A lysyl oxidase-responsive collagen peptide illuminates collagen remodeling in wound healing.

Tissue repair and fibrosis involve the dynamic remodeling of collagen, and accurate detection of these sites is of utmost importance. Here, we use a collagen peptide sensor (1) to visualize collagen formation and remodeling during wound healing in mice and humans. We show that the probe binds selectively to sites of collagen formation and remodeling at different stages of healing. Compared to conventional methods, the peptide sensor localizes preferentially to areas of collagen synthesis and remodeling at the wound edge and not in matured fibrillar collagen. We also demonstrate its applicability for in vivo wound imaging and for discerning differential remodeling in wounds of transgenic mice with altered collagen dynamics. Our findings show the value of 1 as a diagnostic tool to rapidly identify the sites of matrix remodeling in tissue sections, which will aid in the conception of new therapeutic strategies for fibrotic disorders and defective tissue repair.

MMP14 expression and collagen remodelling support uterine leiomyosarcoma aggressiveness.

Fibrillar collagen deposition, stiffness and downstream signalling support the development of leiomyomas (LMs), common benign mesenchymal tumours of the uterus, and are associated with aggressiveness in multiple carcinomas. Compared with epithelial carcinomas, however, the impact of fibrillar collagens on malignant mesenchymal tumours, including uterine leiomyosarcoma (uLMS), remains elusive. In this study, we analyse the network morphology and density of fibrillar collagens combined with the gene expression within uLMS, LM and normal myometrium (MM). We find that, in contrast to LM, uLMS tumours present low collagen density and increased expression of collagen-remodelling genes, features associated with tumour aggressiveness. Using collagen-based 3D matrices, we show that matrix metalloproteinase-14 (MMP14), a central protein with collagen-remodelling functions that is particularly overexpressed in uLMS, supports uLMS cell proliferation. In addition, we find that, unlike MM and LM cells, uLMS proliferation and migration are less sensitive to changes in collagen substrate stiffness. We demonstrate that uLMS cell growth in low-stiffness substrates is sustained by an enhanced basal yes-associated protein 1 (YAP) activity. Altogether, our results indicate that uLMS cells acquire increased collagen remodelling capabilities and are adapted to grow and migrate in low collagen and soft microenvironments. These results further suggest that matrix remodelling and YAP are potential therapeutic targets for this deadly disease.

Dynamics of Fibril Collagen Remodeling by Tumor Cells: A Model of Tumor-Associated Collagen Signatures.

Many solid tumors are characterized by a dense extracellular matrix (ECM) composed of various ECM fibril proteins. These proteins provide structural support and a biological context for the residing cells. The reciprocal interactions between growing and migrating tumor cells and the surrounding stroma result in dynamic changes in the ECM architecture and its properties. With the use of advanced imaging techniques, several specific patterns in the collagen surrounding the breast tumor have been identified in both tumor murine models and clinical histology images. These tumor-associated collagen signatures (TACS) include loosely organized fibrils far from the tumor and fibrils aligned either parallel or perpendicular to tumor colonies. They are correlated with tumor behavior, such as benign growth or invasive migration. However, it is not fully understood how one specific fibril pattern can be dynamically remodeled to form another alignment. Here, we present a novel multi-cellular lattice-free (MultiCell-LF) agent-based model of ECM that, in contrast to static histology images, can simulate dynamic changes between TACSs. This model allowed us to identify the rules of cell-ECM physical interplay and feedback that guided the emergence and transition among various TACSs.

Transition to Chronic Fibrosis in an Animal Model of Retinal Detachment With Features of Proliferative Vitreoretinopathy.

