Anchoring Fibrils Research Articles

Highly Efficient Ex Vivo Correction of COL7A1 through Ribonucleoprotein-Based CRISPR/Cas9 and Homology-Directed Repair to Treat Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and severe genetic skin disease responsible for blistering of the skin and mucosa after minor trauma. RDEB is caused by a wide variety of variants in COL7A1 encoding type VII Collagen, the major component of anchoring fibrils that form key attachment structures for dermal-epidermal adherence. In this study, we achieved highly efficient COL7A1 editing in primary RDEB keratinocytes and fibroblasts from 2 patients homozygous for the c.6508C>T (p.Gln2170∗) variant through CRISPR/Cas9-mediated homology-directed repair. Three guide RNAs targeting the c.6508C>T variant or harboring sequences were delivered together with high-fidelity Cas9 as a ribonucleoprotein complex. Among them, one achieved 73% cleavage activity in primary RDEB keratinocytes and RDEB fibroblasts. Then, we treated RDEB keratinocytes and RDEB fibroblasts with this specific ribonucleoprotein complex and the corresponding donor template delivered as single-stranded oligodeoxynucleotide and achieved up to 58% of genetic correction as well as type VII Collagen rescue. Finally, grafting of corrected 3-dimensional skin onto nude mice induced re-expression and normal localization of type VII Collagen as well as anchoring fibril formation at the dermal-epidermal junction 5 and 10 weeks after grafting. With this promising nonviral approach, we achieved therapeutically relevant specific gene editing that could be applicable to all variants in exon 80 of COL7A1 in primary RDEB cells.

477 Efficient ex-vivo COL7A1 correction of patient's primary keratinocytes and fibroblasts using RNP-based CRISPR/Cas9 and homology-directed repair to treat recessive dystrophic epidermolysis bullosa

RDEB is a severe and life-threatening genetic skin disease responsible for blistering of the skin and mucosa after minor trauma. RDEB is caused by a wide variety of mutations in COL7A1 encoding type VII collagen (C7), the major component of anchoring fibrils (AF) which are critical attachment structures that adhere the epidermis to the dermis. We aimed to achieve highly efficient correction of a null mutation (c.6508C>T, p.Gln2170*) in exon 80 of COL7A1 in a patient’s primary RDEB keratinocytes (KC), fibroblasts (FB) and 3D-skin equivalents (SE) through CRISPR/Cas9-mediated HDR.

The novel mutation in the COL7A1 gene in the patient with dystrophic forms of epidermolysis bullosa

Dystrophic Epidermolysis Bullosa (DEB) is a rare inherited blistering disorder with both autosomal recessive (RDEB) and autosomal dominant (DDEB) mode of inheritance. DEB is characterized by affected dermis-epidermis conjunction, which results from COL7A1 mutations. COL7A1 encodes collagen VII, the major component of anchoring fibrils (AF), which is responsible for dermis-epidermis conjunction. Structural modification of AFs or significant alterations in its numbers could result in DEB. In the current investigation, patients within six families affected by DEB underwent complete clinical evaluation. Moreover, the causative mutations were identified in the COL7A1 gene by next generation sequencing and then confirmed by Sanger sequencing. A pathogenic homozygous and heterozygous glycine substitution mutation were identified in exon 54 of twin siblings of the family I (c.5018G > A, p.G1673E). In family II, a frameshift deletion mutation in exon 75 was identified in a 7-year-old patient (c.6265delC, p.P2090fs). A novel pathogenic frameshift insertion mutation was confirmed in a 6-year-old girl (c.179-180insGAAA, p.F60fs) in family III. These findings expand scientific knowledge of the EB causative mutations, which facilitates the EB classification and lead to more accurate prenatal diagnosis in the families at the high risk of such diseases.

