Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder caused by mutations in the gene encoding fibrillin-1 (FBN1), the main component of extracellular microfibrils. In the aortic wall, these microfibrils maintain structural integrity and sustain hemodynamic load. Pathogenic FBN1 variants are thought to structurally and functionally impair fibrillin-1 microfibrils, leading to progressive aortic aneurysm and dissection, the major causes of morbidity and mortality in MFS. However, the molecular mechanisms whereby these genetic variants translate into structural and mechanical defects are far from being understood.Here we explored the morphology and the nanomechanical characteristics of individual aortic fibrillin-1 microfibrils from MFS patients and non-MFS controls by atomic force microscopy. The topographical assessment revealed a preserved overall pattern and periodicity of the microfibrils, but with morphological irregularities in MFS microfibril beads and interbead segments, consistent with presumed structural fragility. Force spectroscopy revealed a reduction of transverse elastic modulus in patients harboring haploinsufficient FBN1 variants. Nanoindentation analysis was indicative of localized deformation, occurring at markedly lower forces in MFS microfibril beads, suggesting diminished load-bearing capacity.These data provide direct nanoscale evidence of structural and mechanical consequences of FBN1 mutations on human aortic tissue. Altered fibrillin-1 microfibril morphology and reduced stiffness in MFS support a pathogenetic mechanism in which compromised microfibrillar integrity weakens the aortic wall, predisposing it to progressive dilation.
Background: Glaucoma is a leading cause of blindness characterized by progressive degeneration of the optic nerve. Elevated intraocular pressure, typically associated with glaucoma, exerts biomechanical strain on the optic nerve head, where mechanosensitive astrocytes contribute to profibrotic extracellular matrix remodeling. Cross-linking enzymes such as transglutaminase 2 play a role in this process. A unique function of transglutaminase 2 is its ability to catalyze the post-translational modification of proteins through the transamidation of serotonin onto glutamine residues ("serotonylation"). The serotonylation of extracellular matrix proteins, such as fibronectin, could plausibly be involved in extracellular matrix remodeling. Here, we investigated whether optic nerve head astrocytes could serotonylate extracellular proteins in response to a biomechanical insult. Results: Primary human optic nerve head astrocytes subjected to 0-12% cyclic stretch for 24 h exhibited increased extracellular levels of transglutaminase 2 and serotonin. Correspondingly, serotonylation of immunoprecipitated fibronectin samples, detected by immunoblotting, was 2.5-fold higher in stretched cells. Serotonylation of specific glutaminyl residues of fibronectin due to the catalytic effect of transglutaminase 2 was also proven though an in vitro experiment using nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS). Conclusions: These findings demonstrate that fibronectin can be serotonylated by optic nerve head astrocytes and that this modification is enhanced by biomechanical stress. While transglutaminase 2 is classically associated with extracellular matrix cross-linking, these results suggest that serotonylation may represent an additional and unexplored mechanism by which transglutaminase 2 contributes to extracellular matrix remodeling in the glaucomatous optic nerve head.
Hutchinson–Gilford progeria (HGPS) is a rare genetic disorder characterized by clinical features that mimic accelerated aging. The classical form of progeria is caused by a heterozygous pathogenic variant in in the LMNA gene resulting in the truncated lamin A protein progerin that accumulates in the nuclear envelope and exerts toxic effects on connective tissue and bone growth. Although connective tissue abnormalities are a hallmark of the disease, the extracellular matrix (ECM) produced by HGPS fibroblasts has not been systematically characterized. Here, we combined analysis of four RNA-seq datasets with functional and secretome analyses of primary dermal fibroblasts from an infant with HGPS. Transcriptomic analyses identified enrichment of basement membrane–related pathways and consistent dysregulation of the matrisome gene COL4A1. Patient fibroblasts recapitulated canonical HGPS phenotypes, including progerin accumulation, altered nuclear morphology, reduced proliferation, early senescence, and changes in cell mechanics. Secretome proteomics revealed selective downregulation of core basement membrane components, including collagen IV chains with corresponding transcriptional reductions and aberrant collagen IV deposition in vitro. In 3D skin equivalents, HGPS fibroblasts failed to support proper dermo-epidermal basement membrane assembly, a defect mirrored by reduced collagen IV staining in patient skin tissue. Together, these findings identify impaired basement membrane composition and organization as a previously underappreciated feature of HGPS and suggest that ECM remodeling may contribute to cutaneous pathology and potentially broader disease manifestations.
