Cancer-associated fibroblasts-secreted exosomal miR-92a-3p promotes tumor growth and stemness in hepatocellular carcinoma through activation of Wnt/ß-catenin signaling pathway by suppressing AXIN1.

Cancer-associated fibroblasts (CAFs) are a major cellular component in the tumor microenvironment and have been shown to exhibit protumorigenic effects in hepatocellular carcinoma (HCC). This study aimed to delve into the mechanisms underlying the tumor-promoting effects of CAFs in HCC. Small RNA sequencing was conducted to screen differential expressed microRNAs in exosomes derived from CAFs and normal fibroblasts (NFs). The miR-92a-3p expression was then measured using reverse transcriptase quantitative real-time PCR in CAFs, NFs, CAFs-derived exosomes (CAFs-Exo), and NF-derived exosomes (NFs-Exo). Compared to NFs or NF-Exo, CAFs and CAFs-Exo significantly promoted HCC cell proliferation, migration, and stemness. Additionally, compared to NFs or NF-Exo, miR-92a-3p level was notably higher in CAFs and CAFs-Exo, respectively. Exosomal miR-92a-3p was found to enhance HCC cell proliferation, migration, and stemness. Meanwhile, AXIN1 was targeted by miR-92a-3p. Exosomal miR-92a-3p could activate ß-catenin/CD44 signaling in HCC cells by inhibiting AXIN1 messenger RNA. Furthermore, in vivo studies verified that exosomal miR-92a-3p notably promoted tumor growth and stemness through targeting AXIN1/ß-catenin axis. Collectively, CAFs secreted exosomal miR-92a-3p was capable of promoting growth and stemness in HCC through activation of Wnt/ß-catenin signaling pathway by suppressing AXIN1. Therefore, targeting CAFs-derived miR-92a-3p may be a potential strategy for treating HCC.

Osteogenic differentiation of periodontal ligament fibroblasts inhibits osteoclast formation.

One of the deficits of knowledge on bone remodelling, is to what extent cells that are driven towards osteogenic differentiation can contribute to osteoclast formation. The periodontal ligament fibroblast (PdLFs) is an ideal model to study this, since they play a role in osteogenesis, and can also orchestrate osteoclastogenesis.when co-cultured with a source of osteoclast-precursor such as peripheral blood mononuclear cells (PBMCs). Here, the osteogenic differentiation of PdLFs and the effects of this process on the formation of osteoclasts were investigated. PdLFs were obtained from extracted teeth and exposed to osteogenic medium for 0, 7, 14, or 21 out of 21 days. After this 21-day culturing period, the cells were co-cultured with peripheral blood mononuclear cells (PBMCs) for an additional 21 days to study osteoclast formation. Alkaline phosphatase (ALP) activity, calcium concentration, and gene expression of osteogenic markers were assessed at day 21 to evaluate the different stages of osteogenic differentiation. Alizarin red staining and scanning electron microscopy were used to visualise mineralisation. Tartrate-resistant acid phosphatase (TRAcP) activity, TRAcP staining, multinuclearity, the expression of osteoclastogenesis-related genes, and TNF-α and IL-1ß protein levels were assessed to evaluate osteoclastogenesis. The osteogenesis assays revealed that PdLFs became more differentiated as they were exposed to osteogenic medium for a longer period of time. Mineralisation by these osteogenic cells increased with the progression of differentiation. Culturing PdLFs in osteogenic medium before co-culturing them with PMBCs led to a significant decrease in osteoclast formation. qPCR revealed significantly lower DCSTAMP expression in cultures that had been supplemented with osteogenic medium. Protein levels of osteoclastogenesis stimulator TNF-α were also lower in these cultures. The present study shows that the osteogenic differentiation of PdLFs reduces the osteoclastogenic potential of these cells. Immature cells of the osteoblastic lineage may facilitate osteoclastogenesis, whereas mature mineralising cells may suppress the formation of osteoclasts. Therefore, mature and immature osteogenic cells may have different roles in maintaining bone homeostasis.

Single-cell analysis of Crohn's disease: Unveiling heterogeneity and evaluating ustekinumab outcomes.