CRISPR/Cas9-based targeted genome editing for correction of recessive dystrophic epidermolysis bullosa using iPS cells

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in the COL7A1 gene encoding type VII collagen (C7). The spectrum of severity depends on the type of mutation in the COL7A1 gene. C7 is the major constituent of anchoring fibrils (AFs) at the basement membrane zone (BMZ). Patients with RDEB lack functional C7 and have severely impaired dermal-epidermal stability, resulting in extensive blistering and open wounds on the skin that greatly affect the patient's quality of life. There are currently no therapies approved for the treatment of RDEB. Here, we demonstrated the correction of mutations in exon 19 (c.2470insG) and exon 32 (c.3948insT) in the COL7A1 gene through homology-directed repair (HDR). We used the clustered regulatory interspaced short palindromic repeats (CRISPR) Cas9-gRNAs system to modify induced pluripotent stem cells (iPSCs) derived from patients with RDEB in both the heterozygous and homozygous states. Three-dimensional human skin equivalents (HSEs) were generated from gene-corrected iPSCs, differentiated into keratinocytes (KCs) and fibroblasts (FBs), and grafted onto immunodeficient mice, which showed normal expression of C7 at the BMZ as well as restored AFs 2 mo postgrafting. Safety assessment for potential off-target Cas9 cleavage activity did not reveal any unintended nuclease activity. Our findings represent a crucial advance for clinical applications of innovative autologous stem cell-based therapies for RDEB.

From Clinical Phenotype to Genotypic Modelling: Incidence and Prevalence of Recessive Dystrophic Epidermolysis Bullosa (RDEB).

BackgroundRecessive dystrophic epidermolysis bullosa (RDEB) is an inherited genetic disorder characterized by recurrent and chronic open wounds with significant morbidity, impaired quality of life, and early mortality. RDEB patients demonstrate reduction or structural alteration type VII collagen (C7) owing to mutations in the gene COL7A1, the main component of anchoring fibrils (AF) necessary to maintain epidermal-dermal cohesion. While over 700 alterations in COL7A1 have been reported to cause dystrophic epidermolysis bullosa (DEB), which may be inherited in an autosomal dominant (DDEB) or autosomal recessive pattern (RDEB), the incidence and prevalence of RDEB is not well defined. To date, the widely estimated incidence (0.2–6.65 per million births) and prevalence (3.5–20.4 per million people) of RDEB has been primarily characterized by limited analyses of clinical databases or registries.MethodsUsing a genetic modelling approach, we use whole exome and genome sequencing data to estimate the allele frequency of pathogenic variants. Through the ClinVar and NCBI database of human genome variants and phenotypes, DEB Register, and analyzing premature COL7A1 termination variants we built a model to predict the pathogenicity of previously unclassified variants. We applied the model to publicly available sequences from the Exome Aggregation Consortium (ExAC) and Genome Aggregation Database (gnomAD) and identified variants which were classified as pathogenic for RDEB from which we estimate disease incidence and prevalence.ResultsGenetic modelling applied to the whole exome and genome sequencing data resulted in the identification of predicted RDEB pathogenic alleles, from which our estimate of the incidence of RDEB is 95 per million live births, 30 times the 3.05 per million live birth incidence estimated by the National Epidermolysis Bullosa Registry (NEBR). Using a simulation approach, we estimate a mean of approximately 3,850 patients in the US who may benefit from COL7A1-mediated treatments in the US.ConclusionWe conclude that genetic allele frequency estimation may enhance the underdiagnosis of rare genetic diseases generally, and RDEB specifically, which may improve incidence and prevalence estimates of patients who may benefit from treatment.