Tendons are essential connective tissues that transmit mechanical forces from muscles to bones, enabling locomotion and maintaining joint stability. Proper mechanosensation is critical for preserving their extracellular matrix (ECM) integrity. PIEZO1, a mechanosensitive ion channel, has been implicated in regulating tendon architecture under increased mechanical loads, but its role under baseline physiological conditions remains unclear. Here, we generated tendon-targeted Piezo1 conditional knockout (Piezo1 p-t-ko ) mice using a tamoxifen-inducible Scx-CreERT2 system. All in vivo experiments were performed in female mice. PIEZO1 deficiency resulted in significantly reduced Achilles tendon thickness and smaller collagen fibril diameters by transmission electron microscopy. RNA sequencing and qPCR analyses demonstrated downregulation of key ECM-related genes, including Col1a1, Dcn and Fmod, as well as the tendon transcription factors Mkx. Immunostaining further showed reduced signals of MKX, COL1A1 and DCN. Adjacent muscle morphology and transcriptomes were unaltered, supporting the tendon-selective nature of the observed phenotype. Together, these findings suggest that PIEZO1 contributes to collagen fibril architecture and tendon-associated transcriptional programs in female mouse Achilles tendons under baseline physiological conditions.
Pancreatic ductal adenocarcinoma (PDAC) is defined by a dense, collagen-rich stroma that limits therapeutic efficacy and drives metastasis. While collagen reorganization has been studied in resected PDAC tumors, its structure and organization in metastatic PDAC and response to chemotherapy remains poorly characterized. We evaluated collagen structure by Second Harmonic Generation (SHG) imaging of formalin fixed paraffin embedded tissue from pancreatic tumors and liver metastases of 20 PDAC patients that were untreated or treated with FOLFOX or FOLFIRINOX (FOL). Collagen fiber width, length, alignment, and density were assessed at tumor cores, tumor boundaries, and in the adjacent tissue. We document for the first time fundamental differences in these collagen structure features between normal pancreas and normal liver. Surprisingly, the structure of collagen in primary pancreatic cancer was similar to normal pancreas. Liver metastases showed alterations in collagen structure, revealing a transition from a liver-like collagen phenotype (thinner, shorter less dense collagen fibers) at the tumor border to a tumor-core phenotype with thicker, longer, less aligned, and denser collagen fibers. FOL treatment increased length, width, and density at both primary tumors and liver metastases. Overall, these findings reveal organ specific collagen remodeling in PDAC and suggest that chemotherapy with FOL modulates extracellular matrix (ECM) remodeling. These findings expand the scope of PDAC collagen structural characterization and highlight the complexity of matrix targeting strategies in metastatic disease.
Collagens, long regarded as structural molecules, also regulate stress responses and longevity. In this study, we analyzed our RNA sequencing data and publicly available gene expression data to define their role in Caenorhabditis elegans aging. Collagen expression broadly declined with age, with 16 collagen genes consistently downregulated across independent studies, establishing collagen downregulation as a genetic hallmark of aging. In contrast, meta-analysis of 66 datasets (128 comparisons between normal and long-lived animals) showed collagen upregulation in 84% of long-lived conditions, identifying collagen induction as a conserved signature of lifespan extension. Using π-values to integrate fold change and significance of collagen gene expression, we applied K-means clustering and identified Euclidean-based clusters that captured functional, tissue-associated subsets of collagens. Notably, aging-associated collagens were strongly enriched in Euclidean Cluster 1, which overlapped with hypodermal collagens, while Cluster 2 significantly intersects with lifespan-extension and intestine-enriched subsets, and Cluster 3 likely represents structural collagens contributing to cuticle and muscle integrity. These results indicate that collagen genes grouped by expression-based clustering are not randomly distributed but instead reflect tissue-specific patterns and functionality. Together, our findings suggest that collagens are dynamic regulators of aging and longevity in C. elegans. Given the conservation of extracellular matrix biology across species, collagens represent candidate biomarkers and targets for promoting healthy aging in both C. elegans and higher animals.
