Biomechanical Activation of Keloid Fibroblasts Promotes Lysosomal Remodeling and Exocytosis.

Keloids are a severe form of scarring for which the underlying mechanisms are poorly understood, and treatment options are limited or inconsistent. Although biomechanical forces are potential drivers of keloid scarring, the direct cellular responses to mechanical cues have yet to be defined. The aim of this study was to examine the distinct responses of normal dermal fibroblasts and keloid-derived fibroblasts (KDFs) to changes in extracellular matrix stiffness. When cultured on hydrogels mimicking the elasticity of normal or scarred skin, KDFs displayed greater stiffness-dependent increases in cell spreading, F-actin stress fiber formation, and focal adhesion assembly. Elevated actomyosin contractility in KDFs disrupted the normal mechanical regulation of extracellular matrix deposition and conferred resistance on myosin inhibitors. Transcriptional profiling identified mechanically regulated pathways in normal dermal fibroblasts and KDFs, including the actin cytoskeleton, Hippo signaling, and autophagy. Further analysis of the autophagy pathway revealed that autophagic flux was intact in both fibroblast populations and depended on actomyosin contractility. However, KDFs displayed marked changes in lysosome organization and an increase in lysosomal exocytosis, which was mediated by actomyosin contractility. Together, these findings demonstrate that KDFs possess an intrinsic increase in cytoskeletal tension, which heightens the response to extracellular matrix mechanics and promotes lysosomal exocytosis.

Characterisation and correction of polarisation effects in fluorescently labelled fibres.

Many biological structures take the form of fibres and filaments, and quantitative analysis of fibre organisation is important for understanding their functions in both normal physiological conditions and disease. In order to visualise these structures, fibres can be fluorescently labelled and imaged, with specialised image analysis methods available for quantifying the degree and strength of fibre alignment. Here we show that fluorescently labelled fibres can display polarised emission, with the strength of this effect varying depending on structure and fluorophore identity. This can bias automated analysis of fibre alignment and mask the true underlying structural organisation. We present a method for quantifying and correcting these polarisation effects without requiring polarisation-resolved microscopy and demonstrate its efficacy when applied to images of fluorescently labelled collagen gels, allowing for more reliable characterisation of fibre microarchitecture.

Autocrine IL-6 drives cell and extracellular matrix anisotropy in scar fibroblasts.

Fibrosis is associated with dramatic changes in extracellular matrix (ECM) architecture of unknown etiology. Here we exploit keloid scars as a paradigm to understand fibrotic ECM organization. We reveal that keloid patient fibroblasts uniquely produce a globally aligned ECM network in 2-D culture as observed in scar tissue. ECM anisotropy develops after rapid initiation of a fibroblast supracellular actin network, suggesting that cell alignment initiates ECM patterning. Keloid fibroblasts produce elevated levels of IL-6, and autocrine IL-6 production is both necessary and sufficient to induce cell and ECM alignment, as evidenced by ligand stimulation of normal dermal fibroblasts and treatment of keloid fibroblasts with the function blocking IL-6 receptor monoclonal antibody, tocilizumab. Downstream of IL-6, supracellular organization of keloid fibroblasts is controlled by activation of cell-cell adhesion. Adhesion formation inhibits contact-induced cellular overlap leading to nematic organization of cells and an alignment of focal adhesions. Keloid fibroblasts placed on isotropic ECM align the pre-existing matrix, suggesting that focal adhesion alignment leads to active anisotropic remodeling. These results show that IL-6-induced fibroblast cooperativity can control the development of a nematic ECM, highlighting both IL-6 signaling and cell-cell adhesions as potential therapeutic targets to inhibit this common feature of fibrosis.

Loss of RhoE Function in Dermatofibroma Promotes Disorganized Dermal Fibroblast Extracellular Matrix and Increased Integrin Activation.