367 Phase I clinical trial for Recessive Dystrophic Epidermolysis Bullosa using genetically corrected autologous keratinocytes

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is an inherited genetic skin disorder caused by mutations in the COL7A1 gene encoding type VII collagen (C7). The Current therapy is limited to supportive palliation. We report the results from the first Phase I clinical trial of ex vivo gene therapy for the treatment of severe RDEB (NCT01263379). 4 adult, male RDEB subjects enrolled in this trial carried various heterozygous COL7A1 mutations resulting in expression of only truncated C7 protein undetectable by NC2 antibodies and displayed absent, or sparse rudimentary anchoring fibrils (AF) by EM. Autologous RDEB KC isolated from skin biopsies were transduced with GMP grade retrovirus carrying the full-length COL7A1. 6 ∼35cm2 autologous epidermal sheet grafts were manufactured and grafted onto prepared wound beds of each subject (N=24 grafts). The primary endpoints included expression of full-length C7 and restoration of AF at 3 and 6 months, as well as absence of RCR at 3, 6, 12 months post grafting. All 24 grafts were well tolerated by all subjects, and no serious adverse events were reported. At 3 months, 87% (21/24 grafts) and at 6 months, 67% (16/24 grafts) showed significant wound healing, as of 75% healing compared with baseline. Molecular analysis of the skin biopsies obtained from the grafted sites revealed robust C7 expression detected by both NC1 and NC2 C7 antibodies, with morphologically normal appearance and frequency of NC2 reactive AF identified at the basement membrane zone using IEM. These data indicate that COL7A1 ex-vivo gene transfer has a favorable safety profile and capable of cutaneous type VII collagen delivery highlighting the potential of durable cell based RDEB therapy in humans.

From Mesoderm to Mesodermatology: Bone Marrow Mesenchymal Cells Heal Skin Wounds

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by loss-of-function mutations in the COL7A1 gene that result in reduced levels or complete absence of type VII collagen (C7). The disease is characterized by progressively worsening blistering, inflammation, pain, scarring, and contractures. C7 is an extracellular matrix protein that homopolymerizes into anchoring fibrils (AFs) and plays a critical role in adherence of skin layers at the dermal-epidermal junction (DEJ) in cutaneous and mucosal membranes. Multiple cell types can express C7—keratinocytes, fibroblasts (FBs), mesenchymal stromal/stem cells (MSCs, Figure 1), perhaps even hematopoietic cells—but are the cell types functionally and qualitatively similar or equal? In several methodical and incisive studies, injections of allogeneic FBs or MSCs have been used for wound healing in individuals with RDEB,1,2,3,4,5,6 and the study by Kuhl and colleagues reported in this issue adds to our knowledge regarding the use of MSCs as medications for wound healing in RDEB.7

Topical Application of Recombinant Type VII Collagen Incorporates Into the Dermal–Epidermal Junction and Promotes Wound Closure

Patients with recessive dystrophic epidermolysis bullosa (RDEB) have incurable skin fragility, blistering, and skin wounds due to mutations in the gene that codes for type VII collagen (C7) that mediates dermal-epidermal adherence in human skin. In this study, we evaluated if topically applied human recombinant C7 (rC7) could restore C7 at the dermal-epidermal junction (DEJ) and enhance wound healing. We found that rC7 applied topically onto murine skin wounds stably incorporated into the newly formed DEJ of healed wounds and accelerated wound closure by increasing re-epithelialization. Topical rC7 decreased the expression of fibrogenic transforming growth factor-β2 (TGF-β2) and increased the expression of anti-fibrogenic TGF-β3. These were accompanied by the reduced expression of connective tissue growth factor, fewer α smooth muscle actin (α-SMA)-positive myofibroblasts, and less deposition of collagen in the healed neodermis, consistent with less scar formation. In addition, using a mouse model in which skin from C7 knock out mice was grafted onto immunodeficient mice, we showed that applying rC7 onto RDEB grafts with wounds restored C7 and anchoring fibrils (AFs) at the DEJ of the grafts and corrected the dermal-epidermal separation. The topical application of rC7 may be useful for treating patients with RDEB and patients who have chronic skin wounds.

