Molecular Medicine

Introduction: In both humans and experimental animals, obesity is associated with hypothalamic inflammation and neuronal metabolic dysfunction, which contribute to disrupting body energy homeostasis. Although ciliary neurotrophic factor (CNTF) is a neuroprotective cytokine with recognized anti-obesity effects in peripheral metabolic tissues, its role in obesity-associated hypothalamic dysfunction is still poorly characterized. We investigated whether CNTF exerts any direct effects on mHypoE-N41 (N41) hypothalamic neurons and whether it modulates the effects induced by palmitate (PA), an established model of obesity-associated neuronal metabolic dysfunction. Methods: N41 cells were administered CNTF alone or combined with PA to assess whether CNTF affects PA-induced lipotoxic stress. Gene and protein expression was assessed by qRT-PCR, western blotting, and immunofluorescence analysis. Results: In N41 cells, CNTF activated the STAT3, ERK and AMPK signaling pathways. It also upregulated genes involved in mitochondrial metabolism and fatty acid oxidation, including Ucp2, Cpt1 and Pgc1, as well as the proliferation-associated markers Ki-67 and PCNA. PA treatment induced inflammatory and endoplasmic reticulum stress responses and impaired insulin signaling. In PA-treated N41 cells, CNTF significantly attenuated the expression of pro-inflammatory markers Tnf, Mcp1 and Nfkb, restored insulin-induced AKT phosphorylation, and partially reversed PA-induced changes in the expression of metabolic genes Ucp2 and Cpt1. Conclusions: Collectively, these findings indicate that CNTF exerts direct protective metabolic effects on hypothalamic N41 neurons subjected to lipotoxic stress and supports a role for it in the preservation of neuronal metabolic function under obesogenic conditions.

A population-specific missense variant, rs373863828 (G > A, p.Arg457Gln), has been identified in Pacific Islanders and is associated with increased body mass index and lower odds of type 2 diabetes. However, the mechanism underlying these associations is unknown. The variant is located in exon 5 of CREBRF, a gene which is rapidly upregulated in response to nutrient deprivation. This study aimed to assess the effect of rs373863828 on gene expression in adipose tissue from Samoan adults from the Soifua Manuia (''Good Health'') study. Needle subcutaneous adipose (upper buttock) biopsies were obtained in Apia, Samoa, from fasting Samoans without diabetes. RNA isolated from adipose tissue (n=91, 65.9% female) was subjected to bulk paired-end mRNA-sequencing (2x150 base pair). Differential gene expression among the three rs373863828 genotypes (GG, AG, AA) was assessed using a likelihood-ratio test implemented in DESeq2, and overrepresentation analysis for significant differentially expressed genes (DEGs) (padj < 0.1) was performed with clusterProfiler and oRG.DB. Bulk expression was deconvoluted to determine the cell type composition of the adipose tissue. Twenty-two DEGs were associated with rs373863828 genotype including CBX7 (padj=0.026 ) and SELENOM (padj= 0.004), both of which have roles in adipogenesis and metabolism. A single expression profile comprising 13 of the 22 DEGs corresponding to CREBRF genotype was identified, and overrepresentation analysis supported this finding, with terms such as ''keratinocyte differentiation'' and ''skin development'' enriched among significant DEGs. However, PERMANOVA of adipose cell type proportions showed no difference (p > 0.05) among the three genotypes. rs373863828 genotype was associated with expression of 22 genes in whole subcutaneous adipose tissue of this sample of fasting Samoan adults without diabetes. Future work should assess cell- and tissue-specific rs373863828 genotype effects using a combination of single-cell approaches and human cell models with perturbations associated with nutritional stress.