BACKGROUND: The present study aimed to dissect the cellular complexity of Crohn's disease (CD) using single-cell RNA sequencing, focusing on identifying key cell populations and their transcriptional profiles in inflamed tissue. METHODS: We applied scRNA-sequencing to compare the cellular composition of CD patients with healthy controls, utilizing Seurat for clustering and annotation. Differential gene expression analysis and protein-protein interaction networks were constructed to identify crucial genes and pathways. RESULTS: Our study identified eight distinct cell types in CD, highlighting crucial fibroblast and T cell interactions. The analysis revealed key cellular communications and identified significant genes and pathways involved in the disease's pathology. The role of fibroblasts was underscored by elevated expression in diseased samples, offering insights into disease mechanisms and potential therapeutic targets, including responses to ustekinumab treatment, thus enriching our understanding of CD at a molecular level. CONCLUSIONS: Our findings highlight the complex cellular and molecular interplay in CD, suggesting new biomarkers and therapeutic targets, offering insights into disease mechanisms and treatment implications.

Cardiac and skeletal muscle manifestations in the G608G mouse model of Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder resulting from de novo mutations in the lamin A gene. Children with HGPS typically pass away in their teenage years due to cardiovascular diseases such as atherosclerosis, myocardial infarction, heart failure, and stroke. In this study, we characterized the G608G HGPS mouse model and explored cardiac and skeletal muscle function, along with senescence-associated phenotypes in fibroblasts. Homozygous G608G HGPS mice exhibited cardiac dysfunction, including decreased cardiac output and stroke volume, and impaired left ventricle relaxation. Additionally, skeletal muscle exhibited decreased isometric tetanic torque, muscle atrophy, and increased fibrosis. HGPS fibroblasts showed nuclear abnormalities, decreased proliferation, and increased expression of senescence markers. These findings provide insights into the pathophysiology of the G608G HGPS mouse model and inform potential therapeutic strategies for HGPS.

Heterogeneity of cancer-associated fibroblasts and tumor-promoting roles in head and neck squamous cell carcinoma.

Tumor microenvironment (TME) in head and neck squamous cell carcinoma (HNSCC) has a major influence on disease progression and therapy response. One of the predominant stromal cell types in the TME of HNSCC is cancer-associated fibroblasts (CAF). CAF constitute a diverse cell population and we are only at the beginning of characterizing and understanding the functions of various CAF subsets. CAF have been shown to interact with tumor cells and other components of the TME to shape mainly a favourable microenvironment for HNSCC progression, although some studies report existence of tumor-restraining CAF subtypes. The numerous pathways used by CAF to promote tumorigenesis may represent potential therapeutic targets. This review summarizes current knowledge on the origins, subtypes and mechanisms employed by CAF in HNSCC. The aim is to contribute to the understanding on how CAF actively influence the TME and modulate different immune cell types, as well as cancer cells, to establish a conducive setting for cancer growth. Although CAF are currently a promising therapeutic target for the treatment of other types of cancer, there is no significant therapeutic advancement in HNSCC.

Evaluation of antitumor potential of an anti-glypican-1 monoclonal antibody in preclinical lung cancer models reveals a distinct mechanism of action.

Aim: The main objective of this study was to investigate the antitumor effect of a mouse anti-human glypican-1 (GPC1) monoclonal antibody (mAb) on non-small cell lung carcinoma (NSCLC) and associated molecular mechanisms. Methods: The anti-proliferative and anti-migratory activities of anti-GPC1 mAb were examined in A549 and H460 NSCLC cells and LL97A lung fibroblasts. The inhibitory effect of anti-GPC1 mAb on tumor growth was evaluated in an orthotopic lung tumor model. Results: The in vitro study showed that anti-GPC1 mAb profoundly inhibited the anchorage-independent growth of A549 and H460 NSCLC cells and exhibited relatively high cytotoxic activities towards LL97A lung fibroblasts, A549/LL97A and H460/LL97A coculture spheroids. Moreover, anti-GPC1 mAb significantly decreased the expression of phospho-Src (p-Src; Tyr416), p-Akt (Ser473) and ß-catenin in the co-cultured LL97A lung fibroblasts, and the expression of phospho-mitogen-activated protein kinase kinase (p-MEK; Ser217/221) and phospho-90 kDa ribosomal s6 kinase (p-p90RSK; Ser380) in co-cultured A549 cells. When anti-GPC1 mAb was administered to tumor-bearing mice, the inhibitory effect of anti-GPC1 mAb on the orthotopic lung tumor growth was not statistically significant. Nonetheless, results of Western blot analysis showed significant decrease in the phosphorylation of fibroblast growth factor receptor 1 (FGFR1) at Tyr766, Src at Tyr416, extracellular signal-regulated kinase (ERK) at Thr202/Tyr204, 90 kDa ribosomal S6 kinase (RSK) at Ser380, glycogen synthase kinases 3α (GSK3α) at Ser21 and GSK3ß at Ser9 in tumor tissues. These data implicate that anti-GPC1 mAb treatment impairs the interaction between tumor cells and tumor associated fibroblasts by attenuating the paracrine FGFR signal transduction. Conclusions: The relatively potent cytotoxicity of anti-GPC1 mAb in lung fibroblasts and its potential inhibitory effect on the paracrine FGFR signal transduction warrant further studies on the combined use of this mAb with targeted therapeutics to improve therapeutic outcomes in lung cancer.