Purpose: Proliferative vitreoretinopathy (PVR) is the most common cause of failure of surgically repaired rhegmatogenous retinal detachment (RRD). Chemically induced and cell injection PVR models do not fully simulate the clinical characteristics of PVR in the post-RRD context. There is an unmet need for translational models in which to study mechanisms and treatments specific to RRD-PVR. Methods: RRD was induced in adult Dutch Belted rabbits. Posterior segments were fixed or processed for RNA sequencing at 6 hours and 2, 7, 14, and 35 days after induction. Histochemical staining and immunolabeling for glial fibrillary acidic protein, alpha smooth muscle actin, vascular endothelial growth factor receptor 2, CD68, and RPE 65 kDa protein were performed, and labeling intensity was scored. Single cell RNA sequencing was performed. Results: Acute histopathological changes included intravitreal and intraretinal hemorrhage, leukocytic vitritis, chorioretinitis, and retinal rarefaction. Chronic lesions showed retinal atrophy, gliosis, fibrotic subretinal membranes, and epiretinal fibrovascular proliferation. Fibrillar collagen was present in the fibrocellular and fibrovascular membranes in chronic lesions. Moderate to strong labeling of glia and vasculature was detected in chronic lesions. At day 14, most cells profiled by single cell sequencing were identified as Mϋller glia and microglia, consistent with immunolabeling. Expression of several fibrillar collagen genes was upregulated in chronic lesions. Conclusions: Histological and transcriptional features of this rabbit model simulate important features of human RRD-PVR, including the transition to chronic intraretinal and periretinal fibrosis. This animal model of RRD with features of PVR will enable further research on targeted treatment interventions.

Collagen fiber centerline tracking in fibrotic tissue via deep neural networks with variational autoencoder-based synthetic training data generation.

The role of fibrillar collagen in the tissue microenvironment is critical in disease contexts ranging from cancers to chronic inflammations, as evidenced by many studies. Quantifying fibrillar collagen organization has become a powerful approach for characterizing the topology of collagen fibers and studying the role of collagen fibers in disease progression. We present a deep learning-based pipeline to quantify collagen fibers' topological properties in microscopy-based collagen images from pathological tissue samples. Our method leverages deep neural networks to extract collagen fiber centerlines and deep generative models to create synthetic training data, addressing the current shortage of large-scale annotations. As a part of this effort, we have created and annotated a collagen fiber centerline dataset, with the hope of facilitating further research in this field. Quantitative measurements such as fiber orientation, alignment, density, and length can be derived based on the centerline extraction results. Our pipeline comprises three stages. Initially, a variational autoencoder is trained to generate synthetic centerlines possessing controllable topological properties. Subsequently, a conditional generative adversarial network synthesizes realistic collagen fiber images from the synthetic centerlines, yielding a synthetic training set of image-centerline pairs. Finally, we train a collagen fiber centerline extraction network using both the original and synthetic data. Evaluation using collagen fiber images from pancreas, liver, and breast cancer samples collected via second-harmonic generation microscopy demonstrates our pipeline's superiority over several popular fiber centerline extraction tools. Incorporating synthetic data into training further enhances the network's generalizability. Our code is available at https://github.com/uw-loci/collagen-fiber-metrics.

The Role of Epigenomics in Mapping Potential Precursors for Foot and Ankle Tendinopathy: A Systematic Review.

Tendinopathy of the foot and ankle is a common clinical problem for which the exact etiology is poorly understood. The field of epigenetics has been a recent focus of this investigation. The purpose of this article was to review the genomic advances in foot and ankle tendinopathy that could potentially be used to stratify disease risk and create preventative or therapeutic agents. A multi-database search of PubMed, Cochrane, Google Scholar, and clinicaltrials.gov from January 1, 2000 to July 1, 2022 was performed. A total of 18 articles met inclusion and exclusion criteria for this review. The majority of such research utilized case-control candidate gene association to identify different genetic risk factors associated with chronic tendinopathy. Polymorphisms in collagen genes COL5A1, COL27A1, and COL1A1 were noted at a significantly higher frequency in Achilles tendinopathy versus control groups. Other allelic variations that were observed at an increased incidence in Achilles tendinopathy were TNC and CASP8. The extracellular matrix (ECM) demonstrated macroscopic changes in Achilles tendinopathy, including an increase in aggrecan and biglycan mRNA expression, and increased expression of multiple matrix metalloproteinases. Cytokine expression was also influenced in pathology and aberrantly demonstrated dynamic response to mechanical load. The pathologic accumulation of ECM proteins and cytokine expression alters the adaptive response normal tendon has to physiologic stress, further propagating the risk for tendinopathy. By identifying and understanding the epigenetic mediators that lead to tendinopathy, therapeutic agents can be developed to target the exact underlying etiology and minimize side effects.Level of Evidence: Level IV: Systematic Review of Level II-IV Studies.

Quantitative Image Analysis of Fibrillar Collagens Reveals Novel Diagnostic and Prognostic Biomarkers and Histotype-Dependent Aberrant Mechanobiology in Lung Cancer.

