Alexander Nyström

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.

Genetically corrected iPSCs as cell therapy for recessive dystrophic epidermolysis bullosa

iPSCs derived from fibroblasts with mutant type VII collagen were genetically corrected and used as cell therapy in mice with recessive dystrophic epidermolysis bullosa. iPSC-Derived Cell Therapy for Epidermolysis Bullosa Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic condition where even the soft touch of a cotton sheet can cause severe skin blistering and pain. Patients with RDEB often spend their lives in bandages, with no treatment options at present. Now, Wenzel et al. offer a cell therapy that would use induced pluripotent stem cells from patients to treat the wounded areas. The authors took fibroblasts from the tails of mice with or without RDEB (with or without mutated Col7a1) and used these to create iPSC lines. The iPSCs were genetically corrected to express type VII collagen and then differentiated into keratinocytes (skin cells) or fibroblasts. When iPSC-derived fibroblasts were delivered back to the animals with RDEB, only the genetically corrected cells created skin layers that expressed type VII collagen and were resistant to mechanical friction (no blistering). Genetically repaired iPSCs, therefore, offer a viable cell therapy for RDEB if the methods of gene correction can be made safe for use in humans. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in the gene encoding type VII collagen, resulting in fragile skin and mucous membranes that blister easily in response to mechanical stress. Induced pluripotent stem cells (iPSCs) carry the potential to fundamentally change cell-based therapies for human diseases, in particular for RDEB, for which no effective treatments are available. To provide proof of principle on the applicability of iPSCs for the treatment of RDEB, we developed iPSCs from type VII collagen (Col7a1) mutant mice that exhibited skin fragility and blistering resembling human RDEB. Genetically repaired iPSCs could be differentiated into functional fibroblasts that reexpressed and secreted type VII collagen. Corrected iPSC–derived fibroblasts did not form tumors in vivo and could be traced up to 16 weeks after intradermal injection. Moreover, iPSC-based cell therapy resulted in faithful and long-term restoration of type VII collagen deposition at the epidermal-dermal junction of Col7a1 mutant mice. Intradermal injection of genetically repaired iPSC-derived fibroblasts restored the mechanical resistance to skin blistering in mice with RDEB, suggesting that RDEB skin could be effectively and safely repaired using iPSC-based cell therapy.

Cell therapy for basement membrane-linked diseases

For most disorders caused by mutations in genes encoding basement membrane (BM) proteins, there are at present only limited treatment options available. Genetic BM-linked disorders can be viewed as especially suited for treatment with cell-based therapy approaches because the proteins that need to be restored are located in the extracellular space. In consequence, complete and permanent engraftment of cells does not necessarily have to occur to achieve substantial causal therapeutic effects. For these disorders cells can be used as transient vehicles for protein replacement. In addition, it is becoming evident that BM-linked genetic disorders are modified by secondary diseases mechanisms. Cell-based therapies have also the ability to target such disease modifying mechanisms. Thus, cell therapies can simultaneously provide causal treatment and symptomatic relief, and accordingly hold great potential for treatment of BM-linked disorders. However, this potential has for most applications and diseases so far not been realized. Here, we will present the state of cell therapies for BM-linked diseases. We will discuss use of both pluripotent and differentiated cells, the limitation of the approaches, their challenges, and the way forward to potential wider implementation of cell therapies in the clinics.

Cell- and Protein-Based Therapy Approaches for Epidermolysis Bullosa

Dystrophic epidermolysis bullosa (DEB) is a clinically heterogeneous heritable skin fragility disorder characterized by mechanically induced mucocutaneous blistering. On the molecular level DEB is caused by mutations leading to deficiency in collagen VII (CVII), a large extracellular protein building anchoring fibrils that attach the epidermis to the dermis. Severely affected patients suffer from wounds, which heal with excessive scarring causing mutilating deformities of hands and feet. The patients are also predisposed to development of aggressive squamous cell carcinomas at sites of chronic wounds. Currently no available therapies exist for this extremely disabling and stigmatizing disorder. We are developing and evaluating cell- and protein-based therapies for the management of DEB. Dermal fibroblasts are easy to propagate in vitro, they produce CVII, and they have immunomodulating capacities, which makes it possible to use allogeneic fibroblasts for therapy without risking major adverse effects from the hosts immune system. Hence, fibroblasts, and fibroblast-like cells such as mesenchymal stromal cells, are prime candidates for cell-based DEB therapies. An alternative for management of disorders caused by defects in proteins with relatively low turnover rate is to introduce the protein de novo to the tissue by direct application of the protein. CVII is long-lived and expressed in moderate amounts in the skin; this makes injection of collagen VII protein a realistic approach for the treatment of DEB. Here we present methods and protocols that we are using for fibroblast- and recombinant CVII-based therapies of DEB in our model of this disease, the CVII hypomorphic mouse. These protocols are directed towards management of DEB but they can be easily adapted for the treatment of other skin fragility disorders.