Fowl adenovirus serotype 4 (FAdV-4) infection induces inflammatory responses in chicken embryonic cardiac fibroblasts via PI3K/Akt and IκBα/NF-κB signaling pathways.

Fowl adenovirus serotype 4 (FAdV-4) is the main pathogen of the acute infectious disease hepatitis-hydropericardium syndrome (HHS). Previous studies have focused on the mechanisms of FAdV-4 caused liver injury, while studies revealing potential mechanisms of inflammatory injury in FAdV-4-infected chicken cardiac cells remain scare. Here we found that FAdV-4 successfully infected chicken embryonic cardiac fibroblasts (CECF) cells in vitro and significantly upregulated production of inflammatory cytokines including IL-1ß, IL-6, IL-8, and TNF-α, suggesting induction of a strong inflammatory response. Mechanistically, FAdV-4 infection increased expression of phosphorylated Akt in a time-dependent manner, while phosphorylation of Akt and production of pro-inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α were greatly reduced in FAdV-4-infected CECF cells after treatment with LY294002, a potent inhibitor of PI3K, indicating that the inflammatory response induced by FAdV-4 infection is mediated by the PI3K/Akt signaling pathway. Furthermore, FAdV-4 infection increased expression of phosphorylated IκBα, a recognized indicator of NF-κB activation, and treatment with the BAY11-7082, a selective IκBα phosphorylation and NF-κB inhibitor, significantly reduced IκBα phosphorylation and inflammatory cytokines (IL-1ß, IL-6, IL-8, and TNF-α) production in FAdV-4-infected CECF cells, suggesting a critical role of IκBα/NF-κB signaling in FAdV-4-induced inflammatory responses in CECF cells. Taken together, our results suggest that FAdV-4 infection induces inflammatory responses through activation of PI3K/Akt and IκBα/NF-κB signaling pathways in CECF cells. These results reveal potential mechanisms of inflammatory damage in chicken cardiac cells caused by FAdV-4 infection, which sheds new insight into clarification of the pathogenic mechanism of FAdV-4 infection and development of new strategies for HHS prevention and control.

The Combination of Methionine Restriction and Docetaxel Synergistically Arrests Androgen-independent Prostate Cancer But Not Normal Cells.

Background/Aim: Androgen-independent prostate cancer (AIPC) is resistant to androgen-depletion therapy and is a recalcitrant disease. Docetaxel is the first-line treatment for AIPC, but has limited efficacy and severe side-effects. All cancers are methionine-addicted, which is termed the Hoffman effect. Recombinant methioninase (rMETase) targets methionine addiction. The purpose of the present study was to determine if the combination of docetaxel and rMETase is effective for AIPC. Materials and Methods: The half-maximal inhibitory concentrations (IC50) of docetaxel and rMETase alone were determined for the human AIPC cell line PC-3 and Hs27 normal human fibroblasts in vitro. The synergistic efficacy for PC-3 and Hs27 using the combination of docetaxel and rMETase at their IC50s for PC-3 was determined. Results: The IC50 of docetaxel for PC-3 and for Hs27 was 0.72 nM and 0.94 nM, respectively. The IC50 of rMETase for PC-3 and for Hs27 was 0.67 U/ml and 0.76 U/ml, respectively. The combination of docetaxel and rMETase was synergistic for PC-3 but not Hs27 cells. Conclusion: The combination of a relatively low concentration of docetaxel and rMETase was synergistic and effective for AIPC. The present results also suggest that the effective concentration of docetaxel can be reduced by using rMETase, which may reduce toxicity. The present results also suggest the future clinical potential of the combination of docetaxel and rMETase for AIPC.

