Journal of Cellular and Molecular Medicine

Background: Atrial fibrillation (AF) burden is increasingly recognized as a determinant of clinical risk. Currently, AF burden can only be estimated using long-term rhythm monitoring. Bone morphogenetic protein 10 (BMP10) is a protein secreted from cardiac atria associated with AF and AF-related complications. This study evaluated whether BMP10 concentrations are associated with AF burden in a human atrial model: atrial engineered heart tissue (aEHT). Methods: Human induced pluripotent stem cell-derived atrial cardiomyocytes were cast into atrial engineered heart tissues (aEHTs). To mimic AF burden, mature aEHTs were optogenetically-paced at a high rate of 4 Hz, either intermittently for 4 hours every 2 days (~10% burden) or continuously for 24 hours per day (100% burden). After 18 days of high-rate pacing intervention, 7 days of recovery without pacing followed. BMP10 release was quantified by ELISA and contractile function was assessed by video analysis. EHT transcriptional remodeling in response to mimicked AF burden was assessed by RNA sequencing and the effect of recovery was analyzed by qPCR. Results: High-rate optogenetic pacing mimicking AF lead to a dynamic, burden-dependent BMP10 release: BMP10 concentrations in the medium were increased by intermittent optogenetic pacing (~10% burden) and highest under continuous optogenetic pacing (100% burden). BMP10 release declined toward control levels during recovery. Contractile dysfunction was most impaired after continuous pacing and showed only partial recovery within 7 days after pacing cessation. RNA sequencing revealed distinct burden-dependent transcriptional states. Pacing-regulated transcripts were related to BMP/TGF{beta} signaling, atrial identity, calcium handling, contractile phenotype, and electrophysiological remodeling. After recovery, BMP10 mRNA expression remained elevated despite normalization of BMP10 protein release. Conclusions: AF burden dynamically regulates BMP10 release and functional and molecular remodeling in human aEHTs. BMP10 release depicts a secreted protein-based readout of current or recent atrial high-rate stress, whereas persistent transcriptional changes indicate molecular memory of prior AF burden. These findings support BMP10 release as a burden-sensitive AF biomarker

Background and AimsMutations in the desmin (DES) gene cause a variety of cardiomyopathies associated with arrhythmias, yet the electrophysiological consequences of these variants remain largely uncharacterized. The aim of this study was to investigate the pathogenic mechanisms of the de novo DES p.R406W variant, which was identified in a 9-year-old patient who suffered from severe ventricular arrhythmias and sudden cardiac death without overt structural heart disease. MethodsHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the DES p.R406W variant (including the patients line) were compared to isogenic controls. Action potentials (AP) of hiPSC-CMs were recorded using patch-clamp. Furthermore, 3D engineered heart tissues (EHTs) were generated from hiPSC-CMs and their APs were recorded with sharp microelectrodes. Analytical techniques also included transmission electron microscopy (TEM) and integrated transcriptomic and proteomic profiling. Finally, a heterozygous knock-in (KI) mouse model carrying the Des p.R405W ortholog was evaluated through surface ECG, echocardiography and ex vivo cardiac optical mapping. ResultsThe DES p.R406W mutation prolonged AP duration in IM-R406W hiPSC-CMs and EHTs vs Control ones. Multi-omics analysis of EHTs revealed a dysregulation of genes and proteins involved in contractile function, cell adhesion, and electrical activity. TEM imaging revealed changes in Z-disc architecture in mutant tissues. Twenty-week-old Des p.R405W KI mice exhibited ventricular conduction slowing (prolonged QRS) and a high susceptibility to ventricular tachyarrhythmias, likely due to reentrant mechanisms. Mild hypertrophy was also observed, but only in females. ConclusionThe DES p.R406W variant is highly pathogenic, causing electrical and structural remodeling of the myocardium. This study highlights the effectiveness of hiPSC-CMs and EHTs in recapitulating the clinical phenotype of desminopathy, providing a platform for investigating the mechanisms of early-onset cardiac arrhythmias and SCD.

