Novel plasma peptide markers involved in the pathology of CKD identified using mass spectrometric approach Abstract Chronic kidney disease (CKD) may progress to end-stage renal disease (ESRD) at different pace. Early markers of disease progression could facilitate and improve patient management. However, conventional blood and urine chemistry have proven unable to predict the progression of disease at early stages. Therefore, we performed untargeted plasma peptidome analysis to select the peptides involved in progression, which are suitable for long prospective studies in future. The study consists of non-CKD (n = 66) and CKD (n = 106) patients with different stages. We performed plasma peptidomics on these subjects using chromatography and mass spectrometric approaches. Initially, we performed LC-ESI-MS and applied least absolute shrinkage and selection operator logistic regressions to select the peptides that are differentially expressed and we generated a peptidomic score for each subject. Later, we identified and sequenced the peptides with MALDI-MS/MS and also performed univariate and multivariate analyses with the clinical variables and peptidomic score to reveal their association with progression of renal disease. A logistic regression model selected 14 substances showing different concentrations according to renal function, of which seven substances were most likely occur in CKD patients. The peptidomic model had a global P value of < 0.01 with R2 of 0.466, and the area under the curve was 0.87 (95% CI, 0.8149–0.9186; P < 0.0001). The predicted score was significantly higher in CKD than in non-CKD patients (2.539 ± 0.2637 vs − 0.9382 ± 0.1691). The model was also able to predict stages of CKD: the Spearman correlation coefficient of the linear predictor with CKD stages was 0.83 with concordance indices of 0.899 (95% CI 0.863–0.927). In univariate analysis, the most consistent association of peptidomic score in CKD patients was with C-reactive protein, sodium level, and uric acid, which are unanticipated substances. Peptidomic analysis enabled to list some unanticipated substances that have not been extensively studied in the context of CKD but were associated with CKD progression, thus revealing interesting candidate markers or mediators of CKD of potential use in CKD progression management. Key messages • Conventional blood and urine chemistry have proven unable to predict the progression of disease at early stages of chronic kidney disease (CKD). • We performed untargeted plasma peptidome analysis to select the peptides involved in progression. • A logistic regression model selected 14 substances showing different concentrations according to renal function. • These peptides are unanticipated substances that have not been extensively studied in the context of CKD but were associated with CKD progression, thus revealing markers or mediators of CKD of potential use in CKD progression management.

The dichotomous function of interleukin-9 in cancer diseases Abstract The pleiotropic function of the cytokine IL-9 is so far described in many inflammation processes and autoimmune diseases. But its role in cancer immunology is rather diverse as it can have a pro-tumorigenic function as well as anti-tumorigenic characteristics. In various disease models of cancer, this cytokine is involved in different signaling pathways triggering the expression of proteins involved in cell growth, migration, and transformation or repressing cells from the adaptive immune system to reject tumor growth. Additionally, there are even therapeutic approaches for IL-9 in cancer development. This review will give an overview of the various roles of IL-9 in different immune organs and cells and provide an insight in the current state of research in the IL-9-dependent cancer area.

Connecting sex differences, estrogen signaling, and microRNAs in cardiac fibrosis Abstract Sex differences are evident in the pathophysiology of heart failure (HF). Progression of HF is promoted by cardiac fibrosis and no fibrosis-specific therapies are currently available. The fibrotic response is mediated by cardiac fibroblasts (CFs), and a central event is their phenotypic transition to pro-fibrotic myofibroblasts. These myofibroblasts may arise from various cellular origins including resident CFs and epicardial and endothelial cells. Both female subjects in clinical studies and female animals in experimental studies generally present less cardiac fibrosis compared with males. This difference is at least partially considered attributable to the ovarian hormone 17β-estradiol (E2). E2 signals via estrogen receptors to regulate genes are involved in the fibrotic response and myofibroblast transition. Besides protein-coding genes, E2 also regulates transcription of microRNA that modulate cardiac fibrosis. Sex dimorphism, E2, and miRNAs form multi-level regulatory networks in the pathophysiology of cardiac fibrosis, and the mechanism of these networks is not yet fully deciphered. Therefore, this review is aimed at summarizing current knowledge on sex differences, E2, and estrogen receptors in cardiac fibrosis, emphasizing on microRNAs and myofibroblast origins. Key messages • E2 and ERs regulate cardiac fibroblast function. • E2 and ERs may distinctly affect male and female cardiac fibrosis pathophysiology. • Sex, E2, and miRNAs form multi-level regulatory networks in cardiac fibrosis. • Sex-dimorphic and E2-regulated miRNAs affect mesenchymal transition.

