Dipeptidyl Peptidase-4-Mediated Fibronectin Processing Evokes a Profibrotic Extracellular Matrix.

Fibronectin serves as a platform to guide and facilitate deposition of collagen and fibrillin microfibrils. During development of fibrotic diseases, altered fibronectin deposition in the extracellular matrix (ECM) is generally an early event. After this, dysregulated organization of fibrillins and fibrillar collagens occurs. Because fibronectin is an essential orchestrator of healthy ECM, perturbation of its ECM-organizational capacity may be involved in development of fibrosis. To investigate this, we employed recessive dystrophic epidermolysis bullosa as a disease model with progressive, severe dermal fibrosis. Fibroblasts from donors with recessive dystrophic epidermolysis bullosa in 2-dimensional and 3-dimensional cultures displayed dysregulated fibronectin deposition. Our analyses revealed that increase of profibrotic dipeptidyl peptidase-4-positive fibroblasts coincides with altered fibronectin deposition. Dipeptidyl peptidase-4 inhibitors normalized deposition of fibronectin and subsequently of fibrillin microfibrils and collagen I. Intriguingly, proteomics and inhibitor and mutagenesis studies disclosed that dipeptidyl peptidase-4 modulates ECM deposition through the proteolysis of the fibronectin N-terminus. Our study provides mechanistic insights into the observed profibrotic activities of dipeptidyl peptidase-4 and extends the understanding of fibronectin-guided ECM assembly in health and disease.

Identification of PCPE-2 as the endogenous specific inhibitor of human BMP-1/tolloid-like proteinases.

BMP-1/tolloid-like proteinases (BTPs) are major players in tissue morphogenesis, growth and repair. They act by promoting the deposition of structural extracellular matrix proteins and by controlling the activity of matricellular proteins and TGF-ß superfamily growth factors. They have also been implicated in several pathological conditions such as fibrosis, cancer, metabolic disorders and bone diseases. Despite this broad range of pathophysiological functions, the putative existence of a specific endogenous inhibitor capable of controlling their activities could never be confirmed. Here, we show that procollagen C-proteinase enhancer-2 (PCPE-2), a protein previously reported to bind fibrillar collagens and to promote their BTP-dependent maturation, is primarily a potent and specific inhibitor of BTPs which can counteract their proteolytic activities through direct binding. PCPE-2 therefore differs from the cognate PCPE-1 protein and extends the possibilities to fine-tune BTP activities, both in physiological conditions and in therapeutic settings.

Systemic Collagen VII Replacement Therapy for Advanced Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic skin blistering disease associated with progressive multiorgan fibrosis. RDEB is caused by biallelic mutations in COL7A1 encoding the extracellular matrix protein collagen VII (C7), which is necessary for epidermal‒dermal adherence. C7 is not simply a structural protein but also has multiple functions, including the regulation of TGFß bioavailability and the inhibition of skin scarring. Intravenous (IV) administration of recombinant C7 (rC7) rescues C7-deficient mice from neonatal lethality. However, the effect on established RDEB has not been determined. In this study, we used small and large adult RDEB animal models to investigate the disease-modulating abilities of IV rC7 on established RDEB. In adult RDEB mice, rC7 accumulated at the basement membrane zone in multiple organs after a single infusion. Fortnightly IV injections of rC7 for 7 weeks in adult RDEB mice reduced fibrosis of skin and eye. The fibrosis-delaying effect was associated with a reduction of TGFß signaling. IV rC7 in adult RDEB dogs incorporated in the dermal‒epidermal junction of skin and improved disease by promoting wound healing and reducing dermal‒epidermal separation. In both species, IV C7 was well-tolerated. These preclinical studies suggest that repeated IV administration of rC7 is an option for systemic treatment of established adult RDEB.

Increased abundance of Cbl E3 ligases alters PDGFR signaling in recessive dystrophic epidermolysis bullosa.

