In situ study on articular cartilage degeneration in simulated microgravity by HOF-ATR-FTIR spectroscopy.

Fourier transform infrared spectroscopy (FTIRS) can provide rich information on the composition and content of samples, enabling the detection of subtle changes in tissue composition and structure. This study represents the first application of FTIRS to investigate cartilage under microgravity. Simulated microgravity cartilage model was firstly established by tail-suspension (TS) for 7, 14 and 21 days, which would be compared to control samples. A self-developed hollow optical fiber attenuated total reflection (HOF-ATR) probe coupled with a FTIR spectrometer was used for the spectral acquisition of cartilage samples in situ, and one-way analysis of variance (ANOVA) was employed to analyze the changes in the contents of cartilage matrix at different stages. The results indicate that cartilage degenerates in microgravity, the collagen content gradually decreases with the TS time, and the structure of collagen fibers changes. The trends of proteoglycan content and collagen integrity show an initial decrease followed by an increase, ultimately significantly decreasing. The findings provide the basis for the cartilage degeneration in microgravity with TS time, which must be of real significance for space science and health detection.

Endocan may predict the presence of coronary slow flow and coronary artery disease.

OBJECTIVE: Coronary artery disease (CAD) is frequent, but coronary slow flow (CSF) is a less common cardiovascular disease with a significant risk of mortality and morbidity. Endocan is a proinflammatory glycopeptide that has been investigated in cardiovascular diseases as well as some inflammatory diseases in recent years. We planned to compare the levels of endocan in both CAD and CSF in a similar population and examine the relationship of endocan with additional clinical variables. MATERIALS AND METHODS: In the trial, we included 169 consecutive subjects having a coronary angiography indication. According to the results of coronary angiography, 58 people were included in the CAD group, 52 were in the CSF group, and 59 people were in the control group. The control group includes those who did not have any lesions in their epicardial coronary arteries. Thrombolysis in myocardial infarction (TIMI)-frame counts (TFC) were calculated for all patients. RESULTS: Notably, 2.6% of the population in our study had CSF. Both the CAD (555±223 pg/mL) and CSF (559±234 pg/mL) groups had higher endocan levels than the control group (331±252 pg/mL) (p<0.001). There were similar endocan levels between the CAD and CSF groups. Endocan levels were shown to be favorably associated with mean TFC (r=0.267; p0.001). Serum endocan levels (particularly those above 450 pg/mL) and the presence of hyperlipidemia were the most important predictors of both CAD and CSF. CONCLUSION: Endocan levels are higher in CAD and CSF patients than in those with normal coronary arteries.

Identifying optimal serum 1,3-ß-D-Glucan cut-off for diagnosing Pneumocystis Jirovecii Pneumonia in non-HIV patients in the intensive care unit.

BACKGROUND: Serum (1,3)-ß-D-glucan (BDG) detection for diagnosis of Pneumocystis jirovecii pneumonia (PJP) in non-human immunodeficiency virus (HIV) immunocompromised patients lacks intensive care unit (ICU)-specific data. We aimed to assess its performance and determine the optimal cutoff for PJP in ICU population. METHODS: This retrospective study included critically ill non-HIV immunocompromised patients admitted to a medical ICU with suspected pneumonia, undergoing simultaneous microbiological testing for P. jirovecii on lower respiratory tract specimens and serum BDG. Confounders affecting BDG positivity were explored by multivariable logistic regression. Optimal cut-offs were derived from Youden's index for the entire cohort and subgroups stratified by confounders. Diagnostic performance of serum BDG was estimated at different cutoffs. RESULTS: Of 400 patients included, 42% were diagnosed with PJP and 58.3% had positive serum BDG. Serum BDG's area under the receiver operating characteristic curve was 0.90 (0.87-0.93). At manufacturer's 150 pg/ml cut-off, serum BDG had high sensitivity and negative predictive value (94%), but low specificity and positive predictive value (67%). Confounders associated with a positive serum BDG in PJP diagnosis included IVIG infusion within 3 days (odds ratio [OR] 9.24; 95% confidence interval [CI] 4.09-20.88, p < 0.001), other invasive fungal infections (OR 4.46; 95% CI 2.10-9.49, p < 0.001) and gram-negative bacteremia (OR 29.02; 95% CI 9.03-93.23, p < 0.001). The application of optimal BDG cut-off values determined by Youden's index (252 pg/ml, 390 pg/ml, and 202 pg/ml) specific for all patients and subgroups with or without confounders improved the specificity (79%, 74%, and 88%) and corresponding PPV (75%, 65%, and 85%), while maintaining reasonable sensitivity and NPV. CONCLUSIONS: Tailoring serum BDG cutoff specific to PJP and incorporating consideration of confounders could enhance serum BDG's diagnostic performance in the ICU settings.

