Journal of Cellular and Molecular Medicine

BackgroundCalcific aortic valve disease (CAVD) is the most common valvular heart disease in developed countries, yet no pharmacological therapy is available to slow or halt its progression. CAVD is driven by progressive calcification of aortic valve leaflets, in which myeloid cells play a central role. While macrophages have been implicated in CAVD pathogenesis, the contribution of their precursors, monocytes, remains poorly understood. We hypothesized that circulating monocytes acquire a pro-calcific and pro-inflammatory phenotype contributing to valve remodelling and CAVD progression. MethodsWe profiled circulating CD14+ monocytes from healthy volunteers (Vol), patients with CAVD, and without CAVD (NCAVD). Peripheral blood mononuclear cells (PBMCs) were isolated, and monocyte subpopulations were phenotyped by flow cytometry. Transcriptome profiling by RNA sequencing identified disease-associated gene signatures, which were validated by RT-qPCR. The CD14+ monocyte secretome was analysed using multiplex assays. Functional ability of CAVD-derived CD14+ monocytes to induce myofibroblastic transdifferentiation (MT) and osteoblastic differentiation (OD) of human valvular interstitial cells (VICS) was evaluated by immunocytochemistry and quantitative o-cresolphthalein complexone assays. ResultsIn PBMCs, CAVD monocytes displayed a subpopulation shift, with an increased proportion of CD14CD16- classical monocytes and a reduced CD14CD16 non-classical monocyte levels. In CD14+ monocytes, transcriptomic analysis revealed upregulation of inflammation-related (PDK4) and calcification-related (ATP2B1) genes, alongside downregulation of immunomodulatory genes (DDR1, IKBKE). Secretome analysis showed reduced production of immunomodulatory and anti-osteoblastogenic cytokines (IL-4, CCL3) while promoting gene expression of factors promoting MT and OD in VICS. These alterations were associated with a marked monocyte-induced increase in SMA and OPN expression in VICS and a two-fold increase in calcification. ConclusionWe demonstrate for the first time that circulating monocytes from patients with CAVD exhibit enhanced pro-inflammatory and pro-calcific properties that may contribute to CAVD progression. Additionally, we identify dysregulated gene sets within these monocytes that represent potential novel therapeutic targets for CAVD.

Background: Abdominal aortic aneurysms (AAA) are characterized by dilation of the aorta that can lead to aortic rupture and death. The transcriptional co-activators Yes-Associated Protein (YAP) and WW-domain-containing transcriptional co-activator with PDZ-binding motif (TAZ) are mechanosensitive effectors of the highly conserved Hippo signaling pathway. It is hypothesized that cell-specific YAP/TAZ signaling in endothelial cells (EC) plays a pivotal role in mediating AAA formation and rupture. Methods: Single-cell RNA-sequencing in human AAAs was performed and differentially expressed genes (DEGs) were identified in the endothelial cell cluster. YAP/TAZ mRNA and protein expression were also assessed in human AAA and control aortic tissue. Two established murine AAA models were used with male C57BL/6 and EC-CreERT2-YAPfl/fl/TAZfl/fl mice with/without Verteporfin (VPF, YAP/TAZ inhibitor) and XMU-MP-1 (YAP/TAZ activator) treatments. On postoperative days 14 and 28, aortic diameter, histology, cytokine, and MMP2 expressions were evaluated. Results: A significant alteration in EC-specific differentially expressed YAP/TAZ-related genes was observed in which 242 genes were upregulated and 71 genes were downregulated in AAA compared to controls. Human AAA tissue showed a significant increase in YAP and TAZ protein expressions compared to controls. Elastase-treated EC-YAP/TAZ-/- mice showed a significant decrease in AAA diameter compared to littermate controls. Histological quantification revealed preservation of -smooth muscle actin, reduced elastin fiber breaks, and decreased macrophage infiltration in EC-YAP/TAZ-/- mice compared to littermate controls. Importantly, pharmacological inhibition of YAP/TAZ using VPF significantly attenuated AAAs in two experimental murine models. In vitro data demonstrates that VPF inhibits endothelial cell YAP expression, downregulating pathways associated with pathogenic angiogenesis and vascular inflammation. Conclusions: These data suggest that EC-specific YAP/TAZ signaling mediates AAA formation. Pharmacological inhibition of the Hippo pathway can significantly mitigate aortic inflammation and vascular remodeling to decrease the progression of AAAs and prevent aortic rupture.