Epidermolysis bullosa acquisita: Autoimmunity to anchoring fibril collagen

Epidermolysis bullosa acquisita (EBA) is a rare and acquired autoimmune subepidermal bullous disease of skin and mucosa. EBA includes various distinct clinical manifestations resembling genetic dystrophic epidermolysis bullosa (DEB), Bullous pemphigus, Brunsting–Perry pemphigoid, or cicatricial pemphigoid. These patients have autoantibodies against type VII collagen (C7), an integral component of anchoring fibrils (AFs), which are responsible for attaching the dermis to the epidermis. Destruction or perturbation of the normal functioning AFs clinically results in skin fragility, blisters, erosions, scars, milia, and nail loss, all features reminiscent of genetic dystrophic epidermolysis bullosa. These anti-C7 antibodies are “pathogenic” because when injected into a mouse, the mouse develops an EBA-like blistering disease. Currently, treatment is often unsatisfactory; however, some success has been achieved with colchicine, dapsone, photopheresis, plasmapheresis, infliximab, rituximab, and IVIG.

First Use of Allogeneic Stem Cells in the Treatment of Recessive Dystrophic Epidermolysis Bullosa: Demonstration of Biochemical and Clinical Improvement.

Abstract 660Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable, often fatal mucocutaneous blistering skin disease caused by mutations in the type VII collagen (C7) gene, COL7A1. These pathogenic mutations result in severely diminished expression of C7, a collagen localized at the dermal-epidermal junction (DEJ), and absence of anchoring fibrils (AFs) which are C7 containing structures that tether the epidermal basement membrane to the dermal matrix. From birth on, children with RDEB develop painful erosions and blisters on mucosal membranes and skin often resulting in esophageal strictures, mutilating scarring, joint contractures, fusion of fingers and toes and, aggressive squamous cell carcinomas. After first demonstrating that a stem cell enriched fraction of bone marrow (BM) rescued a proportion of RDEB mice from lethality and resulted in a) expression of C7 in skin and mucosal membranes, b) formation of new AFs, and c) resistance to blistering, a ‘first-in-human' phase I-II clinical trial was initiated in October 2007. To date, 7 patients have been treated with stem cells from BM from an HLA matched sibling donor (n=6) or unrelated cord blood (CB) donor (n=1). Follow-up data are reported through August 18, 2009. Conditioning consisted of busulfan 0.8 mg/kg per dose every 6 hours on days–9 to–6, fludarabine 25 mg/m2/day on days–5 to–3, and cyclophosphamide 50 mg/kg/day on days–5 to–2. After infusion of stem cells on day 0, immunoprophylaxis consisted of cyclosporine and mycophenolate mofetil. Patient and graft characteristics are shown in Table 1. Of the 4 patients with adequate follow-up, a progressive increase in C7 deposition by immunofluorescence (IF) at the DEJ, AFs or AF-like structures by electron microscopy, and wound healing with marked reduction in blister formation were documented. Unexpectedly, all patients had substantial chimerism in the skin (11-93%) that persisted over time. In 2 patients with a sex mismatched donor, perivascular localization of the donor cells in the dermis could be discerned using probes to the centromere regions of chromosomes X and Y. In summary, this is the first demonstration that the infusion of BM can ameliorate the severe systemic mucocutaneous manifestations of RDEB and sets the stage for using marrow stem cells in the treatment of a broad spectrum of extracellular matrix disorders.PtDonor (cell dose: NC × 108/kg)Transplant Related ToxicitiesC7 AssessmentAnchoring Fibril AssessmentClinical OutcomeSurvival Days1 15 mo maleHLA 8/8 male sibling BM/CB (3.04; 0.66)NoneIncreased by IF↑ Rudimentary AFsImproved but no change in use of dressingsAlive day 6592 9 mo femaleHLA 8/8 male sibling BMCardiomyo-pathyNot evaluableNot evaluableNot evaluableDied day 03 5.9 maleHLA 5/6 female unrelated CB (0.55)Graft rejectionIncreased by IF and Western↑ Rudimentary AFsNot evaluableDied day 1834 6.3 yo maleHLA 8/8 female sibling BM (3.76)Transient Dialysis ARDSNo change by IF° but WesternNormal AFs observedMarked reduction in blisters and dressingsAlive day 2475 6.2 yo femaleHLA 8/8 male sibling BM (3.07)Transient DialysisIncreased by IF and Western↑ Rudimentary AFsMarked reduction in blisters and dressingsAlive day 1286 6.0 yo femaleHLA 8/8 female sibling BM (3.11)EpistaxispendingpendingEarly reduction in blisteringAlive day 567 14.5 yo femaleHLA 8/8 female sibling BMToo early to evaluatependingpendingToo early to evaluateAlive day -9 Disclosures:No relevant conflicts of interest to declare.