Prostate cancer cancer-associated fibroblasts with stable markers post-androgen deprivation therapy associated with tumor progression and castration resistant prostate cancer.

The specificity and clinical relevance of cancer-associated fibroblasts (CAFs) in prostate cancer (PCa), as well as the effect of androgen deprivation therapy (ADT) on CAFs, remain to be fully elucidated. Using cell lineage diversity and weighted gene co-expression network analysis (WGCNA), we pinpointed a unique CAF signature exclusive to PCa. The specificity of this CAF signature was validated through single-cell RNA sequencing (scRNA-seq), cell line RNA sequencing, and immunohistochemistry. This signature associates CAFs with tumor progression, elevated Gleason scores, and the emergence of castration resistant prostate cancer (CRPC). Using scRNA-seq on collected samples, we demonstrated that the CAF-specific signature is not altered by ADT, maintaining its peak signal output. Identifying a PCa-specific CAF signature and observing signaling changes in CAFs after ADT lay essential groundwork for further PCa studies.

Fibroblasts activation by embryonic signal switching: A novel mechanism of placental growth factor-induced cardiac remodeling.

INTRODUCTION: Cardiac remodeling is defined as cellular interstitial changes that lead dysfunction of the heart after injury. Placental growth factor (PlGF), a member of the VEGF family, has been reported to regulate cardiac hypertrophy in hemodynamic state. We therefore analyze the function of PlGF during cardiac remodeling using cardiac cells and fibroblasts, under Angiotensin II (AngII) stimulation. METHODS: PlGF overexpressed mouse embryonic fibroblasts derived from C57BL/6 mice, were made by deficient retrovirus vector, designated as C57/PlGF. Only retrovirus vector introduced C57 cells (C57/EV) were used as control. After AngII stimulation, wound scratching assay and MTT proliferation assay with or without p38 MAPK inhibitor, SB205580 were performed in retrovirally-introduced C57 cells. Reactive oxygen species (ROS) production, NF-kB activation, IL-6 and TNF-α production were also measured. Then we assessed AngII-induced cell proliferation of mouse cardiac fibroblasts (CFs) and rat primary cardiomyocytes incubating with C57/PlGF conditioned-medium. RESULTS: The PlGF production in C57/PlGF were confirmed by ELISA (1093.48 ± 3.5 pg/ml, ±SE). AngII-induced cell migration, proliferation and H2O2 production were increased in C57/PlGF compared with C57/EV. SB205580 inhibited the AngII-induced cell proliferation in C57/PlGF. In C57/PlGF cells, NF-kB activation was higher, followed by up-regulation of IL-6 and TNF-α production. CFs and cardiomyocytes proliferation increased when stimulated with C57/PlGF conditioned-medium. DISCUSSION: The activation of fibroblast is stimulated by PlGF signaling via p38 MAPK/NF-kB pathway accompanied by elevation of ROS and inflammatory response. Furthermore, these signals stimulate the activation of CFs and cardiomyocytes, indicating that high circulating level of PlGF have a potential to regulate cardiac remodeling.

A mathematical model for wound healing in the reef-building coral Pocillopora damicornis.

Coral reefs, among the most diverse ecosystems on Earth, currently face major threats from pollution, unsustainable fishing practices , and perturbations in environmental parameters brought on by climate change. Corals also sustain regular wounding from other sea life and human activity. Recent reef restoration practices have even involved intentional wounding by systematically breaking coral fragments and relocating them to revitalize damaged reefs, a practice known as microfragmentation. Despite its importance, very little research has explored the inner mechanisms of wound healing in corals. Some reef-building corals have been observed to initiate an immunological response to wounding similar to that observed in mammalian species. Utilizing prior models of wound healing in mammalian species as the mathematical basis, we formulated a mechanistic model of wound healing, including observations of the immune response and tissue repair in scleractinian corals for the species Pocillopora damicornis. The model consists of four differential equations which track changes in remaining wound debris, number of cells involved in inflammation, number of cells involved in proliferation, and amount of wound closure through re-epithelialization. The model is fit to experimental wound size data from linear and circular shaped wounds on a live coral fragment. Mathematical methods, including numerical simulations and local sensitivity analysis, were used to analyze the resulting model. The parameter space was also explored to investigate drivers of other possible wound outcomes. This model serves as a first step in generating mathematical models for wound healing in corals that will not only aid in the understanding of wound healing as a whole, but also help optimize reef restoration practices and predict recovery behavior after major wounding events.

