Journal of Cellular and Molecular Medicine

BackgroundThe etiology and molecular mechanisms of Moyamoya disease (MMD) remain unclear. Exosomes, as carriers of bioactive molecules, may reflect disease-specific alterations and serve as potential biomarkers. This study aimed to investigate disease mechanisms using proteomic profiling of serum-derived exosomes (SDEs) in MMD. Materials and MethodsPeripheral blood each from 15 MMD patients and 15 healthy-controls were used to isolate SDEs via ultracentrifugation. Proteins from pooled SDEs were extracted, digested, and analyzed by LC-MS/MS. Differentially expressed proteins were examined using MetaboAnalyst, DAVID, Enrichr, STRING, and Cytoscape. Key targets were validated at transcript and protein levels using RT-qPCR and ELISA in independent cohorts. ResultsA total of 2,554 proteins were identified, with 213 showing differential expression (118 upregulated, 95 downregulated; p [≤] 0.05). Functional and pathway analyses revealed enrichment in angiogenesis, cytoskeletal remodeling, and endothelial signaling. PRKG2 and MYC were upregulated, while RHOA was downregulated, highlighting their involvement in focal adhesion and PI3K-AKT pathways. Validation confirmed these findings. ConclusionDysregulated proteins were linked to RHOA-ROCK and PI3K-Akt signaling, suggesting their role in driving VSMC phenotypic switching, contributing to vascular occlusion. These findings indicate that altered exosomal-proteins may participate in maladaptive vascular remodeling, although the initial trigger for VSMC transition remains unknown.

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.

Hereditary hemorrhagic telangiectasia (HHT) is a rare vascular disorder caused by pathogenic variants in members of the BMP9/ALK1 signaling hub. In the present study we show that, regardless of whether the alterations are caused by reduced BMP9/ALK1 signaling (pathogenic variants in the ENG or ALK1 genes) or by overactivation of this pathway (such as the SMAD6 pathogenic variants), all are associated with increased endothelial cell (EC) proliferation and high levels of ERK MAPK activation in patient biopsies. We reproduced this phenotype in vitro in ECs lacking SMAD6 or after SMAD1 knockdown using siRNA. Loss of SMAD6 leads to dysregulation of the Notch pathway, with downregulation of phosphatases and consequent overstimulation of ERK. In normal ECs, BMP9 and Notch signaling inhibit ERK activity by upregulating PPP1R3C, a regulatory subunit of the PP1 phosphatase. Notably, BMP9-mediated inhibition of ERK is abolished when cells are transfected with siRNA targeting PPP1R3C. ERK hyperactivation was also observed in an HHT2 mouse model (ALK1-2loxP;Cdh5-CreERT2). Loss of both ALK1 alleles in adult mice leads to vascular failure and hemorrhages in the lung and intestine; these injuries are significantly reduced by treatment with the MEK/ERK inhibitor selumetinib. Overall, our work identifies a key role for ERK activation involved in HHT pathogenesis, suggesting that ERK inhibition may represent a promising therapeutic strategy for these patients. Translational PerspectiveHereditary hemorrhagic telangiectasia (HHTs) is a rare vascular disorder caused by mutations in members of the BMP9/ALK1 signaling hub. In the present study we show that all different forms of HHTs are associated with increased endothelial cell (EC) proliferation, which correlates with high levels of ERK activation in patient biopsies. ERK hyperactivation is also observed in an HHT2 mouse model in which loss of both ALK1 alleles in adult mice leads to vascular failure and hemorrhages in the lung and intestine. These injuries are significantly reduced by treatment with the MEK/ERK inhibitor selumetinib. Overall, our work identifies a key role for ERK activation in HHT pathogenesis, suggesting that ERK/MEK inhibitors may represent a promising therapeutic strategy for these patients.