Association between TGF-ß/BMP signaling pathway polymorphisms and the risk of primary ovarian insufficiency in Korean women.

BACKGROUND: Primary ovarian insufficiency (POI) is one of the leading female infertility diseases in which ovarian function stops before the age of 40. Reports that POI is associated with transforming growth factor (TGF)-ß/bone morphogenetic protein (BMP) signaling pathway-associated genes (e.g., TGF-ß, and BMP15) have been continuous since publication that the TGF-ß superfamily acts as important regulators for ovary and placenta function in humans. Mechanistically, the secretion of follicle-stimulating hormone, progesterone, and estrogen is affected by the TGF-ß superfamily in granulosa cells, which are involved in the development of theca cells, oocytes, and granulosa cells. OBJECTIVE: This study aimed to identify the association between genes related to the TGF-ß/BMP signaling pathway and the risk of POI pathogenesis. METHODS: Possible associations between six gene polymorphisms and POI susceptibility were examined in 139 patients with POI and 345 control subjects. RESULTS: Allele combination of TGFBR1 rs334348 G > A and TGFBR3 rs1805110G > A exhibited association with decreased POI risk (adjusted odds ratio [AOR] = 0.165; 95% confidence interval [CI] 0.032-0.847; P = 0.031). Also, TGFBR1 rs1590 G > T and rs334348 G > A and TGFBR3 rs1805110 G > A allele combination exhibited association with decreased POI risk (OR = 0.553; 95% CI 0.374-0.816; P = 0.003). CONCLUSION: This study suggests that polymorphisms in the TGF-ß signaling pathway genes can be useful biomarkers for POI diagnosis and treatment.

ESM1 facilitates the EGFR/HER3-triggered epithelial-to-mesenchymal transition and progression of gastric cancer via modulating interplay between Akt and angiopoietin-2 signaling.

Gastric cancer (GC) poses global challenges due to its difficult early diagnosis and drug resistance, necessitating the identification of early detection markers and understanding of oncogenic pathways for effective GC therapy. Endothelial cell-specific molecule 1 (ESM1), a secreted glycoprotein, is elevated in various cancers, but its role in GC remains controversial. In our study, ESM1 was elevated in GC tissues, and its concentration was correlated with progression and poorer patient prognosis in independent cohorts. Functionally, ESM1 expression promoted proliferation, anoikis resistance, and motility of GC cells, as well as tumor growth in PDOs and in GC xenograft models. Mechanistically, ESM1 expression triggered the epithelial-to-mesenchymal transition (EMT) of GC cells by enhancing epidermal growth factor receptor (EGFR)/human EGFR 3 (HER3) association and activating the EGFR/HER3-Akt pathway. Additionally, angiopoietin-2 (ANGPT2) was found to be highly correlated with ESM1 and interplayed with Akt to induce the EMT and cancer progression. Use of a signal peptide deletion mutant (ESM1-19del) showed that the secreted form of ESM1 is crucial for its protumorigenic effects by activating the EGFR/HER3-Akt/ANGPT2 pathway to promote the EMT. Patients with high levels of both ESM1 and ANGPT2 had the poorest prognoses. Furthermore, therapeutic peptides successfully inhibited ESM1's induction of the aforementioned signals and motility of GC cells. ESM1's oncogenic role in GC involves activating the EGFR/HER3-Akt/ANGPT2 pathway, presenting a potential therapeutic target for GC.

Treatment with APAC, a dual antiplatelet anticoagulant heparin proteoglycan mimetic, limits early collar-induced carotid atherosclerotic plaque development in Apoe-/- mice.