Dermatofibromas (DFs) are common, benign fibrous skin tumors that can occur at any skin site. In most cases, DFs are solitary and sporadic, but a few are multiple and familial, and the mechanisms leading to these lesions are currently unclear. Using exome sequencing, we have identified a heterozygous variant in a pedigree with autosomal dominant multiple familial DF within RND3 (c.692C>T,p.T231M) that encodes for the small GTPase RhoE, a regulator of the actin cytoskeleton. Expression of T231M-RhoE or RhoE depletion using CRISPR in human dermal fibroblasts increased proliferation and adhesion to extracellular matrix through enhanced ß1 integrin activation and more disorganized matrix. The enzyme PLOD2 was identified as a binding partner for RhoE, and the formation of this complex was disrupted by T231M-RhoE. PLOD2 promotes collagen cross-linking and activation of ß1 integrins, and depleting PLOD2 in T231M-RhoE-expressing cells reduced T231M-RhoE-mediated ß1 integrin activation and led to increased matrix alignment. Immunohistochemical analysis revealed reduced expression of RhoE but increased expression of PLOD2 in the dermis of DF skin samples compared with that of the controls. Our data show that loss of RhoE function leads to increased PLOD2 activation, enhancing integrin activation and leading to a disorganized extracellular matrix, contributing to DF.

Comorbidities of Keloid and Hypertrophic Scars Among Participants in UK Biobank.

Importance: Keloids and hypertrophic scars (excessive scarring) are relatively understudied disfiguring chronic skin conditions with high treatment resistance. Objective: To evaluate established comorbidities of excessive scarring in European individuals, with comparisons across ethnic groups, and to identify novel comorbidities via a phenome-wide association study (PheWAS). Design, Setting, and Participants: This multicenter cross-sectional population-based cohort study used UK Biobank (UKB) data and fitted logistic regression models for testing associations between excessive scarring and a variety of outcomes, including previously studied comorbidities and 1518 systematically defined disease categories. Additional modeling was performed within subgroups of participants defined by self-reported ethnicity (as defined in UK Biobank). Of 502 701 UKB participants, analyses were restricted to 230078 individuals with linked primary care records. Exposures: Keloid or hypertrophic scar diagnoses. Main Outcomes and Measures: Previously studied disease associations (hypertension, uterine leiomyoma, vitamin D deficiency, atopic eczema) and phenotypes defined in the PheWAS Catalog. Results: Of the 972 people with excessive scarring, there was a higher proportion of female participants compared with the 229 106 controls (65% vs 55%) and a lower proportion of White ethnicity (86% vs 95%); mean (SD) age of the total cohort was 64 (8) years. Associations were identified with hypertension and atopic eczema in models accounting for age, sex, and ethnicity, and the association with atopic eczema (odds ratio [OR], 1.68; 95% CI, 1.36-2.07; P < .001) remained statistically significant after accounting for additional potential confounders. Fully adjusted analyses within ethnic groups revealed associations with hypertension in Black participants (OR, 2.05; 95% CI, 1.13-3.72; P = .02) and with vitamin D deficiency in Asian participants (OR, 2.24; 95% CI, 1.26-3.97; P = .006). The association with uterine leiomyoma was borderline significant in Black women (OR, 1.93; 95% CI, 1.00-3.71; P = .05), whereas the association with atopic eczema was significant in White participants (OR, 1.68; 95% CI, 1.34-2.12; P < .001) and showed a similar trend in Asian (OR, 2.17; 95% CI, 1.01-4.67; P = .048) and Black participants (OR, 1.89; 95% CI, 0.83-4.28; P = .13). The PheWAS identified 110 significant associations across disease systems; of the nondermatological, musculoskeletal disease and pain symptoms were prominent. Conclusions and Relevance: This cross-sectional study validated comorbidities of excessive scarring in UKB with comprehensive coverage of health outcomes. It also documented additional phenome-wide associations that will serve as a reference for future studies to investigate common underlying pathophysiologic mechanisms.

