Journal of Cellular and Molecular Medicine

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.

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.

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.

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.

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.

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

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.

Background: Hypoxia develops within growing atherosclerotic lesions, inducing nuclear translocation of hypoxia-inducible factor-1 (HIF1) and metabolic reprogramming. Its role in plaque macrophages and endothelial cells has been studied, but the hypoxic plaque interior is dominated by smooth muscle cell (SMC)-derived cells, for which the role of hypoxia signaling remains unclear. Here, we investigated how loss of Hif1a in SMC lineage cells impacts plaque progression and cell phenotype in murine atherosclerosis. Methods: Atherosclerosis was induced in mice with inducible SMC-specific deletion of Hif1a (Hif1aSMC-KO) and lineage tracing of SMC-derived plaque cells. Plaque size, necrotic core size, calcification, and SMC-derived cell phenotypes were quantified in aortic root sections and gene expression changes mapped by single-cell RNA sequencing. In parallel, a cultured SMC line with or without siRNA-mediated Hif1a knockdown was exposed to hypoxia for assessments of mitochondrial function and reactive oxygen species production. Results: Hif1aSMC-KO mice developed larger plaques, with expanded necrotic cores and increased calcification, compared with littermate controls. SMC-derived plaque cells were more abundant with a higher fraction of Col2a1+ chondromyocytes, and showed elevated markers of proliferation and apoptosis, whereas macrophage and endothelial cell numbers were unaffected. Single-cell RNA sequencing analysis revealed strong dysregulation of mitochondrial genes, including electron transport chain transcripts, along with upregulation of protein folding, proteasome, and oxidative stress response pathways. In cultured SMCs subjected to hypoxia, Hif1a silencing increased cell counts, aggravated mitochondrial proton leak, and led to the accumulation of depolarized, reactive oxygen species-generating mitochondria. Further analysis of SMC-derived cells in plaques from Hif1aSMC-KO mice confirmed increased oxidative stress by 8OHdG staining. Conclusions: HIF1 maintains mitochondrial function and restrains oxidative stress in SMC-derived plaque cells in murine atherosclerosis. Its chronic loss destabilizes redox homeostasis and promotes maladaptive SMC responses, leading to SMC-driven plaque expansion, necrosis, and calcification.

IntroductionIntestinal barrier failure is a key characteristic of both kinds of inflammatory bowel disease (IBD), ulcerative colitis (UC) and Crohns disease (CD). The intestinal barrier, or gut mucosal barrier, is composed of mucus and a tightly interconnected single layer of intestinal epithelial cells (IEC) that line the gut. Together, these components work to contain the gut microbiota within the gut lumen and when they fail, microbes, microbial products, or microbial components cause damage, inflammation, and immune activation in host tissue. We previously reported that High mobility group box 1 (HMGB1) in colonic mucus aggregates bacteria, limits bacterial invasion through mucus, and prevents bacteria from adhering to host tissue. Epithelial surface-associated HMGB1 is decreased in active UC lesions and low levels of HMGB1 are associated with high levels of tissue-adherent bacteria expressing adhesins carrying the molecular target of HMGB1 (ToH1). The study reported here was designed to determine whether HMGB1 defense is also compromised in active lesions from CD patients. MethodsImmunofluorescence microscopy was used to visualize mucus and HMGB1 in tissue from colonic resections performed in CD and non-IBD control patients. ResultsActive CD lesions had areas where the IEC were absent or pulling away from underlying tissue along with areas of increased mucus thickness and goblet cells full of mucus highly positive for alpha-linked-fucose residues. The surface associated HMGB1 was also decreased in active CD lesions. ConclusionTissue from CD patients exhibited cellular and acellular intestinal barrier defects in comparison to control patients. We observed the previously reported loss of IEC barrier integrity and abnormalities in the amount and distribution of mucus in CD lesions. We also report for the first time that CD is associated with decreased HMGB1 defense at the colon surface.

