Epidermal Basement Membrane Research Articles

Laminin peptide YIGSR enhances epidermal development of skin equivalents

Since the use of animal experimentation is restricted with regard to cosmetic materials, alternative in vitro models such as skin equivalents (SEs) are needed. Laminin is one of the major non-collagenous glycoproteins. The pentapeptide YIGSR (Tyr-Ile-Gly-Ser-Arg) is a functional motif of laminin that binds to the laminin receptor. In the present study, we examined whether YIGSR could improve the reconstruction of SEs. YIGSR has no effects on monolayer cell proliferation of CCD25-Sk fibroblasts or HaCaT keratinocytes. Interestingly, YIGSR decreased TGF-β1 levels, although it promoted type Ι collagen synthesis in CCD25-Sk cells. In HaCaT cells, YIGSR decreased the expression of involucrin and loricrin, which are differentiation markers. Furthermore, YIGSR increased levels of proliferating cell nuclear antigen (PCNA), p63, and integrin α6, and decreased involucrin in SE models. In addition, two models containing YIGSR (mixed with dermal equivalents or added into media) did not show any differences in expression levels of PCNA, p63, integrin α6, and involucrin. Therefore, YIGSR is a useful agent for reconstruction of SEs, independent of its method of application. These results indicate that YIGSR stimulates epidermal proliferation and basement membrane formation while inhibiting keratinocyte differentiation of SEs. Taken together, these results indicate that YIGSR promotes the reconstruction of SEs, potentially via decreased TGF-β1 levels and consequent inhibition of epidermal differentiation.

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.

Simulation of melanoblast displacements reveals new features of developmental migration.

ABSTRACTTo distribute and establish the melanocyte lineage throughout the skin and other developing organs, melanoblasts undergo several rounds of proliferation, accompanied by migration through complex environments and differentiation. Melanoblast migration requires interaction with extracellular matrix of the epidermal basement membrane and with surrounding keratinocytes in the developing skin. Migration has been characterized by measuring speed, trajectory and directionality of movement, but there are many unanswered questions about what motivates and defines melanoblast migration. Here, we have established a general mathematical model to simulate the movement of melanoblasts in the epidermis based on biological data, assumptions and hypotheses. Comparisons between experimental data and computer simulations reinforce some biological assumptions, and suggest new ideas for how melanoblasts and keratinocytes might influence each other during development. For example, it appears that melanoblasts instruct each other to allow a homogeneous distribution in the tissue and that keratinocytes may attract melanoblasts until one is stably attached to them. Our model reveals new features of how melanoblasts move and, in particular, suggest that melanoblasts leave a repulsive trail behind them as they move through the skin.

Case of epidermolysis bullosa acquisita with concomitant anti‐laminin‐332 antibodies

Subepidermal autoimmune blistering disease including bullous pemphigoid, pemphigoid gestationis, mucous membrane pemphigoid, anti-laminin-γ1 pemphigoid, linear immunoglobulin A bullous disease and epidermolysis bullosa acquisita (EBA), are all characterized by direct immunofluorescence microscopy or immunoglobulin deposition on the basement membrane zone. Among them, EBA is a rare acquired subepidermal autoimmune blistering disease of the skin and mucous membranes reactive with type VII collagen, a major component of the epidermal basement membrane zone. Anti-laminin-332-type mucous membrane pemphigoid has pathogenic autoantibodies against laminin-332, which is a basement membrane heterotrimeric protein composed of α3, β3 and γ2 laminin chains. We describe a 73-year-old Japanese man presenting with multiple, annular, tense blisters on the lower legs and oral lesions. Despite the severe clinical manifestations, the disease was successfully controlled by combination therapy of oral prednisolone and mizoribine. This case was confirmed to have autoantibodies to both type VII collagen and laminin-332 α3 chain by indirect immunofluorescence of 1 mol NaCl-split normal human skin, various immunoblot analyses and enzyme-linked immunosorbent assays. This case was a rare case of EBA with concomitant anti-laminin-332 antibodies.