A workflow for rapid unbiased quantification of fibrillar feature alignment in biological images.

Measuring the organisation of the cellular cytoskeleton and the surrounding extracellular matrix (ECM) is currently of wide interest as changes in both local and global alignment can highlight alterations in cellular functions and material properties of the extracellular environment. Different approaches have been developed to quantify these structures, typically based on fibre segmentation or on matrix representation and transformation of the image, each with its own advantages and disadvantages. Here we present AFT-Alignment by Fourier Transform, a workflow to quantify the alignment of fibrillar features in microscopy images exploiting 2D Fast Fourier Transforms (FFT). Using pre-existing datasets of cell and ECM images, we demonstrate our approach and compare and contrast this workflow with two other well-known ImageJ algorithms to quantify image feature alignment. These comparisons reveal that AFT has a number of advantages due to its grid-based FFT approach. 1) Flexibility in defining the window and neighbourhood sizes allows for performing a parameter search to determine an optimal length scale to carry out alignment metrics. This approach can thus easily accommodate different image resolutions and biological systems. 2) The length scale of decay in alignment can be extracted by comparing neighbourhood sizes, revealing the overall distance that features remain anisotropic. 3) The approach is ambivalent to the signal source, thus making it applicable for a wide range of imaging modalities and is dependent on fewer input parameters than segmentation methods. 4) Finally, compared to segmentation methods, this algorithm is computationally inexpensive, as high-resolution images can be evaluated in less than a second on a standard desktop computer. This makes it feasible to screen numerous experimental perturbations or examine large images over long length scales. Implementation is made available in both MATLAB and Python for wider accessibility, with example datasets for single images and batch processing. Additionally, we include an approach to automatically search parameters for optimum window and neighbourhood sizes, as well as to measure the decay in alignment over progressively increasing length scales.

Metabolic perturbations in fibrosis disease.

Metabolic changes occur in all forms of disease but their impact on fibrosis is a relatively recent area of interest. This review provides an overview of the major metabolic pathways, glycolysis, amino acid metabolism and lipid metabolism, and highlights how they influence fibrosis at a cellular and tissue level, drawing on key discoveries in dermal, renal, pulmonary and hepatic fibrosis. The emerging influence of adipose tissue-derived cytokines is discussed and brings a link between fibrosis and systemic metabolism. To close, the concept of targeting metabolism for fibrotic therapy is reviewed, drawing on lessons from the more established field of cancer metabolism, with an emphasis on important considerations for clinical translation.

Keloid tissue analysis discredits a role for myofibroblasts in disease pathogenesis.

Myofibroblasts, renowned for their contractility and extracellular matrix production, are widely considered the key effector cells for nearly all scars resulting from tissue repair processes, ranging from normal scars to extreme fibrosis. For example, it is often assumed that myofibroblasts underpin the characteristics of keloid scars, which are debilitating pathological skin scars lacking effective treatments because of a poor understanding of the disease mechanisms. Here, we present primary and published transcriptional and histological evidence that myofibroblasts are not consistently present in primary keloid lesions, and when alpha-smooth muscle actin (αSMA)-positive cells are detected, they are not greater in number or expressing more αSMA than in normal or hypertrophic scars. In conclusion, keloid scars do not appear to require αSMA-positive myofibroblasts; continuing to consider keloids on a quantitative spectrum with normal or hypertrophic scars, with αSMA serving as a biomarker of disease severity, is hindering advancement of understanding and therapy development.

A developmental basis for the anatomical diversity of dermis in homeostasis and wound repair.