Immunomodulation and fibroblast dynamics driving nociceptive joint pain within inflammatory synovium: Unravelling mechanisms for therapeutic advancements in osteoarthritis.

OBJECTIVE: Synovitis is a widely accepted sign of osteoarthritis (OA), characterised by tissue hyperplasia, where increased infiltration of immune cells and proliferation of resident fibroblasts adopt a pro-inflammatory phenotype, and increased the production of pro-inflammatory mediators that are capable of sensitising and activating sensory nociceptors, which innervate the joint tissues. As such, it is important to understand the cellular composition of synovium and their involvement in pain sensitisation to better inform the development of effective analgesics. METHODS: Studies investigating pain sensitisation in OA with a focus on immune cells and fibroblasts were identified using PubMed, Web of Science and SCOPUS. RESULTS: In this review, we comprehensively assess the evidence that cellular crosstalk between resident immune cells or synovial fibroblasts with joint nociceptors in inflamed OA synovium contributes to peripheral pain sensitisation. Moreover, we explore whether the elucidation of common mechanisms identified in similar joint conditions may inform the development of more effective analgesics specifically targeting OA joint pain. CONCLUSION: The concept of local environment and cellular crosstalk within the inflammatory synovium as a driver of nociceptive joint pain presents a compelling opportunity for future research and therapeutic advancements.

Pro-fibrotic effect of the susceptible gene PCSK5 in vascular fibrosis of Takayasu arteritis via TGF-ß and SMAD3 signaling pathway activation.

BACKGROUND: Vascular fibrosis directly causes vascular thickening in Takayasu arteritis (TAK), in which sustained transforming growth factor beta (TGF-ß) activation is critical. Understanding TGF-ß activation regulation and blocking it might yield a therapeutic effect in TAK. Proprotein convertase subtilisin/kexin type 5 (PCSK5) rs6560480 (T/C) is associated with TAK development. In this study, we assessed the association between the PCSK5 rs6560480 genotype and PCSK5 expression in TAK and explored its molecular role in TGF-ß activation and vascular fibrosis development. METHODS: In TAK patients, PCSK5 and TGF-ß expression in plasma and aortic tissue was examined by ELISA and immunohistochemical staining, and PCSK5 rs6560480 was genotyped. The correlation between PCSK5 and extracellular matrix (ECM) expression was examined by Western blotting (WB) and immunohistochemistry staining. Detection by co-immunoprecipitation was performed to detect the interaction between PCSK5 and TGF-ß in adventitial fibroblasts (AAFs). Downstream signaling pathways were detected by WB and validated with appropriate inhibitors. Potential immunosuppressive agents to inhibit the effects of PCSK5 were explored in cell culture and TAK patients. RESULTS: Patients with PCSK5 rs6560480 TT patients had significantly higher PCSK5 levels and more thickened vascular lesions than patients with PCSK5 rs6560480 CT. PCSK5 expression was significantly increased in alpha smooth muscle actin (α-SMA)-positive myofibroblasts in TAK vascular lesions. Overexpressing PCSK5 facilitated TGF-ß and downstream SMAD2/3 activation and ECM expression in AAFs and aorta in in-vitro culture. The mechanistic study supported that PCSK5 activated precursor TGF-ß (pro-TGF-ß) to the mature form by binding the pro-TGF-ß cleavage site. Leflunomide inhibited PCSK5 and pro-TGF-ß binding, decreasing TGF-ß activation and ECM expression, which was also partially validated in leflunomide-treated patients. CONCLUSION: The findings revealed a novel pro-fibrotic mechanism of PCSK5 in TAK vascular fibrosis via TGF-ß and downstream SMAD2/3 pathway activation. Leflunomide might be anti-fibrotic by disrupting PCSK5 and pro-TGF-ß binding, presenting a new TAK treatment approach.

Exploring the mechanism of luteolin improving immune and inflammatory responses in systemic sclerosis based on systems biology and cell experiments.