Healthy tendon is associated with organized and aligned extracellular matrix (ECM) that becomes disorganized with disease, yet the mechanisms and pathogenesis of tendon disease remain poorly understood. Tendons that “wrap-around” joints, such as the flexor digitorum longus (FDL) tendon, contain a unique ECM that shares similarities with diseased tendons, which can be leveraged to study tendon cell biology across distinct loading environments. The pericellular matrix (PCM) is a critical matrix structure that is understudied in tendon and is likely involved in tendon mechanosensation and homeostasis. Two components of the tendon PCM, biglycan and collagen VI, are known regulators of tendon function and are implicated in tendon disease. Given the implication of PCM molecules in regulating tendon properties, this work sought to define the regional development of a murine wrap-around tendon, how biglycan influences these regional properties, and the extent to which these mechanisms involve collagen VI. Gene expression and histological analyses demonstrated regional divergence in FDL tendon properties by P14. While biglycan knockout did not result in broad disruptions to gene expression or the behavior of Scx+ or Col6a1+ cells, several genes were dysregulated with biglycan deficiency. These changes corresponded with inferior mechanical properties in biglycan-deficient tendons. This work defines the development of regional FDL tendon properties and demonstrates that biglycan knockout impacts these regional properties through a collagen VI-independent mechanism. Results from this work provide understanding of biglycan regulation in tendon and lay the foundation for future work researching tendon mechanosensitive mechanisms.
The extracellular matrix (ECM) critically regulates fibroblast behavior during tissue repair and regeneration. However, how culture dimensionality influences fibroblast-mediated ECM remodeling remains unclear. This study investigated the effects of three-dimensional (3D) fibrin hydrogels on the phenotype and remodeling activity of primary human gingival fibroblasts (GFs) compared to conventional two-dimensional (2D) monolayer cultures. Live/dead staining confirmed high GF viability in both conditions, with elongated and branched cell morphologies in 3D fibrin hydrogels, contrasting with spindle-shaped cells in 2D monolayers. Hematoxylin and Eosin, and Masson’s Trichrome staining revealed progressive fibrin degradation and de novo collagen deposition over 21 days of culturing. Gene expression analysis showed that while FN1, COL1A1, and COL3A1 levels remained relatively stable, TGFB1 expression increased significantly from day 7 to day 14 in 3D hydrogels (p < 0.05) and was higher than in 2D cultures at day 21 (p < 0.05). This coincided with a marked upregulation of ACTA2 (p < 0.01), indicating myofibroblast-like differentiation. MMP-2 activity increased significantly over time in both 2D and 3D cultures (p < 0.01 and p < 0.001, respectively). In contrast, PLAU and PLAT expression decreased significantly at days 14 and 21 (p < 0.001 and p < 0.05, respectively), reflecting a temporal shift from fibrinolytic to collagenolytic remodeling. Despite active remodeling, mechanical testing showed no significant changes in hydrogel stiffness or relaxation between day 1 and day 7, or between cell-seeded and acellular gels (p > 0.05), likely due to the small contractile forces generated by the cells relative to the gel’s bulk modulus. Together, these findings demonstrate that 3D fibrin hydrogels provide a biologically active and physiologically relevant microenvironment that supports fibroblast-mediated ECM remodeling, offering a biomimetic model for investigating the mechanobiology of periodontal and peri-implant soft tissue regeneration.
The process of aging is an integral but complex component of life that has been intensely studied for decades, from the molecular level to whole organisms. At the tissue level, bone is one of the most difficult to study due to its composite nature of inorganic and organic phases, but advancements in proteomics are enhancing our understanding of the latter, improving our understanding of skeletal aging through identifying temporal changes across the bone proteome. The relative longevity of extracellular matrix (ECM) proteins can make them more susceptible to accumulating damage modifications over time. In addition, their informational density, including their 2D and 3D structure, protein folding, post translational modifications and proteomic composition, as well as their functional importance, has made them a target of interest in the study of aging and medical conditions such as osteoporosis and arthritis. ECM proteins are also increasingly utilised in forensic science for determining biological sex/age because of their longevity. Following recent developments in peptide location fingerprinting methods that improve capabilities of identifying regional changes in protein structure, this study aimed to identify age-associated regional changes along protein structures in Rattus norvegicus from whole limb LC-MC/MS data. Regional changes in protein structure were identified in a variety of collagenous and non-collagenous ECM proteins, providing evidence for increased remodeling in juvenile rats and a reduced ability in adult rats, alongside damage accumulation in the adult ECM. This research highlights the importance of fibrillar collagen remodeling but is also indicative of potential new roles for osteopontin, thrombin, apolipoproteins and wider ECM regulators such as cartilage oligomeric matrix protein. This demonstrates, in this case, the utility of peptide location fingerprinting as a screening tool to identify biomarker candidates of bone aging between juvenile and adult rats.