Retinal organoids (ROs) model human retinogenesis but remain avascular and lack immune components, limiting their utility for studying vascular retinopathies. We generated vascular organoids (VOrs) from human pluripotent stem cells and evaluated their integration into developing ROs. Dissociated VOr cells (day 12 or day 30) were seeded onto 2D retinal neuroepithelium before 3D transition, followed by long-term culture to day 210. VOr derived cells migrated into ROs and transiently persisted. V30 ROs preserved retinal lamination, major neuronal subtypes and light evoked retinal ganglion cell responses comparable to non vascularised controls, whereas media-only ROs cultured in proangiogenic conditions showed structural disorganisation and reduced neuronal survival. Single-cell RNA sequencing revealed an endogenous, inflammatory endothelial-like population with partial endothelial-to-mesenchymal features and a perivascular fibroblast-like cluster in pro-angiogenic media only RO controls. The endothelial-like cell cluster was significantly enriched for genes upregulated in human retinal endothelial cells exposed to high glucose, indicating a diabetic retinopathy like transcriptional state. Microglia like and border associated macrophage populations were present across all conditions, with developmental shifts in morphology and marker expression. Our findings define conditions under which vascular cells can be incorporated into human ROs without disrupting retinal architecture, while uncovering an intrinsic capacity for inflammatory endothelial activation that partially recapitulates diabetic retinopathy. This work advances the engineering of vascularised, immunocompetent retinal models and highlights endogenous vascular immune programmes that must be considered when modelling retinal disease in vitro.

Peripheral neurons exhibit robust regeneration after nerve injury in adulthood; a process linked to the activation of regeneration-associated genes (RAGs). However, the mechanisms underlying nerve regeneration during early postnatal development remain poorly understood. In a previous work, we observed that motoneurons axotomized at early postnatal stages regenerate without overexpressing RAGs, contrasting with the expected RAG activation observed in postnatal day 30 (P30) mice. To determine whether this phenomenon extends to sensory neurones, we investigated two paradigmatic dorsal root ganglia populations: proprioceptive (PV) and nociceptive (TrpV1) neurones, which exhibit distinct regenerative capacities in adult mice. Following sciatic nerve crush injury at P4, P10, and P30, both neuronal subtypes demonstrated robust axonal regeneration across all postnatal stages, with nociceptors exhibiting significantly superior regeneration compared to proprioceptors. Remarkably, no substantial neuronal death was observed at any developmental timepoint. However, RNA sequencing revealed that P4 and P10 sensory neurones failed to upregulate RAGs following injury, whereas P30 neurones exhibited the expected pro-regenerative transcriptional programme. Pathway enrichment analysis confirmed that classical regeneration-associated signalling pathways were activated exclusively in young adults, whilst developmental and inflammatory pathways predominated in early postnatal stages. Using the conditioning lesion paradigm in primary dorsal root ganglia cultures, we demonstrated that only P30 neurones exhibited enhanced neurite outgrowth following conditioning lesion, whilst P4 and P10 neurones showed no conditioning effect or reduced outgrowth, respectively. These findings indicate that early postnatal sensory neurones possess intrinsic growth capacity independent of RAG activation, suggesting fundamentally distinct regenerative mechanisms across developmental stages.

Abstract Background. Tuberous Sclerosis Complex (TSC) is caused by inactivating mutations in either the TSC1 or TSC2 genes, leading to activation of the mammalian target of rapamycin complex 1 (mTORC1) and unhindered cell growth and proliferation. The epithelium of TSC renal cysts in both mice and humans is composed of proliferating A-intercalated (A-IC) cells. The exact molecular mechanism of kidney cystogenesis in TSC remains speculative. Hypothesis.Superfluous cell proliferation driven by mTORC1 activation increases metabolic demand, causing oxidative stress and excess reactive oxygen species. If unchecked, this overwhelms antioxidant defenses and causes cell death. In TSC kidney cystogenesis, Nuclear Factor Erythroid 2-Related Factor 2 (NFE2L2), also known as NRF2, serves as the "master regulator" of antioxidant and anti-inflammatory responses, enabling cells to survive and proliferate by clearing the toxic environment and supplying nutrients and fuel. Results. RNA-seq and proteomics, along with western blot analysis, showed robust downregulation of Fumarate Hydrase 1 (FH1) and upregulation of NRF2, STAT3, and HIF1 in kidneys from TSC mice with moderate or heavy cyst burden. Confocal microscopy and immunohistochemical staining on kidney sections, and/or western blot studies on nuclear and cytoplasmic fractions, showed nuclear localization of NRF2, STAT3, and HIF1. In cyst-lining cells in TSC mouse models, FH1 downregulation was associated with inactivating succination of KEAP1 in immunoprecipitation experiments, promoting NRF2 nuclear localization in A-IC cells lining the cysts. NRF2 nuclear localization was associated with ectopic induction of the glutamine transporter SLC38A3 (SNAT3) on the basolateral membrane and activation of the NH3/NH4+ transporters RHCG and RHBG in A-IC cells lining the cysts. Twenty-four h urine NH3/NH4+ excretion rates increased significantly in Tsc1 KO vs. WT mice. The activation of NRF2, STAT3, and HIF1 can drive metabolic reprogramming and activate survival genes in proliferating cells. Together with SLC38A3 induction and upregulation of other glutamine and NH3/NH4+ transporters, these factors activate glutaminolysis and aerobic glycolysis, supplying nutrients to proliferating cystic epithelial cells and supporting cyst expansion in TSC. Consistent with this central role for glutaminolysis in kidney cystic epithelium and TSC cystogenesis, we find a significant reduction in kidney cyst burden in Tsc1 KO mice on a glutamine-free diet. Conclusions. NRF2 plays a critical role in antioxidant defense. Along with STAT3 and HIF1, NRF2 is a key player in metabolic reprogramming through glutaminolysis, which supplies nutrients to proliferating cystic epithelial cells and supports cyst expansion in TSC. These findings suggest that inhibiting or inactivating NRF2, alone or in combination with HIF1 or STAT3, may represent a potential treatment strategy for kidney lesions in TSC.