Contemporary aspects on the immunopathogenesis of autoimmune diseases of the epidermal basement membrane in the dog

Autoimmune diseases of the epidermal basement membrane are the result of the immune system self-activation against specific antigens of its essential structural elements. This group of skin diseases is characterized by the destruction of connecting bonds between the membrane zone and dermis, which eventually leads to the dermoepidermal separation and the formation of subepidermal vesicles and bullae. In this article, the immunopathogenesis and the clinical, histopathological and immunohistochemical features of each of these skin diseases are briefly reviewed. The autoimmune diseases of the canine epidermal basement membrane have been recently classified as bullous pemphigoid, mucous membrane pemphigoid, linear IgA dermatosis, epidermolysis bullosa acquisita and bullous systemic lupus erythematosus. Recent advances in immunopathological and molecular techniques have markedly facilitated the understanding of their pathogenesis, thus giving the opportunity for the development of new therapeutic strategies that may improve or eliminate the clinical signs.

Amino Carbonylation of Epidermal Basement Membrane Inhibits Epidermal Cell Function and Is Suppressed by Methylparaben

This study investigated the effect of amino carbonylation (Maillard reaction) on the function of the epidermal basement membrane (BM) by analyzing epidermal cell proliferation and keratinization and stratum corneum barrier function using a three-dimensional human epidermal BM model treated with glyceraldehyde. Intracellular ATP levels were lower in cells cultured on amino-carbonylated epidermal BM as compared to those in normal epidermal BM (control). Moreover, trans-epidermal water loss was increased by culturing on amino-carbonylated BM relative to the control; this was accompanied by downregulation of filaggrin, transglutaminase-1, and serine palmitoyltransferase 2 mRNA levels. p-Hydroxybenzoic acid methyl ester (methylparaben) abrogated the decrease in ATP production and filaggrin expression in human keratinocytes induced by amino-carbonylated collagen. Thus, amino carbonylation of the epidermal BM inhibits moisture retention, keratinization, and ceramide synthesis and disrupts the barrier function of the stratum corneum. These findings suggest that methylparaben can be an effective additive to cosmetics for improving epidermal function that is compromised by amino carbonylation.

Type XVII collagen coordinates proliferation in the interfollicular epidermis.

Type XVII collagen (COL17) is a transmembrane protein located at the epidermal basement membrane zone. COL17 deficiency results in premature hair aging phenotypes and in junctional epidermolysis bullosa. Here, we show that COL17 plays a central role in regulating interfollicular epidermis (IFE) proliferation. Loss of COL17 leads to transient IFE hypertrophy in neonatal mice owing to aberrant Wnt signaling. The replenishment of COL17 in the neonatal epidermis of COL17-null mice reverses the proliferative IFE phenotype and the altered Wnt signaling. Physical aging abolishes membranous COL17 in IFE basal cells because of inactive atypical protein kinase C signaling and also induces epidermal hyperproliferation. The overexpression of human COL17 in aged mouse epidermis suppresses IFE hypertrophy. These findings demonstrate that COL17 governs IFE proliferation of neonatal and aged skin in distinct ways. Our study indicates that COL17 could be an important target of anti-aging strategies in the skin.

T-plastin is essential for basement membrane assembly and epidermal morphogenesis.

The establishment of epithelial architecture is a complex process involving cross-talk between cells and the basement membrane. Basement membrane assembly requires integrin activity but the role of the associated actomyosin cytoskeleton is poorly understood. Here, we identify the actin-bundling protein T-plastin (Pls3) as a regulator of basement membrane assembly and epidermal morphogenesis. In utero depletion of Pls3 transcripts in mouse embryos caused basement membrane and polarity defects in the epidermis but had little effect on cell adhesion and differentiation. Loss-of-function experiments demonstrated that the apicobasal polarity defects were secondary to the disruption of the basement membrane. However, the basement membrane itself was profoundly sensitive to subtle perturbations in the actin cytoskeleton. We further show that Pls3 localized to the cell cortex, where it was essential for the localization and activation of myosin II. Inhibition of myosin II motor activity disrupted basement membrane organization. Our results provide insights into the regulation of cortical actomyosin and its importance for basement membrane assembly and skin morphogenesis.