Background: Gastric carcinoma remains a major cause of cancer related mortality worldwide, with tumor progression increasingly recognized as a consequence of complex interactions within the tumor microenvironment. Hypoxia induced signaling, cancer associated fibroblast (CAF) heterogeneity, and immune checkpoint activation play critical roles in tumor progression and immune evasion. However, their integrated relationship in gastric carcinoma remains insufficiently characterized. Objectives: To evaluate the expression of Hypoxia inducible factor 1 alpha and its association with cancer-associated fibroblast subtypes and Programmed death-ligand 1 expression in gastric carcinoma. Methods: This cross sectional analytical study included 100 histologically confirmed gastric carcinoma cases from Satkhira Medical College. Immunohistochemistry was performed for HIF 1 alpha, smooth muscle actin (SMA), fibroblast activation protein (FAP), and PD L1. CAFs were subclassified into myofibroblastic CAFs (myCAFs) and inflammatory CAFs (iCAFs). Associations between biomarkers and clinicopathological variables were analyzed using chi square test, Spearman correlation, and multivariate logistic regression. Receiver operating characteristic (ROC) curve analysis was used to assess model performance. Result: High HIF 1 alpha expression was observed in 55% of cases and demonstrated significant association with poor differentiation (p = 0.001), advanced tumor stage (p = 0.002), and lymph node metastasis (p = 0.001). iCAF predominance was significantly associated with poor differentiation (p = 0.003), advanced stage (p = 0.004), and nodal metastasis (p = 0.004). High PD L1 expression was significantly associated with poor differentiation (p = 0.03), advanced stage (p = 0.001), and lymph node metastasis (p = 0.002). Multivariate logistic regression identified high HIF 1 alpha expression (OR = 3.8, p = 0.001), iCAF dominance (OR = 4.5, p < 0.001), and advanced tumor stage (OR = 2.9, p = 0.004) as independent predictors of high PD L1 expression. Combined high HIF 1 alpha expression and CAF activation demonstrated the highest rate of PD L1 positivity (76.7%, p < 0.001). ROC curve analysis demonstrated good predictive performance of the model with an area under the curve of 0.81. Conclusion: The present study demonstrates a significant interaction between hypoxia, stromal remodeling, and immune checkpoint activation in gastric carcinoma. High HIF 1 alpha expression and inflammatory CAF predominance are strongly associated with aggressive clinicopathological features and increased PD L1 expression, supporting the existence of a coordinated hypoxia stroma immune axis in gastric carcinoma progression. These findings may have potential implications for prognostic stratification and combined targeted therapeutic strategies.

IntroductionPreeclampsia is characterized by a pro-inflammatory, anti-migratory and anti-angiogenic placental phenotype. Impaired spiral artery remodeling stemming from trophoblast dysfunction is a key pathogenic mechanism. Little is known about the processes that govern trophoblast function normally and in preeclampsia. In preeclampsia, placental Let-7b-5p is reduced. The objectives of this study were to determine the normal function of Let-7b-5p in human trophoblast cells, to examine whether the ssRNA sensors, Toll-like receptor (TLR) 7 and/or TLR8 are mediators of trophoblast Let-7b-5p function, and whether disruption of this pathway promotes a preeclampsia-like phenotype in the trophoblast. MethodsThe human first trimester trophoblast cell line, Sw.71, was transfected with a Let-7b- 5p mimic, a Let-7b-5p inhibitor, or scramble control. Cells were treated with or without the TLR7 inhibitor IRS661 or the TLR8 inhibitor CUCPT9a. Trophoblast migration was measured using a two-chamber assay and interactions with human endometrial endothelial cells (HEECs) was measured using a 3D matrigel model. Trophoblast anti-angiogenic sFlt-1 release was measured by ELISA and sFLT1 mRNA measured by RT-qPCR. ResultsTransfection of trophoblast cells with a Let-7b-5p mimic elevated migration through activation of TLR7 and TLR8, while in a TLR7-dependent manner, the Let-7b-5p mimic negatively regulated sFlt-1 production. Furthermore, inhibition of trophoblast Let-7b-5p reduced migration, elevated FLT1 mRNA expression and sFlt-1 release, and reduced trophoblast-endometrial endothelial cell interactions. ConclusionsThis study highlights a role for TLR7/TLR8-activating Let-7b-5p in promoting normal trophoblast function and endothelial interactions and that disruption in this miR-driven signaling pathway may be relevant to processes driving a pre-eclamptic placental phenotype. HighlightsTrophoblast migration is positively driven by Let-7b-5p activating TLR7 and TLR8 Let-7b-5p, via TLR7, negatively regulates trophoblast anti-angiogenic sFlt-1 production. Inhibition of trophoblast Let-7b-5p reduces trophoblast migration and normal interactions with endometrial endothelial cells, while sFlt-1 production is elevated. TLR7/TLR8-activating Let-7b-5p promotes normal trophoblast function and endothelial interactions and disruption in this miR-driven signaling pathway may promote a preeclamptic placental phenotype.

