Fibroblasts show more potential as target cells than keratinocytes in COL7A1 gene therapy of dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disorder caused by mutations in the type VII collagen gene (COL7A1). Therapeutic introduction of COL7A1 into skin cells holds significant promise for the treatment of DEB. The purpose of this study was to establish an efficient retroviral transfer method for COL7A1 into DEB epidermal keratinocytes and dermal fibroblasts, and to determine which gene-transferred cells can most efficiently express collagen VII in the skin. We demonstrated that gene transfer using a combination of G protein of vesicular stomatitis virus-pseudotyped retroviral vector and retronectin introduced COL7A1 into keratinocytes and fibroblasts from a DEB patient with the lack of COL7A1 expression. Real-time polymerase chain reaction analysis of the normal human skin demonstrated that the quantity of COL7A1 expression in the epidermis was significantly higher than that in the dermis. Subsequently, we have produced skin grafts with the gene-transferred or untreated DEB keratinocytes and fibroblasts, and have transplanted them into nude rats. Interestingly, the series of skin graft experiments showed that the gene-transferred fibroblasts supplied higher amount of collagen VII to the new dermal-epidermal junction than the gene-transferred keratinocytes. An ultrastructural study revealed that collagen VII from gene-transferred cells formed proper anchoring fibrils. These results suggest that fibroblasts may be a better gene therapy target of DEB treatment than keratinocytes.     

Hereditary skin diseases of anchoring fibrils.

Remarkable progress has been made in the last few years in understanding the functions of the anchoring fibrils, polymers of collagen VII, that connect the epidermal basement membrane with the dermal connective tissue. Novel insights into the biology of these fibrils have been gained from studies on dystrophic epidermolysis bullosa (DEB), a group of inherited blistering disorders caused by abnormalities of the anchoring fibrils. Mutations in the COL7A1 gene encoding collagen VII have been disclosed in a number of DEB families, and the mutation analyses and studies on genotype-phenotype correlations in DEB have revealed an unusual complexity of the gene defects and their biological consequences. In analogy to heritable disorders of other collagen genes, predictable phenotypes of COL7A1 mutations causing premature termination codons (PTC) or dominant negative interference have been observed. However, collagen VII seems to be unique among collagens in that many mutations lead to minimal phenotypes, or to no phenotype at all. Furthermore, the mild DEB phenotypes can be severely modulated by a second mutation in individuals compound heterozygous for two different COL7A1 defects. Therefore, not only definition of mutations with diagnostic analyses, but also cell biological, protein chemical and suprastructural studies of the mutated molecules are required for understanding the pathomechanisms underlying DEB.     

Epidermal aspects of type VII collagen: Implications for dystrophic epidermolysis bullosa and epidermolysis bullosa acquisita

Type VII collagen (COL7), a major component of anchoring fibrils in the epidermal basement membrane zone, has been characterized as a defective protein in dystrophic epidermolysis bullosa and as an autoantigen in epidermolysis bullosa acquisita. Although COL7 is produced and secreted by both epidermal keratinocytes and dermal fibroblasts, the role of COL7 with regard to the epidermis is rarely discussed. This review focuses on COL7 physiology and pathology as it pertains to epidermal keratinocytes. We summarize the current knowledge of COL7 production and trafficking, its involvement in keratinocyte dynamics, and epidermal carcinogenesis in COL7 deficiency and propose possible solutions to unsolved issues in this field.     

