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.

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.

Type VII Collagen Deficiency in the Oncogenesis of Cutaneous Squamous Cell Carcinoma in Dystrophic Epidermolysis Bullosa.

Dystrophic epidermolysis bullosa is a rare genetic skin disorder caused by COL7A1 sequence variations that result in type VII collagen deficits and cutaneous and extracutaneous manifestations. One serious complication of dystrophic epidermolysis bullosa is cutaneous squamous cell carcinoma, a leading driver of morbidity and mortality, especially among patients with recessive dystrophic epidermolysis bullosa. Type VII collagen deficits alter TGFß signaling and evoke multiple other cutaneous squamous cell carcinoma progression-promoting activities within epidermal microenvironments. This review examines cutaneous squamous cell carcinoma pathophysiology in dystrophic epidermolysis bullosa with a focus on known oncogenesis pathways at play and explores the idea that therapeutic type VII collagen replacement may reduce cutaneous squamous cell carcinoma risk.

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.

Kidney-Urinary Tract Involvement in Intermediate Junctional Epidermolysis Bullosa.

Importance: Kidney-urinary tract (KUT) manifestations cause substantial morbidity in patients with junctional epidermolysis bullosa (JEB), but the spectrum of disease severity and the clinical course have been poorly characterized. Objective: To examine in a large cohort of patients with intermediate JEB the KUT manifestations, diagnostic and therapeutic procedures, genotype-phenotype correlations, and outcomes as a basis for recommendations, prognosis, and management. Design, Setting, and Participants: In this retrospective, longitudinal case series study, 99 patients with a diagnosis of JEB based on clinical and genetic findings who were treated in a single dermatology department in Freiburg, Germany, were assessed during an 18-year period (January 1, 2003, to December 31, 2021). Clinical, laboratory, and molecular genetic parameters were extracted from patients' medical records. Main Outcomes and Measures: Clinical characteristics, natural history, management of KUT manifestations, and genotype-phenotype correlations of intermediate JEB. Results: Of the 183 patients with JEB, 99 (54%) had intermediate JEB and were included in this cohort. The cohort included 49 female patients and 50 male patients. None of 49 female patients and 15 of 50 male patients had KUT involvement affecting different levels of the urinary tract, resulting in a prevalence of 30% for males; thus, the overall prevalence was 15%. The mean age at onset of KUT manifestations was 6.9 years (range, first weeks of life to 20 years; age was not available for 1 patient). Median follow-up after diagnosis of KUT involvement was 13 years (range, 3 months to 54 years). Patients with laminin 332 or integrin ß4 deficiency had at least 1 missense or splice site genetic variant, leading to residual expression of laminin 332 or integrin α6ß4, respectively. Severity of KUT complications did not correlate with the extent of skin involvement but with the affected protein. Conclusions and Relevance: Physicians and patients with JEB should be aware of the risk for KUT involvement in intermediate JEB, and physicians should apply interdisciplinary and individualized diagnostic and therapeutic procedures for management of these complications. Because this disorder is so rare, multicenter studies are required to make general recommendations.

Case Report: Diagnostic and Therapeutic Challenges in Severe Mechanobullous Epidermolysis Bullosa Acquisita.

Collagen VII is the main constituent of the anchoring fibrils, important adhesive structures that attach the epidermis to the dermal extracellular matrix. Two disorders are caused by dysfunction of collagen VII, both characterized by skin and mucosa fragility, epidermolysis bullosa acquisita (EBA) and dystrophic epidermolysis bullosa (DEB). EBA and DEB share high clinical similarities with significant difference in patients' age of onset and pathogenesis. Our patients presented with severe and recalcitrant mechanobullous EBA with characteristic DIF, IIF and ELISA diagnostics. But in both women recessive COL7A1 variants were also found, in a monoallelic state. Collagen VII from EBA keratinocytes of our cases was significantly more vulnerable to proteolytic degradation than control keratinocytes, hinting that the heterozygous pathogenic variants were sufficient to destabilize the molecule in vitro. Thus, even if the amount and functionality of mutant and normal type VII collagen polypeptides is sufficient to assure dermal-epidermal adhesion in healthy individuals, the functionally-impaired proteins are probably more prone to development of autoantibodies against them. Our work suggests that testing for COL7A1 genetic variants should be considered in patients with EBA, which either have a patient history hinting towards underlying dystrophic epidermolysis bullosa or pose therapeutic challenges.

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.

Canonical TGFß signaling induces collective invasion in colorectal carcinogenesis through a Snail1- and Zeb1-independent partial EMT.

