Matrix Biology Plus

Oncofetal fibronectin (oFN) is a known biomarker of cancer progression and is involved in events such as epithelial mesenchymal transition and multidrug resistance. It is also understood that fibronectin (FN) and its splice variants are differentially arranged by cancer associated fibroblasts (CAFs) to support cancer cell proliferation and migration. Contrastingly, little is understood about the mechanobiology of oFN, a post-translationally modified FN isoform defined by O-glycosylation of a specific threonine residue. To investigate the architectural features of the oFN matrix, CAFs were simulated by culturing human mammary fibroblasts (HMFs) on stiff polyacrylamide gels (20 kPa) in media collected from HCC-1937 (primary site breast adenocarcinoma) and MDA-MB-231 (metastatic breast adenocarcinoma) cells. As a control, HMFs were also cultured on softer gels representing healthy adipose tissue (2 kPa) in media not exposed to cancer cells. After immunocytochemical analysis, tumor conditioned HMFs displayed an expected CAF-like phenotype that was most prominent with MDA-MB-231 conditioned media. Matrices produced by tumor-conditioned HMFs contained more oFN with structural features mostly following that of the overall dense, aligned FN matrix. Cancer cells reseeded on their corresponding decellularized tumor-associated matrices had greater F-actin/oFN colocalization than control matrix. The lack of distinct oFN topographical cues may indicate that oFN O-glycosylation is more directly involved in biochemical signaling than mechanosignaling through matrix architecture. Together, this study established a platform for investigating stromal oFN which revealed that tumor-conditioned media sourced from cancer cells with different metastatic potential influences HMF activation, leading to distinct total FN and thus oFN matrix microarchitecture features.

Seroma is a common complication following abdominoplasty and other procedures involving extensive soft tissue dissection. Although animal seroma models have been reported, the stromal pathology underlying post-abdominoplasty seroma, particularly the role of oxidative stress, remains poorly characterized. Here, we developed a rat abdominal seroma model by surgically separating the subcutaneous tissue from the underlying muscle fascia and investigated the temporal evolution of the associated stromal response. Seroma developed in 9 of 20 operated rats, corresponding to a formation rate of 45.0%. Longitudinal ultrasound demonstrated that fluid accumulation peaked around postoperative day (POD) 7 and largely resolved by POD30. Despite macroscopic fluid resolution, substantial stromal remodeling persisted. Compared with normal abdominal tissue, seroma-associated tissues at POD14 and POD35 exhibited increased collagen deposition, macrophage accumulation, oxidative injury, and vascular remodeling. Additionally, at POD35, macrophage accumulation and 4-hydroxynonenal staining were greater than at POD14, whereas collagen deposition and vascular density remained elevated with no significant differences between postoperative time points. To our knowledge, this study presents the first rat model specifically designed to reproduce post-abdominoplasty seroma and the first temporal characterization of oxidative stress within seroma-associated tissue. These findings reveal that seroma fluid resolution does not coincide with stromal recovery and identify persistent oxidative stress, inflammation, fibrosis, and vascular remodeling as important features of seroma pathology.

Individuals with fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder caused by mutations in the bone morphogenetic protein receptor ACVR1 (also known as ALK2), experience progressive and severely debilitating endochondral heterotopic ossification (HO). Previous studies have identified fibro-adipogenic progenitors (FAPs) as a major cellular source of HO in FOP mouse models. Here, we used mRNA sequencing of FAPs isolated from injured muscle of FOP mice to capture the earliest gene expression changes associated with the transformation of muscle tissues into bone. Whereas the transcriptomes of mutant and wild-type FAPs were indistinguishable prior to injury and at 1 day post injury, dramatic dysregulation of gene expression was observed at 3 and 5 days after injury. Surprisingly, gene expression changes suggested early specification of osteochondral lineages by 3 days post injury, approximately 3 to 4 days before the appearance of histologically identifiable cartilage. We identified dysregulation of genes encoding components of major signaling pathways, cell cycle regulators, extracellular matrix components, and hypoxia response mediators, and showed that these gene expression changes are temporally associated with abnormal population growth of mutant FAPs. We further demonstrated that treatment of FOP mice with either an anti-activin A monoclonal antibody or the ACVR1 kinase inhibitor BLU-782 (IPN60130; fidrisertib) normalized the expression of the majority of dysregulated FAP genes. However, the relative efficacy of the drugs differed, and distinct sets of overlapping genes were normalized by the treatments. The substantial transcriptional changes caused by these drugs in FAPs from normally regenerating muscle were also enumerated and described. These findings define early transcriptional events associated with pathogenic FAP reprogramming and provide a molecular framework for understanding the shared and distinct effects of activin A and ACVR1 inhibition.