Fibrillar collagens are the most abundant extracellular matrix components in non-small cell lung cancer (NSCLC). However, the potential of collagen fiber descriptors as a source of clinically relevant biomarkers in NSCLC is largely unknown. Similarly, our understanding of the aberrant collagen organization and associated tumor-promoting effects is very scarce. To address these limitations, we identified a digital pathology approach that can be easily implemented in pathology units based on CT-FIRE software (version 2; https://loci.wisc.edu/software/ctfire) analysis of Picrosirius red (PSR) stains of fibrillar collagens imaged with polarized light (PL). CT-FIRE settings were pre-optimized to assess a panel of collagen fiber descriptors in PSR-PL images of tissue microarrays from surgical NSCLC patients (106 adenocarcinomas [ADC] and 89 squamous cell carcinomas [SCC]). Using this approach, we identified straightness as the single high-accuracy diagnostic collagen fiber descriptor (average area under the curve = 0.92) and fiber density as the single descriptor consistently associated with a poor prognosis in both ADC and SCC independently of the gold standard based on the TNM staging (hazard ratio, 2.69; 95% CI, 1.55-4.66; P < .001). Moreover, we found that collagen fibers were markedly straighter, longer, and more aligned in tumor samples compared to paired samples from uninvolved pulmonary tissue, particularly in ADC, which is indicative of increased tumor stiffening. Consistently, we observed an increase in a panel of stiffness-associated processes in the high collagen fiber density patient group selectively in ADC, including venous/lymphatic invasion, fibroblast activation (α-smooth muscle actin), and immune evasion (programmed death-ligand 1). Similarly, a transcriptional correlation analysis supported the potential involvement of the major YAP/TAZ pathway in ADC. Our results provide a proof-of-principle to use CT-FIRE analysis of PSR-PL images to assess new collagen fiber-based diagnostic and prognostic biomarkers in pathology units, which may improve the clinical management of patients with surgical NSCLC. Our findings also unveil an aberrant stiff microenvironment in lung ADC that may foster immune evasion and dissemination, encouraging future work to identify therapeutic opportunities.

Persistent homology analysis distinguishes pathological bone microstructure in non-linear microscopy images.

We present a topological method for the detection and quantification of bone microstructure from non-linear microscopy images. Specifically, we analyse second harmonic generation (SHG) and two photon excited autofluorescence (TPaF) images of bone tissue which capture the distribution of matrix (fibrillar collagen) structure and autofluorescent molecules, respectively. Using persistent homology statistics with a signed Euclidean distance transform filtration on binary patches of images, we are able to quantify the number, size, distribution, and crowding of holes within and across samples imaged at the microscale. We apply our methodology to a previously characterized murine model of skeletal pathology whereby vascular endothelial growth factor expression was deleted in osteocalcin-expressing cells (OcnVEGFKO) presenting increased cortical porosity, compared to wild type (WT) littermate controls. We show significant differences in topological statistics between the OcnVEGFKO and WT groups and, when classifying the males, or females respectively, into OcnVEGFKO or WT groups, we obtain high prediction accuracies of 98.7% (74.2%) and 77.8% (65.8%) respectively for SHG (TPaF) images. The persistence statistics that we use are fully interpretable, can highlight regions of abnormality within an image and identify features at different spatial scales.

Regions of homozygosity and a novel variant in Steel syndrome: An added dilemma to diagnosis.

Steel syndrome is an autosomal recessive disorder that is caused by mutations in COL27A1 gene. The majority of reported cases have been of Puerto Rican origin, with few reports from India. The present case adds to the repertoire of homozygous recessive disorders from non-consanguineous Indian families. With the present case, a 4-year-old girl, we wish to signify that although mutations in several genes are known to cause skeletal abnormalities, identification of underlying mutations is important as it not only helps with the ascertainment of diagnosis but also aids in determining the role of surgical interventions which is particularly true for Steel syndrome, where the outcome of surgical intervention is usually dismal.

Multiscale Label-Free Imaging of Fibrillar Collagen in the Tumor Microenvironment.