Adipose-derived stromal cell secretome disrupts autophagy in glioblastoma Abstract Mesenchymal stromal cells (MSCs) are frequently recruited to tumor sites to play a part in the tumor microenvironment (TME). However, their real impact on cancer cell behavior remains obscure. Here we investigated the effects of human adipose-derived stromal cell (hADSC) secretome in autophagy of glioblastoma (GBM), as a way to better comprehend how hADSCs influence the TME. GBM U-87 MG cells were treated with conditioned medium (CM) from hADSCs and autophagic flux was evaluated. hADSC CM treatment blocked the autophagic flux in tumor cells, as indicated by the accumulation of autophagosomes in the cytosol, the high LC3-II and p62/SQSTM1 protein levels, and the lack of increase in the amount of acidic vesicular organelles. These effects were further detected in other GBM cell lines tested and also in co-cultures of hADSCs and U-87 MG. hADSC CM did not compromise lysosomal acidification; however, it was able to activate mTORC1 signaling and, as a consequence, led to a decrease in the nuclear translocation of TFEB, a master transcriptional regulator of lysosomal biogenesis and autophagy, thereby contributing to a defective autophagic process. hADSCs secrete transforming growth factor beta 1 (TGFβ1) and this cytokine is an important mediator of CM effects on autophagy. A comprehensive knowledge of MSC roles in tumor biology is of great importance to shed light on the complex dialog between these cells and to explore such interactions therapeutically. The present results help to elucidate the paracrine effects of MSCs in tumors and bring attention to the potential to be explored in MSC secretome. Key messages hADSC secretome specifically affects the biology of GBM cells. hADSCs block the late steps of autophagic flux in GBM cells. hADSC secretome activates mTORC1 signaling and reduces TFEB nuclear translocation in GBM cells.

Hypoxia drives cardiac miRNAs and inflammation in the right and left ventricle Abstract Alveolar and myocardial hypoxia may be causes or sequelae of pulmonary hypertension (PH) and heart failure. We hypothesized that hypoxia initiates specific epigenetic and transcriptional, pro-inflammatory programs in the right ventricle (RV) and left ventricle (LV). We performed an expression screen of 750 miRNAs by qPCR arrays in the murine RV and LV in normoxia (Nx) and hypoxia (Hx; 10% O2 for 18 h, 48 h, and 5d). Additional validation included single qPCR analysis of miRNA and pro-inflammatory transcripts in murine and human RV/LV, and neonatal rat cardiomyocytes (NRCMs). Differential qPCR-analysis (Hx vs. Nx in RV, Hx vs. Nx in LV, and RV vs. LV in Hx) identified nine hypoxia-regulated miRNAs: let-7e-5p, miR-29c-3p, miR-127-3p, miR-130a-3p, miR-146b-5p, miR-197-3p, miR-214-3p, miR-223-3p, and miR-451. Hypoxia downregulated miR-146b in the RV (p < 0.01) and, less so, in the LV (trend; p = 0.28). In silico alignment showed significant binding affinity of miR-146b-5p sequence with the 3’UTR of TRAF6 known to be upstream of pro-inflammatory NF-kB. Consistently, hypoxia induced TRAF6, IL-6, CCL2(MCP-1) in the mouse RV and LV. Incubating neonatal rat cardiomyocytes with pre-miR-146b led to a downregulation of TRAF6, IL-6, and CCL2(MCP-1). TRAF6 mRNA expression was also increased by 3-fold in the RV and LV of end-stage idiopathic pulmonary arterial hypertension (PAH) patients vs. non-PAH controls. We identified hypoxia-regulated, ventricle-specific miRNA expression profiles in the adult mouse heart in vivo. Hypoxia suppresses miR-146b, thus de-repressing TRAF6, and inducing pro-inflammatory IL-6 and CCL2(MCP-1). This novel hypoxia-induced miR-146b-TRAF6-IL-6/CCL2(MCP-1) axis likely drives cardiac fibrosis and dysfunction, and may lead to heart failure. Key messages Chouvarine P, Legchenko E, Geldner J, Riehle C, Hansmann G. Hypoxia drives cardiac miRNAs and inflammation in the right and left ventricle. • Hypoxia drives ventricle-specific miRNA profiles, regulating cardiac inflammation. • miR-146b-5p downregulates TRAF6, known to act upstream of pro-inflammatory NF-κB. • Hypoxia downregulates miR-146b and induces TRAF6, IL-6, CCL2 (MCP-1) in the murine RV and LV. • The inhibitory regulatory effects of miR-146b are confirmed in primary rat cardiomyocytes (pre-miR, anti-miR) and human explant heart tissue (endstage pulmonary arterial hypertension). • A novel miR-146b-TRAF6-IL-6/CCL2(MCP-1) axis likely drives cardiac inflammation, fibrosis and ventricular dysfunction.