In recessive dystrophic epidermolysis bullosa (RDEB), loss of collagen VII, the main component of anchoring fibrils critical for epidermal-dermal cohesion, affects several intracellular signaling pathways and leads to impaired wound healing and fibrosis. In skin fibroblasts, wound healing is also affected by platelet-derived growth factor receptor (PDGFR) signaling. To study a potential effect of loss of collagen VII on PDGFR signaling we performed unbiased disease phosphoproteomics. Whereas RDEB fibroblasts exhibited an overall weaker response to PDGF, Cbl E3 ubiquitin ligases, negative regulators of growth factor signaling, were stronger phosphorylated. This increase in phosphorylation was linked to higher Cbl mRNA and protein levels due to increased TGFß signaling in RDEB. In turn, increased Cbl levels led to increased PDGFR ubiquitination, internalization, and degradation negatively affecting MAPK and AKT downstream signaling pathways. Thus, our results indicate that elevated TGFß signaling leads to an attenuated response to growth factors, which contributes to impaired dermal wound healing in RDEB.

Dystrophic Epidermolysis Bullosa: Secondary Disease Mechanisms and Disease Modifiers.

The phenotypic presentation of monogenetic diseases is determined not only by the nature of the causative mutations but also is influenced by manifold cellular, microenvironmental, and external factors. Here, heritable extracellular matrix diseases, including dystrophic epidermolysis bullosa (DEB), are no exceptions. Dystrophic epidermolysis bullosa is caused by mutations in the COL7A1 gene encoding collagen VII. Deficiency of collagen VII leads to skin and mucosal fragility, which progresses from skin blistering to severe fibrosis and cancer. Clinical and pre-clinical studies suggest that targeting of secondary disease mechanisms or employment of natural disease modifiers can alleviate DEB severity and progression. However, since many of these mechanisms are needed for tissue homeostasis, informed, selective targeting is essential for safe and efficacious treatment. Here, we discuss a selection of key disease modifiers and modifying processes active in DEB, summarize the still scattered knowledge of them, and reflect on ways forward toward their utilization for symptom-relief or enhancement of curative therapies.

Pro-inflammatory immunity supports fibrosis advancement in epidermolysis bullosa: intervention with Ang-(1-7).

Recessive dystrophic epidermolysis bullosa (RDEB), a genetic skin blistering disease, is a paradigmatic condition of tissue fragility-driven multi-organ fibrosis. Here, longitudinal analyses of the tissue proteome through the course of naturally developing disease in RDEB mice revealed that increased pro-inflammatory immunity associates with fibrosis evolution. Mechanistically, this fibrosis is a consequence of altered extracellular matrix organization rather than that of increased abundance of major structural proteins. In a humanized system of disease progression, we targeted inflammatory cell fibroblast communication with Ang-(1-7)-an anti-inflammatory heptapeptide of the renin-angiotensin system, which reduced the fibrosis-evoking aptitude of RDEB cells. In vivo, systemic administration of Ang-(1-7) efficiently attenuated progression of multi-organ fibrosis and increased survival of RDEB mice. Collectively, our study shows that selective down-modulation of pro-inflammatory immunity may mitigate injury-induced fibrosis. Furthermore, together with published data, our data highlight molecular diversity among fibrotic conditions. Both findings have direct implications for the design of therapies addressing skin fragility and fibrosis.

Diversity of Mechanisms Underlying Latent TGF-ß Activation in Recessive Dystrophic Epidermolysis Bullosa.

Injury- and inflammation-driven progressive dermal fibrosis is a severe manifestation of recessive dystrophic epidermolysis bullosa-a genetic skin blistering disease caused by mutations in COL7A1. TGF-ß activation plays a prominent part in progressing dermal fibrosis. However, the underlying mechanisms are not fully elucidated. TGF-ß is secreted in a latent form, which has to be activated for its biological functions. In this study, we determined that recessive dystrophic epidermolysis bullosa fibroblasts have an enhanced capacity to activate the latent form. Mechanistic and functional assessment demonstrated that this process depends on multiple latent TGF-ß activators, including TSP-1, RGD-binding integrins, matrix metalloproteinases, and ROS, which act in concert, in a self-perpetuating feedback loop to progress fibrosis. Importantly, our study also disclosed keratinocytes as prominent facilitators of fibrosis in recessive dystrophic epidermolysis bullosa. They stimulate microenvironmental latent TGF-ß activation through enhanced production of the above mediators. Collectively, our study provides data on the molecular mechanism behind dysregulated TGF-ß signaling in recessive dystrophic epidermolysis bullosa, which are much needed for the development of evidence-based fibrosis-delaying treatments.

Kindlin-2 Mediates Mechanical Activation of Cardiac Myofibroblasts.