With recent advances in cancer therapeutics, there is a great need for improved imaging methods for characterizing cancer onset and progression in a quantitative and actionable way. Collagen, the most abundant extracellular matrix protein in the tumor microenvironment (and the body in general), plays a multifaceted role, both hindering and promoting cancer invasion and progression. Collagen deposition can defend the tumor with immunosuppressive effects, while aligned collagen fiber structures can enable tumor cell migration, aiding invasion and metastasis. Given the complex role of collagen fiber organization and topology, imaging has been a tool of choice to characterize these changes on multiple spatial scales, from the organ and tumor scale to cellular and subcellular level. Macroscale density already aids in the detection and diagnosis of solid cancers, but progress is being made to integrate finer microscale features into the process. Here we review imaging modalities ranging from optical methods of second harmonic generation (SHG), polarized light microscopy (PLM), and optical coherence tomography (OCT) to the medical imaging approaches of ultrasound and magnetic resonance imaging (MRI). These methods have enabled scientists and clinicians to better understand the impact collagen structure has on the tumor environment, at both the bulk scale (density) and microscale (fibrillar structure) levels. We focus on imaging methods with the potential to both examine the collagen structure in as natural a state as possible and still be clinically amenable, with an emphasis on label-free strategies, exploiting intrinsic optical properties of collagen fibers.

Influence of two natural cross-linkers on microtensile bond strength durability ­ An in vitro study / Influência de dois reticuladores naturais na durabilidade da resistência de união à microtração ­ Um estudo in vitro

Objective: to investigate the effect of two natural cross-linkers on microtensile bond strength (µTBS) and evaluate their influence on the durability of the resin dentin bonds. Material and Methods: the Moringa oleifera and Centella asiatica plant extracts were qualitatively tested with high-performance thin layer chromatography (HPTLC) for the presence of phenols. The phenolic content ranged from 27 to 30 gallic acid equivalents (GAE), µg/mg of dry weight. After etching, two concentrations (5% and 1%) of these two extracts were prepared and used as pretreatment liners on dentin. They were applied for a min. After restoration with resin composite, dentin resin beams were prepared. The study groups were 5% Moringa, 1% Moringa 5% Centella 1% Centella, and control (without cross-linker application). For each group, half of the samples underwent µTBS testing after 24 hours, while the remaining half were immersed in artificial saliva to assess the bond's longevity after 6 months of ageing. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. Results: both 5% and 1% Moringa showed a significant difference (p<0.05) compared to the other groups at both intervals. However, after ageing, the specimens in the control and 1% Centella groups resulted in a significant decrease in µTBS. Conclusion: overall, both concentrations of Moringa (5% and 1%) were effective in stabilising the bond during both intervals.(AU)

Objetivo: investigar o efeito de dois reticuladores naturais na resistência de união (µTBS) à microtração e avaliar sua influência na durabilidade da adesão da resina à dentina. Material e Métodos: extratos das plantas Moringa oleifera e Centella asiatica foram qualitativamente testados através de cromatografia em camada fina de alta performance (HPTLC) para a presença de fenóis. O conteúdo fenólico alcançou entre 27 a 30 equivalentes de ácido gálico (GAE), µg/mg de peso seco. Após o condicionamento, duas concentrações (5% e 1%) dos extratos foram preparadas e utilizadas como forros de pré-tratamento em dentina. Eles foram aplicados por um minuto. Após a restauração com resina composta, palitos de dentina e resina foram preparados. Os grupos foram 5% Moringa, 1% Moringa, 5% Centella, 1% Centella e controle (sem aplicação de reticulador). Para cada grupo, metade das amostras foram submetidas ao teste µTBS após 24 horas, enquanto a outra metade foi imersa em saliva artificial para avaliar a longevidade adesiva após 6 meses de envelhecimento. Foi realizada análise estatística através de ANOVA 1-fator, seguido do teste post hoc de Tukey. Resultados: ambas as concentrações de 5% e 1% de Moringa demonstraram diferença significativa (p<0.05) comparadas aos outros grupos em ambos os intervalos. No entanto, após o envelhecimento, os espécimes dos geupos controle e 1% de Centella resultaram em uma redução significativa de µTBS. Conclusão: no geral, ambas as concentrações de Moringa (5% e 1%) foram efetivas em estabelecer a adesão em ambos os intervalos (AU)

Type XXII Collagen Complements Fibrillar Collagens in the Serological Assessment of Tumor Fibrosis and the Outcome in Pancreatic Cancer.