One goal, different strategies – molecular and cellular approaches for the treatment of inherited skin fragility disorders

Epidermolysis bullosa (EB) is a heterogeneous group of inherited diseases characterized by the formation of blisters in the skin and mucosa. There is no cure or effective treatment for these potentially severe and fatal diseases. Over the past few years, several reports have proposed different molecular strategies as new therapeutic options for the management of EB. From classical vector‐based gene therapy to cell‐based strategies such as systemic application of bone marrow stem cells or local application of fibroblasts, a broad range of molecular approaches have been explored. This array also includes novel methods, such as protein replacement therapy, gene silencing and the use of induced pluripotent stem cells (iPCs). In this review, we summarize current concepts of how inherited blistering diseases might be treated in the future and discuss the opportunities, promises, concerns and risks of these innovative approaches.

Rat Model for Dominant Dystrophic Epidermolysis Bullosa: Glycine Substitution Reduces Collagen VII Stability and Shows Gene-Dosage Effect

Dystrophic epidermolysis bullosa, a severely disabling hereditary skin fragility disorder, is caused by mutations in the gene coding for collagen VII, a specialized adhesion component of the dermal-epidermal junction zone. Both recessive and dominant forms are known; the latter account for about 40% of cases. Patients with dominant dystrophic epidermolysis bullosa exhibit a spectrum of symptoms ranging from mild localized to generalized skin manifestations. Individuals with the same mutation can display substantial phenotypic variance, emphasizing the role of modifying genes in this disorder. The etiology of dystrophic epidermolysis bullosa has been known for around two decades; however, important pathogenetic questions such as involvement of modifier genes remain unanswered and a causative therapy has yet to be developed. Much of the failure to make progress in these areas is due to the lack of suitable animal models that capture all aspects of this complex monogenetic disorder. Here, we report the first rat model of dominant dystrophic epidermolysis bullosa. Affected rats carry a spontaneous glycine to aspartic acid substitution, p.G1867D, within the main structural domain of collagen VII. This confers dominant-negative interference of protein folding and decreases the stability of mutant collagen VII molecules and their polymers, the anchoring fibrils. The phenotype comprises fragile and blister-prone skin, scarring and nail dystrophy. The model recapitulates all signs of the human disease with complete penetrance. Homozygous carriers of the mutation are more severely affected than heterozygous ones, demonstrating for the first time a gene-dosage effect of mutated alleles in dystrophic epidermolysis bullosa. This novel viable and workable animal model for dominant dystrophic epidermolysis bullosa will be valuable for addressing molecular disease mechanisms, effects of modifying genes, and development of novel molecular therapies for patients with dominantly transmitted skin disease.

Analysis of the functional consequences of targeted exon deletion in COL7A1 reveals prospects for dystrophic epidermolysis bullosa therapy

Genetically evoked deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB)-a debilitating disease characterized by chronic skin fragility and progressive fibrosis. Removal of exons carrying frame-disrupting mutations can reinstate protein expression in genetic diseases. The therapeutic potential of this approach is critically dependent on gene, protein, and disease intrinsic factors. Naturally occurring exon skipping in COL7A1, translating collagen VII, suggests that skipping of exons containing disease-causing mutations may be feasible for the treatment of DEB. However, despite a primarily in-frame arrangement of exons in the COL7A1 gene, no general conclusion of the aptitude of exon skipping for DEB can be drawn, since regulation of collagen VII functionality is complex involving folding, intra- and intermolecular interactions. To directly address this, we deleted two conceptually important exons located at both ends of COL7A1, exon 13, containing recurrent mutations, and exon 105, predicted to impact folding. The resulting recombinantly expressed proteins showed conserved functionality in biochemical and in vitro assays. Injected into DEB mice, the proteins promoted skin stability. By demonstrating functionality of internally deleted collagen VII variants, our study provides support of targeted exon deletion or skipping as a potential therapy to treat a large number of individuals with DEB.