Pretibial epidermolysis bullosa: is this case a new subtype with loss of types IV and VII collagen?

Pretibial epidermolysis bullosa (PEB) is an extremely rare subtype of dominant dystrophic epidermolysis bullosa (DDEB), in which recurrent blistering with scarring predominantly involves the pretibial skin. Nail dystrophy, albopapuloid lesions, and hypertrophic scars may also occur. In PEB, immunohistochemical and electron microscopic studies demonstrate the complete or partial loss of the anchoring fibril (AF) in the basement membrane zone, suggesting disturbed synthesis or excessive degradation of collagen VII, the main component of AF. Interestingly, we report a case of PEB with unusual results of joint loss of types IV and VII collagen.

Review of collagen VII sequence variants found in Australasian patients with dystrophic epidermolysis bullosa reveals nine novel COL7A1 variants

Dystrophic epidermolysis bullosa (DEB) is an inherited skin fragility disorder where blistering occurs in the sub-lamina densa zone at the level of anchoring fibrils (AFs) of the dermo-epidermal junction. Both autosomal dominant (DDEB) and recessive (RDEB) result from mutations in the type VII collagen gene (COL7A1). The purpose of this study was to understand the genotype-phenotype correlation in Australian patients with DEB. Skin biopsies from patients were processed for immunofluorescence mapping, the COL7A1 gene was screened for sequence variants. We report 14 Australian families with different forms of dystrophic epidermolysis bullosa (DEB) with 23 different COL7A1 allelic variants, nine of which were novel. Four cases of RDEB-HS combined two premature termination codon (PTC) variants and three other cases of RDEB-HS with combined PTC and spice-site or glycine substitution variants. G2043R, a de novo dominant variant, was also identified in this study. Four "silent" glycine substitutions were found in this study, G2775S, G1673R, G1338V and G2719A. EB17, with combined R2791W and G2210V variants, had a DDEB-Pasini phenotype, in contrast to two family members who had severe DDEB pruriginosa, with the same genotype. In this study, the RDEB variants included nonsense variants, splice site variants, internal deletions or insertions, "silent" glycine substitutions within the triple helix or N or C terminal ends of the triple helix and non-glycine missense variants within the triple helix domain. DDEB usually involves glycine substitutions within the triple helix of COL7A1 although other missense variants or splice-site alterations may underlie some cases.

Transcriptional control of the mouse Col7a1 gene in keratinocytes: basal and transforming growth factor-beta regulated expression.