Following aortic dissection (AD), there is a sustained risk of vascular complications, progressive false lumen aneurysm formation, and rupture. However, no effective therapy exists to prevent these complications, highlighting the need to elucidate the pathophysiology following AD. Elastic fibers are crucial for maintaining aortic wall integrity but are thought to have limited regenerative capacity once disrupted during AD. This study defined that elastic fibers were newly generated in the false lumen wall following AD in humans and mice. In human ADs, new elastic fibers were observed in the false lumen wall 6 months after onset. In mice with descending AD induced by {beta}-aminopropionitrile (BAPN), elastin mRNA was markedly upregulated in the chronic phase following AD, accompanied by elastic fiber formation. These fibers coincided with smooth muscle cell (SMC) markers within the false lumen wall. Of note, lineage tracing studies demonstrated that these cells were not derived from resident SMCs but adventitial progenitor cells. In vitro experiments further demonstrated that adventitial progenitor cells produced elastic fibers while expressing SMC markers. Collectively, these findings suggest that adventitial progenitor cells differentiate into elastogenic SMC-like cells, contributing to false lumen remodeling through de novo elastic fiber formation following AD.

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.

CD239, also known as Lutheran blood group glycoprotein (Lu) or basal cell adhesion molecule (B-CAM), is a transmembrane protein belonging to the immunoglobulin superfamily (IgSF). CD239 serves as a specific receptor for laminin 5, a subunit of laminin-511/-521, which are major components of renal basement membranes. A previous study of another group demonstrated that CD239-null mice are healthy and develop normally. Although no alteration in renal function was observed, most glomeruli in the mutant kidneys exhibited morphological abnormalities. In this study, we investigated the role of CD239 in renal tubules. We examined the distribution of CD239 using renal tubule-specific markers. CD239 was localized to the Henles loop, distal tubule, and collecting duct, but not to the proximal tubule. Next, we analyzed the localization of renal tubular molecules in CD239-null mice. The localization of uromodulin (UMOD) and Na-K-Cl cotransporter (NKCC2) was disrupted in the distal tubules lacking CD239, suggesting that CD239 plays a role in maintaining the polarity of renal epithelial cells. Furthermore, to examine the stability of the distal tubules, CD239-null mice were subjected to chronic kidney disease (CKD) using an adenine-rich diet. Blood analysis revealed that CD239-null mice fed an adenine diet readily developed CKD. Adenine-fed null mice exhibited more marked histological injury along the distal tubules compared to that by controls. These results indicate that CD239 is essential not only for maintaining cellular polarity but also for ensuring the stability of the distal tubules. Although CD239-null humans exhibit no obvious associated pathology, it could be a predisposition to CKD.

BACKGROUNDPatients with inflammatory bowel disease (IBD) are at increased risk of cardiovascular disease, yet the mechanisms linking chronic intestinal inflammation to cardiac dysfunction remain poorly understood. IBD is characterized by profound gut microbiota dysbiosis, which we hypothesize drives systemic immune dysregulation and contributes to cardiac dysfunction. METHODSA chronic colitis mouse model was used to assess gut microbiota dysbiosis, systemic immune cell metabolism, and cardiac remodeling. Cardiac outcomes were evaluated by echocardiography, histology, and molecular analyses. Mechanisms were examined using fecal microbiota transplantation, immune cell depletion, exosome transfer, bone marrow chimeras, RNA-seq, co-immunoprecipitation, confocal microscopy, and siRNA-mediated gene silencing. RESULTSChronic DSS colitis induced cardiac dysfunction, hypertrophy, and fibrosis in mice. These changes were accompanied by sustained gut microbiota dysbiosis, metabolic reprogramming, and mitochondrial dysfunction in circulating immune cells. Fecal microbiota transfer experiments demonstrated that colitis-associated microbiota were sufficient to reprogram systemic immune cells and promote cardiac dysfunction. Immune cell depletion studies identified macrophages as key mediators of colitis-associated cardiac injury. Colitis increased systemic lipopolysaccharide (LPS) translocation, bone marrow chimera experiments demonstrated that hematopoietic TLR4 signaling was required for immune cell metabolic remodeling and cardiac dysfunction during chronic colitis. Transcriptomic analysis identified guanylate-binding protein 2b (GBP2b/GBP1, hereafter referred to as GBP1) as a key downstream effector of LPS-TLR4 signaling. Upon LPS stimulation, GBP1 localized to mitochondria, where it interacted with DRP1 and FIS1 to promote mitochondrial fission, oxidative stress, and enhanced immune cell migration into the heart. In addition, GBP1 was secreted via exosomes, which were taken up by cardiomyocytes and contributed to hypertrophic remodeling, and cardiac dysfunction. CONCLUSIONSThese findings establish the LPS-TLR4-GBP1 axis as a key driver of colitis-associated cardiovascular dysfunction and highlight this pathway as a promising therapeutic target for reducing cardiovascular risk in patients with IBD. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIPatients with inflammatory bowel disease have an increased risk of cardiovascular dysfunction that cannot be fully explained by traditional cardiovascular risk factors. C_LIO_LIGut microbiota dysbiosis and chronic innate immune activation are hallmarks of inflammatory bowel disease, but their direct contribution to cardiac remodeling remains unclear. C_LI What New Information Does This Article Contribute?O_LIChronic colitis-associated gut microbiota dysbiosis induces systemic immune cell metabolic and mitochondrial reprogramming that is sufficient to drive cardiomyocyte hypertrophy and cardiac dysfunction. C_LIO_LIHematopoietic Toll-like receptor 4 signaling links colitis associated gut microbiota to immune metabolic dysfunction and cardiac impairment, establishing a causal gut-immune-heart axis. C_LIO_LIGuanylate-binding protein 2b (GBP2b/GBP1) is identified as a critical downstream effector that promotes mitochondrial fission, oxidative stress, immune cell cardiac infiltration, and exosome-mediated cardiac remodeling. C_LI

