Mechanotransduction through hemidesmosomes during aging and longevity.

Hemidesmosomes are structural protein complexes localized at the interface of tissues with high mechanical demand and shear forces. Beyond tissue anchoring, hemidesmosomes have emerged as force-modulating structures important for translating mechanical cues into biochemical and transcriptional adaptation (i.e. mechanotransduction) across tissues. Here, we discuss the recent insights into the roles of hemidesmosomes in age-related tissue regeneration and aging in C. elegans, mice and humans. We highlight the emerging concept of preserved dynamic mechanoregulation of hemidesmosomes in tissue maintenance and healthy aging.

Histone deacetylase inhibition mitigates fibrosis-driven disease progression in recessive dystrophic epidermolysis bullosa.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a blistering disease caused by mutations in the gene encoding type VII collagen (C7). RDEB is associated with fibrosis, which is responsible for severe complications. The phenotypic variability observed in RDEB siblings suggests that epigenetic modifications contribute to disease severity. Identifying epigenetic changes may help to uncover molecular mechanisms underlying RDEB pathogenesis and new therapeutic targets. OBJECTIVES: To investigate histone acetylation in RDEB skin and to explore histone deacetylase inhibitors (HDACis) as therapeutic molecules capable of counteracting fibrosis and disease progression in RDEB mice. METHODS: Acetylated histone levels were detected in human skin by immunofluorescence and in RDEB fibroblasts by ELISA. The effects of Givinostat and valproic acid (VPA) on RDEB fibroblast fibrotic behaviour were assessed by collagen-gel contraction assay, Western blot and immunocytofluorescence for α-smooth muscle actin, ELISA for released transforming growth factor-ß1 (TGF-ß1). RNA-seq was performed in HDACi- and vehicle-treated RDEB fibroblasts. VPA was systemically administered to RDEB mice, and effects on overt phenotype were monitored. Fibrosis was investigated in the skin using histological and immunofluorescence analyses. Eye and tongue defects were examined microscopically. Mass spectrometry proteomics was performed on skin protein extracts from VPA-treated RDEB and control mice. RESULTS: Histone acetylation decreases in RDEB skin and primary fibroblasts. RDEB fibroblasts treated with HDACis lowered fibrotic traits including contractility, TGF-ß1 release, and proliferation. VPA administration to RDEB mice mitigated severe manifestations affecting eyes and paws. These effects were associated with fibrosis inhibition. Proteomic analysis of mouse skin revealed that VPA almost normalised protein sets involved in protein synthesis and immune response, processes linked to the increased susceptibility to cancer and bacterial infections observed in RDEB patients. CONCLUSIONS: Dysregulated histone acetylation contributes to RDEB pathogenesis by facilitating the progression of fibrosis. Repurposing of HDACi could be considered for disease-modifying treatments of RDEB.

Splicing Modulation via Antisense Oligonucleotides in Recessive Dystrophic Epidermolysis Bullosa.

Antisense oligonucleotides (ASOs) represent an emerging therapeutic platform for targeting genetic diseases by influencing various aspects of (pre-)mRNA biology, such as splicing, stability, and translation. In this study, we investigated the potential of modulating the splicing pattern in recessive dystrophic epidermolysis bullosa (RDEB) patient cells carrying a frequent genomic variant (c.425A > G) that disrupts splicing in the COL7A1 gene by using short 2'-O-(2-Methoxyethyl) oligoribo-nucleotides (2'-MOE ASOs). COL7A1-encoded type VII collagen (C7) forms the anchoring fibrils within the skin that are essential for the attachment of the epidermis to the underlying dermis. As such, gene variants of COL7A1 leading to functionally impaired or absent C7 manifest in the form of extensive blistering and wounding. The severity of the disease pattern warrants the development of novel therapies for patients. The c.425A > G variant at the COL7A1 exon 3/intron 3 junction lowers the efficiency of splicing at this junction, resulting in non-functional C7 transcripts. However, we found that correct splicing still occurs, albeit at a very low level, highlighting an opportunity for intervention by modulating the splicing reaction. We therefore screened 2'-MOE ASOs that bind along the COL7A1 target region ranging from exon 3 to the intron 3/exon 4 junction for their ability to modulate splicing. We identified ASOs capable of increasing the relative levels of correctly spliced COL7A1 transcripts by RT-PCR, sqRT-PCR, and ddPCR. Furthermore, RDEB-derived skin equivalents treated with one of the most promising ASOs exhibited an increase in full-length C7 expression and its accurate deposition along the basement membrane zone (BMZ).