Local invasion is the initial step towards metastasis, the main cause of cancer mortality. In human colorectal cancer (CRC), malignant cells predominantly invade as cohesive collectives and may undergo partial epithelial-mesenchymal transition (pEMT) at the invasive front. How this particular mode of stromal infiltration is generated is unknown. Here we investigated the impact of oncogenic transformation and the microenvironment on tumor cell invasion using genetically engineered organoids as CRC models. We found that inactivation of the Apc tumor suppressor combined with expression of oncogenic KrasG12D and dominant-negative Trp53R172H did not cell-autonomously induce invasion in vitro. However, oncogenic transformation primed organoids for activation of a collective invasion program upon exposure to the prototypical microenvironmental factor TGFß1. Execution of this program co-depended on a permissive extracellular matrix which was further actively remodeled by invading organoids. Although organoids shed some epithelial properties particularly at the invasive edge, TGFß1-stimulated organoids largely maintained epithelial gene expression while additionally implementing a mesenchymal transcription pattern, resulting in a pEMT phenotype that did not progress to a fully mesenchymal state. Notably, while TGFß1 induced pEMT and promoted collective invasion, it abrogated self-renewal capacity of TKA organoids which correlated with the downregulation of intestinal stem cell (ISC) marker genes. Mechanistically, induction of the non-progressive pEMT required canonical TGFß signaling mediated by Smad transcription factors (TFs), whereas the EMT master regulators Snail1 and Zeb1 were dispensable. Gene expression profiling provided further evidence for pEMT of TGFß1-treated organoids and showed that their transcriptomes resemble those of human poor prognosis CMS4 cancers which likewise exhibit pEMT features. We propose that collective invasion in colorectal carcinogenesis is triggered by microenvironmental stimuli through activation of a novel, transcription-mediated form of non-progressive pEMT independently of classical EMT regulators.

Comparison of extracellular matrix enrichment protocols for the improved characterization of the skin matrisome by mass spectrometry.

A striking feature of skin organization is that the extracellular matrix (ECM) occupies a larger volume than the cells. Skin ECM also directly contributes to aging and most cutaneous diseases. In recent years, specific ECM enrichment protocols combined with in silico approaches allowed the proteomic description of the matrisome of various organs and tumor samples. Nevertheless, the skin matrisome remains under-studied and protocols allowing the efficient recovery of the diverse ECM found in skin are still to be described. Here, we compared four protocols allowing the enrichment of ECM proteins from adult mouse back skin and found that all protocols led to a significant enrichment (up to 65%) of matrisome proteins when compared to total skin lysates. The protocols based on decellularization and solubility profiling gave the best results in terms of numbers of proteins identified and confirmed that skin matrisome proteins exhibit very diverse solubility and abundance profiles. We also report the first description of the skin matrisome of healthy adult mice that includes 236 proteins comprising 95 core matrisome proteins and 141 associated matrisome proteins. These results provide a reliable basis for future characterizations of skin ECM proteins and their dysregulations in disease-specific contexts. SIGNIFICANCE: Extracellular matrix proteins are key players in skin physiopathology and have been involved in several diseases such as genetic disorders, wound healing defects, scleroderma and skin carcinoma. However, skin ECM proteins are numerous, diverse and challenging to analyze by mass spectrometry due to the multiplicity of their post-translational modifications and to the heterogeneity of their solubility profiles. Here, we performed the thorough evaluation of four ECM enrichment protocols compatible with the proteomic analysis of mouse back skin and provide the first description of the adult mouse skin matrisome in homeostasis conditions. Our work will greatly facilitate the future characterization of skin ECM alterations in preclinical mouse models and will inspire new optimizations to analyze the skin matrisome of other species and of human clinical samples.

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.

QR-313, an Antisense Oligonucleotide, Shows Therapeutic Efficacy for Treatment of Dominant and Recessive Dystrophic Epidermolysis Bullosa: A Preclinical Study.

Dystrophic epidermolysis bullosa (DEB) is a blistering skin disease caused by mutations in the gene COL7A1 encoding collagen VII. DEB can be inherited as recessive DEB (RDEB) or dominant DEB (DDEB) and is associated with a high wound burden. Perpetual cycles of wounding and healing drive fibrosis in DDEB and RDEB, as well as the formation of a tumor-permissive microenvironment. Prolonging wound-free episodes by improving the quality of wound healing would therefore confer substantial benefit for individuals with DEB. The collagenous domain of collagen VII is encoded by 82 in-frame exons, which makes splice-modulation therapies attractive for DEB. Indeed, antisense oligonucleotide-based exon skipping has shown promise for RDEB. However, the suitability of antisense oligonucleotides for treatment of DDEB remains unexplored. Here, we developed QR-313, a clinically applicable, potent antisense oligonucleotide specifically targeting exon 73. We show the feasibility of topical delivery of QR-313 in a carbomer-composed gel for treatment of wounds to restore collagen VII abundance in human RDEB skin. Our data reveal that QR-313 also shows direct benefit for DDEB caused by exon 73 mutations. Thus, the same topically applied therapeutic could be used to improve the wound healing quality in RDEB and DDEB.