BackgroundMoyamoya disease (MMD) is characterized by progressive arterial stenosis and abnormal collateral formation, but the spatial organization of vessel-wall abnormalities remains incompletely understood. MethodsWe combined Xenium in situ spatial transcriptomics and multiplex immunofluorescence in superficial temporal artery samples from patients with MMD and controls, and performed gain- and loss-of-function experiments in human brain microvascular endothelial cells (HBMECs). Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), tube-formation, Transwell migration, and cell scratch assays were used to assess signaling and endothelial phenotypes. ResultsMMD vascular tissue showed intimal hyperplasia, altered spatial cellular architecture, and enrichment of extracellular matrix- and proteoglycan-related programs, with upregulation of sulfatase 1 (SULF1). In HBMECs, SULF1 knockdown reduced, whereas SULF1 overexpression enhanced, vascular endothelial growth factor A165 (VEGF-A165)-induced vascular endothelial growth factor receptor 2 (VEGFR2), extracellular signal-regulated kinase 1/2 (ERK1/2), and protein kinase B (AKT) phosphorylation, migration, tube formation, and angiogenesis- and adhesion-related gene expression. Heparinase III attenuated the signaling effects associated with SULF1 overexpression. ConclusionThese findings suggest that SULF1-associated extracellular matrix alterations may contribute to local vessel-wall remodeling and enhanced endothelial responsiveness in MMD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/721514v1_ufig1.gif" ALT="Figure 1"> View larger version (81K): org.highwire.dtl.DTLVardef@143db87org.highwire.dtl.DTLVardef@1a9d9org.highwire.dtl.DTLVardef@1362215org.highwire.dtl.DTLVardef@f7c5a3_HPS_FORMAT_FIGEXP M_FIG C_FIG

PurposeOvarian clear cell carcinoma (OCCC) is strongly associated with endometriosis and shows geographic variation in incidence. We investigated whether OCCC and adjacent endometriosis exhibit distinct transcriptional states and whether these patterns differ between United Kingdom (UK) and Japanese cohorts. Experimental DesignWe performed whole-transcriptome spatial profiling on specimens from 16 OCCC cases (8 UK, 8 Japan) in which tumor and endometriosis were both present. Gene expression was analyzed in tumor, endometriosis and stroma. ARID1A status was assessed by immunohistochemistry. ResultsMedian age was 59 years (range 26-82). 13/16 cases (81.3%) had early-stage disease. Tissue compartment rather than cohort of origin was the dominant source of variation across endometriosis and tumor regions. Endometriosis was enriched for inflammatory and immune-related pathways compared to tumor, whilst there was greater representation of chromatin and protein-DNA complex assembly pathways in tumor regions. These patterns were conserved across both cohorts and after stratification by ARID1A status. Mesenchymal-associated gene expression scores also significantly differed across stroma, endometriosis and tumor with clear compartmental separation. Cell type deconvolution analyses showed clear compositional differences between stromal and epithelial disease compartments. ConclusionsOCCC and coexisting endometriosis are transcriptionally distinct, with the dominant contrast being compartmental rather than geographic. ARID1A alone is unlikely to account for the principal spatial transcriptional states identified here. Further analyses will be required to ascertain whether these differences reflect genuine biological differences between OCCC and coexisting endometriosis or represent different stages of endometriosis-associated tumorigenesis. Translational RelevanceOvarian clear cell carcinoma often arises in association with endometriosis, yet the biological transition between these lesions remains poorly understood. Using spatial transcriptomics in matched tumor and adjacent endometriosis from Japanese and UK cohorts, we showed that endometriosis is characterized by inflammatory and antigen-presentation features, whereas tumor regions showed chromatin-organization and oncogenic transcriptional states. These patterns were largely maintained irrespective of ARID1A status and geographic background. In addition, spatial deconvolution suggested differences in local immune composition, with tumor regions showing relatively greater neutrophil- and T cell-associated signals. Together, our data suggest that OCCC and coexisting endometriosis share a spatially linked tissue context, but that tumor regions have distinct transcriptional profile and microenvironment that may be involved in the malignant transformation and inform interpretation of molecular classification in endometriosis-associated OCCC.