BACKGROUND AND AIMS: Mast cell-derived heparin proteoglycans (HEP-PG) can be mimicked by bioconjugates carrying antithrombotic and anti-inflammatory properties. The dual antiplatelet and anticoagulant (APAC) construct administered, either locally or intravenously (i.v.), targets activated endothelium, its adhesion molecules, and subendothelial matrix proteins, all relevant to atherogenesis. We hypothesized that APAC influences cellular interactions in atherosclerotic lesion development and studied APAC treatment during the initiation and progression of experimental atherosclerosis. METHODS: Male western-type diet-fed Apoe-/- mice were equipped with perivascular carotid artery collars to induce local atherosclerosis. In this model, mRNA expression of adhesion molecules including ICAM-1, VCAM-1, P-Selectin, and Platelet Factor 4 (PF4) are upregulated upon lesion development. From day 1 (prevention) or from 2.5 weeks after lesion initiation (treatment), mice were administered 0.2 mg/kg APAC i.v. or control vehicle three times weekly for 2.5 weeks. At week 5 after collar placement, mice were sacrificed, and lesion morphology was microscopically assessed. RESULTS: APAC treatment did not affect body weight or plasma total cholesterol levels during the experiments. In the prevention setting, APAC reduced carotid artery plaque size and volume by over 50 %, aligning with decreased plaque macrophage area and collagen content. During the treatment setting, APAC reduced macrophage accumulation and necrotic core content, and improved markers of plaque stability. CONCLUSIONS: APAC effectively reduced early atherosclerotic lesion development and improved markers of plaque inflammation in advanced atherosclerosis. Thus, APAC may have potential to alleviate the progression of atherosclerosis.

Protective Effects of Vitamin D on Proteoglycans of Human Articular Chondrocytes through TGF-ß1 Signaling.

The extracellular matrix of cartilage primarily constitutes of collagen and aggrecan. Cartilage degradation starts with aggrecan loss in osteoarthritis (OA). Vitamin D (VD) plays an essential role in several inflammation-related diseases and can protect the collagen in cartilage during OA. The present study focused on the role of VD in aggrecan turnover of human articular chondrocytes treated with tumor necrosis factor α (TNF-α) and the possible mechanism. Treatment with different doses of VD and different periods of intervention with TNF-α and TGF-ß1 receptor (TGFßR1) inhibitor SB525334 were investigated. The viability of human chondrocytes and extracellular secretion of TGF-ß1 were measured. The expression of intracellular TGFßR1 and VD receptor was examined. Transcriptional and translational levels of aggrecan and the related metabolic factors were analyzed. The results showed that TNF-α markedly reduced the viability, TGFßR1 expressions and aggrecan levels of human chondrocytes, and increased disintegrin and metalloproteinase with thrombospondin motifs. The alterations were partially inhibited by VD treatment. Furthermore, the effects of VD were blocked by the TGFßR1 inhibitor SB525334 in TNF-α-treated cells. VD may prevent proteoglycan loss due to TNF-α via TGF-ß1 signaling in human chondrocytes.

Strong Activation of ID1, ID2, and ID3 Genes Is Coupled with the Formation of Vasculogenic Mimicry Phenotype in Melanoma Cells.

Gene expression patterns are very sensitive to external influences and are reflected in phenotypic changes. It was previously described that transferring melanoma cells from a plastic surface to Matrigel led to formation of de novo vascular networks-vasculogenic mimicry-that are characteristic to a stemness phenotype in aggressive tumors. Up to now there was no detailed data about the gene signature accompanying this process. Here, we show that this transfer shortly led to extremely strong epigenetic changes in gene expression in the melanoma cells. We observed that on Matrigel numerous genes controlling ribosome biogenesis were upregulated. However, most of the activated genes were inhibitors of the differentiation genes (ID1, ID2, and ID3). At the same time, the genes that control differentiation were downregulated. Both the upregulated and the downregulated genes are simultaneously targeted by different transcription factors shaping sets of co-expressed genes. The specific group of downregulated genes shaping contacts with rDNA genes are also associated with the H3K27me3 mark and with numerous lincRNAs and miRNAs. We conclude that the stemness phenotype of melanoma cells is due to the downregulation of developmental genes and formation of dedifferentiated cells.

The structural basis for the collagen processing by human P3H1/CRTAP/PPIB ternary complex.

