The FASEB Journal

Iron absorption in the small intestine has classically been described by the duodenal DMT1/FPN1 pathway for inorganic non-heme iron, yet emerging evidence suggests that chemically distinct iron forms may use region-specific routes. Nicotianamine (NA), a plant-derived metal chelator, can form NA-iron (NA-Fe) complexes and has been proposed to support intestinal iron absorption through amino acid transporter pathways. However, direct comparisons of transepithelial transfer of inorganic iron and NA-Fe across defined small intestinal regions under controlled epithelial conditions remain limited. Here, we established region-specific 2D epithelial monolayers derived from duodenal and proximal jejunal crypt organoids from male ICR mice cultured on Transwell inserts. Transcriptomic profiling indicated partial retention of regional identity, and barrier integrity was confirmed by junctional marker localization, transepithelial electrical resistance, and low paracellular permeability. We then examined expression and polarized localization of candidate transporters for inorganic iron (Dmt1/Fpn1) and NA-Fe (Pat1/Lat2). Finally, we quantified transepithelial transport using apical loading of isotope-labeled iron (55Fe) or NA-55Fe and measured radioactivity appearing in the basolateral compartment as the primary readout of transepithelial flux. Basolateral appearance of inorganic 55Fe was comparable between duodenum- and proximal jejunum-derived monolayers, whereas NA-55Fe exhibited significantly greater basolateral appearance in proximal jejunum-derived monolayers. These findings demonstrate that organoid derived, region-specific monolayers provide a tractable epithelial platform to evaluate iron form-dependent, region-specific transepithelial transfer and to enable further mechanistic dissection of NA-Fe transport. NEW & NOTEWORTHYNon-heme iron absorption may depend on iron chemical form and intestinal region, but direct epithelial comparisons are scarce. We established duodenum and proximal jejunum derived murine intestinal organoid monolayers on Transwells and quantified transepithelial flux using isotope-labeled iron. Inorganic 55Fe showed no clear regional difference, whereas NA-55Fe displayed greater basolateral appearance in proximal jejunum-derived monolayers. This platform enables mechanistic studies of NA-iron complex transport.

Intervertebral disc degeneration is associated with loss of nucleus pulposus (NP) cell phenotype and extracellular matrix, both processes linked to changes in cytoskeletal contractility and cell shape. Here, we tested whether microenvironment-specific modulation of RhoA signaling can restore NP-like morphology and gene expression in NP cells cultured in 2D and in 3D alginate. In 2D monolayer culture, where cells are spread and mechanically activated, pharmacologic inhibition of RhoA with CT04 reduced RhoA activity, decreased actomyosin contractility gene expression, and shifted morphology toward a smaller, more circular phenotype. Bulk RNA sequencing showed that CT04 treatment increased expression of NP phenotypic and matrix-related genes including ACAN, GDF5, CHST3, and MUSTN1 while decreasing expression of catabolic and fibroblast-associated genes including ADAMTS1/9 and COL1, consistent with enrichment of extracellular matrix pathways. In contrast, RhoA activation with CN03 in 2D culture increased actin and phosphorylated myosin light chain intensity but produced limited phenotypic improvement. In 3D alginate, which minimizes integrin-mediated adhesion, baseline actomyosin markers were reduced relative to 2D culture. In alginate, RhoA activation with CN03 increased the amount of actin, phosphorylated myosin light chain, and actomyosin gene expression, yet also promoted a more compact, circular morphology and increased NP markers, including ACAN and KRT19 with repeated dosing. Across culture conditions, increased cell roundness was consistently associated with increased ACAN expression, indicating strong coupling between cytoskeletal state, morphology, and NP matrix programs. Together, these findings demonstrate that RhoA pathway perturbation can promote NP phenotypic gene expression in both 2D and 3D culture, but the direction of optimal modulation depends on the microenvironment, supporting RhoA signaling as a context-dependent therapeutic target for disc regeneration.