Circulating fragments of type III collagen, measured by PRO-C3, has shown promising results as a tumor fibrosis biomarker. However, the fibrotic tumor microenvironment consists of many other collagens with diverse functions and unexplored biomarker potential. One example hereof is type XXII collagen (COL22). In this study, we investigated the biomarker potential of COL22 by measuring this in serum. An ELISA, named PRO-C22, was developed and measured in two serum cohorts consisting of patients with various solid tumors (n = 220) and healthy subjects (n = 33) (Cohort 1), and patients with pancreatic ductal adenocarcinoma (PDAC) (n = 34), and healthy subjects (n = 20) (Cohort 2). In Cohort 1, PRO-C22 was elevated in the serum from patients with solid tumors, compared to healthy subjects (p < 0.01 to p < 0.0001), and the diagnostic accuracy (AUROC) ranged from 0.87 to 0.98, p < 0.0001. In Cohort 2, the high levels of PRO-C22, in patients with PDAC, were predictive of a worse overall survival (HR = 4.52, 95% CI 1.90−10.7, p = 0.0006) and this remained significant after adjusting for PRO-C3 (HR = 4.27, 95% CI 1.24−10.4, p = 0.0013). In conclusion, PRO-C22 has diagnostic biomarker potential in various solid tumor types and prognostic biomarker potential in PDAC. Furthermore, PRO-C22 complemented PRO-C3 in predicting mortality, suggesting an additive prognostic value when quantifying different collagens.

The Structural Interactions of Molecular and Fibrillar Collagen Type I with Fibronectin and Its Role in the Regulation of Mesenchymal Stem Cell Morphology and Functional Activity.

The observed differences in the structure of native tissue and tissue formed in vitro cause the loss of functional activity of cells cultured in vitro. The lack of fundamental knowledge about the protein mechanism interactions limits the ability to effectively create in vitro native tissue. Collagen is able to spontaneously assemble into fibrils in vitro, but in vivo, other proteins, for example fibronectin, have a noticeable effect on this process. The molecular or fibrillar structure of collagen plays an equally important role. Therefore, we studied the interaction of the molecular and fibrillar structure of collagen with fibronectin. Atomic force and transmission electron microscopy showed that the presence of fibronectin does not affect the native structure and diameter of collagen fibrils. Confocal microscopy demonstrated that the collagen structure affects the cell morphology. Cells are better spread on molecular collagen compared with cells cultured on fibrillar collagen. Fibronectin promotes the formation of a large number of focal contacts, while in combination with collagen of both forms, its effect is leveled. Thus, understanding the mechanisms of the relationship between the protein structure and composition will effectively manage the creation in vitro of a new tissue with native properties.

Mechanical Confinement and DDR1 Signaling Synergize to Regulate Collagen-Induced Apoptosis in Rhabdomyosarcoma Cells.

Fibrillar collagens promote cell proliferation, migration, and survival in various epithelial cancers and are generally associated with tumor aggressiveness. However, the impact of fibrillar collagens on soft tissue sarcoma behavior remains poorly understood. Unexpectedly, this study finds that fibrillar collagen-related gene expression is associated with favorable patient prognosis in rhabdomyosarcoma. By developing and using collagen matrices with distinct stiffness and in vivo-like microarchitectures, this study uncovers that the activation of DDR1 has pro-apoptotic and of integrin ß1 pro-survival function, specifically in 3D rhabdomyosarcoma cell cultures. It demonstrates that rhabdomyosarcoma cell-intrinsic or extrinsic matrix remodeling promotes cell survival. Mechanistically, the 3D-specific collagen-induced apoptosis results from a dual DDR1-independent and a synergistic DDR1-dependent TRPV4-mediated response to mechanical confinement. Altogether, these results indicate that dense microfibrillar collagen-rich microenvironments are detrimental to rhabdomyosarcoma cells through an apoptotic response orchestrated by the induction of DDR1 signaling and mechanical confinement. This mechanism helps to explain the preference of rhabdomyosarcoma cells to grow in and metastasize to low fibrillar collagen microenvironments such as the lung.

Using sequence data to predict the self-assembly of supramolecular collagen structures.

