able to simulate the transport dynamics within the peritoneal membrane.

Proteome-Wide Differential Effects of Peritoneal Dialysis Fluid Properties in an In Vitro Human Endothelial Cell Model.

To replace kidney function, peritoneal dialysis (PD) utilizes hyperosmotic PD fluids with specific physico-chemical properties. Their composition induces progressive damage of the peritoneum, leading to vasculopathies, decline of membrane function, and PD technique failure. Clinically used PD fluids differ in their composition but still remain bioincompatible. We mapped the molecular pathomechanisms in human endothelial cells induced by the different characteristics of widely used PD fluids by proteomics. Of 7894 identified proteins, 3871 were regulated at least by 1 and 49 by all tested PD fluids. The latter subset was enriched for cell junction-associated proteins. The different PD fluids individually perturbed proteins commonly related to cell stress, survival, and immune function pathways. Modeling two major bioincompatibility factors of PD fluids, acidosis, and glucose degradation products (GDPs) revealed distinct effects on endothelial cell function and regulation of cellular stress responses. Proteins and pathways most strongly affected were members of the oxidative stress response. Addition of the antioxidant and cytoprotective additive, alanyl-glutamine (AlaGln), to PD fluids led to upregulation of thioredoxin reductase-1, an antioxidant protein, potentially explaining the cytoprotective effect of AlaGln. In conclusion, we mapped out the molecular response of endothelial cells to PD fluids, and provided new evidence for their specific pathomechanisms, crucial for improvement of PD therapies.

Dual function of HYPONASTIC LEAVES 1 during early skotomorphogenic growth in Arabidopsis.

Seeds germinating underground display a specific developmental programme, termed skotomorphogenesis, to ensure survival of the emerging seedlings until they reach the light. They rapidly elongate the hypocotyl and maintain the cotyledons closed, forming a hook with the hypocotyl in order to protect apical meristematic cells from mechanical damage. Such crucial events for the fate of the seedling are tightly regulated and although some transcriptional regulators and phytohormones are known to be implicated in this regulation, we are still far from a complete understanding of these biological processes. Our work provides information on the diverse roles in skotomorphogenesis of the core components of microRNA biogenesis in Arabidopsis, HYL1, DCL1, and SE. We show that hypocotyl elongation is promoted by all these components, probably through the action of specific miRNAs. Hook development also depends on these proteins however, remarkably, HYL1 exerts its role in an opposite way to DCL1 and SE. Interestingly, we found that a specific HYL1 domain involved in protein-protein interaction is required for this function. Genetic evidences also point to the phosphorylation status of HYL1 as important for this function. We propose that HYL1 help maintain the hook closed during early skotomorphogenesis in a microprocessor-independent manner by repressing the activity of HY5, the transcriptional master regulator that triggers light responses. This work uncovers a previously unnoticed link between components of the miRNA biogenesis machinery, the skotomorphogenic growth, and hook development in Arabidopsis.

A Meta-Analysis of Human Transcriptomics Data in the Context of Peritoneal Dialysis Identifies Novel Receptor-Ligand Interactions as Potential Therapeutic Targets.

Peritoneal dialysis (PD) is one therapeutic option for patients with end-stage kidney disease (ESKD). Molecular profiling of samples from PD patients using different Omics technologies has led to the discovery of dysregulated molecular processes due to PD treatment in recent years. In particular, a number of transcriptomics (TX) datasets are currently available in the public domain in the context of PD. We set out to perform a meta-analysis of TX datasets to identify dysregulated receptor-ligand interactions in the context of PD-associated complications. We consolidated transcriptomics profiles from twelve untargeted genome-wide gene expression studies focusing on human cell cultures or samples from human PD patients. Gene set enrichment analysis was used to identify enriched biological processes. Receptor-ligand interactions were identified using data from CellPhoneDB. We identified 2591 unique differentially expressed genes in the twelve PD studies. Key enriched biological processes included angiogenesis, cell adhesion, extracellular matrix organization, and inflammatory response. We identified 70 receptor-ligand interaction pairs, with both interaction partners being dysregulated on the transcriptional level in one of the investigated tissues in the context of PD. Novel receptor-ligand interactions without prior annotation in the context of PD included BMPR2-GDF6, FZD4-WNT7B, ACKR2-CCL2, or the binding of EPGN and EREG to the EGFR, as well as the binding of SEMA6D to the receptors KDR and TYROBP. In summary, we have consolidated human transcriptomics datasets from twelve studies in the context of PD and identified sets of novel receptor-ligand pairs being dysregulated in the context of PD that warrant investigation in future functional studies.