Visceral white adipose tissue (vAT) is more susceptible than subcutaneous white adipose tissue (scAT) to obesity-associated metaflammation, but the metabolic pathways accompanying this depot specific transition remain incompletely defined. We profiled vAT and scAT from mice fed a control diet or a 60% kcal high-fat diet (HFD) for 1 or 8 weeks. Untargeted metabolomics was integrated with targeted endocannabinoid quantification, gene expression and promoter associated chromatin analyses, adipocyte and stromal vascular fraction (SVF) measurements, cross-omics correlation analysis, and cell-culture perturbations. After 1 week, HFD induced relatively few metabolic changes, although vAT showed early alterations associated with sphingomyelin metabolism. By week 8, both depots exhibited extensive but distinct metabolic remodeling. The KEGG retrograde endocannabinoid-signaling pathway was the highest-ranked enriched pathway in vAT, with arachidonoylglycerol (2AG) among the most strongly altered metabolites. Targeted analysis confirmed selective 2AG accumulation in vAT, accompanied by coordinated changes in genes and promoter-associated chromatin marks related to endocannabinoid synthesis, degradation and receptor expression. Cellular fractionation localized excess 2AG predominantly to vAT adipocytes, whereas Cnr2 was enriched in the SVF and induced by HFD specifically in vAT. Cross omics integration further revealed a denser and predominantly inverse transcript metabolite association network in vAT than in scAT. Using macrophage adipocyte co-culture and secretome profiling, we demonstrated that cannabinoid stimulation shifts activated macrophage outputs away from pro-inflammatory cytokines (Il1b, Ccl2) toward extracellular matrix (ECM) proteoglycan remodeling and intercellular signaling.

Abstract Rationale: Vagal sensory neurons are molecularly and functionally diverse, and they play critical roles in visceral sensation and homeostasis. The vagal system is anatomically divided into jugular and nodose ganglia, which exhibit distinct peripheral innervation patterns and physiological functions. Although recent transcriptomic studies have characterized nodose neuronal subtypes in detail, the molecular profiles of jugular sensory neurons remain poorly defined. Objectives: To define the molecular profiles of genetically identified jugular sensory neurons and determine the transcriptional responses of jugular and nodose airway afferents to allergic airway inflammation. Methods: We performed deep transcriptomic profiling of two genetically defined jugular nociceptor populations, MrgprC11 and MrgprD neurons, and compared them with airway-innervating P2ry1 nodose neurons and Phox2b nodose neurons. We further examined transcriptional changes of MrgprC11 and P2ry1 neurons following OVA-induced allergic airway inflammation. Measurements and Main Results: Transcriptomic analyses revealed distinct molecular signatures of jugular nociceptive neurons and identified subtype-specific genes across diverse functional categories. Following OVA challenge, MrgprC11 and P2ry1 neurons exhibited strikingly divergent transcriptional responses. More than 400 genes that were differentially expressed in both neuronal populations were regulated in opposite directions. GO enrichment analysis suggested that MrgprC11 jugular neurons adopted a metabolically restrained state with reduced translation and bioenergetic activities, whereas P2ry1 nodose neurons exhibited metabolically active state with enhanced translation and bioenergetic activities. Conclusions: These findings provide new insight into vagal sensory neuron diversity and reveal distinct transcriptional adaptations of jugular and nodose airway afferents during allergic inflammation.