Inborn errors of immunity and immunodeficiencies: Antibody-mediated pathology and autoimmunity as a consequence of impaired immune reactions.

B-cell tolerance to self-antigen is an active process that requires the temporal and spatial integration of signals of defined intensity. In common variable immune deficiency disorders, CTLA-4 deficiency, autoimmune lymphoproliferative syndrome, or in collagen VII deficiency, genetic defects in molecules regulating development, activation, maturation, and ECM composition alter the generation of B cells, resulting in immunodeficiency. Paradoxically, at the same time, the defective immune processes favor autoantibody production and immunopathology through impaired establishment of tolerance. The development of systemic autoimmunity in the framework of defective BCR signaling is relatively unusual in genetic mouse models. In sharp contrast, such reduced signaling in humans is clearly linked to pathological autoimmunity. The molecular mechanisms by which tolerance is broken in these settings are only starting to be explored resulting in novel therapeutic interventions. For instance, in CTLA-4 deficiency, homeostasis can be restored by CTLA-4 Ig treatment. Following this example, the identification of the molecular targets causing the reduced signals and their restoration is a visionary way to reestablish tolerance and develop novel therapeutic avenues for immunopathologies.

Matrix metalloproteinases in coronary artery disease and myocardial infarction.

Cardiovascular diseases (CVDs) remain the leading cause of death worldwide. Most cardiovascular deaths are caused by ischaemic heart diseases such as myocardial infarction (MI). Hereby atherosclerosis in the coronary arteries often precedes disease manifestation. Since tissue remodelling plays an important role in the development and progression of atherosclerosis as well as in outcome after MI, regulation of matrix metalloproteinases (MMPs) as the major ECM-degrading enzymes with diverse other functions is crucial. Here, we provide an overview of the expression profiles of MMPs in coronary artery and left ventricular tissue using publicly available data from whole tissue to single-cell resolution. To approach an association between MMP expression and the development and outcome of CVDs, we further review studies investigating polymorphisms in MMP genes since polymorphisms are known to have an impact on gene expression. This review therefore aims to shed light on the role of MMPs in atherosclerosis and MI by summarizing current knowledge from publically available datasets, human studies, and analyses of polymorphisms up to preclinical and clinical trials of pharmacological MMP inhibition.

COL7A1 Editing via RNA Trans-Splicing in RDEB-Derived Skin Equivalents.

Mutations in the COL7A1 gene lead to malfunction, reduction or complete absence of type VII collagen (C7) in the skin's basement membrane zone (BMZ), impairing skin integrity. In epidermolysis bullosa (EB), more than 800 mutations in COL7A1 have been reported, leading to the dystrophic form of EB (DEB), a severe and rare skin blistering disease associated with a high risk of developing an aggressive form of squamous cell carcinoma. Here, we leveraged a previously described 3'-RTMS6m repair molecule to develop a non-viral, non-invasive and efficient RNA therapy to correct mutations within COL7A1 via spliceosome-mediated RNA trans-splicing (SMaRT). RTM-S6m, cloned into a non-viral minicircle-GFP vector, is capable of correcting all mutations occurring between exon 65 and exon 118 of COL7A1 via SMaRT. Transfection of the RTM into recessive dystrophic EB (RDEB) keratinocytes resulted in a trans-splicing efficiency of ~1.5% in keratinocytes and ~0.6% in fibroblasts, as confirmed on mRNA level via next-generation sequencing (NGS). Full-length C7 protein expression was primarily confirmed in vitro via immunofluorescence (IF) staining and Western blot analysis of transfected cells. Additionally, we complexed 3'-RTMS6m with a DDC642 liposomal carrier to deliver the RTM topically onto RDEB skin equivalents and were subsequently able to detect an accumulation of restored C7 within the basement membrane zone (BMZ). In summary, we transiently corrected COL7A1 mutations in vitro in RDEB keratinocytes and skin equivalents derived from RDEB keratinocytes and fibroblasts using a non-viral 3'-RTMS6m repair molecule.

Matrix molecules and skin biology.

An extracellular matrix (ECM) is a prerequisite for multicellular life. It is adapted to tissues and constantly undergoes changes to preserve microenvironmental homeostasis. The ECM acts as a structural scaffold that establishes tissue architecture and provides tensile strength. It has cell-instructive functions by serving as a reservoir and presenter of soluble agents, being directly signaling, integrating transmission of mechanical and biological cues, or serving as a co-factor potentiating signaling. The skin contains a highly developed, mechanically tough, but yet flexible ECM. The tissue-specific features of this ECM are largely attributed by minor ECM components. A large number of genetic and acquired ECM diseases with skin manifestations, provide an illustrative testament to the importance of correct assembly of the ECM for dermal homeostasis. Here, we will present the composition and features of the skin ECM during homeostasis and regeneration. We will discuss genetic and acquired ECM diseases affecting skin, and provide a short outlook to therapeutic strategies for them.