Scleraxis (Scx), a basic helix-loop-helix (bHLH) transcription factor, is a primary marker for tendon and ligament lineages. Consequently, mouse models utilizing Cre recombinase under the control of the Scx locus represents a powerful tool for control of gene expression in tendon. The constitutive ScxCre mouse line is widely used for tendon-specific genetic manipulation. In this study, we demonstrate that ScxCre exhibits undesired significant off-target activity in the male germline, leading to ubiquitous recombination of floxed alleles in all tissues of the resulting offspring. This inheritance of recombined LoxP alleles occurs independently of Cre inheritance, indicating that ScxCre-induces recombination occurs prior to meiosis in diploid germ cells. This off-target activity is not observed in female germline. These findings highlight a critical need for stringent parental sex selection when using ScxCre lines to ensure tissue-specific targeting and avoid unintentional global gene deletion or transgene activation.

BackgroundPhysical loading mediates postnatal growth, homeostasis, and healing of the tendon and its attachment to bone, which is critical for rotator cuff functional integrity. Our prior studies have highlighted the mechano-sensing role of primary cilia; However, the mechanisms through which cilia convert mechanical stimuli into structural functional adaptation under altered loading conditions remain unanswered. MethodsPublicly available scRNA-seq datasets of mechanically loaded human patellar tendon cells were re-analyzed to identify cilia-related transcriptional changes. Tendon-specific cilia knockout mice (ScxCre;Ift88fl/fl) and wild-type controls (Ift88fl/fl) underwent mechanical unloading induced by botulinum toxin A injection, followed by micro-computed tomography, biomechanical testing, histology, qPCR, and immunohistochemistry to evaluate structural, mechanical, and Hedgehog (Hh) signaling responses. Primary tendon fibroblasts from wild-type and cilia-deletion mice were treated with Hh agonist or antagonist to assess Hh signaling responsiveness in vitro. Students t-test for two groups and two-way ANOVA for two groups with two treatments were performed for our statistical analysis. ResultsHere, we find that mechanical force causes changes in cilia- and hedgehog (Hh)-related gene expression in human tendon fibroblasts. Cilia ablation in the enthesis blunts force-driven remodeling of tissue structure and mechanical strength. Cilia deletion also leads to impaired Hh signaling in tendon cells and decreased responsiveness to activation and inactivation of hedgehog signaling. ConclusionsOur results demonstrate loading-regulated ciliary Hh signaling during postnatal growth of the tendon and enthesis and provide proof-of-concept for developing new cilia-targeted mechanical and biological therapies for enthesis repair.

Vitamin D signaling has recognized roles in female reproductive physiology, but its effects at the chromatin level in endometrial stromal cells are still unclear. Here, we investigated how the active form of vitamin D, 1,25-dihydroxyvitamin D3, or calcitriol, influences the accessible chromatin landscape of human endometrial stromal cells. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed on T-HESCs treated with either a vehicle or 1,25(OH)2D3. Ligand treatment increased overall chromatin accessibility, shown by higher ATAC-seq signal intensity, while causing only minor changes in the total number of called peaks. Peak annotation revealed that accessible regions were spread across both promoter-proximal and distal genomic areas. Integrating this data with CUT&RUN and RNA sequencing showed that most vitamin D-responsive cistromic modifications and transcripts were linked to nearby open chromatin, though fewer were associated with regions that were significantly differentially accessible. These results suggest that 1,25(OH)2D3-dependent transcription mainly occurs within a permissive, pre-accessible chromatin environment. This study offers new evidence that active vitamin D influences the epigenomic landscape of human endometrial stromal cells, establishing the chromatin-based molecular response to a chemically-defined VDR ligand, 1,25(OH)2D3, relevant to stromal differentiation and preparation for decidualization. HighlightsO_LIFirst evidence suggesting the direct impact of active vitamin D, 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, enhanced the signal intensity of chromatin accessibility in human endometrial stromal cells C_LIO_LIMost accessible chromatin regions were shared between vehicle and ligand-treated human endometrial stromal cells C_LIO_LI1,25(OH)2D3-responsive transcription occurs largely within pre-accessible chromatin in human endometrial stromal cells C_LIO_LIAssay for transposase-accessible chromatin sequencing (ATAC-seq) defines a chromatin-level pharmacologic response to a chemically defined VDR ligand in human endometrial stromal cells C_LI