Abstract Background Liposarcoma is a rare cancer, comprising four major histological subtypes: well-differentiated, dedifferentiated, myxoid and pleomorphic liposarcoma. Therapeutic options remain limited for advanced disease, and additional biomarkers that may support biological characterization, diagnosis, or risk stratification are needed. Methods Bulk RNA sequencing (RNA-seq) was performed on paired tumour and adjacent non-tumour tissues from an analysable subset of 16 patients (22 tumour specimens) within a clinicopathological cohort of 21 patients. Differential gene expression analysis, principal component analysis, gene set enrichment (GSEA) analysis and single-sample GSEA (ssGSEA) were used to define subtype-specific transcriptional programs and imprinted-gene dysregulation. PEG10 expression was validated by RT-qPCR and RNA fluorescence in situ hybridization (FISH), and its cellular localization was explored by combined FISH and immunofluorescence for P53, vimentin, Ki67, and CD34. PEG10 expression in liposarcoma was further investigated in an external single-cell RNA-sequencing dataset of dedifferentiated liposarcoma. An exploratory survival analysis was performed in the pan-sarcoma TCGA-SARC cohort. Results Transcriptomic profiling identified distinct subtype-specific gene expression patterns and broad dysregulation of imprinted genes in liposarcoma compared with adjacent non-tumour tissue. Among all the imprinted genes, PEG10 showed a marked tumour-to-normal expression difference and good discriminatory performance between tumour and normal tissues in the discovery cohort (AUC=0.867). In the TCGA sarcoma cohort high PEG10 expression was associated with shorter overall survival. Immuno-FISH and single-cell analyses revealed that PEG10 was predominantly detected in tumour and proliferating cell populations. Importantly, PEG10 expression was higher in high-grade tumours. In bulk RNA-seq data, PEG10 expression correlated with hypoxia, extracellular matrix remodelling and invasion ssGSEA signatures. In the external single-cell RNA-seq dataset PEG10high tumours showed a reduced immune cell infiltration. Conclusions This study provides an integrated transcriptomic characterisation of liposarcoma and identifies PEG10 as a candidate tumour-associated biomarker linked to aggressive disease features. The diagnostic and prognostic value of PEG10 requires validation in larger, independent, subtype-balanced liposarcoma cohorts, and its potential therapeutic relevance requires direct functional investigation.

Abstract Endothelial cell (EC) injury is central to the pathogenesis of various renal disorders, highlighting the critical importance of vascular repair for restoring organ health. However, due to limited access to human tissue during active regeneration, the exact reparative processes remain largely unknown. This study aimed to investigate regeneration mechanisms specifically within the glomerular microvasculature by utilizing a selective endothelial cell injury (ECI) model in mice.Lineage tracing identified the origin of regenerating ECs. To uncover the transcriptional dynamics driving these reparative changes, we combined single-cell RNA sequencing and Xenium spatial transcriptomics (ST), mapping distinct glomerular EC (gEC) subpopulations across different phases of injury and recovery. Apold1 emerged as a key regulatory candidate within this process. To functionally validate this gene's specific role during the regenerative phase, Apold1-deficient (Apold1-/-) mice underwent the ECI model. After showing that gECs regenerate primarily from local, pre-existing renal ECs, our transcriptomic analysis identified 10 distinct gEC subpopulations and revealed a complex temporal pattern of regeneration. This process begins with the expression of genes associated with inflammation, followed sequentially by endothelial cell (EC) migration, the activation of immediate early genes, vascular remodeling and cellular proliferation. Within this dynamic process, the EC-specific gene Apold1 was identified as a key regulatory candidate. Apold1-/- mice exhibited impaired endothelial regeneration, characterized by increased endothelial activation and greater immune cell infiltration seven days post-ECI. To our knowledge, this is the first study to elucidate the spatiotemporal mechanisms of glomerular endothelial cell regeneration. By identifying local gECs as the primary source of repair, and stablishing the change in their expression pattern during regeneration, as well as identifying Apold1 as an intrinsic regulator, we have laid the groundwork for future research on renal microvascular disorders.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by T-cell mediated destruction of pancreatic islet {beta}-cells with genetic, environmental, and molecular triggers involved in disease pathogenesis. Patients with T1D have elevated serum levels of microRNA146a (miR146a). Polymorphisms in the miR146a gene that result in reduced expression of miR146a are associated with protection from T1D. We studied physiological regulators of miR146a expression and found that both hyperglycemia and elevated O-GlcNAcylation increased miR146a expression in T cells. Peripheral blood mononuclear cells (PBMCs) from T1D patients showed increased miR146a and O-GlcNAc transferase (OGT) expression, suggesting increased O-GlcNAcylation may promote miR146a expression in T1D patients. To determine the genetic and developmental role of miR146a in T1D, we generated miR146a-knockout (KO) non-obese diabetic (NOD) mice and found that absence of miR146a significantly protected NOD mice from spontaneous autoimmune diabetes. While we found no impact of miR146a knockout on general hematopoietic parameters and immune cell populations, remarkably, immune cell infiltration into the pancreas was significantly attenuated. Protection from autoimmune diabetes in miR146a-KO NOD mice was associated with increased regulatory T (Treg) cells in the spleen and pancreatic lymph node. Mechanistically, absence of miR146a increased NF-{kappa}B c-Rel expression in Treg cells and enhanced c-Rel binding at the Forkhead box protein P3 (FOXP3) promoter, which positively regulated Treg cell development and suppressor function, offering protection from T1D in miR146a-KO NOD mice. Our findings reveal miR146a as a key regulator of Treg cell-mediated immune tolerance through controlling NF-{kappa}B c-Rel-dependent FOXP3 expression and suggest targeting miR146a as a potential strategy to restore peripheral tolerance in T1D.