Background: Cardiac fibrosis is a hallmark of many cardiovascular diseases, driven by sustained fibroblast activation and excessive extracellular matrix deposition, leading to myocardial stiffening and impaired contractility. Current therapies inadequately address this process. This study evaluated the antifibrotic potential of lanifibranor, a balanced pan-peroxisome proliferator-activated receptors (PPARs) agonist, in TGF-beta1-induced cardiac fibrosis. Methods: Human cardiac microtissues, along with 2D and 3D cardiac fibroblast and cardiomyocyte cultures, were used to assess cell viability, structure, metabolism, contractility, and gene expression. Results: Lanifibranor reduced TGF-beta1-induced fibrosis by limiting fibroblast activation and matrix deposition without affecting viability. In fibroblasts, these effects were associated with partial restoration of mitochondrial respiration and reduced focal adhesion maturation. In cardiac microtissues, lanifibranor improved contraction kinetics, decreased profibrotic transcriptional activity, and preserved bioenergetic homeostasis despite altered nucleotide balance. In cardiomyocytes, treatment normalized contractility and calcium handling while maintaining metabolic stability. Conclusions: Lanifibranor attenuates TGF-beta1-driven cardiac fibrosis by combining antifibrotic effects with metabolic and functional improvements in human models.

Background: Lumbar disc herniation, with or without concomitant disc degeneration, is a major cause of lumbar radiculopathy and low back pain, which also a key public musculoskeletal disorder without an exact pathophysiology. Studies have suggested that inflammatory cells and biochemical markers of inflammation also play an important role in lumbar radiculopathy in addition to nerve compression. The aim of the present study was to assess the association of selected circulatory inflammatory markers (CRP, hs-CRP and E-selectin) in patients with lumbar disc herniation without radiological degeneration (LDH) and lumbar disc herniation with radiological degeneration (LDHD). Materials & methods: This case-control study included 208 participants, comprising 104 patients with lumbar disc pathology and 104 controls. Patients were further stratified into LDH (n=67) and LDHD (n=37). Serum CRP, hs-CRP and E-selectin concentrations were measured. Results: Among the patients, 35.6 % presented with LDHD while 64.4 % had only LDH. Significantly increased median hs-CRP (p<0.001) and CRP (p<0.001) were observed in patients groups compared to controls, while CRP showing a consistent independent association across the combined disease (OR=1.68, 95% CI=1.33-2.14, p<0.001), LDHD (OR=1.62, 95% CI=1.16-2.20, p=0.005) and LDH (OR=1.69, 95% CI=1.30-2.20, p<0.001) multivariable models. No significant difference was observed in serum E-selectin between the study groups. Multivariable models incorporating inflammatory and clinical variables demonstrated substantially greater discriminatory performance than individual biomarkers alone. Conclusion: Elevated circulating CRP and hs-CRP concentrations were associated with lumbar disc pathology, with CRP showing a consistent independent association across the combined disease, LDH and LDHD multivariable models, whereas E-selectin showed no significant association. Multivariable models incorporating inflammatory and clinical variables demonstrated greater discriminatory performance than individual biomarkers. These findings support a potential systemic inflammatory component in lumbar disc pathology, although the cross-sectional nature of the measurements does not establish causality or a local inflammatory response within the disc.

BACKGROUND: Subarachnoid hemorrhage (SAH) induces inflammatory responses and subsequent immune cell activation, which may contribute in cerebral vasospasm, microcirculatory impairment and poor neurological outcomes. Although cerebral vasospasm has traditionally been considered a major cause of delayed cerebral ischemia after SAH, therapies targeting angiographic vasospasm have not consistently improved functional outcomes. Early inflammatory responses may contribute to microcirculatory impairment, cerebral vasospasm, and subsequent neurological injury. Herein, we investigated whether interleukin-10 (IL-10), an anti-inflammatory cytokine, improves these outcomes in an experimental SAH model. METHODS: Mice received intramuscular injections of either an adeno-associated virus encoding IL-10 (AAV/IL-10) vector or an AAV expressing green fluorescent protein (AAV/GFP) vector (control). India ink angiography was performed to assess the diameter of the sphenoidal segment of the middle cerebral artery (MCA), the total length of the visible cortical arteries, and cortical staining intensity, as indices of cerebral vasospasm, microcirculatory impairment, and cerebral perfusion, respectively. Perivascular inflammatory cell infiltration and cytokine levels were assessed using immunohistochemistry and ELISA. We also evaluated the therapeutic efficacy of the AAV/IL-10 vector when administered immediately after SAH induction. RESULTS: IL-10 overexpression significantly improved neurological outcomes after SAH and was associated with attenuated cerebral vasospasm and microcirculatory impairment, as well as preservation of cerebral perfusion. It also significantly reduced neutrophil and macrophage infiltration around the internal carotid artery and attenuated SAH-induced elevations in IL-6 and matrix metalloproteinase-3 levels. Mice treated with the AAV/IL-10 vector immediately after SAH induction showed significant improvements in neurological scores and cerebral perfusion. CONCLUSIONS: AAV-mediated IL-10 overexpression improves neurological outcomes after SAH, likely by attenuating inflammatory responses, cerebral vasospasm, and microcirculatory impairment. These findings suggest that IL-10-based anti-inflammatory therapy is a promising therapeutic strategy for SAH.