Prostate cancer (PC) bone metastases (PCBM) are a debilitating disease morbidity that primarily affects the axial skeleton. PCBM are predominantly osteosclerotic with mixed osteosclerotic/osteolytic features, including irregular bone deposition, loss of collagen alignment and increased porosity that mimic features of woven bone formed during development and repair. We hypothesize that progression of PCBM is driven by interactions between tumor cells and the bone microenvironment and is associated with changes in the stromal compartment. We performed tandem mass tag mass spectrometry proteomics on lumbar vertebral specimens from 32 cadaveric PCBM specimens and 3 age-matched controls. Unsupervised clustering segregated specimens into three clusters associated with benign, mixed, and osteosclerotic lesion states, characterized by distinct immune-, tumor-, and osteogenic-associated protein signatures. Histological analysis showed tumor infiltration was accompanied by marrow fibrosis and increased collagen III deposition. We identified a marker signature associated with osteosclerotic remodeling and mapped this signature to independent metastatic PC transcriptomic datasets. The proteomics-derived markers reproduced similar molecular groupings at RNA level, and robustly segregated bone PCBMs from soft tissue metastases PC in RNA data. Together, these findings define molecularly distinct states of tumor-driven changes in PCBM that extend beyond histological classification and may inform biomarker development and therapeutic stratification.

Introduction: Endothelial-mesenchymal transition (EndoMT) is a dynamic endothelial cell-state transition increasingly implicated in fibrosis, vascular dysfunction, inflammation and cancer. Despite expansion of EndoMT-focused transcriptomic research, EndoMT representation remains limited to broader epithelial-mesenchymal transition (EMT) frameworks in standard gene set repositories. Methods: To address the absence of a general-purpose EndoMT gene set for transcriptomic enrichment analysis, we developed an open EndoMT gene set resource and accompanying R package. A literature-informed EndoMT gene set was curated and directionally assigned to EndoMT UP and EndoMT DOWN signatures according to reported or hypothesised expression during partial EndoMT, defined here as an active transitional state rather than a complete mesenchymal endpoint, to improve capture of EndoMT-associated transcriptional changes across different stages of the process. Genes were annotated by marker subtype and implemented as Gene Matrix Transposed files distributed through the R package, together with exported R objects and gene-level metadata. Benchmarking was performed using bulk RNA-seq data sets GSE118446, comprising seven-day IL-1l{beta}/TGF-{beta}2-treated human umbilical vein endothelial cells (HUVECs) and human pulmonary arterial endothelial cells, and GSE342578, comprising three-day TGF-{beta}1/2/3-treated HUVECs; single-cell RNA-seq data set GSE237895, comprising seven-day TGF-{beta}1-treated and SNAI1-overexpressing HUVECs; and endothelial cell and cancer-associated fibroblast populations from human metastatic melanoma single-cell RNA-seq data set GSE72056. Results: In GSE118446, EndoMT UP was positively enriched and EndoMT DOWN negatively enriched in chronically IL-1{beta}/TGF-{beta}2-treated endothelial cells, concordant with experimentally validated EndoMT induction, while partial EndoMT-associated enrichment was detected in single ligand-treatment. In contrast, EndoMT UP was negatively enriched following acute TGF-{beta}1/2/3 treatment, suggesting temporal differences between acute TGF-{beta} stimulation and chronic EndoMT induction. In GSE237895 mesenchymal-like endothelial cells, EndoMT UP was positively enriched and EndoMT DOWN negatively enriched in both TGF-{beta}1-treated and SNAI1-overexpressing models. EndoMT gene sets also discriminated endothelial and mesenchymal/stromal transcriptional states in GSE72056 metastatic melanoma. Marker subtype and Gene Ontology Biological Process analyses provided additional exploratory resolution of EndoMT-associated components beyond MSigDB Hallmark EMT. Discussion: The EndoMT R package provides a dedicated, accessible and interoperable resource for EndoMT-focused transcriptomic enrichment analysis. Benchmarking demonstrates utility for detecting established, partial and divergent EndoMT-associated states, supporting broad applicability across EndoMT biology research.