Oral squamous cell carcinoma (OSCC), a highly aggressive subtype of head and neck cancer, remains a major clinical challenge with limited therapeutic options and poor survival outcomes. The Protein Inhibitor of Activated STAT1 (PIAS1), a SUMO E3 ligase involved in transcriptional regulation, DNA repair, and epithelial-mesenchymal transition (EMT), exhibits cancer type-specific roles, but its function in OSCC has not been well characterized. Here, we investigated the molecular and functional role of PIAS1 in OSCC progression and prognosis. All patient cohorts analysed here comprise HPV-negative disease. Analysis across multiple cohorts revealed that PIAS1 mRNA expression is significantly lower in tumor tissues than in normal oral cavity squamous epithelium, including in a cohort of 60 patient-matched tumor and normal pairs. Within the tumor compartment, protein abundance of PIAS1 correlated positively with patient survival. Functional assays using OSCC cell lines demonstrated that PIAS1 acts in a SUMO E3-dependent manner to suppress TGF{beta}-induced migration and invasion. Transcriptomic profiling of PIAS1-depleted cells revealed alterations in expression of genes with relevance in TGF{beta} signaling and pro-invasive cellular responses. Further analyses showed that 46 of these differentially altered hits intersected with The Cancer Genome Atlas (TCGA)-OSCC datasets, where four of these genes, including INHBA, associated with cell stemness-pathway. Spatial analysis of the mRNA abundance of INHBA within OSCC-derived surgical specimens showed increased abundance at the invasive front, and positively associated with aggressive tumor phenotypes, poor patients' outcomes, and resistance to immunotherapy. Single-cell RNA-seq analyses of INHBA revealed increased levels within tumor cells and cancer-associated fibroblasts (CAFs), suggesting potential tumor-intrinsic and extrinsic (tumor microenvironment) sources and hence actions. Collectively, our findings establish PIAS1 as a tumor suppressor in OSCC that restrains TGF{beta}-driven aggressive behaviour. Furthermore, we identify INHBA as a potential therapeutic target, among other genes characterized by spatially restricted, invasive tumor phenotypes.