Histological changes of the dermal-epidermal junction of superficial scarin rabbit ears induced by ultrapulse CO2 fractional laser

Objective To explore the effects of wound healing and histological changes by utilizing ultrapulse CO2 fractional laser for the treatment of superficial scar in dermal-epidermal junction (DEJ) in rabbit ears. Methods We adopted traditional iodophor treatment and moist exposed burn ointment treatment repairing the wound and utilizing ultra pulse CO2 fractional laser for the treatment of rabbit ears superficial scar. The superficial scars in rabbit ear were induced with ultra pulse CO2 fractional laser. In 1, 2, 4, 8 weeks tissue samples would be taken. Using electron microscopy we observed DEJ ultrastructure and light microscopy to observe the histological changes of the dermal papilla and PAS staining for epidermis basement membrane. Results The wound healing time of moist exposed burn ointment group was shorter than that of iodophor group (P<0.05). The number of hemidesmosomes and anchoring fibers in the moist exposed burn ointment group on the 8th week was more than the 1th week, The number of hemidesmosomes and anchoring fibers in the iodophor group on 8th week was more than 1th week (P<0.05). The number of hemidesmosomes and anchoring fibers in the iodophor group was less than the moist exposed burn ointment group (P<0.05). Conclusions Ultrapulse CO2 fractional laser can cause ultra structural changes in scarring dermal-epidermal junction area, wet wound healing environment promotes the reconstruction of DEJ district organization, and rebuilding the function of the scar skin has the positive significance. Key words: Ultrapulse CO2 Fractional laser; Superficial scar; Dermal-epidermal junction; Regeneration and reconstruction

Dysregulation of mCD46 and sCD46 contribute to the pathogenesis of bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune bullous disease caused by autoantibodies against BP180 in the epidermal basement membrane. Autoantibody-mediated complement activation is an important process in BP pathogenesis. CD46, a crucial complement regulatory protein in the complement activation, has been reported to be involved in several autoimmune diseases. In the present study, we investigated whether CD46 plays a role in BP development. We found that sCD46 expression was significantly increased in the serum and blister fluids of BP patients and correlated with the levels of anti-BP180 NC16A antibody and C3a. Otherwise, the level of mCD46 was decreased in lesions of BP patients, whereas the complement activation was enhanced. We also found that CD46 knockdown in HaCaT human keratinocytes enhanced autoantibody-mediated complement activation. Importantly, exogenous CD46 blocked complement activation in both healthy skin sections and keratinocytes induced by exposure to pathogenic antibodies from BP patients. These data suggest that CD46 deficiency is an important factor in BP pathogenesis and that increasing CD46 levels might be an effective treatment for BP.

Diagnostic Value of Linear Fluorescence Along the Basement Membrane of Sweat Gland Ducts in Bullous Pemphigoid

Linear IgG deposits along the basement membrane of adnexa has been proposed to be useful in the diagnosis of bullous pemphigoid (BP), but no controlled studies have been performed. This study evaluated linear IgG fluorescence of the basement membrane of sweat gland ducts (SGD) and other adnexa in perilesional biopsies from patients with BP (n = 64) and controls (n = 82), using direct immunofluorescence microscopy. Fluorescence intensity was graded semi-quantitatively. Positive SGDs were found in 58 (90.6%) patients with BP and 44 (53.7%) controls, a statistically significant difference (p < 0.0001). The sensitivity of positive SGDs for BP was high (90.6%), but the specificity was low (46.3%). Only strong fluorescence intensity was associated with high specificity. In conclusion, positive SGDs in direct immunofluorescence microscopy are highly sensitive for BP; however, only strong fluorescence has acceptable specificity. Weak positivity of SGDs without linear fluorescence of the epidermal basement membrane may not be sufficiently specific for BP.

Gene Therapy for Epidermolysis Bullosa: Sticky Business

Despite efforts to promote development of therapies for rare diseases, the number of available treatments remains low and progress is too slow to have a significant impact on patients. This does not necessarily reflect a lack of interest in rare disease research, and significant efforts have been made toward the development and clinical testing of molecular therapies for these diseases. The intrinsic challenges of understanding the mechanisms underlying rare diseases render the design of efficacious therapies laborious.

Enhancement of Epidermal Basement Membrane Formation by Synthetic Inhibitors of Extracellular Matrix-degrading Enzymes

Enhancement of Epidermal Basement Membrane Formation by Synthetic Inhibitors of Extracellular Matrix-degrading Enzymes

Analysis of phenotypic heterogeneity of male patients with X-linked Alport syndrome from one family