Phosphate-induced ORAI1 expression and store-operated Ca 2+ entry in aortic smooth muscle cells Abstract Compromised renal phosphate elimination in chronic kidney disease (CKD) leads to hyperphosphatemia, which in turn triggers osteo-/chondrogenic signaling in vascular smooth muscle cells (VSMCs) and vascular calcification. Osteo-/chondrogenic transdifferentiation of VSMCs leads to upregulation of the transcription factors MSX2, CBFA1, and SOX9 as well as tissue-nonspecific alkaline phosphatase (ALPL) which fosters calcification by degrading the calcification inhibitor pyrophosphate. Osteo-/chondrogenic signaling in VSMCs involves the serum- and glucocorticoid-inducible kinase SGK1. As shown in other cell types, SGK1 is a powerful stimulator of ORAI1, a Ca2+-channel accomplishing store-operated Ca2+-entry (SOCE). ORAI1 is stimulated following intracellular store depletion by the Ca2+ sensor STIM1. The present study explored whether phosphate regulates ORAI1 and/or STIM1 expression and, thus, SOCE in VSMCs. To this end, primary human aortic smooth muscle cells (HAoSMCs) were exposed to the phosphate donor β-glycerophosphate. Transcript levels were estimated by qRT-PCR, protein abundance by western blotting, ALPL activity by colorimetry, calcification by alizarin red S staining, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin. As a result, β-glycerophosphate treatment increased ORAI1 and STIM1 transcript levels and protein abundance as well as SOCE in HAoSMCs. Additional treatment with ORAI1 inhibitor MRS1845 or SGK1 inhibitor GSK650394 virtually disrupted the effects of β-glycerophosphate on SOCE. Moreover, the β-glycerophosphate-induced MSX2, CBFA1, SOX9, and ALPL mRNA expression and activity in HAoSMCs were suppressed in the presence of the ORAI1 inhibitor and upon ORAI1 silencing. In conclusion, enhanced phosphate upregulates ORAI1 and STIM1 expression and store-operated Ca2+-entry, which participate in the orchestration of osteo-/chondrogenic signaling of VSMCs. Key messages • In aortic SMC, phosphate donor ß-glycerophosphate upregulates Ca2+ channel ORAI1. • In aortic SMC, ß-glycerophosphate upregulates ORAI1-activator STIM1. • In aortic SMC, ß-glycerophosphate upregulates store-operated Ca2+-entry (SOCE). • The effect of ß-glycerophosphate on SOCE is disrupted by ORAI1 inhibitor MRS1845. • Stimulation of osteogenic signaling is disrupted by MRS1845 and ORAI1 silencing.