Characterization of mutations of the type VII collagen gene (COL7A1) in recessive dystrophic epidermolysis bullosa mitis (M-RDEB) from three Korean patients

In recent years, the molecular basis for the main subtypes of epidermolysis bullosa (EB) has been elucidated with pathogenetic mutations delineated in ten different genes encoding structural components of the dermal-epidermal junction. Both the autosomal dominant and recessive forms of dystrophic EB (DEB) is caused by mutations in the COL7A1 gene. Type VII collagen is a major component of anchoring fibrils, structural elements that stabilize the attachment of the basement membrane to underlying dermis. Recent delineation of the exon-intron organization of the COL7A1 gene provided the basis for the comprehensive design of PCR primer pairs that amplified exons in genomic DNA by placing the primers on the flanking introns. A number of COL7A1 mutations have been reported and some genotype-phenotype correlations are starting to emerge. In this study, we examined mutational analyses from three Korean patients with recessive dystrophic EB (RDEB) mitis. We designed and optimized primers according to the previously reported sequences. Such PCR amplification products can be examined by electrophoretic scanning technique, CSGE heteroduplex analyses. Utilizing heteroduplex analyses, we have identified a number of sequence variants in COL7A1 both in unaffected individuals and in patients with M-RDEB. Mutation detection of the COL7A1 gene revealed six allelic mutations (V6677E, P6685S, Y3749S, P6084S, P6695R and G6697C). We suggest that the full length of type VII collagen polypeptide are synthesized, but those missense mutations, that may affect a critical amino acid, can alter the conformation of the protein and interferes with the assembly and packing of type VII collagen molecules into anchoring fibrils. Immunohistochemical study of skin biopsies by use of anti-type VII collagen antibody showed markedly reduced staining and presence of a dermo/epidermal cleavage. This is the first report of a COL7A1 mutation study in DEB from Korean patients. We hope that these data contribute to the expanding database on COL7A1 mutations in dystrophic epidermolysis bullosa, and further illustrate the extensive diversity of mutational events that led to the RDEB phenotype.     

A de novo glycine substitution mutation in the collagenous domain of COL7A1 in dominant dystrophic epidermolysis bullosa

Abstract Dystrophic epidermolysis bullosa (DEB) is a hereditary mechanobullous disorder characterized by fragility of the skin and mucous membrane due to abnormalities of anchoring fibrils. Both dominant and recessive DEB have been shown to be caused by mutations in COL7A1, the gene encoding type VII collagen which is the major component of anchoring fibrils. De novo mutation in dominant DEB is rare. In this study, we report a novel de novo glycine substitution mutation in COL7A1 in a Chinese female patient presenting with mild DEB. In search of the mutation, we scanned the entire COL7A1 using polymerase chain reaction (PCR) amplification of all exons of COL7A1, followed by heteroduplex analysis and direct sequencing of the PCR products that exhibited heteroduplex pattern. A G-to-A transition at nucleotide position 6082 within exon 73 of COL7A1was detected. The mutation converted a glycine to an arginine (G2028R) within the triple-helical domain of type VII collagen. It was confirmed that the mutation was present only in the proband. Haplotype analyses suggested that the case arose as a de novo occurrence of autosomal dominant DEB. Received: 1 September 1998 / Revised: 23 November 1999 / Accepted: 26 November 1999     

Potential of fibroblast cell therapy for recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin-blistering disorder caused by mutations in the COL7A1 gene that lead to reduced type-VII collagen and defective anchoring fibrils at the dermal-epidermal junction (DEJ). Presently there are no effective treatments for this disorder. Recent mouse studies have shown that intradermal injections of normal human fibroblasts can generate new human type-VII collagen and anchoring fibrils at the DEJ. To assess potential clinical benefits in humans, we gave single intradermal injections of allogeneic fibroblasts to five subjects with RDEB. We noted increased type-VII collagen at the DEJ at 2 weeks and at 3 months following injection and increased anchoring fibrils, although none of these had normal morphology. No adverse effects, clinical or immunopathologic, were noted. We believe the major effect of allogeneic fibroblasts is to increase the recipients' own COL7A1 mRNA levels with greater deposition of mutant type-VII collagen at the DEJ and formation of additional rudimentary anchoring fibrils. Nevertheless, this mutant protein may be partially functional and capable of increasing adhesion at the DEJ. This is the first study demonstrating that intradermal injections of allogeneic fibroblasts have therapeutic potential in human subjects with RDEB.     