Charcot Marie Tooth type 1A (CMT1A) is the most common inherited peripheral neuropathy, caused by duplication of the peripheral myelin protein 22 (PMP22) gene and characterized by dysmyelination and defects in myelin maturation. Hypercellularity is a prominent feature of the disease, although its origin, persistence, and underlying mechanisms remain controversial. Here, we performed a longitudinal analysis of sciatic nerves from wild-type (WT) and CMT1A rats between 2 and 12 months of age. We demonstrated that CMT1A nerves exhibit persistent cellular proliferation extending into adulthood. This phenotype is confirmed by bulk RNA-seq data showing sustained enrichment of proliferation-associated gene signatures. Cell type signature analysis identifies overrepresentation of immature, neural crest related, and epithelial mesenchymal transitional (EMT) transcriptional programs in CMT1A nerves. Among the most upregulated genes, RUNX2 showed strong and prolonged expression at both transcript and protein levels. Consistently with the key role of RUNX2 in the activation of the injury response, CMT1A nerves also showed a marked enrichment in SOX10+JUN+ cells, that only partially overlap with SOX10+RUNX2+ cells, indicating a maladaptive activation of the injury-like Schwann cell (SC) program. Interestingly, we detected robust upregulation of SMOC2, an extracellular matrix protein selectively expressed by endoneurial fibroblasts, pointing to activation of non-canonical cell types and altered extracellular matrix remodeling. Together, these data demonstrate that CMT1A peripheral nerves are characterized by chronic hypercellularity driven by persistent proliferation, EMT, and dysregulated injury transcriptional programs, collectively pointing at progressive and multicellular nerve remodeling as a relevant pathomechanism in the disease.

Background: This systematic review and meta-analysis evaluated the prognostic value of Hippo signaling pathway biomarkers in oral squamous cell carcinoma. Methods: Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, electronic database searches identified 17 eligible studies involving 1,194 patients. Associations between the immunohistochemical expression of Hippo pathway effectors, primarily YAP and TAZ, and clinicopathological characteristics were assessed. Meta-analyses using random-effects models estimated pooled hazard ratios for overall survival. Results: Predominantly nuclear expression of YAP and TAZ was consistently associated with adverse clinicopathological features, including advanced clinical stage, lymph node metastasis, and poorer histological differentiation. Meta-analysis demonstrated that YAP overexpression was associated with worse overall survival (hazard ratio, 1.918; 95% confidence interval, 1.036 to 3.549; p = 0.038). TAZ overexpression was similarly associated with poorer overall survival (hazard ratio, 2.845; 95% confidence interval, 1.682 to 4.811; p < 0.001). Although substantial between-study heterogeneity was observed, the direction of the prognostic association remained consistent across studies. Conclusions: Increased nuclear expression of YAP and TAZ is consistently associated with more aggressive disease and poorer overall survival in oral squamous cell carcinoma. These findings support the prognostic value of Hippo pathway biomarkers, although methodological heterogeneity warrants cautious interpretation.

Background Muscle wasting, systemic inflammation, and functional decline are highly prevalent and detrimental in patients with advanced-stage non small cell lung cancer (NSCLC). Methods: In this cross sectional study, we investigated NSCLC associated muscle remodeling by analyzing skeletal muscle biopsies from patients with NSCLC (n = 18) and matched controls (n = 18) using quantitative proteomics, histology, fluorescence-activated cell sorting, gene expression profiling, and high resolution respirometry. Findings: NSCLC muscle was characterized by type II muscle fiber atrophy, greater collagen deposition, and redistribution of lipids to the extracellular matrix (ECM), together with remodeling of the inflammatory, immune, ECM and mitochondrial proteome. Additionally, mitochondrial respiratory capacity and morphology were altered in patients with NSCLC, which was associated with increased oxidative stress and dysregulated calcium handling. Concomitantly, we detected STAT3 activation and immune cell alterations, which may negatively impact skeletal muscle health in patients with NSCLC. Finally, we identified a shift in fibro-adipogenic progenitors (FAPs), favoring the CD90 subtype. Mechanistically, conditioned media from patient-derived FAPs reduced myotube width in vitro, uncovering a novel mechanism by which altered paracrine signaling from the muscle resident stromal compartment drives atrophy in cancer cachexia. Interpretation: These findings provide human evidence that altered FAP composition, mitochondrial homeostasis, calcium handling, and immune cell landscape accompany muscle wasting in NSCLC, which may inform therapeutic strategies to preserve skeletal muscle health in patients with cancer.