There is a growing trend of applying traditional Chinese medicine (TCM) to treat immune diseases. This study reveals the possible mechanism of luteolin, an active ingredient in the core prescription of TCM, in alleviating systemic sclerosis (SSc) inflammation. Bibliometrics was performed to retrieve the core keywords of SSc inflammation. The key inflammatory indicators in the serum samples of 50 SSc patients were detected by ELISA. Data mining was applied for correlation analysis, association rule analysis, and binary logistic regression analysis on the clinical indicators and medication of 50 SSc patients before and after treatment to determine the core prescription. Network pharmacology was used for identifying candidate genes and pathways; molecular docking was conducted to determine the core monomer components of the prescription, providing a basis for subsequent in vitro molecular mechanism research. The effect of luteolin on SSc-human dermal fibroblasts (HDF) viability and inflammatory factors was evaluated by means of ELISA, RT-PCR, and Western blot. The role of TNF in inflammation was explored by using a TNF overexpression vector, NF-κB inhibitor (PKM2), and SSc-HDF. The involvement of TNF/NF-κB pathway was validated by RT-PCR, Western blot, and immunofluorescence. TCM treatment partially corrected the inflammatory changes in SSc patients, indicating its anti-inflammatory effects in the body. Atractylodes, Yam, Astragalus root, Poria cocos, Pinellia ternata, Salvia miltiorrhiza, Safflower, Cassia twig, and Angelica were identified as the core prescriptions for improving inflammatory indicators. Luteolin was the main active ingredient in the prescription and showed a strong binding energy with TNF and NF-κB. Luteolin exerted anti-inflammatory effects in vitro by reducing inflammatory cytokines in SSc-HDF and inhibiting the activation of TNF/NF-κB. Mechanistically, luteolin inhibited the activation of the TNF/NF-κB pathway in SSc-HDF, as manifested by an increase in extranuclear p-P65 and TNF but a decrease in intranuclear p-P65. Interestingly, the addition of PKM2 augmented the therapeutic function of luteolin against inflammation in SSc-HDF. Our study showed the TCM alleviates the inflammatory response of SSc by inhibiting the activation of the TNF/NF-κB pathway and is an effective therapeutic agent for the treatment of SSc.

Variations in fluid chemical potential induce fibroblast mechano-response in 3D hydrogels.

Mechanical deformation of skin creates variations in fluid chemical potential, leading to local changes in hydrostatic and osmotic pressure, whose effects on mechanobiology remain poorly understood. To study these effects, we investigate the specific influences of hydrostatic and osmotic pressure on primary human dermal fibroblasts in three-dimensional hydrogel culture models. Cyclic hydrostatic pressure and hyperosmotic stress enhanced the percentage of cells expressing the proliferation marker Ki67 in both collagen and PEG-based hydrogels. Osmotic pressure also activated the p38 MAPK stress response pathway and increased the expression of the osmoresponsive genes PRSS35 and NFAT5. When cells were cultured in two-dimension (2D), no change in proliferation was observed with either hydrostatic or osmotic pressure. Furthermore, basal, and osmotic pressure-induced expression of osmoresponsive genes differed in 2D culture versus 3D hydrogels, highlighting the role of dimensionality in skin cell mechanotransduction and stressing the importance of 3D tissue-like models that better replicate in vivo conditions. Overall, these results indicate that fluid chemical potential changes affect dermal fibroblast mechanobiology, which has implications for skin function and for tissue regeneration strategies.

Identification of cellular and noncellular components of mature intact human peripheral nerve.

BACKGROUND AND AIMS: The goal of this study was to define basic constituents of the adult peripheral nervous system (PNS) using intact human nerve tissues. METHODS: We combined fluorescent and chromogenic immunostaining methods, myelin-selective fluorophores, and routine histological stains to identify common cellular and noncellular elements in aldehyde-fixed nerve tissue sections. We employed Schwann cell (SC)-specific markers, such as S100ß, NGFR, Sox10, and myelin protein zero (MPZ), together with axonal, extracellular matrix (collagen IV, laminin, fibronectin), and fibroblast markers to assess the SC's relationship to myelin sheaths, axons, other cell types, and the acellular environment. RESULTS: Whereas S100ß and Sox10 revealed mature SCs in the absence of other stains, discrimination between myelinating and non-myelinating (Remak) SCs required immunodetection of NGFR along with axonal and/or myelin markers. Surprisingly, our analysis of NGFR+ profiles uncovered the existence of at least 3 different novel populations of NGFR+/S100ß- cells, herein referred to as nonglial cells, residing in the stroma and perivascular areas of all nerve compartments. An important proportion of the nerve's cellular content, including circa 30% of endoneurial cells, consisted of heterogenous S100ß negative cells that were not associated with axons. Useful markers to identify the localization and diversity of nonglial cell types across different compartments were Thy1, CD34, SMA, and Glut1, a perineurial cell marker. INTERPRETATION: Our optimized methods revealed additional detailed information to update our understanding of the complexity and spatial orientation of PNS-resident cell types in humans.