Collagen posttranslational processing is crucial for its proper assembly and function. Disruption of collagen processing leads to tissue development and structure disorders like osteogenesis imperfecta (OI). OI-related collagen processing machinery includes prolyl 3-hydroxylase 1 (P3H1), peptidyl-prolyl cis-trans isomerase B (PPIB), and cartilage-associated protein (CRTAP), with their structural organization and mechanism unclear. We determine cryo-EM structures of the P3H1/CRTAP/PPIB complex. The active sites of P3H1 and PPIB form a face-to-face bifunctional reaction center, indicating a coupled modification mechanism. The structure of the P3H1/CRTAP/PPIB/collagen peptide complex reveals multiple binding sites, suggesting a substrate interacting zone. Unexpectedly, a dual-ternary complex is observed, and the balance between ternary and dual-ternary states can be altered by mutations in the P3H1/PPIB active site and the addition of PPIB inhibitors. These findings provide insights into the structural basis of collagen processing by P3H1/CRTAP/PPIB and the molecular pathology of collagen-related disorders.

Chondrosarcoma Co-Culture 3D Model─An Insight to Evaluate Drugs Acting on TAMs.

Chondrosarcoma (CHS), also known as malignant cartilage tumors, is the second most common bone cancer after osteosarcoma. This tumor is particularly chemo- and radioresistant, and the only therapeutic alternative is surgery with wide margins. The tumor immune microenvironment reveals an infiltration of tumor-associated macrophages (TAMs) sometimes approaching 50% of the tumor mass, mainly differentiated into M2-like phenotype and correlated with poor prognosis and metastasis. Thus, macrophage-targeting therapies could have an interest in the management of CHS. To evaluate these strategies, we propose here the development of a three-dimensional (3D) tumoroid co-culture model between two human CHS cell lines (JJ012 and CH2879) and a human leukemia monocytic cell line (THP-1) in a methylcellulose matrix. These two models were compared to the in vivo xenograft models in terms of macrophage phenotypes, proteoglycans, MMP-9, and COX-2 expression. Finally, mifamurtide, an immunomodulator acting on TAMs, was evaluated on the most in vitro relevant model: 3D co-culture CH2879 model. Our results showed that it is now possible to develop 3D models that very accurately mimic what is found in vivo with the possibility of evaluating treatments specific to a tumor cell component.

Nerve-Glial antigen 2: unmasking the enigmatic cellular identity in the central nervous system.

Chondroitin sulfate proteoglycans (CSPGs) are fundamental components of the extracellular matrix in the central nervous system (CNS). Among these, the Nerve-Glial antigen 2 (NG2) stands out as a transmembrane CSPG exclusively expressed in a different population of cells collectively termed NG2-expressing cells. These enigmatic cells, found throughout the developing and adult CNS, have been indicated with various names, including NG2 progenitor cells, polydendrocytes, synantocytes, NG2 cells, and NG2-Glia, but are more commonly referred to as oligodendrocyte progenitor cells. Characterized by high proliferation rates and unique morphology, NG2-expressing cells stand apart from neurons, astrocytes, and oligodendrocytes. Intriguingly, some NG2-expressing cells form functional glutamatergic synapses with neurons, challenging the long-held belief that only neurons possess the intricate machinery required for neurotransmission. In the CNS, the complexity surrounding NG2-expressing cells extends to their classification. Additionally, NG2 expression has been documented in pericytes and immune cells, suggesting a role in regulating brain innate immunity and neuro-immune crosstalk in homeostasis. Ongoing debates revolve around their heterogeneity, potential as progenitors for various cell types, responses to neuroinflammation, and the role of NG2. Therefore, this review aims to shed light on the enigma of NG2-expressing cells by delving into their structure, functions, and signaling pathways. We will critically evaluate the literature on NG2 expression across the CNS, and address the contentious issues surrounding their classification and roles in neuroinflammation and neurodegeneration. By unraveling the intricacies of NG2-expressing cells, we hope to pave the way for a more comprehensive understanding of their contributions to CNS health and during neurological disorders.

Biomimetic Proteoglycans Strengthen the Pericellular Matrix of Normal and Osteoarthritic Human Cartilage.

In osteoarthritis (OA), degradation of cartilage pericellular matrix (PCM), the proteoglycan-rich immediate cell microniche, is a leading event of disease initiation. This study demonstrated that biomimetic proteoglycans (BPGs) can diffuse into human cartilage from both normal and osteoarthritic donors and are preferentially localized within the PCM. Applying immunofluorescence (IF)-guided AFM nanomechanical mapping, we show that this localization of BPGs increases the PCM micromodulus of both normal and OA specimens. These results illustrate the capability of BPGs to integrate with degenerative tissues and support the translational potential of BPGs for treating human OA and other diseases associated with proteoglycan degradation.

