Skin Basement Membrane Research Articles

Recombinant Humanized Collagen Enhances Secreted Protein Levels of Fibroblasts and Facilitates Rats’ Skin Basement Membrane Reinforcement

Collagen and its peptides exhibit remarkable antioxidant activity, superior biocompatibility, and water solubility, making them a significant research focus in skin care. Hence, the recombinant humanized collagen types I, III, and XVII complexed with niacinamide were developed to address damage in human foreskin fibroblasts (HFF-1) caused by ultraviolet radiation and to evaluate basement membrane proteins in a rat skin model. The Cell Counting Kit-8 (CCK-8) assay showed that higher concentrations of the complex increased the survival of damaged cells by approximately 10% and 22%, respectively, compared to the normal group after 16 and 48 h of treatment. Further biochemical analyses using ELISA and immunofluorescence (IF) confirmed that the complex enhanced the expression of collagen type IV, laminin, P63, and transforming growth factor-β (TGF-β) in the damaged cells. Additionally, the complex boosted the activity of the basement membrane in rat skin and stimulated the secretion of integrin, laminin, and perlecan. Overall, the recombinant humanized collagen complex effectively reinforced the skin’s basement membrane.

Weekly Intraperitoneal Injection of Tamoxifen in an Inducible In Vivo Model of Junctional Epidermolysis Bullosa Generates Early and Advanced Disease Phenotypes

Junctional epidermolysis bullosa (JEB) caused by loss of function variants in genes encoding the skin basement membrane proteins Laminin 332 (Lam332), type XVII collagen or integrin α6β4, effects patients from birth with severe blistering eventually leading to scarring and early lethality. Here we have optimised a previously published JEB eKO model with weekly tamoxifen intraperitoneal injections resulting in a more controllable and severe model. Due to the titratable dosing this model now recapitulates both early and advanced stages of the human disease, strengthening its use in therapeutic studies. The gradual loss of Lam-α3 in the skin of the mouse through weekly injections lead to generalised blistering and fibrotic dermal changes in multiple skin sites by week 12 post-tamoxifen. Our findings demonstrate the usefulness of optimising tamoxifen induction in Cre-loxP mouse models of extracellular matrix proteins, an approach that could be applicable to other emerging inducible transgenic disease models to improve their ability to mimic the human disease phenotype.

P16 Fibroblast signalling crosstalk drives junctional epidermolysis bullosa disease progression

Abstract Introduction and aims Junctional epidermolysis bullosa (JEB) is a devastating hereditary skin disorder characterized by severe fragility of skin and mucus membrane, emerges as blistering with little or no trauma. The major classification of JEB includes JEB intermediate and JEB severe; the latter is the most common subtype, which is the result of loss of function mutations in the skin basement membrane protein laminin-332. Lacking this protein impairs basement membrane adhesion of epidermal keratinocytes and leads to deregulated epidermal–dermal crosstalk. The skin dermis is composed of multiple fibroblast subpopulations with different functions in skin homeostasis and repair; however, their specific involvement in skin diseases is largely unknown. Here, we investigate how fibroblast subpopulations, namely papillary and reticular fibroblasts, impact JEB disease progression and skin regeneration. Methods Three-dimensional (3D) organotypic (OT) combining healthy (control) and diseased (JEB) keratinocytes cells with distinct fibroblast subpopulations from different anatomical sites were developed and analysed by immunohistochemistry and RNA-Seq. Results JEB OTs with papillary fibroblasts showed increased epidermal growth, invagination of basal layer and deregulated differentiation compared with reticular fibroblasts. RNA-Seq analysis revealed altered activity in key signalling including immune, cytokine, gap junction, mitogen-activated protein kinase and remodelling of keratinization and ECM proteins, particularly in JEB OT cultured with papillary fibroblast. JEB OTs and cells also demonstrated variation in cell proliferation and activation of transforming growth factor-β1 and immune cell signalling. Currently, we are targeting identified signalling pathways in our two-dimensional and 3D cell culture models and identifying druggable targets, before testing selected candidates in our JEB mouse disease model for further preclinical validation. Conclusions By dissecting the pathological signalling interactions between keratinocytes and distinct fibroblast subpopulations, our goal is to identify new therapeutic target to improve the skin health and regeneration in patients with JEB.