S100A4 promotes inflammation but suppresses lipid accumulation via the STAT3 pathway in chronic ethanol-induced fatty liver Abstract S100A4, a member of the S100 calcium-binding protein family, has been identified in a subpopulation of liver macrophages and promotes liver fibrosis via hepatic stellate cell activation. However, the specific role of S100A4 in alcoholic liver disease (ALD) has not been well investigated. Here, S100A4 knockout (S100A4−/−) mice were used in a chronic-binge ethanol model for studying the role of S100A4 and its related molecular mechanism in ALD. S100A4 expression was increased in ethanol-induced liver tissues of wild-type (WT) mice. Macrophage-derived S100A4 promoted liver inflammation but suppressed lipid accumulation under the ethanol feeding condition. S100A4 deficiency promoted ethanol-induced liver injury and hepatic fat accumulation. Further mechanistic studies found that S100A4 inhibited liver fat accumulation mainly by activating the STAT3 pathway and downregulating lipogenic gene expression, especially that of SREBP-1c. In AML-12 cells, a STAT3 inhibitor abolished STAT3 levels and decreased the expression of SREBP1c. Furthermore, the administration of a neutralizing S100A4 antibody to WT mice significantly promoted ethanol-induced liver injury and fatty accumulation. Thus, S100A4 may represent a potential candidate target for the prevention and treatment of ethanol-induced fatty liver. In this study, we discovered the special role of S100A4 in alcoholic liver disease. S100A4 deficiency attenuated ethanol-induced hepatitis and promoted hepatic fat accumulation in ethanol-induced liver tissues. Further mechanistic studies have found that S100A4 promotes early alcoholic hepatitis mainly by activating the STAT3 pathway and its downstream proinflammatory gene expression. Interestingly, activation of the STAT3 pathway downregulates lipogenic gene expression, especially SREBP-1c. Key messages In this study, we discovered the special role of S100A4 in alcoholic liver disease. S100A4 deficiency attenuated ethanol-induced hepatitis and promoted hepatic fat accumulation in ethanol-induced liver tissues. Further mechanistic studies have found that S100A4 promotes early alcoholic hepatitis mainly by activating the STAT3 pathway and its downstream proinflammatory gene expression. Interestingly, activation of the STAT3 pathway downregulates lipogenic gene expression, especially SREBP-1c.

Substance P induces fibrotic changes through activation of the RhoA/ROCK pathway in an in vitro human corneal fibrosis model Abstract Fibrosis is characterized by hardening, overgrowth, and development of scars in various tissues as a result of faulty reparative processes, diseases, or chronic inflammation. During the fibrotic process in the corneal stroma of the eye, the resident cells called keratocytes differentiate into myofibroblasts, specialized contractile fibroblastic cells that produce excessive amounts of disorganized extracellular matrix (ECM) and pro-fibrotic components such as alpha-smooth muscle actin (α-SMA) and fibronectin. This study aimed to elucidate the role of substance P (SP), a neuropeptide that has been shown to be involved in corneal wound healing, in ECM production and fibrotic markers expression in quiescent human keratocytes, and during the onset of fibrosis in corneal fibroblasts, in an in vitro human corneal fibrosis model. We report that SP induces keratocyte contraction and upregulates gene expression of collagens I, III, and V, and fibrotic markers: α-SMA and fibronectin, in keratocytes. Using our in vitro human corneal fibrosis model, we show that SP enhances gene expression and secretion of collagens I, III, and V, and lumican. Moreover, SP upregulates gene expression and secretion of α-SMA and fibronectin, and increases contractility of corneal fibroblasts during the onset of fibrosis. Activation of the preferred SP receptor, the neurokinin-1 receptor (NK-1R), is necessary for the SP-induced pro-fibrotic changes. In addition, SP induces the pro-fibrotic changes through activation of the RhoA/ROCK pathway. Taken together, we show that SP has a pro-fibrotic effect in both quiescent human keratocytes and during the onset of fibrosis in an in vitro human corneal fibrosis model.