Kinetics and Retention of Polystyrenesulfonate for Proteoglycan Replacement in Cartilage.

Tissue hydration provides articular cartilage with dynamic viscoelastic properties. Early stage osteoarthritis (OA) is marked by loss of proteoglycans and glycosaminoglycans (GAG), lowering fixed charge density, and impairing tissue osmotic function. The most common GAG replacement, chondroitin sulfate (CS), has failed to show effectiveness. Here, we investigated a synthetic polyelectrolyte, poly(styrenesulfonate) (PSS), both as a model compound to investigate polyelectrolyte transport in cartilage, and as a potential candidate to restore bulk fixed charge density in cartilage with GAG loss. Through bovine explants and histology, we determined zonal-based effective diffusion coefficients for three different molecular weights of PSS. Compared to CS, PSS was retained longer in GAG-depleted cartilage in static and compression-based desorption experiments. We explained enhanced solute performance of PSS by its more compact morphology and higher charge density by small-angle X-ray scattering. This study may improve design of GAG mimetic molecules for repairing osmotic function in OA cartilage.

Response to retention hypothesis as a source of targets for arterial wall-directed therapies to prevent atherosclerosis: A critical review.

The subendothelial retention of circulating lipoproteins on extracellular matrix proteins and proteoglycans is one of the earliest events in the development of atherosclerosis. Multiple factors, including the size, type, composition, surrounding pH, and chemical modifications to lipoproteins, influence the electrostatic interactions between relevant moieties of the apolipoproteins on lipoproteins and the glycosaminoglycans of proteoglycans. The length and chemical composition of glycosaminoglycan chains attached to proteoglycan core proteins determine the extent of initial lipoprotein binding and retention in the artery wall. The phenomena of hyperelongation of glycosaminoglycan chains is associated with initial lipid retention and later atherosclerotic plaque formation. This review includes a summary of the current literature surrounding cellular mechanisms leading to GAG chain modification and lipid retention and discusses potential therapeutic strategies to target lipoprotein:proteoglycan interactions to prevent the development and progression of atherosclerosis.

Identification of HTRA1, DPT and MXRA5 as potential biomarkers associated with osteoarthritis progression and immune infiltration.

BACKGROUND: Our study aimed to identify potential specific biomarkers for osteoarthritis (OA) and assess their relationship with immune infiltration. METHODS: We utilized data from GSE117999, GSE51588, and GSE57218 as training sets, while GSE114007 served as a validation set, all obtained from the GEO database. First, weighted gene co-expression network analysis (WGCNA) and functional enrichment analysis were performed to identify hub modules and potential functions of genes. We subsequently screened for potential OA biomarkers within the differentially expressed genes (DEGs) of the hub module using machine learning methods. The diagnostic accuracy of the candidate genes was validated. Additionally, single gene analysis and ssGSEA was performed. Then, we explored the relationship between biomarkers and immune cells. Lastly, we employed RT-PCR to validate our results. RESULTS: WGCNA results suggested that the blue module was the most associated with OA and was functionally associated with extracellular matrix (ECM)-related terms. Our analysis identified ALB, HTRA1, DPT, MXRA5, CILP, MPO, and PLAT as potential biomarkers. Notably, HTRA1, DPT, and MXRA5 consistently exhibited increased expression in OA across both training and validation cohorts, demonstrating robust diagnostic potential. The ssGSEA results revealed that abnormal infiltration of DCs, NK cells, Tfh, Th2, and Treg cells might contribute to OA progression. HTRA1, DPT, and MXRA5 showed significant correlation with immune cell infiltration. The RT-PCR results also confirmed these findings. CONCLUSIONS: HTRA1, DPT, and MXRA5 are promising biomarkers for OA. Their overexpression strongly correlates with OA progression and immune cell infiltration.