Human primary cells can tell body time: Dedicated to Steven A. Brown.

The field of chronobiology has advanced significantly since ancient observations of natural rhythms. The intricate molecular architecture of circadian clocks, their hierarchical organization within the mammalian body, and their pivotal roles in organ physiology highlight the complexity and significance of these internal timekeeping mechanisms. In humans, circadian phenotypes exhibit considerable variability among individuals and throughout the individual's lifespan. A fundamental challenge in mechanistic studies of human chronobiology arises from the difficulty of conducting serial sampling from most organs. The concept of studying circadian clocks in vitro relies on the groundbreaking discovery by Ueli Schibler and colleagues that nearly every cell in the body harbours autonomous molecular oscillators. The advent of circadian bioluminescent reporters has provided a new perspective for this approach, enabling high-resolution continuous measurements of cell-autonomous clocks in cultured cells, following in vitro synchronization pulse. The work by Steven A. Brown has provided compelling evidence that clock characteristics assessed in primary mouse and human skin fibroblasts cultured in vitro represent a reliable estimation of internal clock properties in vivo. The in vitro approach for studying molecular human clocks in cultured explants and primary cells, pioneered by Steve Brown, represents an invaluable tool for assessing inter-individual differences in circadian characteristics alongside comprehensive genetic, biochemical and functional analyses. In a broader context, this reliable and minimally invasive approach offers a unique perspective for unravelling the functional inputs and outputs of oscillators operative in nearly any human tissue in physiological contexts and across various pathologies.

Rare Variant in MRC2 Associated With Familial Supraventricular Tachycardia and Wolff-Parkinson-White Syndrome.

BACKGROUND: Accessory pathways are a common cause of supraventricular tachycardia (SVT) and can lead to sudden cardiac death in otherwise healthy children and adults when associated with Wolff-Parkinson-White syndrome. The goal of this study was to identify genetic variants within a large family with structurally normal hearts affected by SVT and Wolff-Parkinson-White syndrome and determine causality of the gene deficit in a corresponding mouse model. METHODS: Whole exome sequencing performed on 2 distant members of a 3-generation family in which multiple members were affected by SVT or Wolff-Parkinson-White pattern (preexcitation) on ECG identified MRC2 as a candidate gene. Serial electrocardiograms, intracardiac electrophysiology studies, echocardiography, optical mapping studies, and histology were performed on both Mrc2 mutant and WT (wild-type) mice. RESULTS: A rare HET (heterozygous) missense variant c.2969A>G;p.Glu990Gly (E990G) in MRC2 was identified as the leading candidate gene variant segregating with the cardiac phenotype following an autosomal-dominant Mendelian trait segregation pattern with variable expressivity. In vivo electrophysiology studies revealed reentrant SVT in E990G mice. Optical mapping studies in E990G mice demonstrated abnormal retrograde conduction, suggesting the presence of an accessory pathway. Histological analysis of E990G mouse hearts showed a disordered ECM (extracellular matrix) in the annulus fibrosus. Finally, Mrc2 knockdown in human cardiac fibroblasts enhanced accelerated cell migration. CONCLUSIONS: This study identified a rare nonsynonymous variant in the MRC2 gene in individuals with familial reentrant SVT, Wolff-Parkinson-White ECG pattern, and structurally normal hearts. Furthermore, Mrc2 knock-in mice revealed an increased incidence of reentrant SVT and bypass tract formation in the setting of preserved cardiac structure and function.

Exosomes from umbilical cord mesenchymal stromal cells promote the collagen production of fibroblasts from pelvic organ prolapse.

