Collagen-I fibrillogenesis is crucial to health and development, where dysregulation is a hallmark of fibroproliferative diseases. Here, we show that collagen-I fibril assembly required a functional endocytic system that recycles collagen-I to assemble new fibrils. Endogenous collagen production was not required for fibrillogenesis if exogenous collagen was available, but the circadian-regulated vacuolar protein sorting (VPS) 33b and collagen-binding integrin-α11 subunit were crucial to fibrillogenesis. Cells lacking VPS33b secrete soluble collagen-I protomers but were deficient in fibril formation, thus secretion and assembly are separately controlled. Overexpression of VPS33b led to loss of fibril rhythmicity and over-abundance of fibrils, which was mediated through integrin α11β1. Endocytic recycling of collagen-I was enhanced in human fibroblasts isolated from idiopathic pulmonary fibrosis, where VPS33b and integrin-α11 subunit were overexpressed at the fibrogenic front; this correlation between VPS33b, integrin-α11 subunit, and abnormal collagen deposition was also observed in samples from patients with chronic skin wounds. In conclusion, our study showed that circadian-regulated endocytic recycling is central to homeostatic assembly of collagen fibrils and is disrupted in diseases.

Collagen deposition in fibroblasts, the primary collagen-producing cells, is regulated by both macrophages and the circadian rhythm, although how these regulatory processes interact with one another is unknown. Here, we reveal that macrophage-fibroblast interaction enhances collagen deposition which requires a functional circadian rhythm. This response is also dependent on macrophage polarisation status, where M0 (naive) and M1 (pro-inflammatory) macrophages require direct cell-cell contact; whereas M2 (anti-inflammatory) macrophages utilise an additional mechanism through secreted soluble factors that strengthen circadian rhythms and significantly increase collagen deposition in fibroblasts, independent of cell-cell contact. Using mass spectrometry proteomics analysis, we identified PDGFA as a key factor in induction of circadian amplitude in fibroblasts. Crucially, macrophages lacking PDGFA showed diminished ability to modulate fibroblast circadian rhythms and collagen production. Further, in fibroblasts with impaired circadian rhythms, neither macrophages nor macrophage-secreted factors are capable of inducing collagen deposition. These results confirmed that the collagen secretory pathway is also under circadian clock control in lung fibroblasts, and demonstrated that macrophages influence collagen deposition via circadian clock mediated mechanisms. In conclusion, our findings highlight M2-derived PDGFA as a key macrophage-derived factor in extracellular matrix remodelling. ### Competing Interest Statement The authors have declared no competing interest.

Ineffective endometrial matrix remodeling, a key factor in infertility, impedes embryo implantation in the uterine wall. Our study reveals the cellular and molecular impact of human collagenase-1 administration in mouse uteri, demonstrating enhanced embryo implantation rates. Collagenase-1 promotes remodeling of the endometrial ECM, degrading collagen fibers and proteoglycans. This process releases matrix-bound bioactive factors (e.g., VEGF, decorin), facilitating vascular permeability and angiogenesis. Collagenase-1 elevates embryo implantation regulators, including NK cell infiltration and the key cytokine LIF. Remarkably, uterine tissue maintains structural integrity despite reduced endometrial collagen fiber tension. In-utero collagenase-1 application rescues implantation in heat stress and embryo transfer models, known for low implantation rates. Importantly, ex vivo exposure of human uterine tissue to collagenase-1 induces collagen de-tensioning and VEGF release, mirroring remodeling observed in mice. Our research highlights the potential of collagenases to induce and orchestrate cellular and molecular processes enhancing uterine receptivity for effective embryo implantation. This innovative approach underscores ECM remodeling mechanisms critical for embryo implantation.