BackgroundVein graft failure due to intimal hyperplasia and maladaptive remodeling remains a major limitation of peripheral bypass surgery. Although vascular remodeling is recognized as a multilayered process, layer-specific molecular mechanisms that distinguish adaptive from negative remodeling remain incompletely understood. We aimed to investigate the vascular microenvironment of patent and stenotic grafts using spatial transcriptomics. MethodsVein specimens were obtained from three patients undergoing revision surgery. For each patient, a matched set of three samples was collected: unused saphenous vein (Denovo), normally healed vein graft (Non-stenosed), and stenosed vein graft (Stenosed) (n = 3 patients). GeoMx Digital Spatial Profiling with the Human Whole Transcriptome Atlas was used to map gene expression across intima, medial, and adventitial layers. Differential expression, gene ontology, spatial deconvolution, and immunohistochemistry were integrated for analysis. ResultsNon-stenosed and Stenosed grafts shared transcriptional features distinct from Denovo veins, particularly in pathways related to cell proliferation. Non-stenosed grafts showed increased expression of CDKN1A across all vascular layers, whereas Stenosed grafts exhibited enhanced mitogen-activated protein kinase (MAPK) pathway activity, reduced DUSP1-mediated regulation, and increased macrophage accumulation. ECM remodeling showed layer-specific organization, with VCAN and ACAN enriched in the intima and DCN in the adventitia, while Stenosed grafts demonstrated a trend toward collagen-dominant remodeling. Cell deconvolution suggested a shift toward vascular smooth muscle cell (VSMC)-dominant architecture after arterialization, with modest enrichment of synthetic VSMC signatures in stenotic regions. ConclusionsVein graft stenosis appears to be associated with layer-specific alterations in cell cycle regulation, inflammatory signaling, extracellular matrix remodeling, and VSMC phenotype. Spatial transcriptomic analysis reveals molecular heterogeneity not captured by bulk approaches and provides preliminary insight into graft remodeling. These findings may inform future studies to improve long-term graft patency.

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.

1.Cancer-induced bone pain (CIBP) occurs in a majority of patients when primary or metastatic cancer develops within the bone. This pain has a significant impact on quality of life, yet there are limited effective treatment options available. Nerve sprouting is a complex mechanism that has been implicated in CIBP. Netrin-1 is a neuronal guidance molecule that is produced by numerous cell types, including cancer cells. In this study we aimed to determine whether netrin-1 inhibition (with NP137 - a humanized IGg1 monoclonal antibody) could ameliorate nerve sprouting, and nociception by extension, in three models of CIBP - osteosarcoma, metastatic breast cancer, and metastatic prostate cancer. Sustained administration of NP137 failed to produce an anti-nociceptive effect in these models, but a delayed onset was observed in the osteosarcoma model. NP137 did not produce a disease-modifying effect, as micro-computed tomography did not reveal reduced bone destruction in the NP137-treated groups. Additionally, there was no nerve fibre density reduction in any of the groups at the late-stage of the disease, suggesting that nerve sprouting occurs in early- to mid-stage CIBP development. Investigation of NP137 exposure indicated that serum levels of NP137 were comparable between the sham and cancer groups. Our study indicates that netrin-1 may play a role in early-stage CIBP development, but inhibition of this mechanism does not produce robust anti-nociception.

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.

Lymphedema is a chronic disease characterized by impaired lymph drainage and accumulation of protein-rich interstitial fluid, which progresses to develop irreversible fibrosis. Importantly, effective therapies and treatments are lacking to alleviate and mitigate the disease. The pathological inflammation and fibrogenesis underlying lymphedema prompted us to evaluate a preclinical medicine IVA337, a pan-peroxisome proliferator-activated receptor (PPAR) agonist that improves liver fibrosis in patients with metabolic dysfunction-associated steatohepatitis (MASH) by activating three PPAR isoforms (, {beta}/{delta}, {lambda}), which play critical roles in lipid metabolism, anti-inflammation responses, and anti-fibrogenesis. Here, we investigate the therapeutic effects of IVA337 during the early stage of surgery-induced secondary lymphedema in mice and explored the underlying mechanisms. IVA337 administration alleviated lymphedema progression, improved lymphatic drainage, reduced dermal thickness, and resolved lymphatic vessel dilation. Mechanistically, IVA337 suppressed the TGF{beta}/SMAD2/3 signaling pathway, reduced immune cells infiltration, and improves lymphatic vessels integrity. In human dermal lymphatic endothelial cells (HDLECs), IVA337 attenuated TGF{beta} induced SMAD2/3 phosphorylation and preserved the expression of cell junction Claudin5, reduced VE-Cadherin-stained cell-cell gaps. Collectively, our findings demonstrate that IVA337 protects against early stage lymphedema by inhibiting TGF{beta}/SMAD2/3-mediated inflammatory and fibrotic responses. This study provides a potential therapeutic strategy to improve lymphatic function during the early phase of lymphedema and prevent progressive fibrosis in patients with lymphedema and related disorders.