Objective In this study, the phenotype heterogeneity of 2 male patients with X-linked Alport syndrome from one family was analyzed and the likely reasons were discussed by reviewing the literature. Methods The clinical data at the time of diagnosis and during 5 years follow-up of 2-male patients with X-linked Alport syndrome from one family were collected.The α5(IV) chain expression in the epidermal basement membrane was detected by indirect immunofluorescence method.COL4A5 gene mutations in skin fibroblasts and genomic DNA were detected by using reverse transcription polymerase chain reaction and direct sequencing and PCR sequencing methods from skin fibroblasts and genomic DNA, respectively. Results The diagnostic age of patient Ⅲ1 was 14 years old.He had only microscopic hematuria, and proteinuria was negative.A negative α5(Ⅳ) chain staining pattern was detected in his epidermal basement membrane.After 5 years follow-up without drug treatment, he was 19 years old, had persistent microscopic hematuria and normal renal function.The urinary microalbumin was 19.2-31.8 mg/L.The diagnosis age of patient Ⅱ 4 was 29 years old.The hematuria and proteinuria were found at 22 years old.He was treated with tripterygium wilfordii for 1 year.His disease progressed to an end stage of renal disease and he received hemodialysis therapy at 24 years old.He had the renal transplantation surgery at 29 years old, just 2 months before he came to hospital.And his renal function was restored.After 5 years follow-up, his urine examination and renal function were normal.Both patients had a missense mutation c. 3650G>A(p.G1217D)in exon 41 in COL4A5 gene. Conclusions The different phenotypes of 2 male patients from one family with X-linked Alport syndrome were reported.The most possible reason for this is somatic mosaic variants in COL4A5 gene based on literature review.Physicians should be alert to phenotype heterogeneity in male X-linked Alport syndrome despite having the same gene mutation. Key words: Alport syndrome; Hematuria; Proteinuria; Nephritis, hereditary

Characterization and mechanisms of photoageing-related changes in skin. Damages of basement membrane and dermal structures.

Sun-exposed skin is characterized by superficial changes such as wrinkles, sagging and pigmentary changes, and also many internal changes in the structure and function of epidermis, basement membrane (BM) and dermis. These changes (so-called photoageing) are predominantly induced by the ultraviolet (UV) component of sunlight. Epidermis of UV-irradiated skin produced several enzymes such as matrix metalloproteinases (MMPs), urinary plasminogen activator (uPA)/plasmin and heparanase, which degrade dermal collagen fibres and elastic fibres in the dermis, and components of epidermal BM. The BM at the dermal-epidermal junction (DEJ) controls dermal-epidermal signalling and plays an important role in the maintenance of a healthy epidermis and dermis. BM is repetitively damaged in sun-exposed skin compared with unexposed skin, leading to epidermal and dermal deterioration and accelerated skin ageing. UV exposure also induces an increase in vascular endothelial growth factor (VEGF), an angiogenic factor, while thrombospondin-1 (TSP-1), an anti-angiogenic factor, is decreased; these changes induce angiogenesis in papillary dermis with increased migration of elastase-positive leucocytes, leading to dermal elastic fibre damage. Elastic fibres, such as oxytalan fibres in papillary dermis, are associated with not only skin resilience, but also skin surface texture, and elastic fibre formation by fibroblasts is facilitated by increased expression of fibulin-5. Thus, induction of fibulin-5 expression is a damage-repair mechanism, and fibulin-5 is an early marker of photoaged skin. UV-induced skin damage is cumulative and leads to premature ageing of skin. However, appropriate daily skincare may ameliorate photoageing by inhibiting processes causing damage and enhancing repair processes.

Abstract 5161: Development of cutaneous basal cell carcinomas from cutaneous actinic keratoses

Abstract Actinic keratoses (AKs) are extremely common proliferations of epidermal keratinocytes which show cytological atypia and impaired ability to mature as the cells that comprise them migrate from the basal layer of the epidermis to higher levels of the epidermis. A small percentage of AKs are well known to progress to cutaneous squamous cell carcinomas (SCCs). However, progression of AKs to cutaneous basal cell carcinomas (BCCs) has not been convincingly documented despite several attempts, even though BCCs are significantly more common than SCCs in the general US and European populations. Documented cases have been dismissed as actinic keratoses that have simply occurred superimposed upon basal cell carcinomas due to chance occurrence of two common sun induced skin lesions at the same sites. In a university based referral dermatopathology service, one of us (WCL) observed 21 cases of AKs progressing to BCCs during a 29 year period (January 1, 1985 to December 31, 2013) of continuous observation in which 115,898 cases of AKs, 10,188 cases of BCCs and 4,809 cases of SCCs were diagnosed. Only cases in which a continuous progression of cells from those showing changes typical of AKs to those showing changes typical of BCCs was observed were included. During the same interval 308 cases of AKs progressing to SCCs were diagnosed. To further test the hypothesis that AKs can progress to BCCs, cases of AKs that progressed to BCCs were further examined to determine those in which neither the AK nor the BCC occupied the entire skin surface (i.e., length of the epidermal basement membrane) present in the specimen. Of the 21 cases, 13 met this criterion. All were shave biopsies of sun damaged skin. For each biopsy, the proportion of the surface covered by the AK, the proportion occupied by the BCC, and the proportion of the surface overlapped by both lesions were measured and the proportion of overlap was compared to the proportion of overlap expected due to chance. Statistical analysis showed that the observed overlap exceeded the expected overlap by 32 per cent with p &amp;lt; 0.05. We conclude that progression of AKs to BCCs, while not as common as progression of AKs to SCCs, does occur in a small proportion of cases. Citation Format: W Clark Lambert, Claude E. Gagna, Muriel W. Lambert. Development of cutaneous basal cell carcinomas from cutaneous actinic keratoses. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5161.