Extracellular matrix (ECM) remodeling is essential for adaptation to changing mechanical demands, yet the mechanisms linking altered strain to functional outcomes remain poorly understood. This study aimed to define molecular and cellular programs driving the adaptation of tendon to increased (exercise) and decreased (disuse) strain. Male murine flexor tendon explants were cultured in incubator-housed tensile bioreactors and subjected to step changes in cyclic strain. After acclimation at 1% cyclic strain, exercise tendons experienced a step increase to 5% strain, while disuse tendons underwent stress deprivation. Increased strain produced significant mechanical adaptations, including increased elastic modulus and failure stress. Multiscale analyses of matrix organization, tissue composition, protein synthesis, signaling factors, and proteolytic activity revealed the mechanisms underlying these adaptations. Exercise-induced functional improvements were linked to an anabolic remodeling program characterized by TGF-{beta} and IL-6 signaling, small leucine-rich proteoglycan expression, MMP suppression, and enhanced collagen alignment. These findings indicate that regulators of matrix organization and turnover, beyond synthesis alone, are critical for functional adaptation. In contrast, mechanical unloading reduced collagen synthesis and alignment and promoted an MMP-dominant, catabolic phenotype favoring matrix breakdown. This study provides a comprehensive characterization of ECM remodeling, linking defined mechanical perturbations to molecular regulation and emergent structure-function relationships. These findings identify targetable mediators of adaptive remodeling and establish a framework for future studies of maladaptive ECM changes in aging, injury, and disease.

Intestinal epithelial cells are central players in mucosal barrier integrity and host-microbe interactions. Genetic studies have revealed that epithelial dysfunction is a key contributor to the pathogenesis of inflammatory bowel disease. Non-SMC condensin II complex subunit D3 (NCAPD3) is essential for chromatin organization and stability. NCAPD3 also promotes antimicrobial defense and autophagy responses in vitro. NCAPD3 expression is decreased in intestinal epithelial cells from patients with ulcerative colitis; however, it is not known whether loss of NCAPD3 expression drives intestinal barrier dysfunction or is a result of disease-associated inflammation. To investigate this relationship in vivo, a tissue-specific approach was required, as global constitutive knockout of NCAPD3 is embryonic lethal. Therefore, a transgenic mouse line with doxycycline-inducible expression of a short hairpin RNA targeting NCAPD3 restricted to villin-expressing cells was generated (NCAPD3KD mice) to enable the study of NCAPD3 function in the intestinal epithelium. Treatment of NCAPD3KD mice with 9-tert-butyl doxycycline resulted in [~]75% reduction of NCAPD3 protein in EpCAM+ intestinal cells. Short-term epithelial NCAPD3 knockdown did not induce spontaneous colitis but was associated with increased serum amyloid A and a trend towards increased intestinal permeability. Upon dextran sodium sulfate or Salmonella enterica serovar Typhimurium {Delta}AroA challenge, NCAPD3KD mice exhibited exacerbated weight loss, higher disease activity, increased histopathological damage, abnormal colonic cytokines and chemokines, and significantly increased intestinal permeability. These results indicate that NCAPD3 expression in the intestinal epithelium is required for optimal barrier maintenance and antimicrobial defense under chemical or microbial stress. These findings support prior in vitro observations and solidify NCAPD3 as a regulator of intestinal epithelial barrier function and mucosal host defense. Author SummaryNCAPD3 is a multifunctional protein with established roles in chromatin organization, genome stability, mitochondrial function, and antimicrobial defense. Dysregulated NCAPD3 is implicated in human diseases, such as inflammatory bowel disease (IBD) and microcephaly; however, due to its essential role in cellular division, determination of whether NCAPD3 loss drives these pathologies in vivo has been lacking. Using a new transgenic mouse model that selectively reduces NCAPD3 expression in intestinal epithelial cells, our study establishes NCAPD3 as an epithelial regulator of the mammalian intestine that enhances epithelial barrier resilience and antimicrobial defense during stress. Although dispensable for short-term basal homeostasis, NCAPD3 function becomes critical during epithelial injury and enteric infection. Reduced NCAPD3 expression may therefore lower the threshold for inflammatory disease by weakening barrier integrity, amplifying inflammatory cascades, and impairing antimicrobial defenses. These findings position NCAPD3 as a potential modulator of IBD susceptibility and highlight chromatin organization as an important, previously underappreciated layer of intestinal epithelial regulation.