Background: Pulmonary arterial hypertension (PAH) is a serious disease with poor prognosis, especially in infants or preterm babies and there is still no optimal treatment for this disease. Noradrenalin (NE) is a vasoactive mediator which is released by sympathetic ganglion. According to previous studies, NE/1-adrenoreceptors is not only in regulating normal physiologic responses, but also in the pathogenesis of PAH. However, the mechanisms of NE in PAH are not fully understood. Methods: Human PASMC (PASMC) was used in this study. Cell viability assay and Wound healing assay were used to evaluate the proliferation and migration of PASMC. Immunoprecipitation and western blots analysis were used to investigate the mechanisms which involved in NE-induced PASMC proliferation. Results: We investigated that NE could induce human PASMC proliferation and migration. Furthermore, we first find that endothelin 1 (ET-1) signaling pathway plays an important role in NE-induced PASMC proliferation. ET1 is a critical molecular which is known for regulating cell growth and migration. We investigated that NE could increase NE-1 secretion, further enhancing ET-1 bind to its receptors. For further clarifying the downstream signals in NE/ET-1 induced PASMC proliferation, we detected the phosphorylation and expression levels of ERK and JNK. Conclusions: By combining the results from ours and previous studies, we believed that JNK/c-jun pathway may play an important role in NE-induced PASMC proliferation. Key Words: Noradrenaline; Pulmonary Arterial Hypertension; Pulmonary Artery Smooth Muscle Cells; Endothelin-1; JNK/c-Jun Signaling.

Background: The placenta has a unique transcriptomic profile, including microRNAs that are secreted into maternal circulation throughout pregnancy. MicroRNAs are small, non-coding RNA that post-transcriptionally regulate gene expression. Spontaneous preterm birth (sPTB) is associated with substantial differences in both placental pathophysiology and placental gene expression compared to term birth. We aimed to generate microRNA signatures of sPTB and map them to target genes using a microRNA-mRNA network. Methods: This study was conducted within the Conditions Affecting Neurocognitive Development and Learning in Early childhood (CANDLE) study. Placental samples were collected at delivery, and RNA was isolated for mRNA and microRNA sequencing. To investigate sPTB, this study excluded placental samples of participants with iatrogenic indications for PTB or induced labor. We examined differences in microRNA expression in participants who delivered before 37 weeks (N=35) compared to term participants (N=404) in a series of covariate-adjusted linear regression models. We used paired placental microRNA and mRNA expression data from this cohort to validate associations between computationally predicted microRNA-mRNA pairs and establish a microRNA-mRNA network. Results: Expression of 7 microRNAs were increased in sPTB (FDR<0.05) and were inversely correlated with sPTB-associated genes involved in immune signaling. Expression of 12 microRNAs were decreased in sPTB, including 4 members of the maternally expressed chromosome 14 microRNA cluster (miR-376a-3p, miR-376c-3p, miR-377-3p, and miR-381-3p). These microRNAs were predicted to negatively regulate oxidative phosphorylation genes that were increased in sPTB. The associations between miR-376c-3p and miR-377-3p and oxidative phosphorylation were confirmed in microRNA knockdown experiments. Conclusions: This study highlights potential biological mechanisms by which placental microRNA dysfunction might contribute to sPTB and highlights putative sPTB biomarkers that may be detectable in maternal circulation.

AimThe present study investigated the significance of iron in regulating megakaryopoiesis, by a diet-based intervention in an in-vivo model. MethodsMale C57BL/6 mice, aged 4-5 weeks were fed on varying iron diets. Following sacrifice, blood samples collected in EDTA tubes were used to analyse haematological parameters, and iron content of liver and spleen was assessed by biochemical analyses. Megakaryocyte-erythroid progenitors (MEPs) were isolated from bone marrow by magnetic bead-based selection. RNA isolated from bone marrow cells and MEPs were used for gene expression analyses, and RNA Sequencing to identify differentially expressed genes (DEGs) and associated pathways. ResultsMice fed on an iron-deficient diet had reduced hepatic iron content after 5 weeks (p < 0.01), while both the hepatic and spleen iron content increased after 3 weeks in mice on an iron-rich diet (p < 0.05) and developed iron overloading. Hb and RBC counts increased (p < 0.05) in iron-rich mice and decreased in iron-deficient mice (p < 0.05), which also showed elevated platelet counts (p < 0.01). This may be explained by increased expression of Gata1, Tal1 (p < 0.01) Mds1 and Pdpk1 (p < 0.05) in bone marrow cells from iron-deficient mice. MEPs isolated from these mice showed elevated expression of genes associated with megakaryocytic differentiation, platelet functions, and genes encoding TGF-{beta}R1 and Smad 2,3 and 4. ConclusionsIron deficiency may activate TGF-{beta} signalling and downstream Smad-mediated transcriptional programs within MEPs. This may promote a shift in lineage commitment towards megakaryopoiesis through elevated expression of megakaryopoiesis related genes.