Transmembrane collagens-Unexplored mediators of epidermal-dermal communication and tissue homeostasis.

Tissue homeostasis is maintained through constant, dynamic and heterogeneous communication between cells and their microenvironment. Proteins that are at the same time active at the intracellular, cell periphery and deeper extracellular levels possess the ability to, on the individual molecular level, influence the cells and their microenvironment in a bidirectional manner. The transmembrane collagens are a family of such proteins, which are of notable interest for tissue development and homeostasis. In skin, expression of all transmembrane collagens has been reported and deficiency of transmembrane collagen XVII manifests with distinct skin phenotypes. Nevertheless, transmembrane collagens in skin remain understudied despite the association of them with epidermal wound healing and dermal fibrotic processes. Here, we present an overview of transmembrane collagens and put a spotlight on them as regulators of epidermal-dermal communication and as potential players in fibrinogenesis.

Notch-ing up knowledge on molecular mechanisms of skin fibrosis: focus on the multifaceted Notch signalling pathway.

Fibrosis can be defined as an excessive and deregulated deposition of extracellular matrix proteins, causing loss of physiological architecture and dysfunction of different tissues and organs. In the skin, fibrosis represents the hallmark of several acquired (e.g. systemic sclerosis and hypertrophic scars) and inherited (i.e. dystrophic epidermolysis bullosa) diseases. A complex series of interactions among a variety of cellular types and a wide range of molecular players drive the fibrogenic process, often in a context-dependent manner. However, the pathogenetic mechanisms leading to skin fibrosis are not completely elucidated. In this scenario, an increasing body of evidence has recently disclosed the involvement of Notch signalling cascade in fibrosis of the skin and other organs. Despite its apparent simplicity, Notch represents one of the most multifaceted, strictly regulated and intricate pathways with still unknown features both in health and disease conditions. Starting from the most recent advances in Notch activation and regulation, this review focuses on the pro-fibrotic function of Notch pathway in fibroproliferative skin disorders describing molecular networks, interplay with other pro-fibrotic molecules and pathways, including the transforming growth factor-ß1, and therapeutic strategies under development.

Impaired lymphoid extracellular matrix impedes antibacterial immunity in epidermolysis bullosa.

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.

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.

Combinatorial Omics Analysis Reveals Perturbed Lysosomal Homeostasis in Collagen VII-deficient Keratinocytes.

The extracellular matrix protein collagen VII is part of the microenvironment of stratified epithelia and critical in organismal homeostasis. Mutations in the encoding gene COL7A1 lead to the skin disorder dystrophic epidermolysis bullosa (DEB), are linked to skin fragility and progressive inflammation-driven fibrosis that facilitates aggressive skin cancer. So far, these changes have been linked to mesenchymal alterations, the epithelial consequences of collagen VII loss remaining under-addressed. As epithelial dysfunction is a principal initiator of fibrosis, we performed a comprehensive transcriptome and proteome profiling of primary human keratinocytes from DEB and control subjects to generate global and detailed images of dysregulated epidermal molecular pathways linked to loss of collagen VII. These revealed downregulation of interaction partners of collagen VII on mRNA and protein level, but also increased abundance of S100 pro-inflammatory proteins in primary DEB keratinocytes. Increased TGF-ß signaling because of loss of collagen VII was associated with enhanced activity of lysosomal proteases in both keratinocytes and skin of collagen VII-deficient individuals. Thus, loss of a single structural protein, collagen VII, has extra- and intracellular consequences, resulting in inflammatory processes that enable tissue destabilization and promote keratinocyte-driven, progressive fibrosis.

Collagen VII deficient mice show morphologic and histologic corneal changes that phenotypically mimic human dystrophic epidermolysis bullosa of the eye.