Interplay between Cell-Surface Receptors and Extracellular Matrix in Skin.

Skin consists of the epidermis and dermis, which are connected by a specialized basement membrane-the epidermal basement membrane. Both the epidermal basement membrane and the underlying interstitial extracellular matrix (ECM) created by dermal fibroblasts contain distinct network-forming macromolecules. These matrices play various roles in order to maintain skin homeostasis and integrity. Within this complex interplay of cells and matrices, cell surface receptors play essential roles not only for inside-out and outside-in signaling, but also for establishing mechanical and biochemical properties of skin. Already minor modulations of this multifactorial cross-talk can lead to severe and systemic diseases. In this review, major epidermal and dermal cell surface receptors will be addressed with respect to their interactions with matrix components as well as their roles in fibrotic, inflammatory or tumorigenic skin diseases.

The Pseudomonas aeruginosa Lectin LecB Causes Integrin Internalization and Inhibits Epithelial Wound Healing.

The opportunistic bacterium Pseudomonas aeruginosa produces the fucose-specific lectin LecB, which has been identified as a virulence factor. LecB has a tetrameric structure with four opposing binding sites and has been shown to act as a cross-linker. Here, we demonstrate that LecB strongly binds to the glycosylated moieties of ß1-integrins on the basolateral plasma membrane of epithelial cells and causes rapid integrin endocytosis. Whereas internalized integrins were degraded via a lysosomal pathway, washout of LecB restored integrin cell surface localization, thus indicating a specific and direct action of LecB on integrins to bring about their endocytosis. Interestingly, LecB was able to trigger uptake of active and inactive ß1-integrins and also of complete α3ß1-integrin-laminin complexes. We provide a mechanistic explanation for this unique endocytic process by showing that LecB has the additional ability to recognize fucose-bearing glycosphingolipids and causes the formation of membrane invaginations on giant unilamellar vesicles. In cells, LecB recruited integrins to these invaginations by cross-linking integrins and glycosphingolipids. In epithelial wound healing assays, LecB specifically cleared integrins from the surface of cells located at the wound edge and blocked cell migration and wound healing in a dose-dependent manner. Moreover, the wild-type P. aeruginosa strain PAO1 was able to loosen cell-substrate adhesion in order to crawl underneath exposed cells, whereas knockout of LecB significantly reduced crawling events. Based on these results, we suggest that LecB has a role in disseminating bacteria along the cell-basement membrane interface.IMPORTANCEPseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the leading causes of nosocomial infections. P. aeruginosa is able to switch between planktonic, intracellular, and biofilm-based lifestyles, which allows it to evade the immune system as well as antibiotic treatment. Hence, alternatives to antibiotic treatment are urgently required to combat P. aeruginosa infections. Lectins, like the fucose-specific LecB, are promising targets, because removal of LecB resulted in decreased virulence in mouse models. Currently, several research groups are developing LecB inhibitors. However, the role of LecB in host-pathogen interactions is not well understood. The significance of our research is in identifying cellular mechanisms of how LecB facilitates P. aeruginosa infection. We introduce LecB as a new member of the list of bacterial molecules that bind integrins and show that P. aeruginosa can move forward underneath attached epithelial cells by loosening cell-basement membrane attachment in a LecB-dependent manner.

Kallikreins: Essential epidermal messengers for regulation of the skin microenvironment during homeostasis, repair and disease.

As the outermost layer of the skin, the epidermis is playing a major role in organism homeostasis providing the first barrier against external aggressions. Although considered as an extracellular matrix (ECM)-poor subtissue, the epidermal microenvironment is a key regulator of skin homeostasis and functionality. Among the proteins essential for upholding the epidermal microenvironment are the members of the kallikrein (KLK) family composed of 15 secreted serine proteases. Most of the members of these epithelial-specific proteins are present in skin and regulate skin desquamation and inflammation. However, although epidermal products, the consequences of KLK activities are not confined to the epidermis but widespread in the skin. In this review starting with the location and proteolytic activation cascade of KLKs, we present KLKs involvement in skin homeostasis, regeneration and pathology. KLKs have a large variety of substrates including ECM proteins, and evidence suggests that they are involved in the different steps of skin wound healing as discussed here. KLKs are also used as prognosis/diagnosis markers for many cancer types and we are focusing later on KLKs in cutaneous cancers, although their pathogenicity remains to be fully elucidated. Dysregulation of the KLK cascade is directly responsible for skin diseases with heavy inflammatory aspects, highlighting their involvement in skin immune homeostasis. Future studies will be needed to support the therapeutic potential of adjusting KLK activities for treatment of inflammatory skin diseases and wound healing pathologies.