Background Genetic variants in COL4A1 and COL4A2 (encoding collagen IV alpha chain 1/2) occur in genetic and sporadic forms of cerebral small vessel disease (CSVD), a leading cause of stroke, dementia and intracerebral haemorrhage (ICH). However, the molecular mechanisms of CSVD with ICH and COL4A1/COL4A2 variants remain obscure. Methods Vascular function and molecular investigations in mice with a Col4a1 missense mutation and heterozygous Col4a2 knock-out mice were combined with analysis of human brain endothelial cells harboring COL4A1/COL4A2 mutations, and brain tissue of patients with sporadic CSVD with ICH. Findings Col4a1 missense mutations cause early-onset CSVD independent of hypertension, with enhanced vasodilation of small arteries due to endothelial dysfunction, vascular wall thickening and reduced stiffness. Mechanistically, the early-onset dysregulated endothelium-dependent hyperpolarization (EDH) is due to reduced collagen IV levels with elevated activity and levels of endothelial Ca2+-sensitive K+ channels. This results in vasodilation via the Na/K pump in vascular smooth muscle cells. Our data support this endothelial dysfunction preceding development of CSVD-associated ICH is due to increased cytoplasmic Ca2+ levels in endothelial cells. Moreover, cerebral blood vessels of patients with sporadic CSVD show genotype-dependent mechanisms with wall thickening and lower collagen IV levels in those harboring common non-coding COL4A1/COL4A2 risk alleles. Interpretation COL4A1/COL4A2 variants act in genetic and sporadic CSVD with ICH via dysregulated EDH, and altered vascular wall thickness and biomechanics due to lower collagen IV levels and/or mutant collagen IV secretion. These data highlight EDH and collagen IV levels as potential treatment targets. Funding MRC, Wellcome Trust, BHF.

[This corrects the article DOI: 10.1016/j.mbplus.2024.100142.].

Collagen fibrils are the primary supporting scaffold of vertebrate tissues but how they are assembled is unclear. Here, using CRISPR-tagging of type I collagen and SILAC labelling, we elucidate the cellular mechanism for the spatiotemporal assembly of collagen fibrils, in cultured fibroblasts. Our findings reveal multifaceted trafficking of collagen, including constitutive secretion, intracellular pooling, and plasma membrane-directed fibrillogenesis. Notably, we differentiate the processes of collagen secretion and fibril assembly and identify the crucial involvement of endocytosis in regulating fibril formation. By employing Col1a1 knockout fibroblasts we demonstrate the incorporation of exogenous collagen into nucleation sites at the plasma membrane through these recycling mechanisms. Our study sheds light on the assembly process and its regulation in health and disease. Mass spectrometry data are available via ProteomeXchange with identifier PXD036794.

Heavy carbon isotopes in the tendons of people who grew up in the age of nuclear bomb testing have shown that the extracellular matrix (ECM), assembled during development, stays with us for life. However, recent work suggests that type-I collagen in ECM-rich mouse tendon exists in two pools: a permanent matrix, and a more soluble, circadian-regulated matrix. Despite this, the underlying regulation of such distinct pools is not understood. Here, we demonstrate using stable isotope labelling coupled with mass spectrometry proteomics that circadian and permanent matrix pools have significantly different half-lives. Furthermore, the properties of the matrix pools are altered during development and ageing. Tail tendon tissue was harvested from mice fed on a heavy-lysine diet; protein was then extracted for analysis using a sequential two-step protocol. The first, soluble fraction (F1) was found to contain intracellular proteins, and a range of core and associated extracellular matrix proteins, including a pool of type-I collagen shown to be circadian-regulated. The remaining fraction (F2) contained primarily collagens, including type-I collagen which did not show rhythmicity. In adult mice, matrix proteins extracted in the F1 pool had significantly shorter half-lives than F2, including type-I collagen which had half-lives of 4 ± 2 days in F1, compared to 700 ± 100 days in F2. Circadian-regulated matrix proteins were found to have significantly faster turnover than non-circadian in adult mice, but this distinction was lost in older animals. This work identifies protein turnover as the underlying mechanism for the circadian/permanent model of tendon matrix, and suggests a loss of circadian regulation as a characteristic of ECM ageing. ### Competing Interest Statement The authors have declared no competing interest.