Introduction. TAC/SARIFA has been introduced as a new robust and easy-to-evaluate biomarker in several cancer entities, including colorectal cancer. It is defined by direct contact between at least five tumour cells and one adipocyte and is believed to indicate metabolic reprogramming associated with adverse outcome. However, the mechanism that leads to TAC/SARIFA positivity remains unclear. To investigate whether there is an individual component, we conducted a study on double and triple cancers, establishing a within patient design. Methods. We retrospectively analysed a total of 135 cases with 276 colorectal cancers from two academic medical centres. The TAC/SARIFA status was evaluated, as were the basic histopathological factors. The median follow-up time was 120 months. Results. Cases with any TAC/SARIFA positive tumours showed significantly reduced overall survival (62 vs. 88 months; p = 0.011). Analysing the entire cohort, the rates of concordant and discordant cases followed a random distribution. However, restricting the analysis to synchronous pT3/4 cases revealed a significant deviation from a random distribution (p = 0.016). Conclusion. This study reveals significant concordance of TAC/SARIFA status in synchronous locally advanced colorectal double/triple carcinomas, supporting the concept that tumour adipocyte interaction reflects a host related microenvironmental condition linked to metabolic reprogramming rather than a purely tumour intrinsic event.

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

BACKGROUNDAortic dissection (AD) is a life-threatening emergency with high mortality. Although elevated body mass index (BMI) is associated with both AD incidence and mortality, the underlying mechanisms remain unclear. Periaortic adipose tissue (PAAT) increases with BMI, and the PAAT of AD shows marked inflammatory infiltration, suggesting PAAT-driven inflammation may contribute to the development of AD. However, no direct evidence links BMI and PAAT to AD. To further elucidate the obesity-inflammation-AD relationship, we aim to quantify the contributions of BMI, PAAT, and their derived indices to the risk of AD. METHODSThis retrospective multicenter study (June-November 2025) quantified PAAT around the descending thoracic aorta with CT angiography (CTA). Logistic regression analyses were performed to identify AD risk factors. Based on the Boruta algorithm (a machine learning feature selection method) and ROC curve analysis, the variable importance for AD risk was assessed. The dose-response relationship between BMI-Volume-derived metric (BMV) and AD risk was further characterized by quartile stratification and restricted cubic spline (RCS). RESULTSThis study enrolled 376 consecutive participants. After adjusting for potential confounders, BMI, smoking, systolic blood pressure (SBP), diabetes mellitus (DM), TC/HDLC, ApoE, PAAT volume (Volume), PAAT fat attenuation index (FAI), and BMV were identified as independent predictors of AD. Volume was the strongest AD predictor with the highest Z-score. Compared with BMI [AUC 0.627, 95% confidence interval (CI): 0.569-0.687] and Volume (AUC 0.716, 95% CI: 0.662-0.772), BMV showed better discriminatory performance (AUC 0.726, 95% CI: 0.673-0.778). RCS showed an approximately linear positive association between BMV and AD risk (P-overall < 0.001, P-non-linear = 0.09). CONCLUSIONSIn this retrospective multicenter study, BMV, a composite measure integrating systemic and periaortic adipose tissue factor, showed a positive association with AD risk, and improved predictive performance beyond BMI, indicating incremental predictive value, pending external validation. GRAPHIC ABSTRACTA graphic abstract is available for this article. WHAT IS KNOWNO_LIBody-mass index (BMI) appears to be associated with an increased risk of aortic dissection (AD) and higher all-cause mortality, but a definitive consensus remains elusive. C_LIO_LIPAAT has been identified as an independent cardiovascular risk factor, with marked inflammatory cell infiltration observed in the PAAT of PAAT has been identified as an independent cardiovascular risk factor, with marked inflammatory cell infiltration observed in the PAAT of aortic diseases patients. C_LIO_LIPAAT increases with BMI, and its pro-inflammatory function may destabilize the aortic wall, but relationship of BMI-PAAT synergy and AD remains to be demonstrated. C_LI WHAT THE STUDY ADDSO_LIBMI, PAAT volume, and its FAI were independent predictors of AD, with volume ranked as the strongest predictor by the Boruta algorithm. C_LIO_LIThe BMV, a composite metric that integrated systemic and periaortic adipose tissue factor, was positively associated with AD risk and improved predictive performance beyond BMI alone. C_LI