Kidney disease affects over 850 million people and leads to degradation of the kidney cortical compartments and kidney failure. Interstitial fibrosis and tubular atrophy (IFTA) is a universal feature of chronic kidney disease, yet the sequence of tubulointerstitial matrix protein (matrisome) changes that underlie the development and progression of IFTA are not fully understood. In particular, post-translational modifications which may stabilize key matrisome components and result in irreversible and functionally detrimental interstitial fibrosis or tubular basement membrane thickening, have yet to be characterized. Post-translational modifications (e.g., acetylation) have been shown to contribute to kidney diseases, yet they have not been investigated in chronic kidney disease progression. Herein, we established feasibility of matrisome protein quantification in the tubulointerstitium, with concurrent matrisome protein post-translational modification quantification, in archived diagnostic human kidney biopsies, using laser capture microdissection, liquid chromatography tandem mass spectrometry (LC-MS/MS), and bioinformatics analysis. In this pilot study, we focused on two conditions associated with IFTA progression: diabetic nephropathy and kidney transplantation. We examined both healthy and chronically damaged tubulointerstitium in two human kidney biopsies, one representing each condition. We identified signature protein markers of tubulointerstitium (e.g., UMOD, SLC12A1) in abundance, confirming sample composition, and observed uniformity in protein abundance across replicates from the same biopsies. We found increases in certain collagen isoforms (COL1A1, COL6A3) and other ancillary proteins in areas of IFTA. We also successfully quantified post-translational modifications, confirming that hydroxylproline was the most abundant modification in healthy tubulointerstitium from the kidney transplant biopsy, and that hydroxyllysine was also highly abundant and could be confidently quantitated on two collagen isoforms (COL1A1, COL4A2). Lastly, we performed power analysis to determine the required sample size to achieve significant power and false discovery rate. Several proteins exhibited large Hedge effect sizes in the tubulointerstitium (ANPEP, PRODH2), indicating their potential usefulness as fibrosis signatures. Our study is a first step toward comprehensive characterization of tubulointerstitial matrisome changes, including post-translational modifications seen with IFTA progression. Subsequent studies will potentially reveal new molecular markers of key early events in chronic kidney disease progression, and new therapeutic targets for mitigating this process. Data are available via ProteomeXchange identifier PXD079025.

Alterations in the female urinary microbiome have been linked to several several common urinary conditions that increase in prevalence after menopause. Here we use sequencing and metabolomic data to compare the urinary bladder microenvironments between women who are premenopausal, postmenopausal, and postmenopausal using vaginal estrogen. While the urinary microbiome differs between pre- and postmenopausal women, at least 6 weeks of vaginal estrogen is associated with higher urinary lactobacilli and fewer opportunistic pathogens in the urinary postmenopausal microbiome, which closer approximates what is seen in the premenopausal context. Changes in urinary Lactobacillus species are correlated with vaginal pH and the same Lactobacillus species found in the vaginal microbiome tend to also be identified within the urinary bladder microbiome. Urinary metabolites differ by context (e.g., pre-, post-, and post-menopausal using vaginal estrogen), but the majority of metabolites that drive these differences are unannotated. Of the metabolites with shared associations among different Lactobacillus species, L. iners have consistently inverse relationships compared to L. crispatus, L. gasseri, or L. jensenii.