Tendons maintain mechanical function throughout postnatal development whilst undergoing significant microstructural changes. We present a study of postnatal tendon growth and characterise the major changes in collagen fibril architecture in mouse tail tendon from birth to eight weeks by analysing the geometries of cross-sectional transmission electron microscopy images. This study finds that a bimodal distribution of fibril diameters emerges from a unimodal distribution of narrow fibrils as early as the eighth day postnatal, and three distinct fibril populations are visible at around 14 days. The tendons in this study do not show evidence of precise hexagonal packing, even at birth, and the spaces between the fibrils remain constant throughout development. The fibril number in the tissue stabilises around day 28, and the fibril area fraction stabilises around day 26. This study gives coarse-grained insight into the transition periods in early tendon development.

Successful embryo implantation within the uterine wall requires intricate endometrial remodeling. Impaired endometrial receptivity, a common cause of infertility, often results from ineffective remodeling processes. Here, we demonstrate that a single dose of human collagenase-1 administered into the mouse uterus enhances embryo implantation rates. Mechanistically, collagenase-1 induces remodeling of the endometrial extracellular matrix (ECM), leading to the degradation of collagen fibers and proteoglycans. This process releases matrix-bound bioactive factors, such as VEGF, which facilitates local vascular permeability and angiogenesis. Furthermore, collagenase-1 treatment increases NK cell infiltration and elevates levels of the cytokine LIF, a key factor in embryo implantation. Remarkably, the overall structural integrity of the uterine tissue remains uncompromised, even in the presence of reduced tension in endometrial collagen fibers. To assess pre-clinical potential, in-uteri application of collagenase-1 successfully rescued implantation in mouse models subjected to heat stress and embryo transfer, conditions known for their adverse impact on implantation rates. Importantly, ex-vivo exposure of human uterine tissue to collagenase-1 induced collagen de-tensioning and the release of VEGF, demonstrating similar processes observed in the mouse settings, and the potential relevance of this treatment to human conditions. Our findings underscore the immense clinical potential and feasibility of collagenase treatment to enhance uterine receptivity for embryo implantation, offering a controlled and minimally invasive intervention. This innovative approach not only demonstrates the potential to enhance efficiency in livestock breeding but, more importantly, signifies a substantial promise in supporting medical interventions for human reproduction in clinical settings. ### Competing Interest Statement Compositions for remodeling extracellular matrix and methods of use thereof (U.S. patent no. 10722560). Assignee: NanoCell Ltd, Investors: ISa, ISo, EZ. All other authors declare that they have no competing interests. * BM : basement membrane ECM : extracellular matrix ET : embryo transfer fBV : fractional blood volume GFP : green fluorescent protein IVF : in vitro fertilization LIF : leukemia inhibitor factor MMP : matrix metalloproteinase MRI : magnetic resonance imaging PS : permeability surface area SBF-SEM : serial block-face scanning electron microscope SEM : scanning electron microscopy SHG : second-harmonic generation VEGFA : vascular endothelial growth factor A VEGFR2 : vascular endothelial growth factor receptor 2 WOI : window of implantation

Improved soft tissue integration (STI) around dental implants is key for implant success. The formation of an early and long-lasting transmucosal seal around the implant abutment might help to prevent peri-implantitis, one of the major causes of late implant failure. In natural teeth, collagen fibers are firmly inserted and fixed in the cementum of the tooth and emerge perpendicular to the gingival tissue. In contrast, around dental implants, collagen fibers run predominantly parallel to the implant surface, allowing bacterial migration into the peri-implant interface that might lead to peri-implantitis. Previous studies have shown that nanostructured Ti surfaces improve gingival cell response in monolayer cell cultures. Here, we aimed at evaluating the implant-tissue interface using a 3D gingival tissue equivalent (GTE). First, we evaluated the GTE response to a nanostructured (NN) and machined Ti surface after the stimulation with Porphyromonas gingivalis lipopolysaccharide (LPS), to simulate peri-implantitis conditions. Thus, GTE viability, through MTT assay, the release of metalloproteinase-1 (MMP1) and its inhibitor (TIMP1) through ELISA, and the gene expression of extracellular matrix turnover genes by real-time RT-PCR were analyzed. Second, GTE-implant interaction was characterized by serial block face scanning electron microscopy, and collagen-1 orientation at the tissue-implant interface was analyzed by immunofluorescence. While a similar GTE response to LPS stimulation was found for both implant surfaces, a higher proportion of collagen oriented perpendicular to the implant was observed on the NN implant surface. Thus, our results indicate that the nanostructuration of titanium dental implant abutments could allow the correct orientation of collagen fibers and greater soft tissue sealing, while keeping biocompatibility levels and LPS response comparable.