A novel homozygous splice site mutation in COL7A1 in a Chinese patient with severe recessive dystrophic epidermolysis bullosa and squamous cell carcinoma

Background Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils in the dermo–epidermal junction. The development of cutaneous squamous cell carcinoma (SCC) is one of the most serious complications of this disease. We report herein a Chinese patient with the severe generalized subtype of RDEB (RDEB‐sev gen) complicated by SCC. Methods Skin biopsies were examined for histology, basement membrane ultrastructure, and type VII collagen expression. Genomic DNA was extracted from the peripheral blood samples and subjected to polymerase chain reaction amplification and direct automated DNA sequencing. Results Histopathological examination of the patient’s skin revealed an undetectable expression of type VII collagen polypeptides in the basement membrane zone. Mutation analysis identified a novel splice site mutation in intron 64 (IVS64+5g‐>a) of COL7A1 gene, which resulted in an in‐frame deletion of exon 64 in both alleles. Conclusions This report contributes to the expanding database of COL7A1 mutations and emphasizes the need to elucidate the underlying genetic mechanisms associated with the increased incidence of SCC in RDEB patients.     

A new glycine substitution mutation in the COL7A1 gene in a Chinese family with dominant dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is caused by mutations in the COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils. The characteristic genetic lesion in dominant DEB (DDEB) is a glycine substitution in the collagenous domain of the protein. In this study, we identified a Chinese family with a four-generation pedigree of DDEB, in whom a novel glycine substitution mutation in COL7A1 was demonstrated. A heterozygous nucleotide G-->A transition at position 6208 in exon 74 of COL7A1 was detected, which resulted in a glycine to arginine substitution (G2070R) in the triple-helical domain of type VII collagen. This substitution was not found in 110 unrelated normal alleles. This report emphasizes the predominance of glycine substitution mutations in DDEB and contributes to the expanding database on COL7A1 mutations.     

Life before and beyond blistering: The role of collagen XVII in epidermal physiology

Type XVII collagen (COL17) is a transmembranous protein that is mainly expressed in the epidermal basal keratinocytes. Epidermal‐dermal attachment requires COL17 expression at the hemidesmosomes of the epidermal basement membrane zone because congenital COL17 deficiency leads to junctional epidermolysis bullosa and acquired autoimmunity to COL17 induces bullous pemphigoid. Recently, in addition to facilitating epidermal‐dermal attachment, COL17 has been reported to serve as a niche for hair follicle stem cells, to regulate proliferation in the interfollicular epidermis and to be present along the non‐hemidesmosomal plasma membrane of epidermal basal keratinocytes. This review focuses on the physiological properties of COL17 in the epidermis, its role in maintaining stem cells and its association with signalling pathways. We propose possible solutions to unanswered questions in this field.     

A novel missense mutation in the COL7A1 gene causes epidermolysis bullosa pruriginosa

Epidermolysis bullosa (EB) pruriginosa, characterized by severe itching and the presence of nodular prurigo‐like or lichenoid lesions, is a rare clinical type of dystrophic EB. Mutations in the COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils, have been implicated in the pathogenesis of the disorder. In the present study, we screened a Chinese family with EB pruriginosa for COL7A1 mutations by PCR amplification of genomic sequences and direct nucleotide sequencing. The mutation consists of a G→T substitution at nucleotide 6724 in exon 85, which leads to the substitution of glycine by tryptophan at codon 2242. This report adds new variants to the known COL7A1 mutations underlying EB pruriginosa, and provides the basis for genetic counselling and prenatal diagnosis for affected families.     