The excitability of afferents involved in nociceptive signaling reflects the interaction of several co-expressed membrane receptors. Current studies have shown that Toll-like receptor-4 (TLR-4) signaling can exacerbate excitation evoked by transient receptor potential vanilloid type 1 (TRPV1) activity, and this interaction plays a key role in driving and sustaining facilitated pain states. The mechanism by which this potentiated TRPV1 activity secondary to TLR-4 agonism occurs in sensory neurons remains unknown, although intracellular kinase activity is a strong candidate. To address this hypothesized linkage, neuronal cell cultures prepared from dorsal root ganglia (DRG) of male wildtype (WT) and Tlr4-/- mice were used to evaluate calcium transients of neurons after capsaicin administration in culture, pre-treated for 30 minutes with the TLR-4 agonist, lipopolysaccharide (LPS). TRPV1 protein expression at the neuron surface in cultured DRG cells with or without LPS treatment was quantified by flow cytometry assay. The roles of protein kinase A (PKA) and C were assessed using selective inhibitors (KT5720 for PKA and Chelerythrine chloride for PKC) applied to WT-DRG neurons or administered in vivo by intraplantar or intrathecal injection, prior to LPS and capsaicin administration. Behavioral effects of in vivo TRPV1 activation were assessed through paw flinch responses evoked by intraplantar capsaicin injection and by hind paw tactile thresholds measured by von Frey filaments. LPS incubation in cultured DRG neurons enhances the intensity of calcium influx following TRPV1 activation in WT but not Tlr4-/ cells. The augmented calcium influx evoked by capsaicin was prevented by the inhibition of PKA but not PKC. Similarly, mice treated with LPS in the hind paw displayed greater nociceptive responding after capsaicin and increased tactile allodynia. The facilitated component was prevented by the local pre-treatment with the PKA inhibitor. Correspondingly, lumbar spinal blockade of PKA resulted in temporary reversal of hyperalgesia induced by intrathecal LPS injection in mice. Together, these results demonstrate the relevance of TLR-4 in modulating the excitability of nociceptor signaling by regulating TRPV1, thereby influencing pain transmission through PKA signaling.

Background: Endothelial progenitor cells (EPCs) contribute to endothelial repair and neovascularization, and EPC dysfunction is closely associated with oxidative stress-related vascular injury. Forkhead box O3a (FoxO3a) regulates cellular stress responses, whereas miR-34a has been implicated in endothelial dysfunction, senescence, and apoptosis. However, the relationship between FoxO3a and miR-34a-3p in oxidatively injured EPCs remains incompletely defined. Objective: This study investigated the role of FoxO3a in H2O2-induced EPC dysfunction and examined whether miR-34a-3p directly interacts with the FoxO3a 3' untranslated region (3'UTR). Methods: Human umbilical cord blood-derived EPCs were identified by DiI-ac-LDL uptake, FITC-UEA-1 binding, and the expression of EPC-related markers. Oxidative stress was induced by H2O2. Cell viability, apoptosis, and angiogenic capacity were evaluated using CCK-8 assay, Annexin V/7-AAD flow cytometry, and Matrigel tube formation assay, respectively. FoxO3a expression was modulated using adenoviral overexpression or knockdown vectors, and miR-34a was modulated using mimics or antagomir. FoxO3a and miR-34a expression levels were detected by Western blot and qPCR. A dual-luciferase reporter assay was used to verify the interaction between hsa-miR-34a-3p and the FoxO3a 3'UTR. Results: H2O2 reduced EPC viability, increased apoptosis, and impaired tube formation in a concentration-dependent manner. H2O2 increased FoxO3a protein abundance and miR-34a expression, whereas FoxO3a mRNA did not change markedly. FoxO3a overexpression aggravated, whereas FoxO3a knockdown partially alleviated, H2O2-induced EPC dysfunction. Similarly, miR-34a mimics further suppressed EPC viability and tube formation, while miR-34a antagomir exerted a protective effect. Dual-luciferase reporter analysis showed that hsa-miR-34a-3p significantly reduced the activity of the wild-type FoxO3a 3'UTR reporter, while mutation of the predicted binding site abolished this suppression. Conclusion: FoxO3a and miR-34a participate in oxidative stress-induced EPC dysfunction. The dual-luciferase data demonstrate that hsa-miR-34a-3p directly targets the FoxO3a 3'UTR, suggesting the presence of miR-34a-3p-mediated post-transcriptional feedback within the FoxO3a-related stress-response network in EPCs.