Tissue injury and repair following cutaneous exposure of mice to sulfur mustard.

In mouse skin, sulfur mustard (SM) is a potent vesicant, damaging both the epidermis and the dermis. The extent of wounding is dependent on the dose of SM and the duration of exposure. Initial responses include erythema, pruritus, edema, and xerosis; this is followed by an accumulation of inflammatory leukocytes in the tissue, activation of mast cells, and the release of mediators, including proinflammatory cytokines and bioactive lipids. These proinflammatory mediators contribute to damaging the epidermis, hair follicles, and sebaceous glands and to disruption of the epidermal basement membrane. This can lead to separation of the epidermis from the dermis, resulting in a blister, which ruptures, leading to the formation of an eschar. The eschar stimulates the formation of a neoepidermis and wound repair and may result in persistent epidermal hyperplasia. Epidermal damage and repair is associated with upregulation of enzymes generating proinflammatory and pro-growth/pro-wound healing mediators, including cyclooxygenase-2, which generates prostanoids, inducible nitric oxide synthase, which generates nitric oxide, fibroblast growth factor receptor 2, and galectin-3. Characterization of the mediators regulating structural changes in the skin during SM-induced tissue damage and wound healing will aid in the development of therapeutic modalities to mitigate toxicity and stimulate tissue repair processes.

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.

Laminin α5 in the keratinocyte basement membrane is required for epidermal–dermal intercommunication

Laminin α5 is broadly expressed in the epidermal basement membrane (BM) of mature mice and its elimination at this site (Lama5Ker5 mouse) results in hyperproliferation of basal keratinocytes and a delay in hair follicle development, which correlated with upregulation of the dermally-derived laminin α2 and laminin α4 chains in the epidermal BM and of tenascin-C subjacent to the BM. In vitro studies revealed laminin 511 to be strongly adhesive for primary keratinocytes and that loss of laminin α5 does not result in cell autonomous defects in proliferation. Flow cytometry reveals that the loss of laminin α5 resulted in increased numbers of CD45+, CD4+ and CD11b+ immune cells in the skin, which temporo-spatial analyses revealed were detectable only subsequent to the loss of laminin α5 and the appearance of the hyperproliferative keratinocyte phenotype. These findings indicate that immune cell changes are the consequence and not the cause of keratinocyte hyperproliferation. Loss of laminin α5 in the epidermal BM was also associated with changes in the expression of several dermally-derived growth factors involved in keratinocyte proliferation and hair follicle development in adult but not new born Lama5Ker5 skin, including KGF, EGF and KGF-2. In situ binding of FGF-receptor-2α (IIIb)-Fc chimera (FGFR2IIIb) to mouse skin sections revealed decoration of several BMs, including the epidermal BM, which was absent in Lama5Ker5 skin. This indicates reduced levels of FGFR2IIIb ligands, which include KGF and KGF-2, in the epidermal BM of adult Lama5Ker5 skin. Our data suggest an initial inhibitory effect of laminin α5 on basal keratinocyte proliferation and migration, which is exacerbated by subsequent changes in growth factor expression by epidermal and dermal cells, implicating laminin α5 in epidermal–dermal intercommunication.

055 Evidence of epidermal basement membrane transmigration by dendritic cells in dermatitis herpetiformis

055 Evidence of epidermal basement membrane transmigration by dendritic cells in dermatitis herpetiformis