The potential of hydrogel-free tumoroids in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck malignancy, and in particular squamous cell carcinoma (SCC), is responsible for a significant disease burden globally. The lack of an optimal in vitro model system to accurately recapitulate in vivo response to therapy in HNSCC remains a challenge. The development of patient-derived three-dimensional tumour cultures, or tumoroids, has enabled improved modelling of the tumour microenvironment through simulation of important characteristics such as tumour hypoxia, cell-cell interactions and nutrient diffusion characteristics. METHODS: We performed a comprehensive English-language literature review of current methods of tumoroid development utilising Matrigel and Cultrex Basement Membrane Extract 2 (key terms: tumour organoids, tumoroids, hydrogels, Matrigel, Cultrex, squamous cell carcinoma, head and neck)-two common proprietary murine-derived hydrogels containing extracellular matrix proteins. Nascent literature on the establishment of a novel hydrogel-free platform for tumoroid development as distinct from these existing methods was also explored. RESULTS: Whilst useful for facilitating cell-matrix interactions and providing a scaffold for three-dimensional cell growth and organisation, murine-derived cell matrix methods were noted to have notable limitations including temperature sensitivity and the medium forming a barrier to analysis of the supernatant. A novel hydrogel-free method of establishing in vitro tumoroid cultures has been subject to experimentation in colorectal but not head and neck malignancy. The absence of a hydrogel provides for the de novo synthesis of extracellular matrix native to the tumour and self-organisation of cells within this scaffold through the use of ultralow attachment plates. This model demonstrates similar structural and physiological properties to native tissue, whilst enabling more accurate biomimicry of the tumour microenvironment for drug testing. CONCLUSIONS: In the absence of prior experimentation on a hydrogel-free method for establishing HNSCC tumoroids, and comparisons between hydrogel and hydrogel-free models, the future development of a comparative protocol encompassing recruitment, collection, processing and analysis represents a valuable opportunity.

Salivary gland organoid transplantation as a therapeutic option for radiation-induced xerostomia.

BACKGROUND: Xerostomia is a pathological condition characterized by decreased salivation due to salivary gland dysfunction and is frequently attributed to irreversible damage as a side effect of radiation therapy. Stem cell-derived organoid therapy has garnered attention as a promising avenue for resolving this issue. However, Matrigel, a hydrogel commonly used in organoid culture, is considered inappropriate for clinical use due to its undefined composition and immunogenicity. In this study, we aimed to develop a method for culturing collagen-based human salivary gland organoids (hSGOs) suitable for clinical applications and evaluated their therapeutic effectiveness. METHODS: Human salivary gland stem cells were isolated from the salivary gland tissues and cultured in both Matrigel and collagen. We compared the gene and protein expression patterns of salivary gland-specific markers and measured amylase activity in the two types of hSGOs. To evaluate the therapeutic effects, we performed xenogeneic and allogeneic transplantation using human and mouse salivary gland organoids (hSGOs and mSGOs), respectively, in a mouse model of radiation-induced xerostomia. RESULTS: hSGOs cultured in Matrigel exhibited self-renewal capacity and differentiated into acinar and ductal cell lineages. In collagen, they maintained a comparable self-renewal ability and more closely replicated the characteristics of salivary gland tissue following differentiation. Upon xenotransplantation of collagen-based hSGOs, we observed engraftment, which was verified by detecting human-specific nucleoli and E-cadherin expression. The expression of mucins, especially MUC5B, within the transplanted hSGOs suggested a potential improvement in the salivary composition. Moreover, the allograft procedure using mSGOs led to increased salivation, validating the efficacy of our approach. CONCLUSIONS: This study showed that collagen-based hSGOs can be used appropriately in clinical settings and demonstrated the effectiveness of an allograft procedure. Our research has laid the groundwork for the future application of collagen-based hSGOs in allogeneic clinical trials.

Identification of Optic Nerve-Related Biomarkers in Primary Open-Angle Glaucoma Based on Comprehensive Bioinformatics and Mendelian Randomization.

