Johannes S. Kern

A hypomorphic mouse model of dystrophic epidermolysis bullosa reveals mechanisms of disease and response to fibroblast therapy

Dystrophic epidermolysis bullosa (DEB) is a severe skin fragility disorder associated with trauma-induced blistering, progressive soft tissue scarring, and increased risk of skin cancer. DEB is caused by mutations in type VII collagen. In this study, we describe the generation of a collagen VII hypomorphic mouse that serves as an immunocompetent animal model for DEB. These mice expressed collagen VII at about 10% of normal levels, and their phenotype closely resembled characteristics of severe human DEB, including mucocutaneous blistering, nail dystrophy, and mitten deformities of the extremities. The oral blistering experienced by these mice resulted in growth retardation, and repeated blistering led to excessive induction of tissue repair, causing TGF-beta1-mediated contractile fibrosis generated by myofibroblasts and pseudosyndactyly in the extremities. Intradermal injection of WT fibroblasts resulted in neodeposition of collagen VII and functional restoration of the dermal-epidermal junction. Treated areas were also resistant to induced frictional stress. In contrast, untreated areas of the same mouse showed dermal-epidermal separation following induced stress. These data demonstrate that fibroblast-based treatment can be used to treat DEB in a mouse model and suggest that this approach may be effective in the development of clinical therapeutic regimens for patients with DEB.

Mechanisms of Fibroblast Cell Therapy for Dystrophic Epidermolysis Bullosa: High Stability of Collagen VII Favors Long-term Skin Integrity

Here, we report on the first systematic long-term study of fibroblast therapy in a mouse model for recessive dystrophic epidermolysis bullosa (RDEB), a severe skin-blistering disorder caused by loss-of-function of collagen VII. Intradermal injection of wild-type (WT) fibroblasts in >50 mice increased the collagen VII content at the dermal-epidermal junction 3.5- to 4.7-fold. Although the active biosynthesis lasted <28 days, collagen VII remained stable and dramatically improved skin integrity and resistance to mechanical forces for at least 100 days, as measured with a digital 3D-skin sensor for shear forces. Experiments using species-specific antibodies, collagen VII-deficient fibroblasts, gene expression analyses, and cytokine arrays demonstrated that the injected fibroblasts are the major source of newly deposited collagen VII. Apart from transitory mild inflammation, no adverse effects were observed. The cells remained within an area <or=10 mm of the injection site, and did not proliferate, form tumors, or cause fibrosis. Instead, they became gradually apoptotic within 28 days. These data on partial restoration of collagen VII in the skin demonstrate the excellent ratio of clinical effects to biological parameters, support suitability of fibroblast-based therapy approaches for RDEB, and, as a preclinical test, pave way to human clinical trials.

Collagen VII plays a dual role in wound healing

Although a host of intracellular signals is known to contribute to wound healing, the role of the cell microenvironment in tissue repair remains elusive. Here we employed 2 different mouse models of genetic skin fragility to assess the role of the basement membrane protein collagen VII (COL7A1) in wound healing. COL7A1 secures the attachment of the epidermis to the dermis, and its mutations cause a human skin fragility disorder coined recessive dystrophic epidermolysis bullosa (RDEB) that is associated with a constant wound burden. We show that COL7A1 is instrumental for skin wound closure by 2 interconnected mechanisms. First, COL7A1 was required for re-epithelialization through organization of laminin-332 at the dermal-epidermal junction. Its loss perturbs laminin-332 organization during wound healing, which in turn abrogates strictly polarized expression of integrin α6β4 in basal keratinocytes and negatively impacts the laminin-332/integrin α6β4 signaling axis guiding keratinocyte migration. Second, COL7A1 supported dermal fibroblast migration and regulates their cytokine production in the granulation tissue. These findings, which were validated in human wounds, identify COL7A1 as a critical player in physiological wound healing in humans and mice and may facilitate development of therapeutic strategies not only for RDEB, but also for other chronic wounds.