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.

BackgroundMetabolic dysfunction-associated steatohepatitis (MASH) is emerging as a risk factor of cardiometabolic diseases, including the atrial fibrillation (AF) - the most common sustained arrhythmia. Given that the liver is a major source of inflammatory mediators, lipids, and hepatokines under metabolic stress, we hypothesized that hepatocyte-derived factors in MASH may accelerate atrial remodeling and arrhythmogenesis. MethodsAnalysis of the Atherosclerosis Risk in Communities (ARIC) visit 5 cohort was performed to determine the association between the FIB-4 index - a classic indicator of liver fibrosis, and AF risk, with multivariable adjustment for common comorbidities. A murine model of MASH was induced using the GAN (Gubra-Amylin NASH) diet. Programmed intracardiac stimulation and echocardiography were performed to assess AF susceptibility and cardiac function. Calcium imaging, histology, flow cytometry, plasma proteomics, and single-nucleus RNA sequencing (snRNA-seq) analyses were employed to elucidate the role of recruited inflammatory macrophages via hepatocyte-derived osteopontin (OPN) in MASH-induced atrial remodeling. ResultsAnalysis of the ARIC cohort confirmed a higher cumulative incidence of AF and an elevated adjusted hazard ratio (HR) in patients with intermediate and high FIB-4 indices compared to individuals with low FIB-4 scores. MASH mice exhibited increased susceptibility to pacing-induced AF, accompanied by enhanced proarrhythmic calcium release events, atrial enlargement, and fibrosis, independent of ventricular dysfunction. Proteomics and snRNA-seq revealed that the hepatocyte-secreted OPN under MASH conditions promoted the differentiation and recruitment of TGFBR1+ inflammatory macrophages to the atria, leading to gasdermin D (GSDMD) activation - an effector of inflammasome signaling and consequent proarrhythmic atrial remodeling. Activation of the monocyte-derived pro-inflammatory TGFBR1+ macrophages was dependent on the OPN receptor CD44. Furthermore, the MASH-induced atrial fibroinflammatory milieu and enhanced AF susceptibility were mitigated through several strategies, including hepatocyte-specific Spp1 (encoding OPN) deletion, neutralization of circulating OPN, ablation of CD44 or GSDMD. ConclusionsThese findings establish a pathogenic role of the hepatokine osteopontin in driving activation and recruitment of TGFBR1+ inflammatory macrophages into the atria, leading to proarrhythmic atrial remodeling under MASH. Osteopontin-targeted therapy or GSDMD inhibition prevents AF, indicating a novel therapeutic strategy for liver disease-related atrial arrhythmogenesis. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIIn the ARIC cohort, metabolic dysfunction-associated steatohepatitis (MASH) is associated with increased risk of atrial fibrillation (AF) after adjusting for common comorbidities. Elevated levels of circulating osteopontin (encoded by SPP1) predict an increased risk of AF in patients with MASH-induced liver fibrosis. C_LIO_LIMASH enhances hepatocyte secretion of osteopontin, leading to expansion of myeloid cells and recruitment of inflammatory macrophages into atria. This liver-to-atrial inflammatory circuit promotes the development of a substrate conducive to AF, which can be attenuated by hepatocyte-specific Spp1 deletion or neutralizing anti-anti-osteopontin antibody treatment to eliminate the mediator, or ablation of inflammasome effector gasdermin D to correct the atrial response. C_LI What are the clinical implications?O_LIOsteopontin may serve as a biomarker for AF in MASH cohorts. C_LIO_LIAnti-osteopontin therapy through neutralizing antibodies may serve as a novel therapeutic strategy for liver disease-related atrial arrhythmia. C_LI