Purpose: Glaucoma is the primary cause of permanent vision loss worldwide. However, the pathogenesis of primary open-angle glaucoma (POAG), the main type of glaucoma, has not yet been completely understood. Methods: In our study, the POAG cohorts were obtained from the Gene Expression Omnibus (GEO) database (GSE45570). Biomarkers with diagnostic utility for POAG were identified through combining differentially expressed analysis, enrichment analysis, machine learning algorithms, and receiver operating characteristic (ROC) analysis. The regulatory networks (including a competing endogenous RNA (ceRNA) regulatory network and a small molecule compounds-mRNA network) were created. In addition, the Mendelian randomization (MR) analysis was used to identify exposures causally associated with POAG. Finally, the expression of the biomarkers was validated via real-time quantitative polymerase chain reaction (RT-qPCR). Results: The Gene Ontology (GO) items that the differentially expressed genes (DEGs) between POAG and control groups enriched were relevant to light stimulation and DNA methylation. A total of three light stimulation-related biomarkers (RAB8A, PRG3, and SMAD3) were identified, which had diagnostic value for POAG patients. Besides, the ceRNA regulatory network contained 88 nodes and 93 edges, and a small molecule compounds-mRNA network included 66 nodes and 76 edges. The MR results indicated a causal association between DNA methylation GrimAge acceleration and POAG. Additionally, the results of RT-qPCR revealed that the expression trend of RAB8A was consistent with that of GSE45570. Conclusions: Taken together, this study provides three light stimulation-related biomarkers (RAB8A, PRG3, and SMAD3) for the diagnosis of POAG, providing scientifically valuable insights for further studies of POAG. Translational Relevance: Discovering biomarkers that possess diagnostic significance for POAG has the potential to offer new insights into the pathogenesis of POAG and present novel objectives for clinical intervention.

Extracellular matrix substrates differentially influence enteric glial cell homeostasis and immune reactivity.

Introduction: Enteric glial cells are important players in the control of motility, intestinal barrier integrity and inflammation. During inflammation, they switch into a reactive phenotype enabling them to release inflammatory mediators, thereby shaping the inflammatory environment. While a plethora of well-established in vivo models exist, cell culture models necessary to decipher the mechanistic pathways of enteric glial reactivity are less well standardized. In particular, the composition of extracellular matrices (ECM) can massively affect the experimental outcome. Considering the growing number of studies involving primary enteric glial cells, a better understanding of their homeostatic and inflammatory in vitro culture conditions is needed. Methods: We examined the impact of different ECMs on enteric glial culture purity, network morphology and immune responsiveness. Therefore, we used immunofluorescence and brightfield microscopy, as well as 3' bulk mRNA sequencing. Additionally, we compared cultured cells with in vivo enteric glial transcriptomes isolated from Sox10iCreERT2Rpl22HA/+ mice. Results: We identified Matrigel and laminin as superior over other coatings, including poly-L-ornithine, different lysines, collagens, and fibronectin, gaining the highest enteric glial purity and most extended glial networks expressing connexin-43 hemichannels allowing intercellular communication. Transcriptional analysis revealed strong similarities between enteric glia on Matrigel and laminin with enrichment of gene sets supporting neuronal differentiation, while cells on poly-L-ornithine showed enrichment related to cell proliferation. Comparing cultured and in vivo enteric glial transcriptomes revealed a 50% overlap independent of the used coating substrates. Inflammatory activation of enteric glia by IL-1ß treatment showed distinct coating-dependent gene expression signatures, with an enrichment of genes related to myeloid and epithelial cell differentiation on Matrigel and laminin coatings, while poly-L-ornithine induced more gene sets related to lymphocyte differentiation. Discussion: Together, changes in morphology, differentiation and immune activation of primary enteric glial cells proved a strong effect of the ECM. We identified Matrigel and laminin as pre-eminent substrates for murine enteric glial cultures. These new insights will help to standardize and improve enteric glial culture quality and reproducibility between in vitro studies in the future, allowing a better comparison of their functional role in enteric neuroinflammation.

Mineralocorticoid receptor (MR) antagonist eplerenone and MR modulator balcinrenone prevent renal extracellular matrix remodeling and inflammation via the MR/proteoglycan/TLR4 pathway.

Excessive activation of the mineralocorticoid receptor (MR) is implicated in cardiovascular and renal disease. Decreasing MR activation with MR antagonists (MRA) is effective to slow chronic kidney disease (CKD) progression and its cardiovascular comorbidities in animal models and patients. The present study evaluates the effects of the MR modulator balcinrenone and the MRA eplerenone on kidney damage in a metabolic CKD mouse model combining nephron reduction and a 60% high-fat diet. Balcinrenone and eplerenone prevented the progression of renal damages, extracellular matrix remodeling and inflammation to a similar extent. We identified a novel mechanism linking MR activation to the renal proteoglycan deposition and inflammation via the TLR4 pathway activation. Balcinrenone and eplerenone similarly blunted this pathway activation.