Human induced pluripotent stem cells (hiPSCs) are a promising source for cell-based and regenerative therapies. In this study, we developed and optimized a chemically defined differentiation protocol to generate hematopoietic progenitor cells (HPCs) from hiPSCs. We demonstrated that basic fibroblast growth factor (bFGF) plays a crucial role in enhancing HPC differentiation, particularly when applied during both the mesoderm (ME) and hematopoietic endothelial (HE) induction stages. Additionally, we explored the use of P-LM421E8, a recombinant fusion protein of laminin421-E8 fragment with domain 1 (D1) of perlecan possessing heparan sulfate (HS) chains. Our findings indicate that P-LM421E8 effectively supports HPC differentiation, generating stable CD34-high/CD117+ populations comparable to those produced with bFGF treatment. Furthermore, we observed that HPCs generated on P‑LM421E8‑coated surfaces exhibited superior natural killer (NK) cell differentiation potential. Although both P-LM421E8 and bFGF supported HPC differentiation, no significant additive effects were observed when used together. This suggests that P-LM421E8 can effectively support HPC differentiation without the need for exogenous bFGF, possibly through enhanced interaction with endogenous FGFs. The structural properties of P-LM421E8, which facilitate retention and presentation of FGFs via HS chains on its D1, may contribute to its effectiveness in promoting HPC development. Our findings establish P‑LM421E8 as a potent matrix for HPC differentiation and highlight its promise for refining hematopoietic protocols and advancing cell‑based immunotherapies.
Prior analysis of mouse embryos homozygous for a point mutation (p.Ser149Arg) that abrogated secretion of the protease, ADAMTS6, showed that it is essential for cardiovascular and limb development. Because Adamts6S149R/S149R mice do not survive past birth, it is currently not feasible to investigate ADAMTS6 in specific cellular lineages postnatally. Therefore, we generated a conditional allele using CRISPR-Cas9-mediated genome editing to insert unidirectional loxP sites flanking the first coding exon in Adamts6, resulting in a frameshift mutation after loxP recombination. Mice homozygous for the unrecombined floxed allele (Adamts6fl/fl) are viable, fertile, and without overt phenotype. Adamts6del/del embryos, generated upon constitutive recombination induced by a CMV-Cre transgene, also do not survive past birth and have identical defects in the heart and limbs as Adamts6S149R/S149R embryos, demonstrating efficient transgene recombination. In addition to previous defects in cardiovascular and limb development, Adamts6del/del embryos have reduced airway branching, thereby identifying a role for ADAMTS6 in lung maturation. This newly generated Adamts6fl allele makes feasible analysis of ADAMTS6 secreted by cells of different lineages and during specified temporal windows during developmental processes as well as in disease models.
We aim to determine the presence and function of LTBP1 in the corneal stroma. We investigated the temporal and spatial corneal expression of LTBP1, and microfibril-associated glycoprotein 1 (MAGP1), known components of the elastic system and regulators of TGF-β storage and latency. Protein quantification -blotting and proteomics-, immunofluorescence microscopy, mRNA analysis and a luciferase assay were used to study the regulation of transforming growth factor β (TGF-β) by LTBP1 in cornea derived fibroblasts and myofibroblasts. Expression of LTBP1 and MAGP1 was found in adult corneas, and absent or minimal in the scleral stroma. Quantitative polymerase chain reaction and protein blotting analyses showed upregulation of these proteins during early postnatal development and deposition in the corneal matrix with tissue maturation. In vitro, LTBP1 regulated the conversion of fibroblasts to myofibroblasts and maintained TGF-β latent in the matrix. This study shows that LTBP1 and MAGP1, regulators of TGF-β activation and elastic fiber components, are found in adult stroma. LTBP1 is a regulator of TGF-β storage and latency and controls conversion of fibroblast to myofibroblast.