Cell division cycle 42 (Cdc42) is a member of the Rho family of small GTPases, which plays crucial roles in regulating cytoskeletal remodeling, and membrane trafficking. While previous studies implicated Cdc42 in controlling intestinal epithelial homeostasis, the involvement of this small GTPase in the process of intestinal fibrogenesis remains unexplored. Our study was designed to determine whether Cdc42 regulates the fibrogenic activation of intestinal myofibroblasts in vitro. The study was conducted using a CCD-18Co normal human colonic fibroblast cell line, and primary human intestinal myofibroblasts (HIMF) isolated from Crohns disease (CD) patients. CCD-18Co and HIMF cells were stimulated by transforming growth factor-{beta}1 (TGF-{beta}1). Cdc42 was inhibited either genetically, using siRNA-mediated knockdown, or pharmacologically using specific Cdc42 inhibitors, ML141 and CASIN. Genetic and pharmacologic inhibition of Cdc42 markedly reduced TGF-{beta}1 induced expression of the major contractile cytoskeletal proteins, -smooth muscle actin, calponin 1 and L-caldesmon. Furthermore, Cdc42 inhibition significantly attenuated expression of key extracellular matrix (ECM) proteins, fibronectin and collagen I, in activated CCD-18Co cells and HIMF. Interestingly, decreased expression of contractile and ECM proteins in Cdc42-depleted myofibroblasts was not due to downregulation of the TGF-{beta}1 signaling, decreased mRNA transcription or increased lysosomal or proteasomal degradation of these proteins. Such suppressed pro-fibrotic activation of Cdc42-deficient CCD-18Co cells and HIMF involved a selective inhibition of protein translation due to inactivation of the AKT-mammalian target of rapamycin (mTOR) signaling module. These findings highlight Cdc42 as a key regulator of intestinal fibrosis that controls mTOR activation to enhance ECM production and contractile actomyosin cytoskeleton in intestinal myofibroblasts.

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.

Objectives; The role of nuclear receptor subfamily 4, group A, member 3 (NR4A3) in hepatic steatosis, inflammation, and insulin resistance (IR) within the context of metabolic dysfunction-associated steatotic liver disease (MASLD) remains largely underexplored. Consequently, this study aimed to examine NR4A3's impact on MASLD and the potential underlying mechanisms. Methods; We aimed to elucidate the functional role of NR4A3 in MASLD through its knockdown in cell culture and animal models. To establish the cell culture model of MASLD, LO2 cells were treated with free fatty acids (FFAs), while male C57BL/6 mice were fed a high-fat diet (HFD) to create the animal model. NR4A3 knockdown was achieved using specific short hairpin RNA (NR4A3-shRNA) in the mice model and three small interfering RNAs (NR4A3-siRNAs) in the cell culture model. The lipids content, fatty acid synthesis, inflammatory factors, and IR were then assessed with and without NR4A3 knockdown. Furthermore, the underlying mechanism through which NR4A3 exerts its influence was explored by analyzing the interaction between NR4A3 and activating transcription factor 3 (ATF3). Results: In the cell culture experiments, the knockdown of NR4A3 significantly decreased the lipids content, fatty acid synthesis, and inflammatory factors in the LO2 cells treated with FFAs in the NR4A3-shRNA group compared with those in the NC-shRNA control group. In the animal model experiments, NR4A3 knockdown in the HFD male C57BL/6 mice significantly ameliorated HFD-induced hepatic steatosis, inflammation, and IR. Mechanistically, the knockdown of NR4A3 downregulated the expression and transcriptional activity of ATF3, resulting in an impaired ATF3 function. ATF3 overexpression significantly reversed lipid accumulation decline and reduced inflammation after NR4A3 knockdown. Conclusion: The downregulation of NR4A3 alleviates MASLD by modulating ATF3, suggesting this may be a promising therapeutic target.