BACKGROUND: Pelvic organ prolapse (POP) involves pelvic organ herniation into the vagina due to pelvic floor tissue laxity, and vaginal structure is an essential factor. In POP, the vaginal walls exhibit abnormal collagen distribution and decreased fibroblast levels and functions. The intricate etiology of POP and the prohibition of transvaginal meshes in pelvic reconstruction surgery present challenges in targeted therapy development. Human umbilical cord mesenchymal stromal cells (hucMSCs) present limitations, but their exosomes (hucMSC-Exo) are promising therapeutic tools for promoting fibroblast proliferation and extracellular matrix remodeling. AIM: To investigate the effects of hucMSC-Exo on the functions of primary vaginal fibroblasts and to elucidate the underlying mechanism involved. METHODS: Human vaginal wall collagen content was assessed by Masson's trichrome and Sirius blue staining. Gene expression differences in fibroblasts from patients with and without POP were assessed via RNA sequencing (RNA-seq). The effects of hucMSC-Exo on fibroblasts were determined via functional experiments in vitro. RNA-seq data from fibroblasts exposed to hucMSC-Exo and microRNA (miRNA) sequencing data from hucMSC-Exo were jointly analyzed to identify effective molecules. RESULTS: In POP, the vaginal wall exhibited abnormal collagen distribution and reduced fibroblast 1 quality and quantity. Treatment with 4 or 6 µg/mL hucMSC-Exo suppressed inflammation in POP group fibroblasts, stimulated primary fibroblast growth, and elevated collagen I (Col1) production in vitro. High-throughput RNA-seq of fibroblasts treated with hucMSC-Exo and miRNA sequencing of hucMSC-Exo revealed that abundant exosomal miRNAs downregulated matrix metalloproteinase 11 (MMP11) expression. CONCLUSION: HucMSC-Exo normalized the growth and function of primary fibroblasts from patients with POP by promoting cell growth and Col1 expression in vitro. Abundant miRNAs in hucMSC-Exo targeted and downregulated MMP11 expression. HucMSC-Exo-based therapy may be ideal for safely and effectively treating POP.

Cancer-associated fibroblasts (CAFs) gene signatures predict outcomes in breast and prostate tumor patients.

BACKGROUND: Over the last two decades, tumor-derived RNA expression signatures have been developed for the two most commonly diagnosed tumors worldwide, namely prostate and breast tumors, in order to improve both outcome prediction and treatment decision-making. In this context, molecular signatures gained by main components of the tumor microenvironment, such as cancer-associated fibroblasts (CAFs), have been explored as prognostic and therapeutic tools. Nevertheless, a deeper understanding of the significance of CAFs-related gene signatures in breast and prostate cancers still remains to be disclosed. METHODS: RNA sequencing technology (RNA-seq) was employed to profile and compare the transcriptome of CAFs isolated from patients affected by breast and prostate tumors. The differentially expressed genes (DEGs) characterizing breast and prostate CAFs were intersected with data from public datasets derived from bulk RNA-seq profiles of breast and prostate tumor patients. Pathway enrichment analyses allowed us to appreciate the biological significance of the DEGs. K-means clustering was applied to construct CAFs-related gene signatures specific for breast and prostate cancer and to stratify independent cohorts of patients into high and low gene expression clusters. Kaplan-Meier survival curves and log-rank tests were employed to predict differences in the outcome parameters of the clusters of patients. Decision-tree analysis was used to validate the clustering results and boosting calculations were then employed to improve the results obtained by the decision-tree algorithm. RESULTS: Data obtained in breast CAFs allowed us to assess a signature that includes 8 genes (ITGA11, THBS1, FN1, EMP1, ITGA2, FYN, SPP1, and EMP2) belonging to pro-metastatic signaling routes, such as the focal adhesion pathway. Survival analyses indicated that the cluster of breast cancer patients showing a high expression of the aforementioned genes displays worse clinical outcomes. Next, we identified a prostate CAFs-related signature that includes 11 genes (IL13RA2, GDF7, IL33, CXCL1, TNFRSF19, CXCL6, LIFR, CXCL5, IL7, TSLP, and TNFSF15) associated with immune responses. A low expression of these genes was predictive of poor survival rates in prostate cancer patients. The results obtained were significantly validated through a two-step approach, based on unsupervised (clustering) and supervised (classification) learning techniques, showing a high prediction accuracy (≥ 90%) in independent RNA-seq cohorts. CONCLUSION: We identified a huge heterogeneity in the transcriptional profile of CAFs derived from breast and prostate tumors. Of note, the two novel CAFs-related gene signatures might be considered as reliable prognostic indicators and valuable biomarkers for a better management of breast and prostate cancer patients.