Background:Viral myocarditis (MC) is associated with extracellular matrix (ECM) remodeling and involves excessive deposition of collagen and other ECM proteins by cardiac fibroblasts, potentially leading to scarring and ECM stiffening, which may subsequently contribute to impaired cardiac functions. Clarifying whether changes in ECM fiber tension are detectable during either the acute or scarring phase could provide a novel, mechanistically relevant mechanobiological signature. Objectives:Our goal was to ask whether ECM mechano-markers can be identified in the myocardium, beyond excessive collagen fiber deposition, that are associated with the acute infection or the pathological scarring of the human heart, and how this might be associated with the infiltration of macrophages. Methods:Left-ventricular (LV) endomyocardial biopsies were obtained from patients (N = 39) with acute myocarditis MC (N = 21) including COVID-19 (N = 4) patients suspected with myocarditis MC and/or impaired LV function, dilated cardiomyopathy (N = 6), inflammatory dilated cardiomyopathy (N = 12) to specify diagnosis and treatment options. Endomyocardial biopsies were analyzed for viral genomes, immune cells infiltration and ECM remodeling. Fibronectin fiber tension was assessed using the fibronectin-binding tension-sensor (FnBPA5), while collagen fiber deposits were visualized using second harmonic generation (SHG) microscopy. Results:While fibronectin fibers were tensed in healthy hearts, histological staining with our novel tension probe FnBPA5 showed that fibronectin fibers had lost their tension in distinct loci in all patient groups. In the acute inflammatory phase (MC), loci with untensed fibronectin fibers were found in close proximity to infiltrated macrophages. In already dilated hearts, biopsies which presented low densities of infiltrated macrophages, thick collagen I/III fiber bundles as visualized by SHG were found in proximity to untensed fibronectin fibers and myofibroblasts, which together are indicative of fibrotic ECM niches where the inflammation has subsided. Comparison of clinical diagnostic and experimental histological data showed clear correlations between altered ECM niche properties and cardiac function deterioration. Conclusions:Our data suggest that relaxed fibronectin fibers are a recurrent feature of myocarditis and associate with measures of cardiac dysfunction. These findings suggest that fibronectin fiber tension might be a mechanobiological signature that warrants validation in larger, longitudinal cohorts and evaluation of in-vivo measurability.
Following resistance exercise, systemic changes foster improved functionality of tendons and ligaments. Post-exercise, muscle tissue releases exosomes that are thought to facilitate inter-tissue communication. To determine the potential role of exosomes in the exercise-induced adaptations of tendons and ligaments, we modified our engineered human ligament (EHL) model to work with exosome-enriched serum. Treatment of the EHLs with exosomes enriched from fetal bovine serum (fbEXO) resulted in enhanced ligament mechanics and increased collagen content in a dose-response fashion (maximum tensile load [MTL]: 10 %: 0.196 ± 0.138 N, 20 %: 0.278 ± 0.103 N, 40 %: 0.840 ± 0.092 N; r2 = 0.858, P < 0.0001; collagen content: 10 %: 1.073 ± 12.49 µg, 20 %: 86.43 ± 71.65 µg, 40 %: 145.7 ± 84.11 µg; r2 = 0.4735, P = 0.0046). After optimizing an exosome enriched feeding protocol using fbEXO, we confirmed that exosomes enriched from human serum (hsEXO) could sustain EHL function. Subsequently, twelve healthy, recreationally active volunteers (22 ± 3 y, 1,68 ± 0.10 m, 65.6 ± 27.8 kg; 6F/6M) performed a single bout of resistance exercise. Serum samples were collected prior to and 15 min post-exercise, and exosomes were enriched from these samples for treatment of EHLs. EHL function and collagen content did not differ when treated with hsEXO obtained at rest or post-resistance exercise (MTL: 1.30 ± 0.36 vs. 1.20 ± 0.36 N, P = 0.3950; collagen content: 424.6 ± 47.68 vs. 425.2 ± 44.46 µg, P = 0.9663). This model provides a novel way to determine the role of exosomes in connective tissue development and adaptation. The identification of circulating exercise factors that enhance tendon and ligament function remains to be fully elucidated.
Collagen cross-links mediated by the lysyl oxidase and lysyl hydroxylase families of enzymes significantly contribute to the biomechanical strength and rigidity of tissues, influencing cell signaling and the downstream cell phenotype. In the clinic, the proteolytically liberated N-terminal cross-linked peptide of collagen I (NTX) is used as a biomarker of bone and connective tissue turnover, which is altered in several disease processes. Despite the clinical utility of these collagen breakdown products, the majority of the cross-linked peptide species have not been identified in proteomic datasets. Here, we evaluate several parameters for the preparation and identification of these peptides from the collagen I-rich Achilles tendon. Our refined approach, which involves chemical digestion for protein solubilization coupled with mass spectrometry, enables the identification of NTX cross-links in a range of modification states. We then applied a spectral library approach to identify differences in collagen cross-links in bovine pulmonary hypertension. The presented method offers unique opportunities to understand extracellular matrix remodeling events in development, aging, wound healing, and fibrotic disease that modulate collagen architecture through lysyl hydroxylase and lysyl oxidase enzymes.