ObjectiveInvestigate how Ca2+/calmodulin dependent protein kinase kinase 2 (CaMKK2) orchestrates a catabolic shift in chondrocytes during early osteoarthritis (OA). MethodsCartilage, osteochondral plugs and chondrocytes were collected from patients undergoing total hip arthroplasty or non-OA donors. SOX9 levels were assessed via immunoblotting or immunohistochemistry (IHC). Sox9 levels were also assessed by IHC in knee joints from wild-type (WT) and Camkk2-/- mice that underwent sham or destabilization of medial meniscus (DMM), with or without STO-609 (0.033 mg/kg) treatment. Co-immunoprecipitation followed by mass spectrometry was performed to identify CaMKK2 interacting proteins in chondrocytes. Kinase assays were analyzed by immunoblotting and phosphosites identified by mass spectrometry. Proteasome function was assessed in murine and human chondrocytes lacking or expressing kinase-active or kinase-inactive CaMKK2. ResultsInhibition or loss of CaMKK2 increased SOX9, whereas the expression of kinase-active, not inactive, CaMKK2 reduced Sox9 in human and mouse OA cartilage. Proteomic analysis of CaMKK2 immunoprecipitates revealed the presence of ubiquitin E3 ligase Ubr4 and the 19S proteasome regulatory particle (RP). CaMKK2 kinase activity was dispensable for its interactions with Ubr4, 19S RP, and Sox9-ubiquitin conjugates, and kinase-inactive CaMKK2 attenuated Sox9 degradation in chondrocytes. Further, CaMKK2 phosphorylated the 19S RP ATPase Psmc5 on Ser136, and an intact kinase increased proteasome activity in chondrocytes. ConclusionsOur findings identify CaMKK2 as a dual-function regulator of chondrocyte UPS with a scaffolding role to assemble UPSUbr4-19S RP around polyubiquitinated proteins such as Sox9, and a catalytic role to enhance proteasome function, potentially through Psmc5 phosphorylation, thereby linking chondrocyte inflammatory signaling to Sox9 degradation and cartilage degeneration.

Human embryo implantation, occurring approximately one week after fertilization, remains poorly understood due to ethical and technical limitations of in vivo investigation. To overcome these barriers, and model this critical developmental event, encompassing peri- and early post-implantation stages, we used an in vitro embryo attachment model composed of donor-derived endometrial epithelial cells forming an open-faced endometrial layer (OFEL) and human stem cell-derived blastoids recapitulating human day 5 blastocysts in peri-implantation model. Following attachment, developmental progression was further investigated on laminin-coated substrates to capture early post-implantation dynamics. Despite its central role as the primary endocrine signal of early pregnancy, human chorionic gonadotropin (hCG) remains largely uncharacterized in this context. Here, we describe the transcriptomic profile of blastoid-endometrial co-cultures relative to OFEL alone, identifying CGA and CGB3/5/8 as among the most strongly upregulated genes following blastoid attachment to hormonally stimulated OFEL. Consistent with these findings, immunoassays and luteinizing hormone/choriogonadotropin receptor (LHCGR) activation assays of conditioned media confirmed the secretion of heterodimeric, biologically active hCG and its free subunits in co-cultures, but not in endometrial layers alone. Notably, the hyperglycosylated hCG heterodimer was the predominant isoform detected. Co-culture with the endometrial component significantly increased hCG secretion compared with blastoids cultured alone, an effect further enhanced by hormonal priming in the peri-implantation model. Collectively, these findings indicate that a hormonally primed endometrial environment not only promotes blastoid attachment but also amplifies embryonic hCG production and bioactivity, underscoring the importance of maternal endocrine cues in early embryo-endometrium communication. Furthermore, our peri- and early post-implantation models recapitulate key aspects of reciprocal endocrine signaling between embryonic and endometrial tissues, providing a tractable experimental framework to investigate embryo-endometrium crosstalk.