Impaired lymphoid extracellular matrix impedes antibacterial immunity in epidermolysis bullosa

Significance We describe a unique role for the lymphoid extracellular matrix in maintaining systemic innate immunity. Our findings are based on studies of the genetic skin disorder recessive dystrophic epidermolysis bullosa in which affected individuals display dramatically increased bacterial colonization of skin and mucosa. We show that the increased colonization is a consequence of loss of the protein at fault—collagen VII—from the lymphoid extracellular matrix. Our study describes an intrinsic innate immune defect resulting from a genetic connective tissue disease. Hence, the data will have broad implications for deciphering the hitherto-underestimated regulatory role of the extracellular matrix in lymphoid organs and for the understanding of pathomechanisms in disorders of the extracellular matrix. Genetic loss of collagen VII causes recessive dystrophic epidermolysis bullosa (RDEB), a skin fragility disorder that, unexpectedly, manifests also with elevated colonization of commensal bacteria and frequent wound infections. Here, we describe an unprecedented systemic function of collagen VII as a member of a unique innate immune-supporting multiprotein complex in spleen and lymph nodes. In this complex, collagen VII specifically binds and sequesters the innate immune activator cochlin in the lumen of lymphoid conduits. In genetic mouse models, loss of collagen VII increased bacterial colonization by diminishing levels of circulating cochlin LCCL domain. Intraperitoneal injection of collagen VII, which restored cochlin in the spleen, but not in the skin, reactivated peripheral innate immune cells via cochlin and reduced bacterial skin colonization. Systemic administration of the cochlin LCCL domain was alone sufficient to diminish bacterial supercolonization of RDEB mouse skin. Human validation demonstrated that RDEB patients displayed lower levels of systemic cochlin LCCL domain with subsequently impaired macrophage response in infected wounds. This study identifies an intrinsic innate immune dysfunction in RDEB and uncovers a unique role of the lymphoid extracellular matrix in systemic defense against bacteria.

The role of TGFβ in wound healing pathologies

Wound healing is one of the most complex processes in multicellular organisms, involving numerous intra- and intercellular signalling pathways in various cell types. It involves extensive communication between the cellular constituents of diverse skin compartments and its extracellular matrix. Miscommunication during healing may have two distinct damaging consequences: the development of a chronic wound or the formation of a hypertrophic scar/keloid. Chronic wounds are defined as barrier defects that have not proceeded through orderly and timely reparation to regain structural and functional integrity. Several growth factors are involved in wound healing, of which transforming growth factor beta (TGFβ) is of particular importance for all phases of this procedure. It exerts pleiotropic effects on wound healing by regulating cell proliferation, differentiation, extracellular matrix production, and modulating the immune response. In this review we are presenting the role of TGFβ in physiological and pathological wound healing. We show that the context-dependent nature of the TGFβ signaling pathways on wound healing is the biggest challenge in order to gain a therapeutically applicable comprehensive knowledge of their specific involvement in chronic wounds.

Injury- and inflammation-driven skin fibrosis: The paradigm of epidermolysis bullosa

Genetic or acquired destabilization of the dermal extracellular matrix evokes injury- and inflammation-driven progressive soft tissue fibrosis. Dystrophic epidermolysis bullosa (DEB), a heritable human skin fragility disorder, is a paradigmatic disease to investigate these processes. Studies of DEB have generated abundant new information on cellular and molecular mechanisms at play in skin fibrosis which are not only limited to intractable diseases, but also applicable to some of the most common acquired conditions. Here, we discuss recent advances in understanding the biological and mechanical mechanisms driving the dermal fibrosis in DEB. Much of this progress is owed to the implementation of cell and tissue omics studies, which we pay special attention to. Based on the novel findings and increased understanding of the disease mechanisms in DEB, translational aspects and future therapeutic perspectives are emerging.