Collagen fibrils are the major constituents of the extracellular matrix, which provides structural support to vertebrate connective tissues. It is widely assumed that the superstructure of collagen fibrils is encoded in the primary sequences of the molecular building blocks. However, the interplay between large-scale architecture and small-scale molecular interactions makes the ab initio prediction of collagen structure challenging. Here, we propose a model that allows us to predict the periodic structure of collagen fibers and the axial offset between the molecules, purely on the basis of simple predictive rules for the interaction between amino acid residues. With our model, we identify the sequence-dependent collagen fiber geometries with the lowest free energy and validate the predicted geometries against the available experimental data. We propose a procedure for searching for optimal staggering distances. Finally, we build a classification algorithm and use it to scan 11 data sets of vertebrate fibrillar collagens, and predict the periodicity of the resulting assemblies. We analyzed the experimentally observed variance of the optimal stagger distances across species, and find that these distances, and the resulting fibrillar phenotypes, are evolutionary well preserved. Moreover, we observed that the energy minimum at the optimal stagger distance is broad in all cases, suggesting a further evolutionary adaptation designed to improve the assembly kinetics. Our periodicity predictions are not only in good agreement with the experimental data on collagen molecular staggering for all collagen types analyzed, but also for synthetic peptides. We argue that, with our model, it becomes possible to design tailor-made, periodic collagen structures, thereby enabling the design of novel biomimetic materials based on collagen-mimetic trimers.

A Biomimetic High Throughput Model of Cancer Cell Spheroid Dissemination onto Aligned Fibrillar Collagen.

Cell dissemination during tumor development is a characteristic of cancer metastasis. Dissemination from three-dimensional spheroid models on extracellular matrices designed to mimic tissue-specific physiological microenvironments may allow us to better elucidate the mechanism behind cancer metastasis and the response to therapeutic agents. The orientation of fibrillar collagen plays a key role in cellular processes and mediates metastasis through contact-guidance. Understanding how cells migrate on aligned collagen fibrils requires in vitro assays with reproducible and standardized orientation of collagen fibrils on the macro-to-nanoscale. Herein, we implement a spheroid-based migration assay, integrated with a fibrillar type I collagen matrix, in a manner compatible with high throughput image acquisition and quantitative analysis. The migration of highly proliferating U2OS osteosarcoma cell spheroids onto an aligned fibrillar type I collagen matrix was quantified. Cell dissemination from the spheroid was polarized with increased invasion in the direction of fibril alignment. The resulting area of cell dissemination had an aspect ratio of 1.2 ± 0.1 and an angle of maximum invasion distance of 5° ± 44° relative to the direction of collagen fibril alignment. The assay described here can be applied to a fully automated imaging and analysis pipeline for the assessment of tumor cell migration with high throughput screening.

Steel syndrome: Report of three patients, including monozygotic twins and review of clinical and mutation profiles.

Steel syndrome (MIM# 615155) is an autosomal recessive skeletal disorder, characterized by dislocations of the hips and radial heads, carpal coalition, short stature, facial dysmorphism, and scoliosis. Until date 47 patients have been reported. However, disease causing variants have been identified only in twenty Puerto Rican and nine non-Puerto Rican families. Here we report two monozygotic twins and a boy from two families with novel missense variants, c.295G > A p.(Ala99 Thr), c.3056C > A p.(Pro1019His) and c.2521G > A p.(Gly841Arg) in COL27A1. We describe for the first time, cleft palate and delayed carpal bone ossification as features of Steel syndrome. We reviewed clinical features in all mutation-proven Steel syndrome patients. Short stature and dislocation/subluxation of hip joint are consistently observed. Other features include dislocated radial heads, scoliosis, lordosis, carpal coalition, facial dysmorphism, hearing loss, bilateral fifth finger clinodactyly, knee deformities and developmental delay. Seven missense variants and eight null variants are reported in COL27A1 until date. We also looked into the genotype-phenotype correlation in Puerto Rican and non-Puerto Rican patients.

Tyrosine-sulfated dermatopontin shares multiple binding sites and recognition determinants on triple-helical collagens with proteins implicated in cell adhesion and collagen folding, fibrillogenesis, cross-linking, and degradation.

Dermatopontin (DPT), a small extracellular matrix protein that stimulates collagen fibrillogenesis, contains sulfotyrosine residues but neither its level of sulfation nor its binding sites on fibrillar collagens are known. Here, we discovered that DPT is present in a relatively high mass concentration (~ 0.02%) in porcine corneal stroma, from which we purified five DPT charge variants (A-E) containing up to six sulfations. The major variant (C), containing four sulfotyrosine residues, was used to locate binding sites for DPT on triple-helical collagens II and III using the Collagen Toolkits. DPT-binding loci included the triple helix crosslinking sites and collagenase cleavage site. We find that strong DPT-binding sites on triple-helical collagen comprise an arginine-rich, positively-charged sequence that also contains hydrophobic residues. This collagen-binding signature of DPT is similar to that of the chaperone HSP47. Thus, we propose that DPT assumes the role of HSP47 as a collagen chaperone during and after the secretion. Peptide II-44, harbouring the conserved collagenase cleavage site, shows the strongest DPT-binding of the Collagen Toolkit II peptides. Substituting any of the three arginine residues (R) with alanine in the sequence GLAGQRGIVGLOGQRGER of II-44 resulted in almost complete loss of DPT binding. Since osteogenesis imperfecta, spondyloepiphyseal dysplasia, and spondyloepimetaphyseal dysplasia congenita are associated with missense mutations that substitute the corresponding arginine residues in collagens alpha-1(I) and alpha-1(II), we suggest that disrupted DPT binding to fibrillar collagens may contribute to these connective tissue disorders. In conclusion, the present work provides a cornerstone for further elucidation of the role of DPT.