The dermis has disparate embryonic origins; abdominal dermis develops from lateral plate mesoderm, dorsal dermis from paraxial mesoderm and facial dermis from neural crest. However, the cell and molecular differences and their functional implications have not been described. We hypothesise that the embryonic origin of the dermis underpins regional characteristics of skin, including its response to wounding. We have compared abdomen, back and cheek, three anatomical sites representing the distinct embryonic tissues from which the dermis can arise, during homeostasis and wound repair using RNA sequencing, histology and fibroblast cultures. Our transcriptional analyses demonstrate differences between body sites that reflect their diverse origins. Moreover, we report histological and transcriptional variations during a wound response, including site differences in ECM composition, cell migration and proliferation, and re-enactment of distinct developmental programmes. These findings reveal profound regional variation in the mechanisms of tissue repair. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Dissecting Fibroblast Heterogeneity in Health and Fibrotic Disease.

PURPOSE OF REVIEW: Fibroblasts, the major cell population in all connective tissues, are best known for their role in depositing and maintaining the extracellular matrix. Recently, numerous specialised functions have been discovered revealing unpredicted fibroblast heterogeneity. We will discuss this heterogeneity, from its origins in development to alterations in fibrotic disease conditions. RECENT FINDINGS: Advances in lineage tracing and single-cell transcriptional profiling techniques have revealed impressive diversity amongst fibroblasts in a range of organ systems including the skin, lung, kidney and heart. However, there are major challenges in assimilating the findings and understanding their functional significance. Certain fibroblast subsets can make specific contributions to healthy tissue functioning and to fibrotic disease processes; thus, therapeutic manipulation of particular subsets could be clinically beneficial. Here we propose that four key variables determine a fibroblast's phenotype underpinning their enormous heterogeneity: tissue status, regional features, microenvironment and cell state. We review these in different organ systems, highlighting the importance of understanding the divergent fibroblast properties and underlying mechanisms in tissue fibrosis.

Cartilage-like composition of keloid scar extracellular matrix suggests fibroblast mis-differentiation in disease.

Following wound damage to the skin, the scarring spectrum is wide-ranging, from a manageable normal scar through to pathological keloids. The question remains whether these fibrotic lesions represent simply a quantitative extreme, or alternatively, whether they are qualitatively distinct. A three-way comparison of the extracellular matrix (ECM) composition of normal skin, normal scar and keloids was performed using quantitative discovery-based proteomics. This approach identified 40 proteins that were significantly altered in keloids compared to normal scars, and strikingly, 23 keloid-unique proteins. The major alterations in keloids, when functionally grouped, showed many changes in proteins involved in ECM assembly and fibrillogenesis, but also a keloid-associated loss of proteases, and a unique cartilage-like composition, which was also evident histologically. The presence of Aggrecan and Collagen II in keloids suggest greater plasticity and mis-differentiation of the constituent cells. This study characterises the ECM of both scar types to a depth previously underappreciated. This thorough molecular description of keloid lesions relative to normal scars is an essential step towards our understanding of this debilitating clinical problem, and how best to treat it.

Spatial and Single-Cell Transcriptional Profiling Identifies Functionally Distinct Human Dermal Fibroblast Subpopulations.

Previous studies have shown that mouse dermis is composed of functionally distinct fibroblast lineages. To explore the extent of fibroblast heterogeneity in human skin, we used a combination of comparative spatial transcriptional profiling of human and mouse dermis and single-cell transcriptional profiling of human dermal fibroblasts. We show that there are at least four distinct fibroblast populations in adult human skin, not all of which are spatially segregated. We define markers permitting their isolation and show that although marker expression is lost in culture, different fibroblast subpopulations retain distinct functionality in terms of Wnt signaling, responsiveness to IFN-γ, and ability to support human epidermal reconstitution when introduced into decellularized dermis. These findings suggest that ex vivo expansion or in vivo ablation of specific fibroblast subpopulations may have therapeutic applications in wound healing and diseases characterized by excessive fibrosis.

The Gastric Ganglion of Octopus vulgaris: Preliminary Characterization of Gene- and Putative Neurochemical-Complexity, and the Effect of Aggregata octopiana Digestive Tract Infection on Gene Expression.