Background: Patients receiving anti-TNF treatment for chronic inflammatory disease display impaired antibody responses, but it remains unclear how immune memory formation is affected. We evaluated antibody responses and memory B cells (Bmem) after COVID-19 booster vaccination in inflammatory bowel disease (IBD) patients receiving anti-TNF treatment. Methodology: Blood was sampled at baseline, 1, and 6 months after WH1/BA.5 bivalent or XBB.1.5 monovalent vaccination from 27 IBD patients receiving intravenous anti-TNF and 44 controls. Neutralizing antibodies were measured using an infectious virus assay. SARS-CoV-2 spike receptor binding domain (RBD)-specific serum IgG was quantified by ELISA, and RBD-specific Bmem were immunophenotyped by flow cytometry using recombinant proteins from ancestral, Omicron BA.1, BA.5, XBB.1.5, and JN.1 variants. Results: Serum IgG to vaccine RBD and neutralizing antibodies in patients increased pre to 1 month post-vaccination, but were lower than controls. Ancestral-, BA.5- and XBB.1.5-specific Bmem increased after vaccination but were significantly lower in patients than controls. Within RBD-specific Bmem, frequencies of recently activated CD21lo cells were increased after vaccination, and were higher in patients than controls. Fewer antigen-specific Bmem in patients expressed IgG4, and more expressed IgG3 or IgD following vaccination. Following vaccination, more RBD-specific Bmem recognized multiple viral variants. However, patients had fewer Bmem that could bind to subvariants than controls. Conclusion: Antibody and Bmem responses to COVID-19 booster vaccination in anti-TNF-treated IBD patients displayed reduced capacity, durability and cross-reactivity, suggesting impaired immune memory for protection against breakthrough infection. This supports the recommendation for annual booster vaccination to prevent severe disease and viral spread.

Preeclampsia is a pregnancy complication characterized by hypertension, proteinuria, and end-organ dysfunction. Abnormal placentation leading to reduced placental perfusion may contribute to its development. Previous studies demonstrated that the activation of the apelin receptor (APJ) system has hypotensive, renoprotective, and antioxidant effects in preeclamptic rat models. Apelin and elabela (ELA) can stimulate the proliferation of trophoblast cells, suggesting a role in embryonic development. However, the mechanisms underlying the actions of apelin or ELA in trophoblast cells are not well understood, particularly in hypoxic settings. The immortalized HTR-8/SVneo trophoblastic cells were treated with cobalt chloride (CoCl2) at 0.2 mM for 24 hours to mimic hypoxic conditions. RT-qPCR, ELISA or Western blotting was used to measure mRNA or protein levels of apelin, elabela, and the components of IL-6 signaling in cell lysates or conditioned media. The exposure to CoCl2 increased total apelin and elabela content approximately 2-fold in the conditioned media but did not affect APJ levels. CoCl2 upregulated proinflammatory cytokine concentrations: soluble fms-like tyrosine kinase 1 (sFlt-1), soluble gp130 (sgp130), interleukin-6 (IL-6), and sIL-6 receptor (IL-s6R). Both apelin and elabela downregulated IL-6 mRNA but had no effect on sFlt-1 mRNA. Apelin attenuated sgp130, while ELA decreased the membrane form of IL-s6R. Apelin also decreased the pSTAT3/STAT3 ratio. CoCl2-induced hypoxia upregulated the pro-inflammatory milieu in HTR-8/SVneo cells. Local activation of this peptidergic system may be a compensatory response of the trophoblast cells to hypoxia as exogenous apelin and elabela treatment ameliorated the hypoxia-induced pro-inflammatory milieu.

Introduction: Osteoarthritis (OA) is a degenerative joint condition characterized by chronic pain and the need for pain management. Locally targeting the endocytotic AP2 complex in nociceptors presents a potential strategy for providing sustained pain relief in individuals with OA. Objective: We investigated whether pain behavior associated with OA can be mitigated by genetically silencing the AP2alpha2 subunit of the AP2 complex in nociceptors and by pharmacologically inhibiting the AP2 complex through the intraarticular administration of a small lipidated decoy peptide. Method: Monoiodoacetate (MIA) was employed to induce knee joint OA in mice and rats. Pain behavior was assessed using dynamic weight-bearing and von Frey filaments. Upon confirmation of established OA pain behavior, in vivo AP2alpha2 genetic knockdown in mice was achieved through sciatic nerve transfection of a targeting AP2alpha2 short hairpin RNA (shRNA). To pharmacologically target endocytosis, a single intraarticular injection of peptide was administered into the arthritic knee of rats. The injection contained either the AP2 inhibitor peptide or a scrambled peptide control. Results: Pain behavior was significantly reduced after both genetic and pharmacological disruption of AP2-driven endocytosis. Animals treated with the Ap2 inhibitor peptide exhibited reduced pain behavior throughout the 28-day assay period. Following the completion of behavioral testing, arthritic knee joints and contralateral healthy knee joints were subsequently collected to assess the impact of the treatment on disease progression. Micro-computed tomography analysis revealed a preservation of bone volume in the arthritic joints that received the AP2 inhibitor peptide treatment, in contrast to the scrambled peptide group. Conclusion: These findings demonstrate that the inhibition of nociceptor endocytosis by a small lipidated peptide presents a promising approach to provide sustained relief from joint pain in individuals with arthritis.