Anchoring fibrils at the cutaneous basement membrane zone of the stratified squamous epithelia are essential to maintaining skin integrity, as absence of these structures leads to the chronic blistering disease, dystrophic epidermolysis bullosa. Type VII collagen, the major component of anchoring fibrils, is synthesized primarily by basal keratinocytes and to a lesser degree by dermal fibroblasts. To elucidate the transcriptional control elements of the type VII collagen gene (Col7a1), 3 kb of 5' flanking sequence of the mouse gene was cloned, sequenced, and fused to the chloramphenicol acetyltransferase reporter gene. Promoter deletion analyses revealed that 560 bp of Col7a1 5' flanking sequence was sufficient and necessary for basal level of transcription in cultured murine keratinocytes. Mutagenesis of DNA sequences with similarity to consensus binding sites for transcription factors, including Sp1/Sp3, AP2, AP1, and Smads, within the p-560Col7a1 promoter/chloramphenicol acetyltransferase construct, coupled with DNA binding assays, revealed the importance of these sites for basal Col7a1 expression. The effect of transforming growth factor beta, an activator of Col7a1 expression in keratinocytes and dermal fibroblasts, was examined using the same Col7a1 promoter/chloramphenicol acetyltransferase constructs. These analyses demonstrated that transforming growth factor beta1 stimulation of Col7a1 transcription is dependent on a putative interaction between Smads and AP1. Interestingly, the Smad-like binding site was essential for both basal and transforming growth factor beta1 stimulated Col7a1 transcription. Collectively, these findings attest to the complex regulation of Col7a1 transcription in epidermal keratinocytes.

Biology of anchoring fibrils: lessons from dystrophic epidermolysis bullosa.

Anchoring fibrils are adhesive suprastructures that ensure the connection of the epidermal basement membrane with the dermal extracellular matrix. The fibrils represent polymers of collagen VII, the major structural fibril component, but may also contain other proteins. Remarkable progress has been made in the last few years in understanding the functions of skin basement membrane components including the anchoring fibrils. Novel insights into the biology of the anchoring fibrils have been gained from experimental studies on dystrophic epidermolysis bullosa (DEB), a group of inherited blistering disorders caused by mutations in the gene for collagen VII, COL7A1. Mutation analyses of DEB families have disclosed more than 100 COL7A1 gene defects so far, but the unusual complexity of the mutation constellations and their biological consequences are only beginning to emerge. In analogy to heritable disorders of other collagen genes, predictable phenotypes of COL7A1 mutations causing premature termination codons or dominant negative interference have been observed. However, collagen VII seems to represent a remarkable exception among collagens in that many mutations, including heterozygous glycine substitutions and deletions, lead to minimal phenotypes, or to no phenotype at all. In contrast to fibrillar collagens, structural abnormalities of collagen VII molecules in anchoring fibrils appear to be tolerated to a certain extent. However, the mild DEB phenotypes can be severely modulated by a second aberration in individuals compound heterozygous for two different COL7A1 mutations. Therefore, not only definition of mutation(s) but also cell biological, protein chemical and suprastructural studies of the mutated molecules yield novel insight into the molecular pathomechanisms underlying disease.

Three homozygous PTC mutations in the collagen type VII gene of patients affected by recessive dystrophic epidermolysis bullosa: analysis of transcript levels in dermal fibroblasts.

The Hallopeau-Siemens variant of recessive dystrophic epidermolysis bullosa (HS-RDEB) is a severe inherited skin disease characterized by the absence of collagen type VII (COLVII) and anchoring fibrils (AF), caused by mutations in collagen type VII gene (COL7A1). Mutations leading to the formation of premature termination codons (PTCs) of translation are the characteristic genetic lesions in HS-RDEB patients; many PTC mutations have been found to be associated with a marked reduction or complete absence of COLVII mRNA. In this article, we report homozygosity for three different mutations in the COL7A1 of HS-RDEB patients. One mutation, the R2685X, falling in exon 109, is a novel mutation, whereas the other two, the 425A-->G falling in exon 3 and the 497insA in exon 4, have been previously identified in compound heterozygosity with different mutations in other unrelated RDEB patients. Haplotype analysis in three Italian families carrying the 497insA mutation suggested a common origin of this mutation and indicated that this is an ancestral Italian mutation. All these mutations generate PTCs and are associated with the absence of COLVII expression, as detected by immunofluorescence analysis of the patient's skin. Evaluation of the levels of the mutated COLVII mRNAs in cultured skin fibroblasts of the patients and of their parents showed that all the mutated transcripts were expressed at consistent levels. Therefore, our results indicate that a marked mRNA reduction is not a constant feature associated with PTC mutations in COL7A1.