Background: Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related mortality, with an increasing incidence and dismal 10% survival at 5 years despite best available care. PDAC is characterized by low tumour cellularity, recurrent driver mutation alterations, and substantial inter and intra-tumoural heterogeneity. The influence of individual genetic features such as sex and ancestry on tumour heterogeneity remains poorly understood. Methods: We assembled a cohort of PDAC across five datasets for a total of 7,527 tumours. Measures of genomic instability were assessed across sex and ancestry on a global level (SNV density and percent genome altered by copy number events), and at a gene level for recurrent events. KRAS variant frequencies, ploidy and mRNA abundance were also evaluated. Ancestry was assigned using ADMIXTURE from germline genotypes where available, and patient reported otherwise. Results: We observed ancestry-specific mutation patterns in global point mutation density, with higher SNV burden in African PDAC tumours and lower SNV burden in Asian PDAC tumours as compared to European tumours. We also identified an ancestry bias in the driver gene, GNAS, in which driver SNVs in GNAS was detected more frequently in those of Asian ancestry, and less frequently in those of African ancestry. No sex- or ancestry-associations were found for KRAS, but individual KRAS variants showed a trend towards ancestry-related variation. Conclusions: These findings demonstrate that ancestry contributes to the genomic heterogeneity of PDAC, shaping both global and gene-specific mutation patterns. This work underscores the importance of including diverse ancestries in PDAC molecular profiling efforts which may inform the development of tailored molecular diagnostics and targeted therapies.

Developmental programs establish tissue identity and organization, but how their reactivation contributes to adult tissue responses remains poorly understood. Here, we identify a functional contribution of apolipoprotein A-I (APOA1), which marks a late fetal colonic epithelial state that reappears after injury. Spatial and temporal profiling localized Apoa1 transcription predominantly to epithelial cells at the tips of developing proximal colonic folds. Apoa1 loss delayed fold maturation and altered the abundance and composition of developing enteroendocrine populations, linking this apolipoprotein to mucosal architecture and epithelial differentiation. APOA1 protein closely interacted with WNT5A during development and injury, while Apoa1 deficiency increased Wnt5a expression and altered its epithelial protein distribution. Following colonic injury, single-cell and spatial profiling identified genotype-associated transcriptional changes involving stress responses, barrier and immune functions, and metabolism, with a shared component across males and females. These changes accompanied reduced enteroendocrine representation and increased representation of inflammation-associated macrophages. Finally, regional and compartmental expression analyses, together with hindgut explant perturbation, identified HOXB7 transcription factor as a regulator of developmental Apoa1 expression. Together, these findings establish functional relevance for a component of a reactivated late fetal epithelial program, connecting APOA1 to colonic maturation and the epithelial and inflammatory organization of injured tissue.

Myocardin (MYOCD) is the principal trigger-switch for vascular smooth muscle cell (VSMC) growth arrest and the cyto-contractile program of differentiation. While much is known about MYOCD-dependent VSMC differentiation, limited information exists as to its transcriptional regulation. Here, we report moderate mRNA expression of Myeloid Ecotropic viral Integration Site 1 (MEIS1) in both human and mouse VSMCs. MEIS1 mRNA is reduced in parallel with myocardin mRNA and protein under conditions of VSMC de-differentiation. Loss- and gain-of-Meis1 expression studies in cultured human VSMCs and the mouse carotid artery support MEIS1 as an activator of myocardin and VSMC differentiation. CRISPR-mediated knockout of the endogenous Meis1 gene in vitro and elicited lower levels of VSMC contractile markers and an increase in growth rate. Similarly, Femoral artery wire injury, in mice lacking Meis1 showed and increased of neointima formation. Mechanistically, we identified a conserved MEIS1 response element in the proximal promoter of myocardin, that appears to contribute to the promoter activity, suggesting a potential regulatory interaction. These results suggest a previously unrecognized role for the homeodomain protein MEIS1 in the activation of myocardin and the quiescent, VSMC differentiated state.

Glaucoma is an optic neuropathy characterized by progressive retinal degeneration. Population genetic analyses describe an association of variants in angiopoietin-like protein 7 (ANGPTL7) with lower intraocular pressure (IOP) and decreased glaucoma susceptibility. In addition to its reported role in the trabecular meshwork (TM), we demonstrate that ANGPTL7 is also expressed within disease-relevant cell types of the human posterior eye, specifically prelaminar optic nerve head astrocytes (ONHA). Contrasting with previously published findings, we observe no difference in IOP between Angptl7-/- mice and wild-type littermates. Despite absence of an IOP phenotype, loss of ANGPTL7 protects from IOPinduced retinal ganglion cell (RGC) damage in a rodent glaucoma neurodegeneration model. ANGPTL7 may signal through LAIR1-positive immune cells, potentially contributing to pro-inflammatory signaling and phenotypic modulation of resident immune cells, with evidence suggesting that oligomerization of ANGPTL7 is necessary for its activity. Comprehensively, our data highlight an unappreciated role of ANGPTL7 in modulating neuroinflammation in the posterior eye, contributing to glaucoma progression.