P2Y12 receptor (P2RY12), mainly expressed on platelets, is known for its central role in hemostasis. P2RY12 activation is also involved in cancer development through platelet adhesion to cancer cells supporting immune-evasion, promoting tumor angiogenesis and metastasis, among others. P2RY12 is known as an actionable target, and P2RY12 antagonists are in clinical use for cardiovascular diseases. However, very little data are available regarding the protein expression of P2RY12 in lung carcinomas. We performed immunohistochemical staining for P2RY12 in a cohort of non-small cell lung cancer (NSCLC) samples comprising 320 adenocarcinomas (LUAD) and 158 squamous cell carcinomas (LUSC). Results were evaluated using a dual approach combining microscopic assessment and digital image analysis (QuPath). Results were correlated with clinical-pathological data. We found significantly higher P2RY12 protein expression in LUSC compared to LUAD (p<0.001) via eyeballing (absent/low expression in 21.7% (34/158) and moderate/high expression in 78.3% (124/158) of LUSC cases versus absent/low expression in 98.4% (315/320) and moderate/high expression in 1.6% (5/320) of LUAD cases). Digital analysis yielded similar results. High P2RY12 expression was associated with a significantly better 5-year overall survival rate for the entire cohort (p=0.0048) as well as for the LUAD (p=0.015) and LUSC (p=0.05) subgroups. Furthermore, P2RY12 showed excellent discriminatory performance for classifying carcinomas as LUAD or LUSC, with an AUC of 0.916 in ROC-analysis. High P2RY12 expression is linked to a better prognosis and might serve as a promising novel prognostic biomarker for NSCLC. Its assessment could be implemented in future routine diagnostic workup. At the same time, the data suggest that P2RY12 could also serve as a diagnostic marker for LUSC.

BackgroundThe CXCR2 receptor pathway plays a major role in inflammatory and invasive angiogenesis in human disease. ObjectiveWe evaluated AZD5069, a selective CXCR2 antagonist, as an angiogenesis inhibitor in human cell culture. MethodsHuman Umbilical Venous Endothelial Cells (HUVECs), Human Aortic Endothelial Cells (HAECs), and Human Pulmonary Artery Endothelial Cells (HPAECs) were cultured with standard in vitro techniques. AZD5069 (0, 8, 16, 32, 64, 128, 256 M) was evaluated as an angiogenesis inhibitor with fluorescent-labeled 5-Ethynyl-2-deoxyuridine (EdU) uptake to quantify endothelial cell proliferation, scratch assay to quantify endothelial cell migration, and Geltrex assay to quantify endothelial cell tubule and hub formation. AZD5069 cytotoxicity was evaluated with in situ terminal deoxynucleotidyl transferase 2-Deoxyuridine triphosphate- 5 (dUTP) nick-end labeling (TUNEL) to quantify apoptosis and membrane-impermeable cyanine dye uptake to quantify necrotic cell death. ResultsAZD5069 significantly reduced HUVEC, HAEC, and HPAEC proliferation, migration, tubule count, total tubule length, and node count with a dose-response. AZD5069 did not cause apoptosis nor necrotic cell death. ConclusionsAZD5069 inhibited angiogenesis without cytotoxicity in human endothelial cell culture. The endothelial cell CXCR2 receptor pathway may be a novel target for anti-angiogenesis therapy. AZD5069 may have clinical utility in cardiovascular, oncologic, and inflammatory disease. Condensed AbstractThe CXCR2 receptor pathway plays a major role regulating angiogenesis in inflammation and cancer. The CXCR2 receptor pathway has been evaluated in humans as a target for therapy in inflammatory disease and cancer but not as a therapeutic approach to block pathologic angiogenesis. AZD5069 is a clinical stage, direct CXCR2 antagonist. In human endothelial cell culture, AZD5069 inhibited angiogenesis without causing apoptosis or necrotic cell death. The endothelial cell CXCR2 receptor pathway may be a novel target for anti-angiogenesis therapy. AZD5069 may have clinical utility as a novel angiogenesis blocker in human disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/731993v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1ac8fb4org.highwire.dtl.DTLVardef@ea89forg.highwire.dtl.DTLVardef@607f94org.highwire.dtl.DTLVardef@157cec4_HPS_FORMAT_FIGEXP M_FIG Visual Abstract: AZD5069, a selective CXCR2 antagonist, significantly reduced endothelial cell proliferation, migration, and vascular tubule formation without causing necrotic or apoptotic cell death. The endothelial cell CXCR2 receptor pathway may be a novel target for anti-angiogenesis therapy. AZD5069 may have clinical utility in human cardiovascular, oncologic, and inflammatory disease with pathologic, dysregulated, or excessive angiogenesis. C_FIG