The circadian clock in tendon regulates the daily rhythmic synthesis of collagen-I and the appearance and disappearance of small-diameter collagen fibrils in the extracellular matrix. How the fibrils are assembled and removed is not fully understood. Here, we first showed that the collagenase, membrane type I-matrix metalloproteinase (MT1-MMP, encoded by Mmp14), is regulated by the circadian clock in postnatal mouse tendon. Next, we generated tamoxifen-induced Col1a2-Cre-ERT2::Mmp14 KO mice (Mmp14 conditional knockout (CKO)). The CKO mice developed hind limb dorsiflexion and thickened tendons, which accumulated narrow-diameter collagen fibrils causing ultrastructural disorganization. Mass spectrometry of control tendons identified 1195 proteins of which 212 showed time-dependent abundance. In Mmp14 CKO mice 19 proteins had reversed temporal abundance and 176 proteins lost time dependency. Among these, the collagen crosslinking enzymes lysyl oxidase-like 1 (LOXL1) and lysyl hydroxylase 1 (LH1; encoded by Plod2) were elevated and had lost time-dependent regulation. High-pressure chromatography confirmed elevated levels of hydroxylysine aldehyde (pyridinoline) crosslinking of collagen in CKO tendons. As a result, collagen-I was refractory to extraction. We also showed that CRISPR-Cas9 deletion of Mmp14 from cultured fibroblasts resulted in loss of circadian clock rhythmicity of period 2 (PER2), and recombinant MT1-MMP was highly effective at cleaving soluble collagen-I but less effective at cleaving collagen pre-assembled into fibrils. In conclusion, our study shows that circadian clock-regulated Mmp14 controls the rhythmic synthesis of small diameter collagen fibrils, regulates collagen crosslinking, and its absence disrupts the circadian clock and matrisome in tendon fibroblasts.

The circadian clock in mammals temporally coordinates physiological and behavioural processes to anticipate daily rhythmic changes in their environment. Chronic disruption to circadian rhythms (e.g., through ageing or shift work) is thought to contribute to a multitude of diseases, including degeneration of the musculoskeletal system. The intervertebral disc (IVD) in the spine contains circadian clocks which control ∼6% of the transcriptome in a rhythmic manner, including key genes involved in extracellular matrix (ECM) homeostasis. However, it remains largely unknown to what extent the local IVD molecular clock is required to drive rhythmic gene transcription and IVD physiology. In this work, we identified profound age-related changes of ECM microarchitecture and an endochondral ossification-like phenotype in the annulus fibrosus (AF) region of the IVD in the Col2a1 - Bmal1 knockout mice. Circadian time series RNA-Seq of the whole IVD in Bmal1 knockout revealed loss of circadian patterns in gene expression, with an unexpected emergence of 12-hour ultradian rhythms, including FOXO transcription factors. Further RNA sequencing of the AF tissue identified region-specific changes in gene expression, evidencing a loss of AF phenotype markers and a dysregulation of ECM and FOXO pathways in Bmal1 knockout mice. Consistent with an up-regulation of FOXO1 mRNA and protein levels in Bmal1 knockout IVDs, inhibition of FOXO1 in AF cells suppressed their osteogenic differentiation. Collectively, these data highlight the importance of the local molecular clock mechanism in the maintenance of the cell fate and ECM homeostasis of the IVD. Further studies may identify potential new molecular targets for alleviating IVD degeneration.