Mesenchymal‐epithelial interactions regulate gene expression of type VII collagen and kalinin in keratinocytes and dermal‐epidermal junction formation in a skin equivalent model

Anchoring fibrils constituted primarily of type VII collagen and anchoring filaments composed of kalinin are essential structural elements of the dermal-epidermal junction and critical for its stability. The role of fibroblasts in the production of these structural elements and the formation of the dermal-epidermal junction was studied by using a living skin equivalent model. This model had been modified such that keratinocytes and fibroblasts were allowed direct contact. After 2 weeks, immunohistochemical studies showed the linear deposition of type VII collagen and kalinin, as well as type IV collagen and alpha6 integrin at the dermal-epidermal junction. By electron microscopy, anchoring fibrils, a continuous lamina densa, and numerous hemidesmosomes were noted. Reverse transcriptase-polymerase chain reaction analysis showed an increased expression of both type VII collagen and kalinin genes in keratinocytes when they were in direct contact with fibroblasts. These results suggest that fibroblasts synthesize an extracellular matrix which favors keratinocyte adhesion and the formation of a dermal-epidermal junction by increasing the production and the further arrangement of dermal-epidermal junction components.

The molecular genetics of dystrophic epidermolysis bullosa

EPIDERMOLYSIS BULLOSA (EB) is a heterogeneous group of inherited skin diseases characterized by blistering of the skin and mucous membranes after minor trauma.^1-3The genes responsible for the three groups of EB (EB simplex, junctional EB, and dystrophic EB [DEB]) have recently been identified, and some molecular defects within these genes have been characterized.^4,5Specifically, EB simplex has been shown to arise from mutations in the keratin 5 and 14 genes^6-10; the genes encoding nicein/kalinin/epiligrin are the candidate genes in junctional EB¹¹; and both dominant and recessive forms of DEB (DDEB and RDEB, respectively) have been closely linked to the type VII collagen gene (COL7A1)^12-16that encodes the major component of anchoring fibrils (AFs).^17,18Biochemistry and molecular biology of basement membrane zone components involved in human diseases have been reviewed by Marinkovich¹⁹in this issue of theArchives. This editorial focuses on the

Distribution of type-VII collagen in xenografted human carcinomas.

The distribution of type-VII collagen, the main molecular component of the anchoring fibrils (AF) attaching the basal lamina (BL, lamina densa of the basement membrane) to the surrounding connective tissue, was investigated in four xenografted human carcinomas of the hypopharynx (H-Stg 1), the lung (L 261), the sigmoid colon (CA 1), and the rectum (R 85). The studies were performed with a recently prepared, affinity-purified and highly specific antibody to type-VII collagen by using the indirect immunofluorescence and the APAAP (alkaline phosphatase anti-alkaline phosphatase) techniques. For comparison, the localization of the intrinsic BL components laminin and type-IV collagen were additionally analyzed in all four carcinomas. It was shown that type-VII collagen usually colocalized to laminin and type-IV collagen and was deposited at the borderline between carcinoma cell clusters and the surrounding strands of connective tissue in a similar, but more diffuse and less continuous distribution than both intrinsic BL components. In the squamous cell carcinoma H-Stg 1 and the adenocarcinoma L261, type-VII collagen was additionally accumulated in enlarged extracellular spaces between carcinoma cells, away from the contact zone to the connective tissue and again colocalized to laminin and type-IV collagen. Numerous carcinoma cells of both xenografts showed remarkable intracytoplasmic immunoreactivity for the antibody to type-VII collagen. Even in the case of the gastrointestinal carcinomas CA 1 and R 85, faint immunoreactivity for type-VII collagen was found at the contact zone between the mucosal epithelium and the surrounding connective tissue. These results confirm that epithelial carcinoma cells are obviously involved with the synthesis of the main molecular component of AF usually attaching the BL to the adjacent connective tissue and hint at a possible correlation between the localization of type-VII collagen and the observed pattern of the BL. However, it cannot be decided whether there is a direct causal relation between both phenomena or whether they are both the consequence of an independent but common cause, such as abnormal cellular differentiation of carcinoma cells. In no case, can the discontinuities in the distribution of type-VII collagen be explained by active tumor cell invasion since xenografted human carcinomas neither invade nor metastasize.