ObjectiveTo investigate and clarify the role of Ginsenoside Ro (GRo) in diabetic cardiomyopathy (DiaCM) and to elucidate the molecular mechanism by which GRo ameliorates DiaCM. Methods[circled1] The construct of type 2 diabetic mouse model. The bought C57BL/6 male mice were housed in a specific pathogen-free (SPF) animal facility and randomly divided into control, STZ (model), STZ + GRo, and control+GRo groups. The STZ (model) and STZ + GRo groups were fed a high-fat and high-glucose diet combined with intraperitoneal injection of streptozotocin (STZ). The control and control + GRo groups were fed a normal diet, while the control + GRo and STZ + GRo groups were treated with GRo via oral gavage. Then, all groups were evaluated for cardiac function and structure by small animal echocardiography and histological staining including hematoxylin and eosin (HE) and Massons trichrome staining to screen and confirm diabetic cardiomyopathy in mice. Finally, immunofluorescence staining of cilia in mouse heart tissue was performed to determine whether GRo inhibits abnormal ciliary growth. [circled2] The construct of cell models. First, the CCK-8 (Cell Counting Kit-8) assay was used to separately evaluate the cytotoxicity of GRo and the combination of TGF-{beta}1 and PA in myocardial fibroblasts and cardiomyocytes. Subsequently, mouse myocardial fibroblast lines (MCFs) were treated with transforming growth factor-beta 1 (TGF-{beta}1), and H9c2 cardiomyocytes were treated with palmitic acid (PA). Both cell types then received the GRo treatment. [circled3] Molecular and cellular testing. Firstly, we measured serum levels of cardiac injury markers (CK-MB, MYO, and TNNI3), glutathione (GSH), and malondialdehyde (MDA). Secondly, we examined the expression of myocardial fibrosis-related genes (Col1a1, etc.), myocardial hypertrophy markers (Nppa, etc.), cilia-specific genes (Pkd1, etc.), and oxidative stress-related genes (Nrf2, etc.) in both animal and cell samples by Western blotting and RT-qPCR. Finally, we used immunofluorescence staining of myocardial fibroblasts to detect cilia length and phalloidin staining of cardiomyocytes to measure their cross-sectional area. [circled4] The correlation mechanism. Firstly, the cilia-specific inhibitory drug HIP-4 was used to disrupt cilia homeostasis by inhibiting cilia growth. Secondly, small activating RNA (saRNA) was used to upregulate the Pkd1 gene to verify whether GRo exerts its anti-fibrotic effects through the inhibition of PC1. Results[circled1] Animal level. A diabetic cardiomyopathy mouse model was successfully established by combining STZ injection with a high-fat and high-glucose diet, and treatment with GRo significantly ameliorated the associated symptoms. [circled2] Cellular level. We successfully established a myocardial fibrosis model by treating myocardial fibroblasts with TGF-{beta}1, and a myocardial hypertrophy model by treating cardiomyocytes with PA. Immunofluorescence staining demonstrated that GRo significantly decreased cilia length in the fibrosis model, while phalloidin staining showed that GRo significantly attenuated the increase in cardiomyocyte cross-sectional area. [circled3] Molecular level. Compared with the model group, GRo treatment significantly reduced serum levels of cardiac injury markers (CK-MB, MYO and TNNI3), glutathione (GSH) and malondialdehyde (MDA). Western blotting and RT-qPCR analyses of both animal and cell samples revealed that GRo markedly alleviated indicators of myocardial fibrosis and hypertrophy, while also suppressing cilia-specific genes and oxidative stress-related genes. Overall, GRo significantly ameliorated the markers associated with myocardial fibrosis and hypertrophy, and inhibited cilia-specific protein expression as well as oxidative stress parameters. [circled4] The correlation mechanism. The cilia-specific drug hedgehog pathway inhibitor 4 (HPI-4) was used to revealed that cilia homeostasis is closely linked to myocardial fibrosis and shortened cilia inhibit the fibrosis progression. Furthermore, upregulation of the Pkd1 gene by small activating RNA demonstrated that PC1 overexpression abrogates the therapeutic effect of GRo. Finally, GRo can alleviate DiaCM.

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.