We identify the focal adhesion protein kindlin-2 as player in a novel mechanotransduction pathway that controls profibrotic cardiac fibroblast to myofibroblast activation. Kindlin-2 is co-upregulated with the myofibroblast marker α-smooth muscle actin (α-SMA) in fibrotic rat hearts and in human cardiac fibroblasts exposed to fibrosis-stiff culture substrates and pro-fibrotic TGF-ß1. Stressing fibroblasts using ferromagnetic microbeads, stretchable silicone membranes, and cell contraction agonists all result in kindlin-2 translocation to the nucleus. Overexpression of full-length kindlin-2 but not of kindlin-2 missing a putative nuclear localization sequence (∆NLS kindlin-2) results in increased α-SMA promoter activity. Downregulating kindlin-2 with siRNA leads to decreased myofibroblast contraction and reduced α-SMA expression, which is dependent on CC(A/T)-rich GG(CArG) box elements in the α-SMA promoter. Lost myofibroblast features under kindlin-2 knockdown are rescued with wild-type but not ∆NLS kindlin-2, indicating that myofibroblast control by kindlin-2 requires its nuclear translocation. Because kindlin-2 can act as a mechanotransducer regulating the transcription of α-SMA, it is a potential target to interfere with myofibroblast activation in tissue fibrosis.

Epidermolysis bullosa.

Epidermolysis bullosa (EB) is an inherited, heterogeneous group of rare genetic dermatoses characterized by mucocutaneous fragility and blister formation, inducible by often minimal trauma. A broad phenotypic spectrum has been described, with potentially severe extracutaneous manifestations, morbidity and mortality. Over 30 subtypes are recognized, grouped into four major categories, based predominantly on the plane of cleavage within the skin and reflecting the underlying molecular abnormality: EB simplex, junctional EB, dystrophic EB and Kindler EB. The study of EB has led to seminal advances in our understanding of cutaneous biology. To date, pathogenetic mutations in 16 distinct genes have been implicated in EB, encoding proteins influencing cellular integrity and adhesion. Precise diagnosis is reliant on correlating clinical, electron microscopic and immunohistological features with mutational analyses. In the absence of curative treatment, multidisciplinary care is targeted towards minimizing the risk of blister formation, wound care, symptom relief and specific complications, the most feared of which - and also the leading cause of mortality - is squamous cell carcinoma. Preclinical advances in cell-based, protein replacement and gene therapies are paving the way for clinical successes with gene correction, raising hopes amongst patients and clinicians worldwide.

The Status Quo of Rare Diseases Centres for the Development of a Clinical Decision Support System - A Cross-Sectional Study.

Clinical decision support systems (CDSS) help to improve the diagnostics and treatment of rare diseases (RD). As one of four funded consortia of the Medical Informatics Initiative supported by the Federal Ministry of Education and Research (BMBF, Germany), MIRACUM develops a clinical decision support system (CDSS) for RD based on distributed data of ten university hospitals. The CDSS will be developed at the Rare Diseases Centres (RDC) of the MIRACUM consortium. Since it is essential to deliver decision support at the right time and place in the clinician's workflow, this study aimed to capture relevant information of the RDCs regarding patient admission and diagnostic process. Additionally, we investigated how patient documentation and digitalisation is performed at the centres. Therefore, we conducted a cross-sectional survey involving experts in the RDs domain to capture relevant information for the further development of a CDSS in RD. For each centre, one expert on RDs participated in the study (n=8). The survey identified several challenges regarding the reuse of patient data, e.g. the paper-based documentation of a patientâAZs medical history and coding of diagnoses using ICD-10. However, we noticed a relevant use of current software diagnosis support and a similarly performed diagnostic process in all RDC. Further studies are needed to get more detailed insights and to define specific requirements.

Skin proteomics - analysis of the extracellular matrix in health and disease.

INTRODUCTION: The skin protects the human body from external insults and regulates water and temperature homeostasis. A highly developed extracellular matrix (ECM) supports the skin and instructs its cell functions. Reduced functionality of the ECM is often associated with skin diseases that cause physical impairment and also have implications on social interactions and quality of life of affected individuals. AREAS COVERED: With a focus on the skin ECM we discuss how mass spectrometry (MS)-based proteomic approaches first contributed to establishing skin protein inventories and then facilitated elucidation of molecular functions and disease mechanisms. EXPERT OPINION: MS-based proteomic approaches have significantly contributed to our understanding of skin pathophysiology, but also revealed the challenges in assessing the skin ECM. The numerous posttranslational modifications of ECM proteins, like glycosylation, crosslinking, oxidation, and proteolytic maturation in disease settings can be difficult to tackle and remain understudied. Increased ease of handling of LC-MS/MS systems and automated/streamlined data analysis pipelines together with the accompanying increased usage of LC-MS/MS approaches will ensure that in the coming years MS-based proteomic approaches will continue to play a vital part in skin disease research. They will facilitate the elucidation of molecular disease mechanisms and, ultimately, identification of new druggable targets.