The endometrium, the inner lining of the uterus, is a dynamic tissue that undergoes precise molecular and structural changes to achieve a receptive state capable of supporting embryo implantation. Although the uterine environment was long considered sterile, molecular studies have detected microbial signals and bioactive compounds that may influence endometrial function. Endometrial epithelial organoids (EEOs) provide a three-dimensional in vitro model that recapitulates the architecture, polarity, and hormonal responsiveness of native endometrial tissue. This study aimed to elucidate how bacterial-derived compounds, including D-lactate (D-lac), commonly associated with Lactobacillus communities, and lipopolysaccharides (LPS), a component of Gram-negative bacteria, affect the transcriptomic profile of the endometrial epithelium under a hormonally induced receptive state. EEOs were exposed to different concentrations of these compounds, and relative metabolic activity was monitored through resazurin-based assays, revealing no significant alterations across the conditions tested. Transcriptomics analysis of hormonally stimulated EEOs, mimicking the mid-secretory phase, revealed that D-lac modulated genes related to epithelial development, tissue remodelling and growth regulation, whereas LPS influenced genes associated with inflammatory signalling and immune response. While key markers of receptivity remained largely stable, small transcriptional changes suggest that microbial signals may modulate the functional balance of the receptive endometrium. These findings highlight a modulatory role of microbial signals on endometrial epithelial function and demonstrate that EEOs are a robust platform for exploring host-microbe interactions in the uterus, offering new insights into the mechanisms underlying uterine receptivity.

The human endometrium undergoes cyclic, hormone-driven remodeling that establishes a transient window of receptivity required for embryo implantation, placentation, and maintenance of pregnancy. Decidualization of endometrial stromal cells is a central component of this process and can be induced in vitro using cAMP alone or in combination with ovarian steroid hormones (EPC: estradiol, progesterone, and cAMP). Although cAMP activates the core decidual transcriptional program, whether hormone supplementation induces a more physiologically relevant response remains unclear, particularly in 3D endometrial organoid (Endo-organoid) models which have emerged as a new alternative methodology (NAM). Here, we compared morphological and transcriptomic responses of human endometrial stromal cell-derived Endo-organoids undergoing decidualization induced by cAMP or EPC stimulation. EPC-treated Endo-organoids exhibited enhanced structural remodeling and more advanced morphological transformation compared with cAMP-treated organoids. RNA-seq analysis revealed substantial overlap in canonical decidual gene expression between the two conditions, but EPC induced broader transcriptional and pathway-level changes, including enrichment of metabolic, stress-response, and differentiation-related processes. Together, these findings demonstrate that while cAMP activates the core decidual program, EPC elicits a broader and more physiologically relevant decidualization response in 3D human Endo-organoids, providing guidance for optimizing Endo-organoids to study endometrial receptivity, implantation, and early pregnancy success.

The human fallopian tube plays a critical role in reproductive biology, yet the structural organization and immune repertoire of this tissue remain incompletely characterized. Here, we performed an in-depth analysis of human fallopian tube tissue from women of reproductive age across three distinct anatomical regions (isthmus, ampulla, and fimbriae) across the menstrual cycle. Using antibody-based imaging for EPCAM, CD8A, and CD20 together with automated image analysis, the epithelial thickness and spatial distribution of T and B lymphocytes was assessed. No significant differences in epithelial thickness were observed between proliferative and secretory phases within any tubal region. In contrast, significant regional differences were identified, with the epithelium being thickest in the isthmus and thinnest in the ampulla. Both CD8A+ T lymphocytes and CD20+ B lymphocytes were detected throughout the fallopian tube, and a strong correlation between T and B lymphocyte abundance was observed across patients. Spatial analysis further revealed that both lymphocyte populations were preferentially localized within the mucosal compartment adjacent to the lumen. Notably, intraepithelial B lymphocytes were identified throughout the fallopian tube. Together, these findings provide new insight into epithelial organization and immune cell distribution in the human fallopian tube, highlighting the complexity of the tubal immune microenvironment and its potential relevance for reproductive biology.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely recognized to potentially interfere with skeletal muscle regeneration. However, current knowledge is based almost exclusively on non-aspirin NSAIDs. Aspirin (ASA) differs from other NSAIDs in its ability to irreversibly acetylate cyclooxygenase-2 (COX-2), thereby redirecting its activity toward a lipoxygenase (LOX)-like function that enables the production of unique ASA-triggered specialized pro-resolving lipid mediators (AT-SPMs). Despite this, the potential impact of ASA on musculoskeletal tissue repair remains poorly understood. This study directly compared the effect of ASA against non-ASA NSAIDs on in vitro myogenesis and in vivo skeletal muscle injury and regeneration. Unlike non-ASA NSAIDs, including indomethacin (INDO), celecoxib, and SC-236, which markedly impaired C2C12 myotube formation at concentrations near their pharmacological ranges, ASA only interfered with myogenesis at overtly supraphysiological concentrations. In mice, an oral dose of 3 mg/kg/day INDO following barium chloride-induced muscle injury reduced regenerating myofiber cross-sectional area and impaired the recovery of muscle force-generating capacity. In contrast, a potency-matched oral treatment with 30 mg/kg/day ASA hastened the resolution of cellular inflammation, promoted myonuclear accretion, and improved recovery of absolute muscle strength. The beneficial effects of ASA on inflammatory resolution and muscle strength--but notably not myonuclear accretion--were reversed in mice co-treated with ASA + INDO. These findings demonstrate that, unlike non-ASA NSAIDs, ASA does not impair skeletal muscle regeneration and may promote a favorable early inflammatory environment for repair via unique COX-dependent pro-resolving and COX-independent anabolic mechanisms.