Hypoxia-inducible factor prolyl hydroxylase (HIF-PH) inhibitors are widely used for the treatment of renal anemia; however, their effects on intraocular vascular endothelial growth factor (VEGF) expression remain unclear. In this study, we examined the effects of all five HIF-PH inhibitors --roxadustat, daprodustat, vadadustat, enarodustat, and molidustat--on Vegfa expression in the retina in mice. C57BL/6J mice were orally administered each inhibitor. Six hours after administration, the kidney, retina, and liver were collected, and transcription levels were quantified by real-time quantitative reverse transcription PCR. Renal Epo transcription was significantly increased by molidustat (P < 0.01), roxadustat (P < 0.01), and enarodustat (P < 0.05). Retinal Vegfa transcription was significantly increased by four inhibitors (P < 0.01), with molidustat showing no significant effect. In the liver, Vegfa transcription was increased by daprodustat (P < 0.05) and vadadustat (P < 0.01). Furthermore, renal Epo and retinal Vegfa transcription levels showed a moderate positive correlation with a marginal trend toward statistical significance (r = 0.37, P = 0.08). These findings indicate that HIF-PH inhibitors differentially regulate hypoxia-responsive genes across tissues and suggest that retinal VEGF upregulation should be considered when evaluating the safety of these agents.

Nephrons are the functional units of the kidney; within each nephron, the glomerulus is the initial site of selective filtration that allows removal of waste products while preserving proteins in the bloodstream. Each glomerulus consists of a network of capillaries surrounded by specialized epithelial cells, podocytes, which mediate selective filtration. Abnormalities in glomerular structure impair renal function, resulting in proteinuria and kidney disease. Although several microscopy-based approaches exist to characterize glomerular architecture and structural abnormalities, quantitative analysis is often limited by labor-intensive image segmentation. In this study we present a semi-automated approach for segmentation and analysis of glomerular architecture from three-dimensional confocal microscopy data. Using mTmG transgenic mice that express membrane-associated EGFP in podocytes and membrane-associated tdTomato across all other cell types, we reconstruct podocyte processes and glomerular capillaries from volumetric renal images. This semi-automated approach reduces manual segmentation effort and supports more efficient, standardized analysis of glomerular architecture in three-dimensional confocal microscopy datasets.

BackgroundBiliary atresia (BA) is a severe neonatal liver disease characterized by progressive fibrosis and bile duct obliteration. ObjectiveAlthough immune dysregulation is implicated in the pathogenesis of BA, the specific mechanisms driving bile duct injury remain incompletely understood. This study aimed to characterize tertiary lymphoid structures (TLSs) within extrahepatic biliary remnants (EBRs), identify their cellular mediators, and evaluate the therapeutic potential of targeting IL-21 receptor signaling. DesignWe performed integrated bulk RNA sequencing, single-cell RNA sequencing, spatial transcriptomics, multiplex immunohistochemistry, and flow cytometry on clinical samples from BA patients and non-BA cholestatic controls. TLS maturation was assessed by CD23 immunohistochemistry in EBRs from 148 BA patients and correlated with clinical parameters. Anti-IL-21R antibody treatment was evaluated in a rhesus rotavirus-induced BA mouse model, with treatment initiated on day 4 post-infection. ResultsTLSs were identified in BA EBRs with significantly higher prevalence than in matched liver tissues. Mature TLSs containing CD23 germinal centers were associated with elevated serum matrix metalloproteinase-7, more advanced hepatic fibrosis, and localized autoantibody deposition on injured bile ducts. Single-cell profiling revealed expanded CD4+ T peripheral helper (Tph) cells expressing IL-21 and CXCL13 within TLS-containing EBRs. Tph cells were enriched in peripheral blood of BA patients compared to non-BA cholestatic controls (P = 0.0025), and serum IL-21 was significantly elevated (P < 0.0001). Post-infection IL-21R blockade in the mouse model reduced jaundice incidence, improved weight gain, prevented extrahepatic biliary obstruction, and significantly improved long-term survival. ConclusionTLSs in BA extrahepatic biliary remnants harbor expanded Tph cells associated with IL-21-mediated B cell activation and bile duct injury. IL-21R blockade ameliorated disease in a murine BA model, identifying the IL-21/IL-21R axis as a potential therapeutic target warranting further investigation. Key MessagesO_ST_ABSWhat is already known on this topicC_ST_ABSImmune dysregulation contributes to biliary atresia (BA), with documented lymphocyte infiltration and defective B cell tolerance. However, the cellular mechanisms linking local immune activation to bile duct injury are unclear, and the roles of organized lymphoid structures and specific CD4 T cell subsets in orchestrating local humoral responses have not been characterized. What this study addsThis study demonstrates that mature tertiary lymphoid structures in extrahepatic biliary remnants are associated with disease severity markers and localized bile duct injury in BA. We identify T peripheral helper cells as an expanded IL-21-producing CD4 T cell population within these structures, and show that post-infection IL-21 receptor blockade prevents biliary obstruction and improves survival in a murine BA model. How this study might affect research, practice or policyThese findings identify the IL-21/IL-21R signaling axis as a candidate therapeutic target in BA warranting further preclinical and translational investigation. TLS maturation status in biliary remnants and serum autoantibody levels may serve as potential biomarkers of disease severity, meriting prospective evaluation in clinical cohorts.