Proteomic Profiling of Fibroblasts Isolated from Chronic Wounds Identifies Disease-Relevant Signaling Pathways.

Chronic skin wounds accompany many prevalent age-related diseases and are a major cause of morbidity and mortality. Both keratinocytes and fibroblasts contribute to the pathomechanisms in chronic skin wounds. Dysregulated pathways in the epidermis have been extensively studied, but little is known of the influence of dermal fibroblasts on chronic wounding. We isolated fibroblasts from chronic wounds, propagated them in vitro, and analyzed them using proteomic profiling in combination with functional characterization of the proteomic changes. Chronic wound-associated fibroblasts exhibit a unique proteome profile characteristic of lysosomal dysfunction and dysregulated TGFß signaling. They display a decreased propensity for cell proliferation and migration, combined with an enhanced ability to contract the extracellular matrix. With these properties, chronic wound-associated fibroblasts actively contribute to pathological inabilities to close wounds and represent potential targets for pharmacological interference for changing cellular phenotypes.

Skin Fragility: Perspectives on Evidence-based Therapies.

The term skin fragility disorders describes a group of conditions in which the structural integrity of the skin is compromised and its resistance to external shear forces diminished. Skin fragility can have different causes, ranging from genetic variations to inflammatory or physical phenomena. The genetic skin fragility disorders, collectively called epidermolysis bullosa, serve as a paradigm for the study of causes and mechanisms of skin fragility. Recent biomedical research has revealed substantial genetic heterogeneity of the epidermolysis bullosa group, delivered ample new knowledge on its pathophysiology, and facilitated the design of evidence-based therapeutic strategies. The therapy development process extends from in vitro testing to preclinical validation in animal models, and clinical trials. This article reviews different approaches to curative and symptom-relief therapies, and appraises their status and perspectives for clinical implementation.

EGFR inhibition for metastasized cutaneous squamous cell carcinoma in dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is a hereditary skin fragility disorder, characterized by trauma-induced blistering followed by soft tissue fibrosis. One of the most feared complications is the early development of aggressive cutaneous squamous cell carcinomas (SCC). For patients with locally advanced or metastasized SCCs treatment with cetuximab, a monoclonal antibody against epidermal growth factor receptor (EGFR), has been proposed and so far, treatment of five DEB patients with cetuximab has been published. With this report, we extend the spectrum of EB patients treated with cetuximab by adding two additional patients. Taking together all DEB cases treated with cetuximab, we propose that cetuximab should be administered as early as possible, since it seems to be more efficient and is accompanied by rather mild adverse effects. We also show that EGFR is frequently expressed in DEB-associated SCCs, although there were noticeable differences in the level of expression, which may influence responsiveness to EGFR-targeting therapies. Although only limited experiences with targeted cancer treatments in EB exist, such reports highlight the treatments' effects in this specific cohort and assist our therapeutic decisions.

Newer Treatment Modalities in Epidermolysis Bullosa.

The term epidermolysis bullosa (EB) refers to a group of hereditary skin blistering diseases. The group is clinically and genetically heterogeneous, but all EB forms are associated with mechanically induced skin blistering and fragility. The causative gene mutations of most EB types are known. The current international consensus classification contains four main types: EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB), and Kindler syndrome (KS). The classification is based on the morphological level of blister formation. In EBS, the split is intra-epidermal, in JEB along the basement membrane and in DEB below the basement membrane. In Kindler syndrome, the dermal-epidermal junction is disorganized, and blisters can occur on all three levels. Each major EB type has further subtypes which may differ in terms of their genetic, biological or clinical characteristics. Traditionally, EB treatments have been symptomatic, but increasing understanding of disease etio-pathogenesis is facilitating development of novel evidence-based therapy approaches. First gene- and cell-based therapies are being tested at preclinical level and in clinical trials. New knowledge on secondary disease mechanisms has led to development and clinical testing of urgently needed symptom-relief therapies using small molecules and biologicals.