Secreted protein acidic and rich in cysteine (SPARC) and a disintegrin and metalloproteinase with thrombospondin type 1 motif (ADAMTS1) increments by the renin-angiotensin system induce renal fibrosis in deoxycorticosterone acetate-salt hypertensive rats.

Secreted protein acidic and rich in cysteine (SPARC), an extracellular matrix (ECM) protein, was recently shown to induce collagen deposition through the production of a disintegrin and metalloproteinase with thrombospondin type 1 motif (ADAMTS1) in the aging heart. ADAMTS1 regulates ECM turnover by degrading ECM components, and its excessive activation contributes to various pathological states, including fibrosis. The present study investigated the pathophysiological regulation and role of SPARC and ADAMTS1 in renal fibrosis using uninephrectomized rats treated with deoxycorticosterone acetate (DOCA, 40 mg/kg/week, subcutaneously) and salt (1% in drinking water). The administration of DOCA and salt gradually and significantly elevated systolic blood pressure during the 3-week treatment period, induced proteinuria, decreased creatinine clearance, and increased NADPH oxidase-derived superoxide production, malondialdehyde concentrations, and monocyte chemoattractant protein-1 and osteopontin expression in the kidneys. Glomerulosclerosis, fibrillar collagen deposition, and transforming growth factor-ß expression increased in a time-dependent manner, and SPARC and ADAMTS1 expression showed a similar pattern to these changes. The angiotensin II type-1 receptor blocker losartan suppressed the overexpression of SPARC and ADAMTS1, and an in vitro exposure to angiotensin II induced the production of both SPARC and ADAMTS1 in renal fibroblast NRK-49F cells. Knockdown of the SPARC gene with small interfering RNA reduced all forms (the 110-kDa latent and 87- and 65-kDa bioactive forms) of ADAMTS1 expression as well as collagen production. These results suggest that SPARC is induced by the renin-angiotensin system and may be a fibrogenic factor, at least in part, by producing ADAMTS1 in hypertensive renal disease.

Tenomodulin knockout mice exhibit worse late healing outcomes with augmented trauma-induced heterotopic ossification of Achilles tendon.

Heterotopic ossification (HO) represents a common problem after tendon injury with no effective treatment yet being developed. Tenomodulin (Tnmd), the best-known mature marker for tendon lineage cells, has important effects in tendon tissue aging and function. We have reported that loss of Tnmd leads to inferior early tendon repair characterized by fibrovascular scaring and therefore hypothesized that its lack will persistently cause deficient repair during later stages. Tnmd knockout (Tnmd-/-) and wild-type (WT) animals were subjected to complete Achilles tendon surgical transection followed by end-to-end suture. Lineage tracing revealed a reduction in tendon-lineage cells marked by ScleraxisGFP, but an increase in alpha smooth muscle actin myofibroblasts in Tnmd-/- tendon scars. At the proliferative stage, more pro-inflammatory M1 macrophages and larger collagen II cartilaginous template were detected in this group. At the remodeling stage, histological scoring revealed lower repair quality in the injured Tnmd-/- tendons, which was coupled with higher HO quantified by micro-CT. Tendon biomechanical properties were compromised in both groups upon injury, however we identified an abnormal stiffening of non-injured Tnmd-/- tendons, which possessed higher static and dynamic E-moduli. Pathologically thicker and abnormally shaped collagen fibrils were observed by TEM in Tnmd-/- tendons and this, together with augmented HO, resulted in diminished running capacity of Tnmd-/- mice. These novel findings demonstrate that Tnmd plays a protecting role against trauma-induced endochondral HO and can inspire the generation of novel therapeutics to accelerate repair.