ObjectiveTo 1) determine the expression and distribution of all PDE4 isozymes (A-D) along the length of the anterior urethra, 2) culture fibroblasts and epithelial cells from healthy and strictured urethras, 3) investigate an in vitro model of anterior urethral stricture disease (aUSD), and 4) assess the therapeutic potential of phosphodiesterase-4 (PDE4) inhibitors and testosterone compared to paclitaxel. MethodsThe presence and relative abundance of PDE4 isozymes (A-D) was confirmed using immunohistochemistry on 5 male cadaveric urethras. Human urethral fibroblasts (FBs) were cultured from healthy control urethras of patients undergoing vaginoplasty (n=3) and from idiopathic bulbar urethral strictures (L2S1E2) of patients undergoing urethroplasty (n=3). Epithelial cells (ECs) were cultured from a healthy control urethra and two urethral strictures. To investigate a model of aUSD, Control FBs were stimulated with TGF{beta}1 and compared to Stricture FBs on assays of cell proliferation and expression of genes relevant to aUSD pathophysiology. To test therapeutics, Stricture FBs were treated with the PDE4 inhibitor, roflumilast, testosterone (T), or paclitaxel and compared to Control FBs on the previously mentioned assays and cell viability. ResultsPDE4- A, B, and D were detected along the length of the urethra. Expression levels did not differ between urethral regions. TGF{beta}1 altered proliferation and gene expression in a dose-dependent manner. Roflumilast and T preserved cell viability and proliferation and decreased expression of genes positively associated with auSD. ConclusionUrethral FBs and ECs can be cultured from healthy and strictured surgical specimens, enabling in vitro research. PDE4 inhibitors and T may be non-cytotoxic alternatives or additions to paclitaxel for aUSD. HighlightsO_LIPDE4 isozymes A, B, and D are expressed in adult anterior urethras C_LIO_LIPDE4 is expressed equally from proximal bulbar to meatal urethra C_LIO_LIEpithelial cells and fibroblasts can be cultured from healthy and stricture urethra C_LIO_LITGF{beta}1 may not be an optimal method to model aUSD in vitro C_LIO_LIUnlike paclitaxel, roflumilast and testosterone are not toxic to urethral cells C_LI

BACKGROUNDSmooth muscle cells (SMCs) comprise the majority of cells in human atherosclerotic lesions and are thought to be a major source of cholesterol-overloaded foam cells in human and mouse atheromas. However, the transcriptomic profile, specific markers, and biologic itinerary of SMC foam cells relative to macrophage foam cells remain poorly defined. METHODSSingle-cell RNA sequencing (scRNA-seq) was performed on fresh coronary artery segments from heart transplant recipients with early- to intermediate-stage atherosclerosis. Gene expression in a putative SMC foam cell cluster was compared with cultured SMCs loaded with aggregated low-density lipoprotein (agLDL) or cholesterol-methyl-{beta}-cyclodextrin (Chol-M{beta}CD). Candidate markers distinguishing SMC from macrophage foam cells were validated using additional publicly-available scRNA-seq datasets, Xenium spatial transcriptomics, and immunofluorescence microscopy of human coronary atheromas. Pathway analysis was performed using Gene Set Enrichment Analysis Hallmark gene sets. RESULTSA distinct SMC foam cell cluster derived from fibromyocytes ("lipomyocytes") was identified using markers induced by in vitro cholesterol loading. agLDL loading reproduced the lipomyocyte transcriptional profile, whereas Chol-M{beta}CD induced an inflammatory phenotype colocalizing with macrophages rather than lipomyocytes. Lipomyocytes highly expressed SERPINE1, encoding plasminogen activator inhibitor-1 (PAI-1), and CFH, encoding complement factor H, which were validated in human coronary lesions by spatial transcriptomics and immunofluorescence microscopy. Compared with macrophage foam cells, lipomyocytes demonstrated distinct pathway activation, including enrichment of extracellular matrix, coagulation and angiogenesis pathways. CONCLUSIONSSMC foam cells, or lipomyocytes, represent a distinct foam cell phenotype with unique markers and biologic programs that differ from macrophage foam cells during atherosclerotic plaque development. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LISmooth muscle cell (SMC) foam cells, or lipomyocytes, arise from fibromyocytes and exhibit a transcriptomic profile that is markedly distinct from that of macrophage foam cells. C_LIO_LIIn vitro loading of SMCs with aggregated LDL recapitulates the gene expression profile of SMC foam cells in human coronary atheromas, whereas loading with cyclodextrin-bound cholesterol does not. C_LIO_LIPlasminogen activator inhibitor 1 (PAI-1, encoded by SERPINE1) and Complement Factor H are specific markers of SMC foam cells and are not expressed by macrophage foam cells. C_LI What Are the Clinical Implications?O_LISMCs contribute a substantial proportion, and potentially the majority, of foam cells in atherosclerotic lesions. C_LIO_LIDefining the biological trajectory of SMC foam cells within plaques is critical for understanding their roles in plaque progression, rupture and thrombosis, and for establishing their relevance as a distinct therapeutic target to reduce major cardiovascular events. C_LI