Objective: To determine how fibronectin fragment (Fn-f), a matrikine that induces an osteoarthritis (OA)-like phenotype, reshapes chondrocyte states and transcriptional programs at single-cell resolution. Design: Primary human ankle chondrocytes from two cadaveric donors without OA were treated with PBS or purified recombinant Fn-f for 18 hours and profiled by single-cell RNA sequencing. Transcriptional states were characterized by marker genes and functional enrichment, compared with classifications used for OA chondrocytes, and evaluated for Fn-f-dependent changes in abundance and transcriptional programs. Results: Five transcriptionally distinct populations were identified with features that matched previous classifications of effector, inflammatory, pre-hypertrophic/fibrochondrocyte, reparative, and homeostatic chondrocytes. Fn-f markedly redistributed cells among these states, enriching inflammatory chondrocytes while reducing effector and reparative populations. Within transcriptional states, Fn-f differentially altered matrix/cartilage, inflammatory, and stress/immediate-early programs. These responses did not uniformly correspond to assigned subtype identities, demonstrating that transcriptional programs can be dynamically deployed across chondrocyte states. Conclusions: Controlled perturbations of healthy chondrocytes complement observational OA atlases by resolving stimulus-dependent changes in chondrocyte state abundance and transcriptional programs. Discordance between subtype annotations and dynamic functional programs further highlights the need for harmonized definitions of chondrocytes across phenotypic states.

Key plasma protein, including albumin, complement system proteins, and coagulation factors circulate in biochemically and functionally distinct isoforms, or proteoforms. Despite the importance of these proteoforms being widely accepted, they are challenging to resolve systematically, and as a consequence, we still lack a more complete picture of the total proteoform diversity of the human plasma proteome. Here, we generated 3,114 intact protein fractions by extensively separating plasma according to isoelectric point and molecular weight. We then analysed these fractions by liquid chromatography data-independent acquisition mass spectrometry (DIA-MS) upon tryptic digestions, and combined the resultant data into a multidimensional, proteoform resolved atlas of the human plasma proteome. We report that 5,077 unequivocally detected plasma proteins circulate at least 179,049 distinct proteoforms, of which we detect 44,083, with a median of 11 proteoforms per protein, at high confidence. Substantially increasing the number of experimentally confirmed proteoforms, our atlas implies that the canonical protein forms account for less than half of the total protein mass in plasma. We detect the greatest proteoform diversity among the most abundant plasma proteins and those functioning in nutrient transport, complement and coagulation. Finally, our data clarifies discrepancies between the Olink and SomaScan proteomic platforms, as according to our atlas, these techniques provide correlated values for relatively abundant proteins with low to medium proteoform diversity, but struggle in the quantification of low-abundant proteins and on those which exist in many proteoforms. In sum, our data reveals extensive proteoform diversity of the human plasma proteome, and implies that proteoform diversity represents an enormous untapped reservoir for biomedical research and biomarker discovery.

Aging is associated with impaired skin wound healing, but the molecular mechanisms underlying this decline remain incompletely understood. In particular, how aging affects the temporal regulation of mechanosensitive signaling during tissue repair is poorly understood. In this study, we investigated age-associated changes in cutaneous wound healing, with a focus on Yes-associated protein (YAP)-associated mechanosensitive responses, and examined whether ICE mice, an inducible model of epigenetic aging, reproduce age-associated impairment in wound repair. Splinted full-thickness excisional wounds were generated in young and naturally aged mice, and wound closure, gene expression, and active YAP were evaluated over time. Aged mice exhibited delayed wound closure, particularly during the mid-to-late phases of repair. Expression of the fibrosis-related genes Tgfb1, Col1a1, and Col3a1, and the YAP-associated genes Ctgf, Cyr61, and Arhgap18 did not differ significantly between young and aged wounds at the examined time points. In contrast, Ankrd1 was selectively elevated in aged wounds at postoperative day 14. Active YAP immunofluorescence showed age-associated differences in staining patterns during wound healing, indicating altered regulation of mechanosensitive signaling with aging. Furthermore, ICE mice exhibited delayed wound closure compared with Cre control mice, reproducing a major functional feature observed in naturally aged mice. These findings suggest that age-associated impairment of wound healing is characterized not by uniform activation or suppression of YAP-related signaling, but by altered temporal regulation of mechanosensitive responses together with selective Ankrd1 elevation. ICE mice could provide a useful experimental model for investigating age-associated defects in cutaneous wound healing.