Interleukin-6 (IL-6), produced by skeletal muscle and extramuscular tissues, regulates skeletal muscle function through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. However, the interaction between intrinsic (locally produced) IL-6 and extrinsic (circulating) IL-6 in skeletal muscle remains unclear. We investigated whether and how intrinsic expression of IL-6 in cultured primary human myoblasts influences their response to extrinsic stimulation with recombinant human IL-6 (rhIL-6). Using gene silencing, we found that suppression of intrinsic IL-6 enhanced rhIL-6-induced phosphorylation of STAT1 and STAT3. Silencing STAT3 also increased rhIL-6-induced STAT1 phosphorylation, but silencing STAT1 had no effect on STAT3 phosphorylation. Pretreatment of myoblasts with neutralising anti-IL-6 antibodies increased phosphorylation of STAT1 and STAT3 induced by 50 ng/mL rhIL-6, whereas pretreatment with 5 ng/mL rhIL-6 reduced this response. Despite increased JAK/STAT signalling, IL-6 silencing decreased glucose and oleic acid uptake and oxidation under both basal and rhIL-6-stimulated conditions. Collectively, our results imply that intrinsic IL-6 restrains activation of the JAK/STAT pathway by extrinsic IL-6, but acts synergistically with it to promote myoblast energy metabolism.

Angiotensin-converting enzyme 2 (ACE2) is expressed in adipocytes, yet the mechanisms regulating its intracellular trafficking remain unclear. Here, we investigated whether ACE2 trafficking is coordinated with insulin-responsive vesicle dynamics mediated by the glucose transporter GLUT4 in 3T3L1 adipocytes. Subcellular localization analyses revealed that adipocyte differentiation promotes partial incorporation of ACE2 into insulin-responsive GLUT4-associated vesicular compartments, whereas ACE2 displayed a diffuse distribution in fibroblasts lacking a mature GLUT4 trafficking system. Reconstitution of insulin-responsive GLUT4 vesicle formation through exogenous expression of Sortilin and AS160 in fibroblasts was sufficient to partially recruit ACE2 into perinuclear GLUT4-positive compartments, indicating dependence on canonical GLUT4 vesicle machinery. NanoBiT assays demonstrated a regulated association between ACE2 and GLUT4 that was modestly enhanced by acute insulin stimulation but reduced following prolonged insulin exposure. Insulin stimulation also produced a slight increase in ACE2 surface exposure, while association with GLUT4 was accompanied by reduced ACE2 shedding, suggesting that recruitment into distinct trafficking routes may alter ACE2 accessibility to shedding machinery in adipocytes. Structural modeling further suggested that ACE2 and GLUT4 can form a membrane-compatible complex. Together, these findings indicate that ACE2 trafficking is coordinated with insulin-responsive GLUT4 vesicle dynamics, revealing a previously unrecognized association between metabolic signaling and ACE2 cellular dynamics in adipocytes, with potential implications for metabolic dysfunction and ACE2-associated disease processes.