O20 Dissecting junctional epidermolysis bullosa heterogeneity

Abstract Introduction and aims Epidermolysis bullosa (EB) is a family of rare, incurable, inherited disorders that are characterized by extreme skin and mucosal fragility. One of the most severe forms is junctional EB (JEB). JEB is caused by mutations in genes encoding the skin basement membrane proteins laminin 332 (α3, β3, γ2), type XVII collagen or integrin α6β4. Despite similarities between the disease pathology of JEB and other forms of EB there is a dramatic reduction in life expectancy for patients with JEB. Patients with JEB rarely survive beyond the first year of life. To investigate this disparity, we have optimized a previously published conditional tamoxifen induced knockout JEB mouse model (Lama3flx/flxK14CreERT). This mouse model recapitulates all key features of JEB, such as thickening of the epidermis and visible blistering owing to the loss of laminin α3 at the basement membrane. Methods Single-cell (sc)RNA-Seq analysis was conducted on pooled skin samples from a healthy (control) and JEB mouse model (n = 3 per genotype). Immunofluorescent staining was conducted to confirm scRNA-Seq analysis including markers for skin differentiation (K14, transglutaminase 1, loricrin), proliferation (Ki67), basement membrane (laminin α3, laminin β3, laminin γ2, collagen VII) and immune markers (F4/80, Ly6-G/C, CD3, CD79). Results We can see large differences in cellular populations across the two conditions. With increased T- and B-immune-cell subpopulations in the JEB model compared with the healthy control. Utilizing CellChatDB we identified disease-specific signalling pathways that identify activated T- and B-cell populations interacting with JEB macrophage populations via proinflammatory pathways. Conclusions We have utilized an in vivo mouse model to gain insight into the molecular and cellular differences in a JEB diseased phenotype. Identifying changes in cellular pathways could be used as a targeted approach to identify a beneficial therapeutic treatment for JEB.

O19 Improvement of a junctional epidermolysis bullosa mouse model

Abstract Introduction and aims Junctional epidermolysis bullosa (JEB) is the most severe of the epidermolysis bullosa (EB) disorders and is caused by mutations in genes encoding the skin basement membrane proteins laminin 332, type XVII collagen or integrin α6β4. Affected individuals experience blistering from birth leading to scarring, granulation tissue and susceptibility to infection. In JEB, generalized severe, there is 50% mortality in the first 2 years of life, often owing to failure to thrive or overwhelming infection. A number of animal models for JEB exist; however, because of severe blistering on the entire surface of the skin, these mice die soon after birth, reducing their experimental utility. In 2017, the first adult inducible knockout mouse model of JEB was published. In this mouse model, the disruption of the Lama3 gene is specifically induced in epidermal basal keratinocytes after birth. These mice remain viable, which allows us to study the longer-term consequences of the loss of laminin α3. However, the mice did not develop the severe phenotype observed in patients with JEB. Methods We have optimized this previously published methodology of a conditional knockout JEB mouse model (Lama3flx/flxK14CreERT) to now include more regular tamoxifen injections, which Results in a more controlled and severe phenotype of JEB. Results Widespread skin blistering develops after 12 weeks of tamoxifen treatment with an impaired skin barrier (transepidermal water loss) and a loss of epidermal lipids. Immunofluorescent staining was conducted to assess skin differentiation (K14, transglutaminase 1, loricrin), proliferation (Ki67), basement membrane proteins (laminin α3, laminin β3, laminin γ2, collagen VII) and immune markers (F4/80, Ly6-G/C, CD3, CD79). Conclusions These mice recapitulate all key features of human JEB with loss of laminin α3 at the basement membrane, epidermal thickening and visible blisters. This allows us to delve deeper into the functional and downstream pathways affected by the loss of laminin α3 and discover why the prognosis of patients with JEB is so severe.

Biomimetic tri-layered artificial skin comprising silica gel-collagen membrane-collagen porous scaffold for enhanced full-thickness wound healing

Full-thickness wounds are severe cutaneous damages with destroyed self-healing function, which need efficient clinical interventions. Inspired by the hierarchical structure of natural skin, we have for the first time developed a biomimetic tri-layered artificial skin (TLAS) comprising silica gel-collagen membrane-collagen porous scaffold for enhanced full-thickness wound healing. The TLAS with the thickness of 3–7 mm displays a hierarchical nanostructure consisting of the top homogeneous silica gel film, the middle compact collagen membrane, and the bottom porous collagen scaffold, exquisitely mimicking the epidermis, basement membrane and dermis of natural skin, respectively. The 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide/N-Hydroxysuccinimide-dehydrothermal (EDC/NHS-DHT) dual-crosslinked collagen composite bilayer, with a crosslinking degree of 79.5 %, displays remarkable biocompatibility, bioactivity, and biosafety with no risk of hemolysis and pyrogen reactions. Notably, the extra collagen membrane layer provides a robust barrier to block the penetration of silica gel into the collagen porous scaffold, leading to the TLAS with enhanced biocompatibility and bioactivity. The full-thickness wound rat model studies have indicated the TLAS significantly facilitates the regeneration of full-thickness defects by accelerating re-epithelization, collagen deposition and migration of skin appendages. The highly biocompatible and bioactive tri-layered artificial skin provides an improved treatment for full-thickness wounds, which has great potential in tissue engineering.