Effect of cellular senescence on the response of human peritoneal mesothelial cells to TGF-ß.

Transforming growth factor ß (TGF-ß) is implicated in both mesothelial-to-mesenchymal transition (MMT) and cellular senescence of human peritoneal mesothelial cells (HPMCs). We previously showed that senescent HPMCs could spontaneously acquire some phenotypic features of MMT, which in young HPMCs were induced by TGF-ß. Here, we used electron microscopy, as well as global gene and protein profiling to assess in detail how exposure to TGF-ß impacts on young and senescent HPMCs in vitro. We found that TGF-ß induced structural changes consistent with MMT in young, but not in senescent HPMCs. Of all genes and proteins identified reliably in HPMCs across all treatments and states, 4,656 targets represented overlapping genes and proteins. Following exposure to TGF-ß, 137 proteins and 46 transcripts were significantly changed in young cells, compared to 225 proteins and only 2 transcripts in senescent cells. Identified differences between young and senescent HPMCs were related predominantly to wound healing, integrin-mediated signalling, production of proteases and extracellular matrix components, and cytoskeleton structure. Thus, the response of senescent HPMCs to TGF-ß differs or is less pronounced compared to young cells. As a result, the character and magnitude of the postulated contribution of HPMCs to TGF-ß-induced peritoneal remodelling may change with cell senescence.

In-Depth Analysis of the Extracorporeal Proteome Adsorbed to Dialysis Membranes during Hemodialysis.

Used hemodialysis membranes (HD-M) are a valuable reservoir of biological information. Proteins bind to HD-M, but whether this process depends on the type of membrane or patient factors or selectively affects specific protein classes has not been adequately elucidated. State-of-the-art proteomics techniques are capable of identifying and quantifying this therapy-specific subproteome to enable the analysis of disease- or membrane-induced pathophysiologies. We demonstrate the feasibility of the deep proteomic characterization of the extracorporeal proteome adsorbed to HD-M. A shotgun proteomics approach using nano-flow liquid chromatography coupled to mass-spectrometry identified 1648 unique proteins eluted by a chaotropic buffer from the HD-M of eight patients. In total, 995 proteins were present in all eluates; a more stringent approach showed that a core proteome of 310 proteins could be identified independently in all samples. Stability of the dialyzer proteome was demonstrated by a >90% re-identification rate on longitudinal samples of a single patient. The core proteome showed an overrepresentation of pathways of hemostasis and the immune system, and showed differences in membrane materials (polysulfone vs. helixone). This study demonstrates that optimized conditions combined with high-performance proteomics enable the in-depth exploration of the subproteome bound to HD-M, yielding a stable core proteome that can be exploited to study patient-specific factors and improve hemodialysis therapy.

Understanding Cell Model Characteristics-RNA Expression Profiling in Primary and Immortalized Human Mesothelial Cells, and in Human Vein and Microvascular Endothelial Cells.

In vitro studies are essential in pre-clinical research. While choice of cell lines is often driven by handling and cost-effectiveness, in-depth knowledge on specific characteristics is scant. Mesothelial cells, which interact with endothelial cells, are widely used in research, including cancer and drug development, but have not been comprehensively profiled. We therefore performed RNA sequencing of polarized, primary peritoneal (HPMC) and immortalized pleural mesothelial cells (MeT-5A), and compared them to endothelial cells from umbilical vein (HUVEC) and cardiac capillaries (HCMEC). Seventy-seven per cent of 12,760 genes were shared between the 4 cell lines, 1003 were mesothelial and 969 were endothelial cell specific. The transcripts reflected major differences between HPMC and MeT-5A in DNA-related processes, extracellular matrix, migration, proliferation, adhesion, transport, growth factor- and immune response, and between HUVEC and HCMEC in DNA replication, extracellular matrix and adhesion organization. Highly variable shared genes were related to six clusters, cell tissue origin and immortalization, but also cell migration capacity, cell adhesion, regulation of angiogenesis and response to hypoxia. Distinct, cell type specific biological processes were further described by cellular component-, molecular function- and Reactome pathway analyses. We provide crucial information on specific features of the most frequently used mesothelial and endothelial cell lines, essential for appropriate use.