Epithelial origin of cutaneous anchoring fibrils.

Anchoring fibrils are essential structural elements of the dermoepidermal junction and are crucial to its functional integrity. They are composed largely of type VII collagen, but their cellular origin has not yet been confirmed. In this study, we demonstrate that the anchoring fibrils are primarily a product of epidermal keratinocytes. Human keratinocyte sheets were transplanted to a nondermal connective tissue graft bed in athymic mice. De novo anchoring fibril formation was studied ultrastructurally by immunogold techniques using an antiserum specific for human type VII procollagen. At 2 d after grafting, type VII procollagen/collagen was localized both intracellularly within basal keratinocytes and extracellularly beneath the discontinuous basal lamina. Within 6 d, a subconfluent basal lamina had developed, and newly formed anchoring fibrils and anchoring plaques subjacent to the xenografts were labeled. Throughout the observation period of the experiment, the maturity, population density, and architectural complexity of anchoring fibrils beneath the human epidermal graft continuously increased. Identical findings were obtained using xenografts cultivated from cloned human keratinocytes, eliminating the possibility of contributions to anchoring fibril regeneration from residual human fibroblasts. Immunolabeling was not observed at the mouse dermoepidermal junction at any time. These results demonstrate that the type VII collagen of human cutaneous anchoring fibrils and plaques is secreted by keratinocytes and can traverse the epidermal basal lamina and that the fibril formation can occur in the absence of cells of human dermal origin.

Hemidesmosomes, Collagen VII, and Intermediate Filaments in Basal Cell Carcinoma

We have undertaken an analysis of hemidesmosomes (HD) and their associated structures, intermediate filaments (IF) and anchoring fibrils (AF), in various types of basal cell carcinoma (BCC). Using a combination of electron microscopy and immunofluorescence microscopy we show that there is a correlation between the loss of HD and tumor type (i.e., in solid and infiltrative BCC hemidesmosomes are present, sometimes in reduced numbers), while there appears to be a lack of hemidesmosomes in cells of sclerosing specimens. Moreover, even though there is a loss of cytoplasmic constituents of the HD in sclerosing forms of BCC, this is not the case with regard to collagen VII, a component of AF, which are normally associated with the extracellular side of the HD. Collagen VII is localized to the basement membrane zone of tumor cells in the absence of the cytoplasmic constituents of HD. Furthermore, deposits of collagen VII occur in the connective tissue close to tumor cell populations in all but one of the BCC specimens we analyzed. In addition to modifications in HD and AF in BCC tissue, there are changes in the cytoskeletal elements of both tumor cells and the normal appearing epidermis that overlies tumor areas. In sclerosing BCC microfilaments are commonly observed along the basal portions of tumor cells where they abut the connective tissue. IF are often found interacting with these microfilaments. Indirect immunofluorescence analysis of tumor tissue using a monoclonal keratin antibody preparation, AE1, which in normal epidermis stains basal cells, reveals that AE1 antibodies only weakly stain tumor cells. Moreover, in the epidermis that overlies tumor cell regions AE1 antibodies stain suprabasal cells and not basal cells. This change in staining pattern generated by AE1 antibodies appears to depend upon the proximity of tumor cells. These results are discussed in relation to the organization of the HD and its associated AF and IF. The possibility that HD, IF, and AF antibody preparations may be of diagnostic use is raised.