BackgroundKnee osteoarthritis (KOA) is a prevalent degenerative joint disease in which synovial inflammation and fibrosis are closely linked to pain, stiffness, and functional limitation. Growing evidence suggests that metabolic dysregulation, particularly in lipid metabolism, is involved in KOA pathogenesis, but the underlying mechanisms remain incompletely defined. MethodsSprague Dawley rats underwent bilateral anterior cruciate ligament transection to establish a KOA model; sham-operated rats served as controls. RNA sequencing of synovial tissues was performed to identify differentially expressed genes (DEGs) and enriched pathways, followed by GO/KEGG and GSEA analyses. In vivo, adeno-associated virus vectors were used to overexpress or knock down PPAR-{gamma} and phosphoenolpyruvate carboxykinase 1 (PCK1) via intra-articular injection. Ex vivo, primary rat fibroblast-like synoviocytes (FLSs) were stimulated with IL-1{beta} and transfected with PPAR-{gamma} or PCK1 siRNA/overexpression plasmids. synovitis and fibrosis were evaluated by HE, Masson, and Sirius Red staining, immunofluorescence, ELISA, RT-qPCR, and Western blotting. ResultsRNA-seq revealed 621 up-regulated and 228 down-regulated genes in KOA synovium versus sham, with DEGs significantly enriched in PPAR signaling, adipocytokine, and AMPK pathways. Metabolism-related genes including Fabp5, Plin1, Adipoq, Lep, and Pck1 were up-regulated. GSEA indicated downregulation of PPAR-{gamma} signaling in KOA synovium. In vivo and ex vivo, PPAR-{gamma} expression was reduced in KOA, whereas PCK1, FABP5, and ADIPOQ were increased. PPAR-{gamma} overexpression alleviated synovial inflammation, collagen I deposition, and fibrosis, and suppressed FABP5, ADIPOQ, and PCK1 expression; PPAR-{gamma} knockdown produced the opposite effects. Functional studies showed that PCK1 overexpression aggravated synovial inflammatory cell infiltration and fibrosis, elevated IL-1{beta}, IL-18, and TGF-{beta}, and decreased TIMP1 levels in serum, synovial tissue, and FLSs supernatants, whereas PCK1 silencing reversed these changes. ConclusionsThe PPAR-{gamma}/PCK1 metabolic axis modulates synovitis and fibrosis in KOA. Downregulation of PPAR-{gamma} and consequent upregulation of PCK1 promote synovitis and fibrotic remodeling. These findings identify the PPAR-{gamma}/PCK1 pathway as a potential therapeutic target for KOA.

Background: Severe aortic stenosis is associated with systemic and splanchnic hemodynamic disturbances that may alter gut microbial metabolism and host inflammatory responses. Objectives: We aimed to determine whether TAVI remodels the gut microbiome-derived metabolome and whether post-procedural SCFA dynamics are associated with the inflammatory cytokine response. Methods: We conducted a prospective paired single-center study of patients undergoing elective TAVI at Bern University Hospital. Stool and blood samples were collected before and three months after the procedure. Gut microbial composition was profiled by full-length 16S rRNA sequencing, circulating short-chain fatty acids (SCFAs) by targeted metabolomics, and inflammatory mediators by multiplex cytokine analysis, and integrated with hemodynamic and clinical data. Results: Forty patients were enrolled. Following TAVI, microbial richness declined without significant restructuring of overall community composition. In contrast, circulating SCFA profiles were significantly remodeled, driven by selective reductions in butyrate and isovalerate. A greater decline in circulating butyrate was inversely associated with IL-18 elevation (rho=0.668, p<0.001, n=36), independent of aortic valve calcification burden, hemodynamic improvement, and cardiovascular medications. Baseline isovalerate was nominally associated with 1-month adjudicated adverse events (AUC 0.77; exploratory). Conclusions: TAVI is associated with selective changes in gut microbiome-derived metabolic output rather than broad alterations in microbial community structure. Declining circulating butyrate identifies a gut-metabolite-immune axis linked to IL-18 dynamics and represents a potential biomarker of inflammatory recovery following valve intervention.