Understanding the pathological significance of protein glycosylation and translating disease-associated glycan alterations into diagnostic and therapeutic opportunities require integrated analysis of glycans, their carrier glycoproteins, and spatial context. Here, we present an expanded multimodal glycoprotein analysis framework incorporating lectin-assisted imaging mass cytometry (Lectin-IMC) for stepwise discovery and spatial validation of disease-associated glyco-niches. Disease-associated glycans identified by laser microdissection-assisted lectin microarray (LMD-LMA) tissue glycome mapping are spatially evaluated by Lectin-IMC in relation to cell types and tissue microenvironments, and candidate carrier glycoproteins identified by MS-based glycoproteomics are subsequently incorporated for higher-order evaluation of glycan-protein-cell type relationships. Multiplex panel design is guided by LMA-based assessment of lectin-lectin interactions and biologically informed probe selection. Using a dilated cardiomyopathy model, we constructed a five-lectin panel centered on Wisteria floribunda agglutinin (WFA), which recognizes fibrosis-associated asialo N-glycans identified previously. Glycan-dependent WFA detection was validated by competitive inhibition and PNGase F treatment, and WFA-reactive glycans were spatially associated with fibrotic regions containing ACTA2VIM myofibroblast-like cells. Among six extracellular matrix glycoprotein candidates identified in WFA-binding fractions, periostin showed the strongest spatial correspondence with WFA-positive regions by pixel-based quantitative analysis and was further associated with ACTA2VIMWFA fibrotic microenvironments. The optimized Lectin-IMC panel was also transferable to a human FFPE cardiomyopathy specimen. Collectively, Lectin-IMC provides intuitive spatial visualization and interpretability of glycan-protein-cell type relationships and serves as an on-tissue spatial validation and prioritization layer within an iterative multimodal framework in which spatially defined glyco-niches can guide subsequent proteomic or glycoproteomic discovery.

Purpose: The neural retina and retinal pigment epithelium/choroid (RPE/Cho) are metabolically coupled. Metabolic dysfunction of the RPE/Cho contributes to photoreceptor death in age-related macular degeneration (AMD). This study aimed to determine how aging, a major risk factor for AMD, alters the proteome and metabolic pathways in the retina and RPE/Cho. Methods: We performed quantitative proteomics on neural retina and RPE/Cho from young (8-week) and old (73-week) C57BL/6J mice. The differentially expressed proteins between old and young, as well as between the retina and RPE/Cho, were visualized using volcano plots and heatmaps, and functional analyses were assessed by Gene Ontology enrichment tools. Results: Aging altered the expression of 85 retinal proteins and 272 RPE/Cho, with 16 shared between tissues. In the aged retina, proteins associated with NADP(H) metabolism, neurodegeneration, and immunity were mostly upregulated, while proteins in lipid metabolism, transcription, and epigenetic regulation decreased. In the aged RPE/Cho, all 21 significantly altered lysosomal enzymes and transport proteins increased, together with complement components, and photoreceptor outer segment proteins. These changes accompanied increases in proteins associated with lipid metabolism and protection against lipid peroxidation, while several glutathione metabolism enzymes decreased. Seven shared proteins involved in lipid and NADP(H) metabolism, changed in opposite directions between retina and RPE/Cho. Comparison of the retinal and RPE/Cho proteomes identified 3073 differentially abundant proteins, of which 2452 maintained tissue-specific differences across both age groups, while approximately 17% showed age-dependent shifts in tissue enrichment. These shifts involved proteins associated with metabolism, extracellular matrix, visual function, lysosomal pathways, and innate immunity. Within the metabolic proteome, several glycolytic and fatty acid oxidation proteins showed greater relative enrichment in RPE/Cho with age, whereas glucose transporters and mitochondrial complexes shifted toward the retina. Conclusions: Overall, these findings show that aging differentially remodels the retina and RPE/Cho proteome, accompanied by altered metabolic specialization between the two tissues.