Chronic colitis due to an epithelial barrier defect: the role of kindlin-1 isoforms†

Kindlin‐1 is an epithelium‐specific phosphoprotein and focal adhesion adaptor component. Mutations in the corresponding gene (KIND1) cause Kindler syndrome (KS), which is manifested by skin blistering, poikiloderma, photosensitivity and carcinogenesis. Some patients also exhibit gastrointestinal symptoms, but it has remained unclear whether these represent a feature of Kindler syndrome or a coincidence. We examined kindlin‐1 in human gastrointestinal epithelia and showed that it is involved in the aetiopathology of Kindler syndrome‐associated colitis. Kindlin‐1 expression was assessed by indirect immunofluorescence, western blot and RT–PCR. Kindlin‐1 is expressed in oral mucosa, colon and rectum. Both the full‐length 74 kDa kindlin‐1 protein and a 43 kDa isoform were detected in CaCo2 cells, the latter resulting from alternative splicing. In the first months of life, patients (homozygous for null mutations) had severe intestinal involvement with haemorrhagic diarrhoea and showed morphological features of severe ulcerative colitis. Later in childhood, histopathology demonstrated focal detachment of the epithelium in all segments of the colon, chronic inflammation and mucosal atrophy. These findings define an intestinal phenotype for Kindler syndrome as a consequence of a primary epithelial barrier defect. The different clinical intestinal manifestations in Kindler syndrome patients may be explained by partial functional compensation of kindlin‐1 deficiency by the intestinal isoform or by the presence of truncated mutant kindlin‐1. Copyright

Global remodelling of cellular microenvironment due to loss of collagen VII

The mammalian cellular microenvironment is shaped by soluble factors and structural components, the extracellular matrix, providing physical support, regulating adhesion and signalling. A global, quantitative mass spectrometry strategy, combined with bioinformatics data processing, was developed to assess proteome differences in the microenvironment of primary human fibroblasts. We studied secreted proteins of fibroblasts from normal and pathologically altered skin and their post‐translational modifications. The influence of collagen VII, an important structural component, which is lost in genetic skin fragility, was used as model. Loss of collagen VII had a global impact on the cellular microenvironment and was associated with proteome alterations highly relevant for disease pathogenesis including decrease in basement membrane components, increase in dermal matrix proteins, TGF‐β and metalloproteases, but not higher protease activity. The definition of the proteome of fibroblast microenvironment and its plasticity in health and disease identified novel disease mechanisms and potential targets of intervention.

Forty-two novel COL7A1 mutations and the role of a frequent single nucleotide polymorphism in the MMP1 promoter in modulation of disease severity in a large European dystrophic epidermolysis bullosa cohort.

Background  Dystrophic epidermolysis bullosa (DEB) is a severe genetic skin blistering disorder caused by mutations in the gene COL7A1, encoding collagen VII. Recently, the MMP1 promoter single nucleotide polymorphism (SNP) rs1799750, designated as 1G 2G, was shown to be involved in modulation of disease severity in patients with recessive DEB (RDEB), and was proposed as a genetic modifier.

Losartan ameliorates dystrophic epidermolysis bullosa and uncovers new disease mechanisms

Genetic loss of collagen VII causes recessive dystrophic epidermolysis bullosa (RDEB)—a severe skin fragility disorder associated with lifelong blistering and disabling progressive soft tissue fibrosis. Causative therapies for this complex disorder face major hurdles, and clinical implementation remains elusive. Here, we report an alternative evidence‐based approach to ameliorate fibrosis and relieve symptoms in RDEB. Based on the findings that TGF‐β activity is elevated in injured RDEB skin, we targeted TGF‐β activity with losartan in a preclinical setting. Long‐term treatment of RDEB mice efficiently reduced TGF‐β signaling in chronically injured forepaws and halted fibrosis and subsequent fusion of the digits. In addition, proteomics analysis of losartan‐ vs. vehicle‐treated RDEB skin uncovered changes in multiple proteins related to tissue inflammation. In line with this, losartan reduced inflammation and diminished TNF‐α and IL‐6 expression in injured forepaws. Collectively, the data argue that RDEB fibrosis is a consequence of a cascade encompassing tissue damage, TGF‐β‐mediated inflammation, and matrix remodeling. Inhibition of TGF‐β activity limits these unwanted outcomes and thereby substantially ameliorates long‐term symptoms.