Cellular senescence contributes to the progression of many age related musculoskeletal diseases. Cellular senescence is a biological state that arises from replicative exhaustion and various cellular stressors, including elevated oxidative stress, mitochondrial dysfunction, mechanical overload, and chronic exposure to pro-inflammatory cytokines and proteases. Although senolytic agents show promise for eliminating senescent cells, their translation has been hindered by the lack of physiologically relevant and scalable in vitro screening methods. In the present study, we developed a standardized, physiologically relevant senescence-induction model and validated a metabolic activity assay as a rapid, scalable method for screening senolytic compounds. We used primary human intervertebral disc cells (IVD) as an example, but the workflow applies to many other cell types. To mimic inflammatory and oxidative stress, we used a combination of TLR-2 activation (Pam2CSK4) and tert-butyl hydroperoxide (tBHP), a potent ROS generator. Senescence induction was validated by quantifying {beta}-galactosidase fluorescence intensity, {beta}-gal enzymatic activity, and the expression of the p16 senescence marker across 3 IVD cell types: nucleus pulposus (NP), inner annulus fibrosus (iAF), and outer annulus fibrosus (oAF) cells. The combined Pam2CSK4 + tBHP exposure generated a robust senescent phenotype across all 3 IVD cell types, with oAF cells exhibiting the strongest increases in {beta}-gal fluorescence, {beta}-gal enzymatic activity, and p16 expression. We then used oAF cells to evaluate if the metabolic activity assay (Alamar Blue) could be used to determine both cytotoxicity of senolytic drugs in non-senescent cells and senolytic activity in a mixed population of senescent and non-senescent cells. We validate the method by comparing metabolic activity results with {beta}-gal enzymatic activity and p16 expression in induced and noninduced cells following exposure to three known senolytics (o-Vanillin, RG-7112, and ABT-199). The metabolic activity assay reliably identified a therapeutic window in which the three senolytics were non-toxic to non-senescent cells while selectively reducing metabolic activity in a mixed population of senescent and non-senescent cells. The reductions in metabolic activity in the mixed population correlated with decreases in SA {beta}-gal enzymatic activity and p16 expression, validating metabolic activity as a sensitive and scalable senolytic readout.

BackgroundTraditional immunohistochemistry (IHC) with chromogen detection has limited multiplex capacity, detecting at most 4 protein markers per tissue section simultaneously, thereby restricting comprehensive spatial analysis of valuable human biopsies. We developed and validated a robust serial IHC (sIHC) staining method to detect multiple antigens on a single kidney biopsy slide, maximizing data yield for diagnosing and studying complex kidney diseases. MethodsFormalin-fixed, paraffin-embedded kidney biopsy sections were subjected to repeated IHC/imaging cycles with antibody removal using an optimized sodium dodecyl sulfate-glycerol buffer stripping protocol. Images were then co-registered, and analysis was performed using a variety of methodologies, including color deconvolution, cell segmentation, and spatial clustering. ResultsThis optimized sIHC method successfully detected up to 20 antigens on a single slide. Combining image analysis and artificial intelligence software, for example with HALO (Indica Labs), the assay assembles high-dimensional images and enables quantitative histology and single-cell spatial analysis. Using this advanced method, we were able to identify rare cell populations, such as double-negative T cells, that are challenging to detect conventionally. ConclusionWe have developed a validated, high-capacity sIHC protocol that uses standard IHC procedures with commercially available, clinically validated off-the-shelf antibodies. This method is a valuable, cost-effective tool for obtaining extensive, high-dimensional single-cell-resolved spatial data from limited pathology samples, such as a human kidney biopsy.

At the onset of intervertebral disc degeneration, within the disc core, the pH and osmolarity decrease, and the residing notochordal cells (NCs) gradually transition towards nucleus pulposus cells (NPCs). How these microenvironmental cues shape the NCs extracellular vesicles (EV)-enriched secretome, and thus EV-mediated communication with NPCs during this transition, remains poorly understood. To study this, we collected the secretome from pig NC-rich tissue cultured for 4 days in either healthy or degenerate disc media to mimic these changes. In both conditions, NC-rich tissues were largely comparable at the histological and biochemical levels. Despite, tissues released glycosaminoglycans (GAGs), depleting the extracellular matrix. Surprisingly, degenerative media did not differentially release inflammatory regulators, though it reduced PGE2 release. We asked whether this extended to EV-enriched secretome media (SM_EV+), and found that the degenerative media reduced the number of EVs without altering their morphology or size. We then determined NC-EV association of inflammatory and matrix regulators. NC-EV isolation enriched MMP1, IL6 and IL10 and depleted soluble GAGs. Conversely, EV-depletion (SM_EV-) removed most GAGs without affecting MMP1, IL6, and IL10, suggesting that they contribute to the NC-EV soft corona. Functionally, healthy SM_EV+ improved GAG production by NPCs, but attenuated TBXT expression. Degenerate SM_EV+ did not elicit detectable EV-specific effects. These findings suggest that, in health, secretome-mediated communication from NCs to NPCs is only partially EV-mediated. At the onset of IVD degeneration, low pH and osmolarity impair the release of NC-EVs and negate the EV-specific beneficial matrix-anabolic effects on NPCs, contributing to the NC-to-NPC transition.