Lithium preserves peritoneal membrane integrity by suppressing mesothelial cell αB-crystallin.

Life-saving renal replacement therapy by peritoneal dialysis (PD) is limited in use and duration by progressive impairment of peritoneal membrane integrity and homeostasis. Preservation of peritoneal membrane integrity during chronic PD remains an urgent but long unmet medical need. PD therapy failure results from peritoneal fibrosis and angiogenesis caused by hypertonic PD fluid (PDF)-induced mesothelial cytotoxicity. However, the pathophysiological mechanisms involved are incompletely understood, limiting identification of therapeutic targets. We report that addition of lithium chloride (LiCl) to PDF is a translatable intervention to counteract PDF-induced mesothelial cell death, peritoneal membrane fibrosis, and angiogenesis. LiCl improved mesothelial cell survival in a dose-dependent manner. Combined transcriptomic and proteomic characterization of icodextrin-based PDF-induced mesothelial cell injury identified αB-crystallin as the mesothelial cell protein most consistently counter-regulated by LiCl. In vitro and in vivo overexpression of αB-crystallin triggered a fibrotic phenotype and PDF-like up-regulation of vascular endothelial growth factor (VEGF), CD31-positive cells, and TGF-ß-independent activation of TGF-ß-regulated targets. In contrast, αB-crystallin knockdown decreased VEGF expression and early mesothelial-to-mesenchymal transition. LiCl reduced VEGF release and counteracted fibrosis- and angiogenesis-associated processes. αB-crystallin in patient-derived mesothelial cells was specifically up-regulated in response to PDF and increased in peritoneal mesothelial cells from biopsies from pediatric patients undergoing PD, correlating with markers of angiogenesis and fibrosis. LiCl-supplemented PDF promoted morphological preservation of mesothelial cells and the submesothelial zone in a mouse model of chronic PD. Thus, repurposing LiCl as a cytoprotective PDF additive may offer a translatable therapeutic strategy to combat peritoneal membrane deterioration during PD therapy.

Peritoneal Dialysis Fluid Supplementation with Alanyl-Glutamine Attenuates Conventional Dialysis Fluid-Mediated Endothelial Cell Injury by Restoring Perturbed Cytoprotective Responses.

Long-term clinical outcome of peritoneal dialysis (PD) depends on adequate removal of small solutes and water. The peritoneal endothelium represents the key barrier and peritoneal transport dysfunction is associated with vascular changes. Alanyl-glutamine (AlaGln) has been shown to counteract PD-induced deteriorations but the effect on vascular changes has not yet been elucidated. Using multiplexed proteomic and bioinformatic analyses we investigated the molecular mechanisms of vascular pathology in-vitro (primary human umbilical vein endothelial cells, HUVEC) and ex-vivo (arterioles of patients undergoing PD) following exposure to PD-fluid. An overlap of 1813 proteins (40%) of over 3100 proteins was identified in both sample types. PD-fluid treatment significantly altered 378 in endothelial cells and 192 in arterioles. The HUVEC proteome resembles the arteriolar proteome with expected sample specific differences of mainly immune system processes only present in arterioles and extracellular region proteins primarily found in HUVEC. AlaGln-addition to PD-fluid revealed 359 differentially abundant proteins and restored the molecular process landscape altered by PD fluid. This study provides evidence on validity and inherent limitations of studying endothelial pathomechanisms in-vitro compared to vascular ex-vivo findings. AlaGln could reduce PD-associated vasculopathy by reducing endothelial cellular damage, restoring perturbed abundances of pathologically important proteins and enriching protective processes.