Molecular mechanisms of phenotypic variability in junctional epidermolysis bullosa

Background Junctional epidermolysis bullosa (JEB), a group of hereditary skin fragility disorders, is associated with a wide variety of phenotypes, although all forms are characterised by trauma induced skin blistering and tissue separation at the dermal–epidermal junction zone. A subgroup, coined JEB-other, is associated with mutations in the COL17A1 gene encoding collagen XVII or, more rarely, with mutations in the laminin 332 genes LAMA3, LAMB3, or LAMC2. The objective of this study is comprehensive genotype–phenotype analysis in JEB-other patients with COL17A1 mutations and elucidation of disease mechanisms underlying different skin phenotypes. Methods and results COL17A1 mutations and their clinical and cellular consequences were systematically analysed in 43 patients with JEB-other. Cell culture, RT-PCR, and protein biochemistry were applied to assess the effects of splice site mutations—that is, the nature and amounts of transcripts and polypeptides synthesised and their association with the phenotypic outcome. 34 distinct COL17A1 mutations were disclosed, 12 of them novel. mRNA and protein analyses demonstrated that patients with only about 12–14% of the physiological collagen XVII levels had mild cutaneous involvement and a long life span. Conclusions In contrast to complete null phenotypes, presence of minor amounts of collagen XVII protein in JEB skin is associated with mild phenotypic manifestations. The data have significant implications for design of molecular therapies for JEB, since they suggest that already a low extent of collagen XVII restoration will improve skin stability and alleviate symptoms.

S2k guidelines for the treatment of pemphigus vulgaris/foliaceus and bullous pemphigoid.

(1) Department of Dermatology and Allergology, Philipps-Universität Marburg, Germany (2) Department of Dermatology, University Hospital Erlangen, Erlangen, Germany (3) Helios Hospital Wuppertal, Department of Dermatology, Allergology and Dermatosurgery, Wuppertal, Germany (4) Department of Dermatology and Venereology, University of Cologne (5) Department of Dermatology, Medical Center, University of Freiburg, Germany (6) Dermatologist in Private Practice, Fulda, Germany (7) Department of Dermatology and Allergology, University Hospital Ulm, Germany (8) Pediatric Immunology and Rheumatology, University Hospital and Outpatient Clinic for Pediatrics, Leipzig, Germany (9) Department of Dermatology, Allergology, and Venereology, Campus Luebeck, University Hospital Schleswig-Holstein (UKSH), Luebeck, Germany (10) Department of Dermatology, Venereology, and Allergology, University Hospital Wuerzburg, Wuerzburg, Germany (11) Division of Evidence-based Medicine (dEBM), Department of Dermatology, Charité University Hospital Berlin, Germany (12) Shire Deutschland GmbH, Berlin, Germany (13) Department of Dermatology and Venereology, University Hospital Munich (LMU), Munich, Germany (14) Allergy Center, Department of Dermatology, Charité University Hospital Berlin, Germany

Overexpression of the Flii gene increases dermal-epidermal blistering in an autoimmune ColVII mouse model of epidermolysis bullosa acquisita.

Epidermolysis bullosa (EB) is a severe genetic skin fragility syndrome characterized by blister formation. The molecular basis of EB is still largely unknown and wound healing in patients suffering from EB remains a major challenge to their survival. Our previous studies have identified the actin remodelling protein Flightless I (Flii) as an important mediator of wound repair. Here we identify Flii as a novel target involved in skin blistering. Flii expression was significantly elevated in 30 patients with EB, most prominently in patients with recessive dystrophic EB (RDEB) who have defects in production of type VII collagen (ColVII). Using an autoimmune ColVII murine model of EB acquisita (EBA) and an immunocompetent‐ColVII‐hypomorphic genetic mouse model of RDEB together with murine Flii alleles, we investigated the contribution of Flii to EB. Overexpression of Flii produced severe blistering post‐induction of EBA, while decreased Flii reduced blister severity, elevated integrin expression, and improved ColVII production. Flii+/− blistered skin showed reduced α‐SMA, TGF‐β1, and Smad 2/3 expression, suggesting that decreasing Flii may affect fibrosis. In support of this, Flii‐deficient fibroblasts from EBA mice were less able to contract collagen gels in vitro; however, addition of TGF‐β1 restored collagen contraction, suggesting an interplay between Flii and TGF‐β1. Elevated Flii gene and protein expression was further observed in the blisters of ColVII hypomorphic mice, a murine model of RDEB, suggesting that reducing Flii in blistered skin could be a potential new approach for treating patients with EB. Copyright