Implant-associated bone infection involves a complex interplay between pathogenic stimuli and host cell responses, yet analysis in preclinical models has typically relied on qualitative or semi-quantitative measures. We aimed to establish a quantified evaluation framework to define host-pathogen relationships in a preclinical implant infection model. Staphylococcus aureus-coated stainless-steel implants were inserted trans-cortically in mouse tibiae and bone changes recorded longitudinally by in vivo micro-CT. An automated segmentation task list was developed to independently isolate and quantify cortical, periosteal-reactive, and trabecular bone compartments. RGB trichrome histomorphometry was used to quantify bone matrix integrity, osteocyte lacunar geometry, and osteoclastic activity. Droplet digital PCR was used to determine absolute bacterial and host genome copy number. Infected implants produced marked reductions in trabecular bone volume fraction, number, and bone mineral density (BMD), together with decreased cortical bone volume fraction and increased cortical porosity, accompanied by significant elevations in periosteal bone volume fraction. Histologically, infected bone exhibited increased eroded surface indicative of osteoclastic resorption, extensive degraded bone matrix and pathological remodelling of osteocyte lacunae towards circularity, consistent with an osteocytic osteolysis response. Infection-induced changes to cortical bone structure correlated mostly with host cell rather than bacterial load; however, cortical BMD negatively correlated with the bacterial:host genome ratio. This multifaceted, quantified framework reveals distinct pathobiological effects of implant-associated infection on trabecular, cortical, and periosteal bone compartments, bone matrix and osteocyte and osteoclast populations, consistent with reports in human patients, suggesting that major pathological changes are driven by the host bone cell response to infection.

This study aims to investigate the cardioprotective effects of 4-Hydroxybenzaldehyde (4-HBA) against isoproterenol (ISO)-induced cardiac fibrosis and to elucidate the underlying mechanisms. In vivo, cardiac fibrosis was induced in C57BL/6 mice by subcutaneous injection of ISO, and the mice were treated with 4-HBA or a TGF-{beta} inhibitor. Assessments using echocardiography, histopathology, and Western blotting demonstrated that 4-HBA significantly alleviated ISO-induced cardiac dysfunction, reduced collagen deposition, and attenuated apoptosis in mice. Mechanistically, 4-HBA inhibited TGF-{beta} expression and Smad2/3 phosphorylation. In vitro, ISO was applied to cardiomyocytes (HL-1) and cardiac fibroblasts (MCFs), with or without 4-HBA or TGF-{beta} inhibitor intervention. The results showed that 4-HBA suppressed HL-1 apoptosis and fibroblast proliferation, and significantly reduced the expression of extracellular matrix genes, TGF-{beta} levels, and Smad2/3 phosphorylation in MCFs. These findings indicate that 4-HBA reduces myocardial injury while targeting the TGF-{beta}/Smad2/3 pathway to attenuate cardiac fibrosis, highlighting its potential as a therapeutic agent for fibrotic cardiomyopathy.This study aims to investigate the cardioprotective effects of 4-Hydroxybenzaldehyde (4-HBA) against isoproterenol (ISO)-induced cardiac fibrosis and to elucidate the underlying mechanisms. In vivo, cardiac fibrosis was induced in C57BL/6 mice by subcutaneous injection of ISO, and the mice were treated with 4-HBA or a TGF-{beta} inhibitor. Assessments using echocardiography, histopathology, and Western blotting demonstrated that 4-HBA significantly alleviated ISO-induced cardiac dysfunction, reduced collagen deposition, and attenuated apoptosis in mice. Mechanistically, 4-HBA inhibited TGF-{beta} expression and Smad2/3 phosphorylation. In vitro, ISO was applied to cardiomyocytes (HL-1) and cardiac fibroblasts (MCFs), with or without 4-HBA or TGF-{beta} inhibitor intervention. The results showed that 4-HBA suppressed HL-1 apoptosis and fibroblast proliferation, and significantly reduced the expression of extracellular matrix genes, TGF-{beta} levels, and Smad2/3 phosphorylation in MCFs. These findings indicate that 4-HBA reduces myocardial injury while targeting the TGF-{beta}/Smad2/3 pathway to attenuate cardiac fibrosis, highlighting its potential as a therapeutic agent for fibrotic cardiomyopathy.