The gastric ganglion is the largest visceral ganglion in cephalopods. It is connected to the brain and is implicated in regulation of digestive tract functions. Here we have investigated the neurochemical complexity (through in silico gene expression analysis and immunohistochemistry) of the gastric ganglion in Octopus vulgaris and tested whether the expression of a selected number of genes was influenced by the magnitude of digestive tract parasitic infection by Aggregata octopiana. Novel evidence was obtained for putative peptide and non-peptide neurotransmitters in the gastric ganglion: cephalotocin, corticotrophin releasing factor, FMRFamide, gamma amino butyric acid, 5-hydroxytryptamine, molluscan insulin-related peptide 3, peptide PRQFV-amide, and tachykinin-related peptide. Receptors for cholecystokininA and cholecystokininB, and orexin2 were also identified in this context for the first time. We report evidence for acetylcholine, dopamine, noradrenaline, octopamine, small cardioactive peptide related peptide, and receptors for cephalotocin and octopressin, confirming previous publications. The effects of Aggregata observed here extend those previously described by showing effects on the gastric ganglion; in animals with a higher level of infection, genes implicated in inflammation (NFκB, fascin, serpinB10 and the toll-like 3 receptor) increased their relative expression, but TNF-α gene expression was lower as was expression of other genes implicated in oxidative stress (i.e., superoxide dismutase, peroxiredoxin 6, and glutathione peroxidase). Elevated Aggregata levels in the octopuses corresponded to an increase in the expression of the cholecystokininA receptor and the small cardioactive peptide-related peptide. In contrast, we observed decreased relative expression of cephalotocin, dopamine ß-hydroxylase, peptide PRQFV-amide, and tachykinin-related peptide genes. A discussion is provided on (i) potential roles of the various molecules in food intake regulation and digestive tract motility control and (ii) the difference in relative gene expression in the gastric ganglion in octopus with relatively high and low parasitic loads and the similarities to changes in the enteric innervation of mammals with digestive tract parasites. Our results provide additional data to the described neurochemical complexity of O. vulgaris gastric ganglion.

Mechanisms of wound closure following acute arm injury in Octopus vulgaris.

BACKGROUND: Octopoda utilise their arms for a diverse range of functions, including locomotion, hunting, defence, exploration, reproduction, and grooming. However the natural environment contains numerous threats to the integrity of arms, including predators and prey during capture. Impressively, octopoda are able to close open wounds in an aquatic environment and can fully regenerate arms. The regrowth phase of cephalopod arm regeneration has been grossly described; however, there is little information about the acute local response that occurs following an amputation injury comparable to that which frequently occurs in the wild. METHODS: Adult Octopus vulgaris caught in the Bay of Naples were anaesthetised, the distal 10 % of an arm was surgically amputated, and wounded tissue was harvested from animals sacrificed at 2, 6, and 24 h post-amputation. The extent of wound closure was quantified, and the cell and tissue dynamics were observed histologically, by electron microscopy, as well as using ultrasound. RESULTS: Macroscopic, ultrasonic and ultrastructural analyses showed extensive and significant contraction of the wound margins from the earliest time-point, evidenced by tissue puckering. By 6 h post amputation, the wound was 64.0 ± 17.2 % closed compared to 0 h wound area. Wound edge epithelial cells were also seen to be migrating over the wound bed, thus contributing to tissue repair. Temporary protection of the exposed tip in the form of a cellular, non-mucus plug was observed, and cell death was apparent within two hours of injury. At earlier time-points this was apparent in the skin and deeper muscle layers, but ultimately extended to the nerve cord by 24 h. CONCLUSIONS: This work has revealed that O. vulgaris ecologically relevant amputation wounds are rapidly repaired via numerous mechanisms that are evolutionarily conserved. The findings provide insights into the early processes of repair preparatory to regeneration. The presence of epithelial, chromatophore, vascular, muscle and neural tissue in the arms makes this a particularly interesting system in which to study acute responses to injury and subsequent regeneration.