Ferroptosis is linked to various diseases, but the role of transferrin (TF) in endometriosis (EM) remains unclear. Expression levels of ferroptosis-related proteins, including transferrin (TF), transferrin receptor (TFRC), and glutathione peroxidase 4 (GPX4), were analyzed by western blotting. Compared to normal endometrial stromal cells, eutopic and ectopic endometrial stromal cells from EM patients exhibited significantly enhanced proliferative and migratory abilities, accompanied by a marked reduction in glutathione (GSH) levels in both eutopic and ectopic tissues. TF and TFRC expression was upregulated in ectopic endometrium relative to normal controls, while GPX4 expression was downregulated. To evaluate the functional role of TF, siRNA-mediated knockdown was performed in endometrial stromal cells, with knockdown efficiency confirmed by western blotting. Functional assays demonstrated that TF knockdown not only suppressed cell proliferation (CCK-8 and clonogenic assays) and migration (wound healing assay) but also significantly increased apoptosis rate (flow cytometry with Annexin V-FITC/PI staining).These findings implicate TF in the pathogenesis and progression of endometriosis, likely through modulating endometrial stromal cell proliferation, migration, and apoptosis.

In eutherian mammals, blastocyst implantation is often associated with a quasi-inflammatory reaction in the endometrium, which is resolved with the establishment of the definitive placenta. This is understandable in the case of invasive placentation, since implantation entails a nidatory injury to the maternal tissue due to the invading blastocyst. Quasi-inflammatory processes have also been documented in pregnant pigs, even though the blastocyst only attaches to, rather than invades into, the endometrium of the uterus. In this study, we asked what processes in early porcine pregnancy lead to the resolution of attachment-associated inflammation. In generic wound healing the transition from a pro- to an anti-inflammatory state is caused by a corresponding transition from M1 to M2 polarized macrophages following efferocytosis by macrophages of apoptotic neutrophils. In order to determine whether this scenario applies to the pregnancy-related resolution of inflammation in the porcine uterus, we produced a series of bulk transcriptome samples spanning days (D) 13 to 25 of gestation. This time span corresponds to the transition from pre- to post-attachment stages of pregnancy. We found slower changes in the transcriptome between D20 and D25 than prior to D20, suggesting a turning point in pregnancy-related reprogramming. The turning point at D20 corresponds to the time of firm attachment of trophectoderm to uterine luminal epithelium and the cessation of IFNG signaling from the blastocyst. This transition coincides with increased expression of RNAs of genes implicated in resolution of inflammation and M2 polarization such as ARG1, MRC1/CD206, CD86, TGFb1 and IL10, as well as a significant increase in expression of HGPD, the enzyme that metabolizes prostaglandins. While immunoreactivity for ARG1 was found in putative macrophages in the sub-epithelial stratum compactum, other markers of M2 polarized macrophages were localized to non-immune cells: MRC1 was found on fibroblast-like stromal cells, CD86 on trophoblast cells, and IL10 in luminal and glandular epithelia. These results suggest that intrauterine immune regulation is decoupled from that of the rest of the body by engaging non-immune cell types as anti-inflammatory mediators during the peri-attachment period of pregnancy.

Rotator cuff tendinopathy is highly prevalent in aging populations, yet the mechanisms leading to age-dependent tendon degeneration are not well understood. In addition to tensile loading, tendons are subjected to compressive forces at certain anatomical sites (e.g., Achilles, rotator cuff), where altered adaptive responses may contribute to degenerative remodeling. The objective of this study was to investigate age-related differences in tendon responses to dynamic compressive loading using an ex vivo model. Murine flexor tendon explants from young and aged animals were cultured in a biaxial bioreactor and subjected to different levels of dynamic compressive loading. We then observed changes in metabolic activity, matrix composition, matrix biosynthesis, matrix structure, and gene expression. Young tendons exposed to moderate levels of compression maintained homeostasis, whereas high compression induced a robust adaptive response characterized by increased glycosaminoglycan accumulation, elevated collagen content, and upregulation of remodeling-associated genes including collagen I, decorin, and MMP-9, as well as inflammatory and apoptotic markers. In contrast, aged tendons demonstrated a qualitatively different response, with transcriptional downregulation of key remodeling markers alongside elevated secretion of matrix-degrading enzymes and pro-inflammatory cytokines, indicative of a maladaptive mechanobiological response even at low compressive levels. These findings reveal that impaired mechanosensitivity and a lower threshold for injury may predispose chronically loaded tissues to degenerative pathology associated with excessive compressive loading.