Background and AimsPerianal fistulizing Crohns disease (PFCD) is a challenging complication with poorly understood pathogenesis and limited treatment options, largely due to the lack of clinically relevant animal models. Interferon-gamma (IFN-{gamma}) signaling is hyperactivated in human PFCD. We aimed to establish mouse models recapitulating human PFCD and to evaluate IFN-{gamma} as a new therapeutic target. MethodsPerianal fistulas were established in three mouse models with concurrent Crohns disease-like intestinal inflammation: wild-type (WT) mice with 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced proctocolitis, Il10-/-mice, and TNF{Delta}69AU/+ mice. A modified MAGNIFI-CD index was developed for longitudinal fistula assessment in mice. Transcriptomic analysis and flow cytometry were conducted on mouse fistula tissue. Re-analysis of single-cell and spatial transcriptomics of human PFCD tissues was performed. Therapeutic benefits of anti-TNF-, upadacitinib, and IFN-{gamma} pathway antagonists were evaluated in the PFCD models. ResultsAll three PFCD models sustained chronic perianal fistula tracts for at least 5 weeks after wire removal. All three models closely recapitulate the pathological and molecular features of PFCD in patients, as confirmed by clinical examination, MRI, histopathology, immunostaining, flow cytometry, and transcriptomics. IFN-{gamma} signaling emerged as a central and conserved pathway across all three mouse models and human PFCD. Targeting of the IFN-{gamma} pathway promptly improved fistula healing with mitigation of IFN-{gamma} signaling, inflammation, and epithelial-to-mesenchymal transition (EMT). Moreover, combining IFN-{gamma} and TNF- blockade demonstrated augmented therapeutic efficacy compared to anti-TNF- monotherapy. ConclusionsThese PFCD mouse models and imaging tools provide first reliable and clinically relevant platforms for mechanistic studies and therapeutic evaluation. IFN-{gamma} signaling represents a potential therapeutic target warranting clinical investigation.

IntroductionThe quality of preclinical studies for rare diseases, such as Duchenne muscular dystrophy (DMD), relies on the availability of comprehensive natural disease history data. In addition to the classic BL10-mdx mouse, in recent years, the D2-mdx model has increasingly been used as an alternative model due to its reportedly more severely impaired phenotype. To improve our understanding of disease progression in these two DMD models, we conducted a comprehensive natural history study. Materials and MethodsThis involved a cross-sectional analysis of key in vivo and ex vivo outcome measures performed in two independent laboratories, using the same study setup in compliance with TREAT-NMD Standard Operating Procedures (SOPs), while also taking advantage of site-specific expertise. Globally, largely comparable results were obtained across the two study sites. ResultsBody composition showed pronounced differences between the strains, with BL10-mdx mice displaying a hypertrophic and D2-mdx mice displaying an atrophic phenotype. Dystrophic mice of each strain exhibited significant alterations of disease-relevant indices related to muscle functionality and integrity, mostly worsening with age, in comparison to their wildtypes. Cardiac function was affected earlier and more severely in D2-mdx mice. DiscussionNotably, for some parameters, genetic-background related differences were observed, emphasizing the need to include control groups with matching genetic backgrounds in experimental designs. ConclusionsCollectively, our natural history study provides benchmark data for these two mdx mouse strains to guide model selection for preclinical DMD studies, allowing accurate data interpretation. HighlightsO_LIDistinct body composition phenotypes: BL10-mdx mice exhibit pseudohypertrophy while D2-mdx mice display pronounced atrophy. C_LIO_LIEarlier cardiac dysfunction in D2-mdx: D2-mdx mice develop reduced ejection fraction and stroke volume from 28 weeks, while BL10-mdx only at 52 weeks. C_LIO_LIGenetic background-dependent variations: Intrinsic deficits in wildtype D2 mice demonstrate that genetic background influences outcome measures independent of dystrophic pathology. C_LIO_LIComparable ex vivo muscle physiology: Despite divergent in vivo phenotypes, isolated muscle contractile parameters show similar impairment in both dystrophic models. C_LIO_LIMulti-site standardized validation: Cross-sectional study at two independent laboratories following harmonized TREAT-NMD Standard Operating Procedures. C_LI