A persistent challenge in cartilage tissue engineering is identifying cell sources capable of regenerating cartilage with native-like properties. Variability in chondrogenic potential among human- and animal-derived cells complicates graft development and disease modeling. Here we benchmarked ten chondrogenic cell sources under standardized culture conditions using hyaluronan-alginate biomaterials and implementing a functionality index combining histological and mechanical metrics referenced to native tissue. Mesenchymal stem cells and fetal chondroprogenitors demonstrated superior regenerative capacity, achieving 103% and 95% of native cartilage functionality, respectively. In contrast, adult articular chondrocytes from healthy and osteoarthritic donors produced fibrocartilaginous tissue with functionality scores below 50%. These findings provide a quantitative framework to guide cell source selection and optimize cartilage graft standardization for clinical applications and physiologically relevant disease models, addressing regulatory demands for product characterization.

Macrophages, alongside mammary epithelial cells, are major contributors to the innate immune response triggered by bacterial invasion of the mammary gland. To investigate breed-specific immune mechanisms, we stimulated monocyte-derived macrophages from Holstein and Normande cattle with Escherichia coli and Streptococcus uberis, two major mastitis pathogens. We monitored bacterial survival using [methode] and profiled gene expression via RNA-seq. E. coli exhibited significantly higher survival 6 hours post-infection compared to S. uberis (p < 0.05). Moreover, E. coli induced a robust transcriptional response, with 4403 differentially expressed genes (FDR < 0.05, |log2FC| > 1), versus only 813 for S. uberis. Qualitative differences were also observed: genes such as CCL2 and CSF1 were uniquely induced by S. uberis. Additionally, macrophages from Holstein and Normande cattle showed distinct expression profiles: 185, 45, and 174 genes were differentially expressed at steady state or after E. coli or S. uberis stimulation, respectively. These genes were enriched in immune-related pathways. Overall, our results demonstrate a stronger response to E. coli, although S. uberis specifically induces a subset of genes. The observed breed-specific differences may reflect underlying immune mechanisms that could influence disease susceptibility, warranting further functional validation.

Current treatment of orthopedic ligament injury relies principally on non-regenerative materials and surgical repair rather than strategies that promote intrinsic tissue regeneration. This study evaluated whether biologic augmentation with XWRAP, a human amniotic membrane allograft, combined with a cryopreserved amniotic-fluid-derived biologic containing micronized human amnion membrane, promotes regeneration of ligament architecture in a rabbit medial collateral ligament (MCL) gap-defect model. Standardized 4-mm mid-substance MCL gap defects were created in New Zealand White rabbits (n=3) and either treated with the amniotic membrane/amniotic-fluid biologic combination or left untreated, with intact contralateral or adjacent MCL serving as native controls; ligaments were evaluated by transmission electron microscopy at 2 and 6 weeks post-procedure, with collagen fibril diameter quantified from calibrated high-magnification micrographs. Treated defects developed significantly larger mean collagen fibril diameters than untreated defects at both time points (75 vs. 50 nm at 2 weeks and 88 vs. 62 nm at 6 weeks; p<0.0001 for each comparison), reaching approximately 90% of native, uninjured ligament fibril diameter (98 nm) by 6 weeks. Treated defects also developed a broader, bimodal fibril-diameter distribution resembling native ligament architecture, whereas untreated defects remained characterized by a narrow, unimodal population of small-diameter fibrils typical of scar-associated repair. These findings indicate that biologic augmentation with XWRAP combined with an amniotic-fluid-derived biologic promotes a regenerative, rather than purely reparative, healing response following ligament injury, progressively restoring collagen fibril architecture toward that of native tissue in this preclinical model.