Introduction: Podocyte injury is central to the pathogenesis of most glomerulonephritides (GN) and causes segmental glomerulosclerotic lesions that predict progression in IgA Nephropathy (IgAN). Recent advances in high-resolution microscopy and AI-assisted image analysis have enabled detailed quantification of podocyte foot process (FP) morphology. However, whether nanoscale podocyte morphometrics can predict disease progression or treatment response in GN has not been investigated. Aim: To evaluate whether nanoscale podocyte morphometric parameters predict clinical characteristics, disease progression, and treatment response in GN, with a focus on IgAN. Method: Podocyte morphometrics were analyzed in kidney biopsies from patients with GN using high-resolution microscopy and the deep learning-based tool Automatic Morphometric Analysis of Podocytes (AMAP). Four morphometric parameters were quantified: slit diaphragm length (SDL), FP area, FP circularity and FP perimeter. These parameters were correlated with clinical characteristics, conventional electron microscopy (EM) findings and longitudinal follow-up data. Results: The study included 37 patients with GN from Danderyd University Hospital (Stockholm, Sweden), with IgAN representing the largest diagnostic subgroup (n = 19). The median follow-up for the cohort was 3.0 years. SDL correlated significantly with urine albumin-to-creatinine ratio (uACR; p = 0.021), whereas conventional EM measurements did not (p = 0.22). Within the IgAN subgroup, lower SDL was associated with a steeper decline in eGFR, higher FP area with increased long-term proteinuria, and higher FP circularity with improvement in uACR during the first year. The association between lower SDL and eGFR decline remained as a trend in IgAN patients not treated with corticosteroids (p = 0.068) but was absent in the treatment group (p = 0.59). Conclusion: In this proof-of-concept study, nanoscale podocyte morphometrics demonstrated greater sensitivity than conventional EM in quantifying podocyte injury and predicting progression in IgAN. These findings suggest that high-resolution morphometrics may improve risk stratification in IgAN but require validation in larger, independent cohorts before clinical implementation.

BackgroundSingle-nephron glomerular filtration rate (GFR) represents a nephron-level functional index that may reveal key pathophysiological mechanisms driving progression in patients with diabetic nephropathy. However, its clinical relevance remains incompletely understood. This cross-sectional study assessed single-nephron estimated GFR (eGFR) across different chronic kidney disease (CKD) stages in patients with advanced diabetic nephropathy. MethodsNephron number was estimated as the number of nonglobally sclerotic glomeruli per kidney using computed tomography-derived cortical volume combined with biopsy stereology. Single-nephron eGFR was calculated by dividing eGFR by the nephron number of both kidneys. Patients were stratified according to CKD stage at kidney biopsy. Associations between CKD stages and single-nephron eGFR were evaluated using multivariable linear regression models adjusted for age, sex, urinary protein excretion, and eGFR. ResultsThe study included 105 patients with biopsy-proven diabetic nephropathy and overt proteinuria (median age 59 years, 83% male, HbA1c 6.6%, 57% had nephrotic range proteinuria). The percentage of globally sclerotic glomeruli, mesangial expansion score, and prevalence of nodular lesions increased significantly with advancing CKD stage. Median nephron number declined from 529,178 to 224,458 per kidney, whereas glomerular volume remained constant. Single-nephron eGFR decreased markedly with CKD stage and remained significantly inversely associated with CKD stage after adjustment for clinicopathologic covariates (P for trend <0.001). ConclusionIn overt diabetic nephropathy, single-nephron eGFR decreased with advancing CKD stage, despite relatively preserved glomerular volume. At this stage of disease, structural alterations specific to diabetic nephropathy may impair effective single-nephron filtration capacity.