Human Peritoneal Mesothelial Cells Display Phagocytic and Antigen-Presenting Functions to Contribute to Intraperitoneal Immunity.

Mesothelial cells lining the peritoneal cavity are strategically positioned to respond to and counter intraperitoneal infections, cancer cells, and other challenges. We have investigated human peritoneal mesothelial cells (HPMCs) for phagocytic activity, expression of surface Major Histocompatibility Complex (MHC) class II and accessory molecules involved in antigen presentation, and the ability to present recall antigens to T cells. Phagocytosis of dextran, latex beads, and Escherichia coli was observed by flow cytometry, and internalization was visualized using confocal and electron microscopy. Flow cytometry and/or cellular enzyme-linked immunosorbent assay showed constitutive expression of ICAM-1, LFA-3, and B7-1, but not B7-2 or MHC class II. Interferon-gamma induced MHC II and ICAM-1 expression in a dose- and time-dependent manner. Importantly, HPMCs induced autologous CD3 T-lymphocyte proliferation (H incorporation) after pulse with recall antigen. Human peritoneal mesothelial cells equipped with phagocytic and antigen-presenting machinery are anticipated to have an integral role in intraperitoneal immune surveillance.

Wound repair: a showcase for cell plasticity and migration.

A skin wound requires several cell lineages to exhibit considerable plasticity as they migrate towards and over the site of damage to contribute to repair. The keratinocytes that re-epithelialize the tissue, the dermal fibroblasts and potentially other mesenchymal stem cell populations that repopulate damaged connective tissue, the immune cells that counter infections, and endothelial cells that re-establish blood supply and facilitate the immune response - all of these cells are 'dynamic' in that they are activated by immediate wound cues, they reprogram to adopt cell behaviours essential for repair including migration, and finally they must resolve. In adult tissues, repair is unique in its requirement for dramatic cell changes and movements otherwise associated only with development and disease.

The biomechanical and histological sequelae of common skin banking methods.

Human skin allografts are used worldwide as an adjunct for the healing of burns when autograft skin is not available or not indicated. Allograft skin comes from human cadaveric donors, and so must be preserved until use. This study forms the first investigation to compare the mechanical and histological integrity of human split-thickness skin grafts preserved by either glycerolisation or cryopreservation (with or without the cryoprotectant DMSO). Stress relaxation was used to assess mechanical properties, whilst histological analysis allowed for evaluation of structural integrity. Preservation of tissue, whether by freezing or glycerolisation, altered the relaxation behaviours of skin. Young's modulus upon initial loading significantly decreased for skin frozen without cryoprotectant, but remained unchanged for skin frozen with cryoprotectant and skin preserved with glycerol. After 1.5h of stress relaxation, both fresh skin and skin frozen without DMSO displayed similar relaxation rates. Samples frozen with DMSO or preserved with glycerol had increased relaxation rate and had not reached load equilibrium within this time. To understand the structural basis for the biomechanical changes, samples were histologically assessed. All preservation protocols resulted in a similar degree of visible damage, but cryopreservation appeared particularly damaging to the extracellular matrix, whereas glycerolisation caused dramatic separation of the epidermis from the underlying dermis. The mechanical property alterations reveal that preservation results in laxity, which clinically could hinder contact dependent healing properties, but alternatively may increase capacity for coverage. The structural changes confirm that preservation techniques do not conserve grafts in an in vivo state.

The 24th annual meeting of the European Tissue Repair Society (ETRS) in Edinburgh, Scotland.

From the 10th to 12th of September 2014, in the midst of the Scottish Independence debate, the European Tissue Repair Society descended on Edinburgh for their 24th Annual Meeting. In the beautiful and historic setting of the Royal College of Surgeons of Scotland, Professors David Thomas (Chair), Phil Stephens, Chris Lloyd, and their teams from Cardiff hosted an educational and inspiring program.