Purpose: Lymphatic system plays a critical role in fluid regulation and immune response. The conjunctiva of the ocular surface is endowed with a rich lymphatic network, and it provides an ideal site to study lymphatic structure and function. The purpose of this study is to investigate potential morphological and functional changes of conjunctival lymphatics with aging, a time-dependent biological deterioration process. Methods: Young and aged fluorescently labeled transgenic mice of Prox-1, the master control gene for lymphatic determination, were used in the study. For morphological assessment, conjunctival lymphatic vessels were examined in vivo by our advanced live imaging system. For functional analysis, lymphatic drainage efficiencies were measured by fluorescently labeled tracer injection. Results: Compared to young mice, both vascular branching points and intraluminal valves were significantly reduced in conjunctival lymphatic vessels of aged mice. Moreover, lymphatic functional deterioration and drainage deficiencies, such as fluid leakage and reflux, were also detected in the aged condition. Conclusions: Conjunctival lymphatic system undergoes morphological as well as functional changes with aging. Further investigation into this phenomenon may provide novel insights into lymphatic and age-related diseases inside and outside the eye.

CCCTC-binding factor (CTCF) is frequently mutated in endometrial cancer, resulting in genetic haploinsufficiency that contributes to tumour progression. We previously showed that depletion of CTCF disrupted cell polarity in KLE endometrial cancer spheroids; however, the implications for gene dysregulation and endometrial cancer pathophysiology remains poorly understood. ZNF185, an actin-associated and LIM domain-containing protein involved in cytoskeletal remodelling, was identified as a dysregulated target following CTCF haploinsufficiency. In this study, shRNA-mediated knockdown of CTCF was used to model haploinsufficiency in endometrial cancer cells, leading to the identification of a previously unrecognised isoform of ZNF185, named ZNF185B. Unlike the full-length protein, ZNF185B lacked co-localisation with F-actin and exhibited a diffuse cytoplasmic distribution, and ZNF185B was significantly upregulated in CTCF-depleted endometrial cancer cells and in an auxin-inducible degron model in a dose-dependent manner. Functional studies demonstrated that depletion of ZNF185 expression reduced endometrial cancer cell proliferation and clonogenic potential. Together, these findings identify ZNF185B as a novel isoform negatively regulated by CTCF protein dosage and establish ZNF185 as a requirement for endometrial cancer cell proliferation. Our results suggest that dysregulated ZNF185 expression is a crucial downstream consequence of CTCF haploinsufficiency and may contribute to tumour progression in endometrial cancer.