Dominant dystrophic epidermolysis bullosa caused by a novel G2037R mutation and by a known G2028R mutation in the type VII collagen gene (COL7A1)

An autosomal dystrophic epidermolysis bullosa (DDEB) is a hereditary mechanobullous disease characterized by blistering of the skin and the mucous membrane. DDEB is caused by a heterozygous mutation in the COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils, and phenotypically classified into several types. We experienced two boys with DDEB and examined the mutation analyses of the COL7A1 genes of the two patients and their fathers to clarify the relationship between the genotypes and phenotypes, that is, the mutation sites of COL7A1 gene and the clinical types of DDEB. The case 1 and 2 patients and their fathers revealed a heterozygous nucleotide G to A transition at position 6109 and 6082 in 73 exon of COL7A1, which resulted in a glycine to arginine substitution (G2037R and G2028R), respectively. G2037R found in the case 1 patient was a novel mutation. There was no clear relationship recognized between the two mutation sites in the COL7A1 gene and the clinical variations.     

Role of p38 MAPK in transforming growth factor beta stimulation of collagen production by scleroderma and healthy dermal fibroblasts

Transforming growth factor beta has been implicated as a mediator of excessive extracellular matrix deposition in scar tissue and fibrosis, including systemic sclerosis. To further characterize the mechanism of collagen gene expression in systemic sclerosis and healthy skin fibroblasts, we examined the role of p38 MAPK signaling in collagen gene regulation by transforming growth factor beta. Treatment of dermal fibroblasts with transforming growth factor beta resulted in a prolonged activation of p38 MAPK. Furthermore, a specific inhibitor of p38 suppressed transforming growth factor beta stimulation of collagen type I mRNA and the alpha2(I) collagen promoter activity. To further probe the role of p38 in collagen regulation by transforming growth factor beta, we utilized an expression vector containing p38alpha cDNA. Ectopic expression of p38alpha enhanced COL1A2 promoter activity and potentiated transforming growth factor beta stimulation of this promoter. The p38 response element in the COL1A2 promoter overlapped with the previously characterized transforming growth factor beta response element. Consistent with these observations, collagen type I mRNA and protein levels were increased in transforming-growth-factor-beta-stimulated fibroblasts transduced with an adenoviral vector expressing p38alpha. To determine the possible role of p38 in abnormal collagen production by systemic sclerosis fibroblasts, p38 protein levels were compared in systemic sclerosis and healthy skin fibroblasts. Both cell types exhibited similar total levels of p38 MAPK and similar kinetics of p38 activation in response to transforming growth factor beta. In conclusion, this study demonstrates a costimulatory role for p38 MAPK in transforming growth factor beta induction of the collagen type I gene. Expression levels and activation status of p38 are not consistently elevated in systemic sclerosis fibroblasts suggesting that the p38 MAPK pathway is not dysregulated in systemic sclerosis fibroblasts.     

Differential contribution of dermal resident and bone marrow-derived cells to collagen production during wound healing and fibrogenesis in mice

Recent studies show that bone marrow (BM)-derived cells migrating into a dermal wound promote healing by producing collagen type I. However, their contribution to the repair process has not been fully verified yet. It is also unclear whether BM-derived cells participate in dermal fibrogenesis. We have addressed these issues using transgenic mice that harbor tissue-specific enhancer/promoter sequences of α2(I) collagen gene linked to either enhanced green fluorescent protein (COL/EGFP) or the luciferase (COL/LUC) reporter gene. Following dermal excision or subcutaneous bleomycin administration, a large number of EGFP-positive collagen-producing cells appeared in the dermis of COL/EGFP reporter mice. When wild-type mice were transplanted with BM cells from transgenic COL/EGFP animals and subjected to dermal excision, no EGFP-positive BM-derived collagen-producing cells were detected throughout the repair process. Luciferase assays of dermal tissues from COL/LUC recipient mice also excluded collagen production by BM-derived cells during dermal excision healing. In contrast, a limited but significant number of CD45-positive collagen-producing cells migrated from BM following bleomycin injection. These results indicate that resident cells in the skin are the major source of de novo collagen deposition in both physiological and pathological conditions, whereas BM-derived cells participate, in part, in collagen production during dermal fibrogenesis.