Purpose. To determine which proposed keratoconus (KC) mechanisms carry its genetic susceptibility and how it relates to corneal structure, and which describe its corneal tissue. Methods. Six mechanism modules and two attribution sets (transforming growth factor-{beta} [TGF-{beta}] and canonical type-2 atopy) were tested for enrichment in three independent European KC genome-wide association studies (GWAS) and in GWAS of central corneal thickness (CCT) and corneal resistance factor (CRF), with colocalization at matrix loci. Tissue expression was pooled across nine arms from eight studies with control-source meta-regression; activator protein-1 (AP-1)/immediate-early regulatory activity was scored in 107 donors, spatial expression in 21, and serum responses in human and rabbit stromal cells. Results. Extracellular matrix (ECM) was the only module enriched in all three KC sources (P [&le;] 5.46 x 10-) and for CCT, CRF, and thickness-independent CRF (P [&le;] 2.07 x 10-). COL5A1 shared causal variants with CCT in all three sources (posterior probability 0.985-0.991), and FNDC3B and COL1A1 with CRF or its thickness-independent component (0.961-0.986). Pooled, KC corneas had lower general and corneal-local inflammatory and stress/immediate-early expression (log2 fold-change -0.97, -0.98, -0.82; P [&le;] 0.049). In whole-cornea and stromal tissue, ECM expression reversed between postmortem-donor and pathological controls (log2 fold-change +0.36, -2.34), whereas inflammatory and TGF-{beta} programs were lower against both. AP-1/immediate-early regulatory activity was lower in four donor cohorts (Hedges' g -1.50) and stress/immediate-early expression in two spatial cohorts; serum raised immediate-early readouts in human and rabbit cells. Conclusions. KC combines matrix-mechanical genetic susceptibility with a low-inflammatory, low-immediate-early corneal state whose matrix expression direction depends on the control source. Because this susceptibility is genetically shared with corneal thickness and resistance, interventions on the inherited disease are judged by corneal structure and mechanics.

Clinical translation of mesenchymal stromal cell (MSC) therapies derived from primary tissue sources is limited by manufacturing inconsistencies driven by donor-to-donor variability, which reduces predictability of clinical response. Induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) offer a promising donor-independent, clonal alternative strategy that is also more amenable to engineering, but whether engineered iMSCs reproduce the potency attributes of primary MSCs remains unclear. Here, we benchmarked a doxycycline-inducible, hTERT-immortalized iMSC line engineered to overexpress TGFB1 (TGFB1-iMSCs), a key regulator of macrophage polarization, against multiple adipose tissue-derived MSC (MSC(AT)) donors across predefined immunomodulatory and angiogenic potency attributes. TGFB1-iMSCs were smaller and more circular with comparable or higher proliferative rates than primary MSC(AT) donors. Interestingly, they had a distinct angiogenic signature (EDIL3, EDN1, PDGFA), and nine differentially expressed microRNAs were predicted to target secretory trafficking and growth factor receptor signalling. Functionally, however, TGFB1-iMSCs secreted less VEGF and showed intermediate HUVEC tube formation, but matched or exceeded all primary MSC(AT) donors in a monocyte-macrophage transwell immunomodulatory assay. Notably, in a murine destabilization of the medial meniscus (DMM) model of post-traumatic osteoarthritis, a single intra-articular injection of TGFB1-iMSCs, but not MSC(AT), reduced total synovial macrophage numbers and lowered the MHCII/CD206 ratio at eight weeks, although it did not reduce cartilage degeneration, synovitis, or fibrosis. Together, these findings show that TGFB1-engineered iMSCs have a macrophage-directed potency profile distinct from that of primary MSC(AT). Immortalized iMSCs therefore provide a reproducible platform for mechanistically dissecting engineered MSC potency and its effects on the immune environment.