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.

Treatment of keratinocytes with 4-phenylbutyrate in epidermolysis bullosa: Lessons for therapies in keratin disorders.

BACKGROUND: Missense mutations in keratin 5 and 14 genes cause the severe skin fragility disorder epidermolysis bullosa simplex (EBS) by collapsing of the keratin cytoskeleton into cytoplasmic protein aggregates. Despite intense efforts, no molecular therapies are available, mostly due to the complex phenotype of EBS, comprising cell fragility, diminished adhesion, skin inflammation and itch. METHODS: We extensively characterized KRT5 and KRT14 mutant keratinocytes from patients with severe generalized EBS following exposure to the chemical chaperone 4-phenylbutyrate (4-PBA). FINDINGS: 4-PBA diminished keratin aggregates within EBS cells and ameliorated their inflammatory phenotype. Chemoproteomics of 4-PBA-treated and untreated EBS cells revealed reduced IL1ß expression- but also showed activation of Wnt/ß-catenin and NF-kB pathways. The abundance of extracellular matrix and cytoskeletal proteins was significantly altered, coinciding with diminished keratinocyte adhesion and migration in a 4-PBA dose-dependent manner. INTERPRETATION: Together, our study reveals a complex interplay of benefits and disadvantages that challenge the use of 4-PBA in skin fragility disorders.

ADAM9 contributes to vascular invasion in pancreatic ductal adenocarcinoma.

A disintegrin and a metalloprotease (ADAM)-9 is a metzincin cell-surface protease with strongly elevated expression in solid tumors, including pancreatic ductal adenocarcinoma (PDAC). In this study, we performed immunohistochemistry (IHC) of a tissue microarray (TMA) to examine the expression of ADAM9 in a cohort of >100 clinically annotated PDAC cases. We report that ADAM9 is prominently expressed by PDAC tumor cells, and increased ADAM9 expression levels correlate with poor tumor grading (P = 0.027) and the presence of vasculature invasion (P = 0.017). We employed gene expression silencing to generate a loss-of-function system for ADAM9 in two established PDAC cell lines. In vitro analysis showed that loss of ADAM9 does not impede cellular proliferation and invasiveness in basement membrane. However, ADAM9 plays a crucial role in mediating cell migration and adhesion to extracellular matrix substrates such as fibronectin, tenascin, and vitronectin. This effect appears to depend on its catalytic activity. In addition, ADAM9 facilitates anchorage-independent growth. In AsPC1 cells, but not in MiaPaCa-2 cells, we noted a pronounced yet heterogeneous impact of ADAM9 on the abundance of various integrins, a process that we characterized as post-translational regulation. Sprout formation of human umbilical vein endothelial cells (HUVECs) is promoted by ADAM9, as examined by transfer of cancer cell conditioned medium; this finding further supports a pro-angiogenic role of ADAM9 expressed by PDAC cancer cells. Immunoblotting analysis of cancer cell conditioned medium highlighted that ADAM9 regulates the levels of angiogenic factors, including shed heparin-binding EGF-like growth factor (HB-EGF). Finally, we carried out orthotopic seeding of either wild-type AsPC-1 cells or AsPC-1 cells with silenced ADAM9 expression into murine pancreas. In this in vivo setting, ADAM9 was also found to foster angiogenesis without an impact on tumor cell proliferation. In summary, our results characterize ADAM9 as an important regulator in PDAC tumor biology with a strong pro-angiogenic impact.

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.

Epidermolysis bullosa: Molecular pathology of connective tissue components in the cutaneous basement membrane zone.

Epidermolysis bullosa (EB), a group of heritable skin fragility disorders, is characterized by blistering, erosions and chronic ulcers in the skin and mucous membranes. In some forms, the blistering phenotype is associated with extensive mutilating scarring and development of aggressive squamous cell carcinomas. The skin findings can be associated with extracutaneous manifestations in the ocular as well as gastrointestinal and vesico-urinary tracts. The phenotypic heterogeneity reflects the presence of mutations in as many as 20 different genes expressed in the cutaneous basement membrane zone, and the types and combinations of the mutations and their consequences at the mRNA and protein levels contribute to the spectrum of severity encountered in different subtypes of EB. This overview highlights the molecular genetics of EB based on mutations in the genes encoding type VII and XVII collagens as well as laminin-332. The mutations identified in these protein components of the extracellular matrix attest to their critical importance in providing stability to the cutaneous basement membrane zone, with implications for heritable and acquired diseases.