BACKGROUND: Absence of collagen VII causes blistering of the skin, eyes and many other tissues. This disease is termed dystrophic epidermolysis bullosa (DEB). Corneal fibrosis occurs in up to 41% and vision loss in up to 64% of patients. Standard treatments are supportive and there is no cure. The hypomorphic mouse model for DEB shows production of collagen VII at 10% of wild type levels in skin and spleen, but the eyes have not been described. Our purpose is to characterize the corneas to determine if this is an appropriate model for study of ocular therapeutics. METHODS: Western blot analysis (WB) and immunohistochemistry (IHC) were performed to assess presence and location of collagen VII protein within the hypomorphic mouse cornea. Additional IHC for inflammatory and fibrotic biomarkers transforming growth factor-beta-1 (TGF-ß1), alpha-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), proteinase 3, tenascin C and collagen III were performed. Clinical photographs documenting corneal opacification were assessed and scored independently by 2 examiners. Histology was then used to investigate morphologic changes. RESULTS: IHC and WB confirmed that hypomorphic mice produce less collagen VII production at the level of the basement membrane when compared with wild-types. IHC showed anomalous deposition of collagen III throughout the stroma. Of the 5 biomarkers tested, TGF-ß1 showed the strongest and most consistently staining. Photographs documented corneal opacities only in mice older than 10 weeks, opacities were not seen in younger animals. Histology showed multiple abnormalities, including epithelial hyperplasia, ulceration, fibrosis, edema, dysplasia, neovascularization and bullae formation. CONCLUSIONS: The collagen VII hypomorphic mouse shows reduced collagen VII production at the level of the corneal basement membrane. Corneal changes are similar to pathology seen in humans with this disease. The presence of anomalous stromal collagen III and TGF-ß1 appear to be the most consistent and strongest staining biomarkers in diseased mice. This mouse appears to mimic human corneal disease. It is an appropriate model for testing of therapeutics to treat EB ocular disease.

RNA-based therapies for genodermatoses.

Genetic disorders affecting the skin, genodermatoses, constitute a large and heterogeneous group of diseases, for which treatment is generally limited to management of symptoms. RNA-based therapies are emerging as a powerful tool to treat genodermatoses. In this review, we discuss in detail RNA splicing modulation by antisense oligonucleotides and RNA trans-splicing, transcript replacement and genome editing by in vitro-transcribed mRNAs, and gene knockdown by small interfering RNA and antisense oligonucleotides. We present the current state of these therapeutic approaches and critically discuss their opportunities, limitations and the challenges that remain to be solved. The aim of this review was to set the stage for the development of new and better therapies to improve the lives of patients and families affected by a genodermatosis.

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.

High Local Concentrations of Intradermal MSCs Restore Skin Integrity and Facilitate Wound Healing in Dystrophic Epidermolysis Bullosa.

Dystrophic epidermolysis bullosa (DEB) is an incurable skin fragility disorder caused by mutations in the COL7A1 gene, coding for the anchoring fibril protein collagen VII (C7). Life-long mechanosensitivity of skin and mucosal surfaces is associated with large body surface erosions, chronic wounds, and secondary fibrosis that severely impede functionality. Here, we present the first systematic long-term evaluation of the therapeutic potential of a mesenchymal stromal cell (MSC)-based therapy for DEB. Intradermal administration of MSCs in a DEB mouse model resulted in production and deposition of C7 at the dermal-epidermal junction, the physiological site of function. The effect was dose-dependent with MSCs being up to 10-fold more potent than dermal fibroblasts. MSCs promoted regeneration of DEB wounds via normalization of dermal and epidermal healing and improved skin integrity through de novo formation of functional immature anchoring fibrils. Additional benefits were gained by MSCs' anti-inflammatory effects, which led to decreased immune cell infiltration into injured DEB skin. In our setting, the clinical benefit of MSC injections lasted for more than 3 months. We conclude that MSCs are viable options for localized DEB therapy. Importantly, however, the cell number needed to achieve therapeutic efficacy excludes the use of systemic administration.

Epidermal Basement Membrane in Health and Disease.

Skin, as the organ protecting the individual from environmental aggressions, constantly meets external insults and is dependent on mechanical toughness for its preserved function. Accordingly, the epidermal basement membrane (BM) zone has adapted to enforce tissue integrity. It harbors anchoring structures created through unique organization of common BM components and expression of proteins exclusive to the epidermal BM zone. Evidence for the importance of its correct assembly and the nonredundancy of its components for skin integrity is apparent from the multiple skin blistering disorders caused by mutations in genes coding for proteins associated with the epidermal BM and from autoimmune disorders in which autoantibodies target these molecules. However, it has become clear that these proteins not only provide mechanical support but are also critically involved in tissue homeostasis, repair, and regeneration. In this chapter, we provide an overview of the unique organization and components of the epidermal BM. A special focus will be given to its function during regeneration, and in inherited and acquired diseases.