Marfan syndrome is a multisystem connective tissue disorder with age-dependent and sex-dependent phenotypic variability. This study characterized early cardiovascular, valvular, transmitral flow, and skeletal changes in Fbn1C1041G/+ mice. Male and female wild-type and Fbn1C1041G/+ mice were examined from 4 to 20 weeks of age. Transthoracic echocardiography was used to measure aortic root and ascending aortic diameters, anterior mitral leaflet length, and mitral inflow Doppler parameters. Lateral X-ray imaging was used to quantify kyphosis angle. Fbn1C1041G/+ mice of both sexes developed progressive aortic dilation, with earlier and broader aortic root involvement in males and delayed annular dilation in females. Anterior mitral leaflet elongation was detected from 8 weeks in males and at 20 weeks in females. In contrast, reduced E-wave velocity and E/A ratio were present from 4 weeks in both sexes, without changes in A-wave velocity. Kyphosis was detectable earlier in females than in males. These findings show that early Fbn1-related phenotypes follow organ-specific and sex-dependent temporal patterns, supporting age-resolved and sex-stratified assessment in preclinical Marfan syndrome studies.

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: Diabetic kidney disease (DKD) remains a leading cause of end-stage renal disease worldwide, characterized by progressive structural and metabolic alterations secondary to chronic hyperglycemia. While numerous type 1 and type 2 rodent models have been developed to study the pathophysiology of DKD, no single model perfectly recapitulates the full clinical spectrum of human disease. The selection of an optimal model depends deeply on the specific research objective, as phenotypic expression and histopathological severity vary significantly across different strains and induction methods. The present study provides a comparative analysis of the renal histological of three widely utilized murine models: the chemically induced streptozotocin (STZ) model and the genetic Akita (type 1) and db/db (type 2) models. Methods: Male STZ-induced (28 weeks post-induction), heterozygous Akita (28 weeks old), and db/db mice at two different age intervals (18-21 and 16-24 weeks old) were assessed. Renal injury was quantified using four light-microscopic parameters: glomerulomegaly, mesangial hypercellularity, tubular vacuolization and arteriolar hyalinosis. Due to observed discrepancies between metabolic and structural findings in the db/db strain, transmission electron microscopy (TEM) was employed for subcellular characterization. Results: All models exhibited significant hyperglycemia and albuminuria. At the light-microscopic level, STZ and Akita mice demonstrated consistent and pronounced renal lesions. In contrast, db/db mice despite increasing albuminuria and obesity, light microscopy revealed heterogeneous and inconsistent histopathological changes. However, TEM analysis of db/db mice kidneys successfully captured early ultrastructural injury, including irregular glomerular basement membrane (GBM) thickening and focal podocyte foot process effacement, which were undetectable by light microscopy. Conclusions: Our findings indicate that the Akita and STZ-induced models exhibit prominent structural alterations detectable by conventional light microscopy, whereas the db/db model requires ultrastructural evaluation by TEM to reliably confirm renal injury. This study underscores the limitation of routine histology in certain type 2 diabetes models and highlights the complementary value of TEM for accurate histopathological characterization. Collectively, the alternative histopathological markers identified herein offer sensitive and readily accessible indices for monitoring early-to-moderate DKD progression, providing a more robust framework for preclinical model selection and therapeutic evaluation in future studies.

Platelets are increasingly recognized as active regulators of inflammation beyond their canonical hemostatic functions. Although platelets rapidly accumulate in the injured myocardium after myocardial infarction (MI), the mechanisms by which they coordinate the inflammatory response remain poorly understood. Glycoprotein A repetitions predominant (GARP) is a membrane receptor that presents latent transforming growth factor-{beta}1 (TGF-{beta}1) on activated platelets and supports its activation. Given the central role of TGF-{beta}1 in inflammation and tissue repair, we hypothesized that platelet GARP-dependent activation of TGF-{beta}1 regulates inflammatory resolution and repair after MI. Using mice with megakaryocyte- and platelet-specific Garp deletion, we demonstrate that loss of platelet GARP selectively impaired generation of bioactive TGF-{beta}1 without altering platelet reactivity. Following permanent coronary artery ligation, platelet-specific Garp deficiency markedly increased mortality from ventricular rupture and exacerbated adverse left ventricular remodeling, independent of initial infarct size. Transcriptomic and histological analyses revealed heightened endothelial cell activation, increased leukocyte recruitment, delayed inflammatory resolution, and defective extracellular matrix deposition in the absence of platelet GARP. Mechanistically, platelet GARP-dependent TGF-{beta}1 signaling restrained endothelial activation after MI. Together, these findings identify platelet GARP-mediated activation of TGF-{beta}1 as a critical platelet-intrinsic counter-regulatory checkpoint that limits endothelial-driven inflammation and promotes infarct stabilization. Our study reveals an unexpected protective immunoregulatory function of platelets in cardiac repair after ischemic injury.