Molecular changes in solitary fibrous tumor progression Abstract Solitary fibrous tumors (SFTs) are NAB2-STAT6 fusion-associated neoplasms. There are several subtypes of NAB2-STAT6 fusions, but their clinical significances are still unclear. Moreover, the mechanisms of malignant progression are also poorly understood. In this study, using 91 SFT cases, we examined whether fusion variants are associated with clinicopathological parameters and also investigated the molecular mechanism of malignant transformation using whole-exome sequencing. We detected variant 1b (NAB2ex4-STAT6ex2) in 51/91 (56%) cases and variants 2a/2b (NAB2ex6-STAT6ex16/17) in 17/91 (19%) cases. The NAB2-STAT6 fusion variant types were significantly associated with their primary site (P < 0.001). In addition, a TERT promoter mutation was detected in 7/73 (10%) cases, and it showed a significant association with malignant SFTs (P = 0.003). To identify molecular changes during malignant progression, we selected an index patient to obtain parallel tissue samples from the primary and metastatic tumors. In the metastatic tissue, 10 unique molecular alterations, including those in TP53 and APAF1, were detected. In vitro functional experiments showed that APAF1 depletion increased the tumor potency of cells expressing NAB2-STAT6 fusion protein under treatment with staurosporine. We found that TP53 immunopositivity (P = 0.006) and loss of APAF1 immunoreactivity (P < 0.001) were significantly associated with malignant SFTs. Our study suggests that dysfunction of TP53 and APAF1 leads to impaired apoptotic function, and eventually contributes toward malignant SFT transformation. Key messages We firstly found that the TERT promoter mutation was strongly associated with malignant SFTs (P = 0.003) and the representative 1b (NAB2ex4-STAT6ex2) or 2a (NAB2ex6-STAT6ex16) fusion variants similarly contribute to tumorigenicity. We also found that TP53 immunopositivity (P = 0.006) and loss of APAF1 immunoreactivity (P < 0.001) were significantly associated with malignant SFTs. Our study suggests that dysfunction of TP53 and APAF1 leads to impaired apoptotic function, and eventually contributes toward malignant SFT transformation.

Bone allografts combined with adipose-derived stem cells in an optimized cell/volume ratio showed enhanced osteogenesis and angiogenesis in a murine femur defect model Abstract Critical sized defects, especially in long bones, pose one of the biggest problems in orthopedic surgery. By definition, these defects do not heal without further treatment. Different therapeutic options range from autologous bone grafts, for example, free vascularized bone grafts, to commercially available bone allografts. Disadvantages of these bone allografts are related to reduced osteogenesis, since they are solely composed of cell-free bone matrix. The purpose of this study was to investigate the cell seeding efficiency of human adipose-derived stem cells (hASCs) on human bone allografts in vitro and furthermore analyze these optimized seeded allografts in a critical sized defect model in vivo. Cancellous human bone allografts were colonized with human ASCs in vitro. Cell seeding efficiency was evaluated by Cell Counting Kit-8 assay. Thereafter, optimized hASC-seeded bone scaffolds were examined in a murine femur defect model, stabilized with the MouseExFix system. Subsequently, x-ray analysis and histology were performed. Examination of cell seeding efficiency revealed an optimum starting population of 84,600 cells per 100 mm3 scaffold. In addition, scaffolds seeded with hASCs showed increased osteogenesis compared with controls. Histological analysis revealed increased remodeling and elevated new bone formation within hASC-seeded scaffolds. Moreover, immunohistochemical stainings revealed increased proliferation, osteogenesis, and angiogenesis. In this study, we systemically optimized cell/volume ratio of two promising components of tissue engineering: hASCs and human bone allografts. These findings may serve as a basis for future translational studies. Key messages Bone tissue engineering. Mesenchymal stem cells derived from human adipose tissue (hASCs). Optimal cell/volume ratio of cell-seeded scaffolds. Increased osteogenesis and angiogenesis in vivo.