Overuse injury in tendon tissue (tendinopathy) is a frequent and costly musculoskeletal disorder and represents a major clinical problem with unsolved pathogenesis. Studies in mice have demonstrated that circadian clock-controlled genes are vital for protein homeostasis and important in the development of tendinopathy. We performed RNA sequencing, collagen content and ultrastructural analyses on human tendon biopsies obtained 12 hours apart in healthy individuals to establish whether human tendon is a peripheral clock tissue and we performed RNA sequencing on patients with chronic tendinopathy to examine the expression of circadian clock genes in tendinopathic tissues. We found time-dependent expression of 280 RNAs including 11 conserved circadian clock genes in healthy tendons and markedly fewer (23) differential RNAs with chronic tendinopathy. Further, the expression of COL1A1 and COL1A2 was reduced at night but was not circadian rhythmic in synchronised human tenocyte cultures. In conclusion, day-to-night changes in gene expression in healthy human patellar tendons indicate a conserved circadian clock as well as the existence of a night-reduction in collagen I expression. KEY POINTS: Tendinopathy is a major clinical problem with unsolved pathogenesis. Previous work in mice have shown that a robust circadian rhythm is required for collagen homeostasis in tendons. The use of circadian medicine in the diagnosis and treatment of tendinopathy has been stifled by the lack of studies on human tissue. Here, we establish that the expression of circadian clock genes in human tendons is time dependent, and now we have data to corroborate that circadian output is reduced in diseased tendon tissues. We consider our findings to be of significance in advancing the use of the tendon circadian clock as a therapeutic target or preclinical biomarker for tendinopathy. Abstract figure legend The primary goal of this study was to establish whether human tendon tissue is a circadian clock. Limited by obtaining only two patellar biopsies from each subject, we designed a study taking the biopsies 12 hours apart, once in the day and once in the night, and analysed the gene expression by RNAseq. We found 280 time-dependent RNAs between 9 AM and 9 PM biopsies and found night reduction in genes involved in collagen I fibrillogenesis by RT-qPCR. We also took samples to examine the presence of a chronomatrix by transmission electron microscopy but the differences were not statistically significant. In chronic patellar tendinopathy biopsies we found fewer time-dependent genes and no night-reduction in expression of genes involved in collagen I fibrillogenesis. Our data suggests that tendinopathy may be accompanied by a dampened circadian rhythm. This article is protected by copyright. All rights reserved.

Collagen is a key structural component of multicellular organisms and is arranged in a highly organized manner. In structural tissues such as tendons, collagen forms bundles of parallel fibers between cells, which appear within a 24-h window between embryonic day 13.5 (E13.5) and E14.5 during mouse embryonic development. Current models assume that the organized structure of collagen requires direct cellular control, whereby cells actively lay down collagen fibrils from cell surfaces. However, such models appear incompatible with the time and length scales of fibril formation. We propose a phase-transition model to account for the rapid development of ordered fibrils in embryonic tendon, reducing reliance on active cellular processes. We develop phase-field crystal simulations of collagen fibrillogenesis in domains derived from electron micrographs of inter-cellular spaces in embryonic tendon and compare results qualitatively and quantitatively to observed patterns of fibril formation. To test the prediction of this phase-transition model that free protomeric collagen should exist in the inter-cellular spaces before the formation of observable fibrils, we use laser-capture microdissection, coupled with mass spectrometry, which demonstrates steadily increasing free collagen in inter-cellular spaces up to E13.5, followed by a rapid reduction of free collagen that coincides with the appearance of less-soluble collagen fibrils. The model and measurements together provide evidence for extracellular self-assembly of collagen fibrils in embryonic mouse tendon, supporting an additional mechanism for rapid collagen fibril formation during embryonic development.