Key PointsO_LIPerfused human kidney MPS revealed CsA-associated sublethal tubular stress that was not detected by conventional 2D viability assays or by KIM-1 release in 3D MPS. C_LIO_LIAt matched exposure, VCS preserved mitochondria and activated ER chaperones and iron detoxification, with no p21 arrest compared to CsA. C_LIO_LIMechanistic separation supports VCSs nephroprotection potential and early mechanism-based biomarkers to guide CNI choice. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/737071v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@e5e01dorg.highwire.dtl.DTLVardef@1dc9167org.highwire.dtl.DTLVardef@1ce22f8org.highwire.dtl.DTLVardef@5a053a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG BackgroundCalcineurin inhibitors (CNIs) are indispensable for transplantation immunosuppression, yet cyclosporine A (CsA) produces renal toxicity. Voclosporin (VCS), a CsA analog, is proposed to be less nephrotoxic, but mechanisms remain unclear. MethodsPrimary human proximal tubule epithelial cells (PTECs) were exposed to CsA or VCS in 2D monolayers and perfused 3D kidney microphysiological system (MPS). Viability was assessed in 2D cultures by MTS, mitochondrial membrane potential ({Delta}{Psi}m) by TMRM flow cytometry, and soluble injury and inflammatory biomarkers in MPS effluents by ELISA and MSD multiplex assays. RNA sequencing of 3D-cultured PTECs was used to identify differentially expressed genes and pathways. ResultsIn 2D PTECs, neither drug reduced viability. In 3D MPS effluents, KIM-1 did not distinguish CsA from VCS, whereas the MSD biomarker panel showed larger aggregate deviation with CsA. Confocal tomography showed CsA-associated mitochondrial fragmentation, whereas VCS preserved reticular mitochondrial architecture. TMRM flow cytometry showed a treatment-dependent difference in TMRM-positive cells, with VCS yielding the highest TMRM-positive fraction and exceeding CsA, supporting preservation of {Delta}{Psi}m relative to CsA. RNA-seq identified 1188 CsA-specific and 185 VCS-specific differentially expressed genes, with 304 shared. Pathway analysis indicated CsA enrichment of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress, p21-associated G2/M checkpoint arrest, and transcriptional signatures consistent with ferroptosis priming, while VCS mainly induced ER chaperone and ER-associated degradation gene programs without activating canonical UPR sensors and showed limited cell-cycle suppression. ConclusionsA physiologically relevant 3D kidney MPS revealed sublethal tubular stress from CsA that is masked in 2D culture, including mitochondrial depolarization, proteostatic stress, and ferroptosis priming. At matched exposure, VCS preserved mitochondrial function and proteostasis while eliciting a narrower, adaptive ER quality control response. These data support VCS as a nephron-sparing immunosuppressant and 3D MPS as a mechanism-based platform for evaluating renal safety of drugs and nominating early sub-lethal tubular injury biomarkers.

The platelet P2Y1 receptor (P2Y1R) is necessary for inflammation, signalling via Rho-GTPase pathways to elicit functions that are distinct from aggregation (PLC-dependent canonical signalling pathway). Whether these distinct platelet inflammatory functions can be selectively suppressed to preserve hemostasis through the rational design of P2Y1R antagonists has not been explored. In silico molecular docking analysis examined biased nucleotide interactions within the P2Y1R binding pocket. The identified possible key amino acid residues guided rational design to synthesize compounds for pathway selective inhibition, evolving from nucleotide to non-nucleotide structures. The nucleotide analogue KMR-82-13 was predicted to engage distinct regions of the binding pocket and selectively inhibited platelet chemotaxis while preserving aggregation. These findings informed the design of a non-nucleotide compound KSN-159-27, aiming to retain key KMR-82-13-like interactions while improving drug-like properties. Docking and molecular dynamics simulation supported a stable but dynamic binding mode for KSN-159-27 within the P2Y1R pocket, consistent with pathway-selective inhibition. KSN-159-27 displayed characteristics of a pathway selective inverse agonist at P2Y1R towards G12/13-mediated pathways, but not those associated by Gq activation in P2Y1R-transfected HEK293T cells. KSN-159-27 showed functionally selective inhibition for platelet P2Y1R-mediated functions. In vivo, KSN-159-27 suppressed inflammatory cell recruitment, whilst preserving bleeding time and ADP-induced thromboembolic responses, in contrast to the neutral P2Y1R antagonist MRS2500. This first demonstration for the rational design of a pathway selective inverse agonist at platelet P2Y1Rs has significant implications for novel therapeutic strategies developed to safely target platelet activation during inflammation, in contrast to current anti-platelet drugs used in the prevention of thrombosis. Key PointsO_LIBiased inverse platelet P2Y1R agonists selectively supress inflammation whilst preserving hemostasis and the ability of platelets to aggregate. C_LIO_LIBiased inverse agonism selectively inhibited P2Y1R G12/13 (Rho-GTPAse functions) but not Gq activities (PLC functions). C_LI