Osteoarthritis (OA) and systemic sclerosis (SSc) are diseases with distinct etiologies but similar disease phenotypes, most prominently fibrosis, a process governed by the pathological activation of fibroblasts. Mechanisms underpinning fibrosis in these diseases are widely studied, though have yet to be directly compared. We employed single-cell RNA-seq and high-resolution spatial transcriptomics to describe the functional plasticity and the range of fibroblast activation states in both diseases. We identified disease-enriched fibroblast subsets conserved in both diseases, including fibrogenic myofibroblast-like cells, chemokine- and cytokine-expressing inflammatory fibroblasts, and lining-like fibroblasts. These subsets exhibited convergent fibrotic programs also observed in idiopathic pulmonary fibrosis, as well as overlapping osteochondral transcriptional programs. Xenium 5K spatial transcriptomics revealed that conserved, disease-associated fibroblasts reside in highly similar anatomic niches in OA synovium and SSc skin. These consisted of a fibrotic ECM-rich niche harboring myofibroblast-like cells and an inflammatory niche containing inflammatory fibroblasts adjacent to infiltrating immune cells. Disease-associated transcriptional remodeling converged on shared functional pathways and common putative upstream molecular regulators. These findings identify conserved cellular states, spatial niches, and regulatory programs across OA and SSc, suggesting that fibrotic disease is underpinned by shared pathogenic stromal states and candidate targets for therapeutic strategies are applicable across both diseases.

Background: Platelets are critical drivers of thrombo-inflammatory responses in different cardiovascular diseases. Abdominal aortic aneurysm (AAA) is a progressive, life-threatening vascular disorder mainly characterised by chronic inflammation, extracellular matrix degradation, and the formation of a platelet-rich intraluminal thrombus (ILT). Experimental and clinical evidence identified platelets as main players in AAA pathology as evidenced by elevated platelet activation and procoagulant activity that critically contribute to AAA progression. Methods: The present study investigated the contribution of glycoprotein (GP)Ib alpha, the von Willebrand factor (VWF)-binding subunit of the platelet GPIb-IX-V complex, to AAA initiation and progression in experimental AAA using the ePPE mouse model and in patients. Results: Genetic ablation of platelet GPIb alpha significantly attenuated early aneurysm expansion in experimental AAA, indicating a critical role for GPIb alpha during the initial stages of aneurysm development. This initial effect was compensated at later time points showing no differences in aneurysm progression between groups. Notably, genetic deletion of GPIb alpha induced a constitutively hyperactive platelet phenotype already in naive mice that was further amplified during experimental AAA. This elevated platelet hyperactivity was mainly due to increased GPVI activation of platelets 28 days post-surgery. To assess the clinical relevance, spatial profiles of human ILT specimens from patients with AAA were analysed. In the ILT, we detected a highly compartmentalised distribution of GPIb alpha and VWF with pronounced enrichment within the luminal layer. In parallel, circulating VWF activity as well as platelet surface expression of GPIb alpha were significantly increased in patients with AAA. Conclusion: Collectively, these findings identify a dysregulated GPIb alpha-VWF axis in human AAA pathology, mainly characterised by enhanced platelet GPIb alpha surface expression and increased activity of circulating VWF.

Aged skeletal muscle has decreased ability to rebound from physiological stressors such as disuse atrophy. We previously observed that older adults during recovery following muscle disuse were characterized by rapid skeletal muscle immune cell expansion, cellular senescence and collagen deposition, responses that were biased toward older women. Here we used single-nucleus RNA sequencing and complementary in vitro muscle primary cell experiments in young (YF; 22[plusmn]3y; n=8) and older females (OF; 66[plusmn]5y; n=9) to investigate age-mediated cellular function and intercellular communication during recovery from disuse atrophy. Compared with YF, OF exhibited a markedly greater transcriptional response at 7d-recovery (YF: 1,548 DEGs, OF: 7,999 DEGs), driven primarily by slow- (YF: 96 DEGs, OF: 984 DEGs) and fast-twitch myonuclei (YF: 194 DEGs, OF: 1,234 DEGs), satellite cells (YF: 548 DEGs, OF: 2,784 DEGs) and FAPs (YF: 326 DEGs, OF: 2,102 DEGs). Satellite cells from OF demonstrated collagen signatures including elevated THBS1 expression and enrichment of TGF-[beta] signaling. Concurrently, OF FAPs exhibited increased expression of fibroblast activation marker ADAMTS14. Furthermore, NicheNet analyses unmasked altered FAP-satellite cell communication in OF. Complementary In vitro experiments revealed that myogenic progenitor cells collected at 7d-recovery from OF (vs YF) donors displayed impaired myogenic differentiation and a cellular senescence-associated phenotype, while fibroblasts exhibited greater myofibroblast-like activation. Conditioned media derived from OF fibroblasts at 7d-recovery further increased cellular senescence and impaired myogenic potential (vs YF fibroblast conditioned media). Collectively, these findings suggest that recovery from disuse atrophy in older females is characterized by altered FAP-satellite cell communication and intrinsic function which may contribute to poor muscle remodeling.