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.

(Objective) In our previous research, we identified latent chronic pancreatitis in the normal tissue surrounding pancreatic cancer. We also discovered the presence of Enterococcus faecalis (E. faecalis), a type of intestinal bacterium, in the pancreatic fluid and tissue of pancreatic cancer patients, suggesting it may be one of the factors contributing to the development of latent chronic pancreatitis. In this study, we performed pathological analyses to investigate its characteristics and investigate a possibility of E. faecalis infection. (Methods) Pathological analyses were performed, using 16 cases of pancreatic cancer and intraductal papillary mucinous neoplasia (IPMN) involving lesions in the pancreas tail. The involvement of E. faecalis was investigated with immunohistochemical analysis and serological methods. (Results) All cases exhibited inflammatory changes in pancreatic tissue without a clinical diagnosis of chronic pancreatitis, along with macrophage infiltration. These changes did not significantly differ according to preoperative treatment. DNA encoding E. faecalis 16s ribosomal RNA was detected in many cases, however, a positive immunostaining to E. faecalis was observed in only a few cases. Serum capsular polysaccharide (CPS) antibody levels exceeding the mean values were observed in patients with established chronic pancreatitis, while the level was not correlated with E. faecalis immunostaining. (Conclusion) These results suggest the E. faecalis infection is involved in the early stage of the progression of chronic latent pancreatitis and the diagnostic technology incorporating novel multi-biomarkers may be useful for identifying high-risk individuals for future pancreatic cancer development.

Non-small cell lung cancer (NSCLC) is the most common form of lung cancer accounting for most cancer-related deaths worldwide. Despite substantial recent advances in targeted therapies and immunotherapy, the prognosis for advanced-stage disease remains comparably poor, which is why the identification of novel molecular biomarkers as well as therapeutic targets influencing tumor development, progression, and metastasis remain important. This study focusses on SERPINB13, a serine-protease inhibitor expressed in selected tissues that is dysregulated in several tumor entities. However, its role in NSCLC still remains largely unclear. We analyzed SERPINB13 transcription in a cohort of non-small cell lung cancer (NSCLC) cases including both lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) by transcriptome profiling. Epigenetic modifications were assessed via Methylation BeadChips. Additionally, SERPINB13 protein expression was assessed by immunohistochemistry (IHC) in an independent cohort of NSCLC comprising 126 LUSC patients. Correlation analyses were performed to associate SERPINB13 expression with key clinico-pathological parameters, including overall survival and extent of tumor-infiltrating immune cells. To functionally investigate the regulatory influence of peripheral blood mononuclear cells (PBMCs) on SERPINB13 expression in LUSC tumor cells in vitro, we utilized the SERPINB13-expressing LUSC cell line LUDLU-1. Here, gene transcription was analyzed by quantitative real-time PCR (RT-qPCR), confirmed by Western blot on the protein level. Transcriptome analysis revealed a significant upregulation of SERPINB13 in lung squamous cell carcinoma (LUSC) compared to lung adenocarcinoma (LUAD), highlighting a subtype-specific expression pattern. This differential expression was further associated with a distinct epigenetic DNA methylation signature at the SERPINB13 loci in LUSC, suggesting transcriptional regulation via hypomethylation. IHC analysis demonstrated that high SERPINB13 protein expression is significantly associated with prolonged overall survival in LUSC. Notably, SERPINB13 expression was enriched in immune-inflamed ("hot") tumors, characterized by elevated infiltrating lymphocytes and immune activation. Mechanistically, co-culture experiments with PBMCs induced SERPINB13 expression in a LUSC cell line in a dose- and time-dependent manner in the absence of direct cell contact. This suggests that soluble factors secreted by immune cells might play a key role in regulating SERPINB13 expression in the tumor microenvironment. Taken together, SERPINB13 is a novel prognostic indicator in LUSC that is modulated by Immune cells. Further studies are necessary to decipher the crosstalk of Immune cells on the Serpin B13 expressing tumor cells in depth, with regard to a possible interventional strategy. immunomodulatory potential strategies and personalized therapeutic approaches in NSCLC.