Aims: Mature, contractile vascular smooth muscle cells (vSMC) in muscular blood vessels can de-differentiate into synthetic vSMC, while human pluripotent stem cells (hPSC) differentiate into proliferating, fetal-like vSMC that can be induced to form contractile vSMC. Given the complicated embryonic origins and conflicting views on vSMC heterogeneity, our aim was to establish how closely hPSC-vSMC of a known germ layer lineage and defined phenotype are representative of vSMC obtained from aorta (AoSMC). Methods and Results: We generated paraxial mesoderm (PM)-derived synthetic and contractile vSMC from hPSC lines and performed phenotypic and mechanical comparisons with AoSMC. Synthetic hPSC-vSMC and cultured AoSMC are proliferative, sensitive to contact inhibition and exhibit phenotype switching. Contractile hPSC-vSMC are non-proliferative, capable of phenotype switching and have elevated levels of contractile proteins (CNN1, TAGLN, ACTA2). These contractile proteins also displayed density-dependent, cell-to-cell heterogeneity in both AoSMC and hPSC-vSMC. Single-cell traction force measurements on micropost array detectors (mPADs) yielded comparable forces per micropost in the nN range across cell types. Quantitative 3D force measurements on humanized smooth muscle cell microtissues (SMTs) developed similar forces per cell. Larger forces were generated by MEKi-treated hPSC-vSMC and AoSMC versus controls. Conclusions: We have systematically studied the properties of hPSC-vSMC and performed comprehensive phenotypic and mechanical comparisons with AoSMC. These results demonstrate that synthetic hPSC-vSMC generated in vitro and cultured AoSMC derived from tissues are comparable and mechanically similar, but results with hPSC-vSMC are more reproducible.

Hepatic stellate cells (HSCs) are the key cellular drivers of liver fibrosis. During liver injury and chronic inflammation HSCs adopt an activated phenotype and secrete fibrotic extracellular matrix (ECM) components such as collagen 1. Mechanical cues derived from the fibrotic ECM drive and support the activation of HSCs, via mechanisms that involve integrins and the mechano-sensitive transcriptional regulator YAP1. It is not yet well understood how external mechanical cues are translated into a molecular response that alters YAP1 nuclear shuttling. There is evidence that suggests the PDZ and LIM domain protein (PDLIM) 5 can regulate YAP1 shuttling in human epithelial cells. We therefore investigated whether PDLIM5 is expressed in HSCs and contributes to YAP1 associated HSC mechano-activation. PDLIM5 protein was detected in HSCs in fibrotic human and mouse liver. PDLIM5 transcript and protein were expressed by primary human and mouse HSCs and by the immortalised HSC LX-2 cell line. PDLIM5 localised with actin stress fibres suggesting a role in HSC adhesion. Co-immunoprecipitation and proximity ligation in LX-2 cells support an association between PDLIM5 and YAP1. We used pharmacological (paclitaxel) and genetic (siRNA and CRISPRi) approaches to inhibit PDLIM5 in HSCs. Inhibiting PDLIM5 reduced YAP1 nuclear localisation and fibrotic gene (COL1A1, ACTA2) expression in LX-2 cells. Overall, these data support a role for PDLIM5 in regulating YAP1 localisation and fibrogenic gene expression in HSCs.