Dermal stiffness governs the topography of the epidermis and the underlying basement membrane in young and old human skin.

The epidermis is a stratified epithelium that forms the outer layer of the skin. It is composed primarily of keratinocytes and is constantly renewed by the proliferation of stem cells and their progeny that undergo terminal differentiation as they leave the basal layer and migrate to the skin surface. Basal keratinocytes rest on a basement membrane composed of an extracellular matrix that controls their fate via integrin-mediated focal adhesions and hemidesmosomes which are critical elements of the epidermal barrier and promote its regenerative capabilities. The distribution of basal cells with optimal activity provides the basement membrane with its characteristic undulating shape; this configuration disappears with age, leading to epidermal weakness. In this study, we present an in-depth imaging analysis of basal keratinocyte anchorage in samples of human skin from participants across the age spectrum. Our findings reveal that skin aging is associated with the depletion of hemidesmosomes that provide crucial support for stem cell maintenance; their depletion correlates with the loss of the characteristic basement membrane structure. Atomic force microscopy studies of skin and invitro experiments revealed that the increase in tissue stiffness observed with aging triggers mechanical signals that alter the basement membrane structure and reduce the extent of basal keratinocyte anchorage, forcing them to differentiate. Genomic analysis revealed that epidermal aging was associated with mechanical induction of the transcription factor Krüppel-like factor 4. The altered mechanical properties of tissue being a new hallmark of aging, our work opens new avenues for the development of skin rejuvenation strategies.

Emerging Gene Therapeutics for Epidermolysis Bullosa under Development.

The monogenetic disease epidermolysis bullosa (EB) is characterised by the formation of extended blisters and lesions on the patient's skin upon minimal mechanical stress. Causal for this severe condition are genetic mutations in genes, leading to the functional impairment, reduction, or absence of the encoded protein within the skin's basement membrane zone connecting the epidermis to the underlying dermis. The major burden of affected families justifies the development of long-lasting and curative therapies operating at the genomic level. The landscape of causal therapies for EB is steadily expanding due to recent breakthroughs in the gene therapy field, providing promising outcomes for patients suffering from this severe disease. Currently, two gene therapeutic approaches show promise for EB. The clinically more advanced gene replacement strategy was successfully applied in severe EB forms, leading to a ground-breaking in vivo gene therapy product named beremagene geperpavec (B-VEC) recently approved from the US Food and Drug Administration (FDA). In addition, the continuous innovations in both designer nucleases and gene editing technologies enable the efficient and potentially safe repair of mutations in EB in a potentially permanent manner, inspiring researchers in the field to define and reach new milestones in the therapy of EB.

Using proteomics to compare the molecular structures of sulfide and permeate-depilated sheepskins

An environmentally friendly method using real or artificial bovine milk permeate to both depilate and preserve sheepskins has been reported which completely and cleanly removed the wool from the hair follicle and had no detrimental effects on the skin. A proteomic analysis, assessing the relative abundance of proteins in matched permeate-depilated and chemically depilated (sulfide) sheepskins, showed variations in the levels of specific collagen types in the skin's basement membrane and other proteins associated with the follicles. These findings were corroborated by biochemical analyses of matched permeate depilated and raw skin samples, and provide clues to the mechanism of non-invasive and complete depilation. They also support the observation that permeate-depilated skins were smoother than their sulfide-depilated counterparts and resulted in leather with a superior surface.Graphical abstract

Elastin is Related to Functions of Melanocytes in Normal and Abnormal Skin

Context: Functions of melanocytes are supported by many growth factors and cytokines derived from epidermal and dermal cells, especially keratinocytes and fibroblasts. In addition to these skin cells, elastin fibers are thought to be important for regulating melanocyte functions in normal and abnormal skin. However, the role of elastin has not been fully investigated. Recently, the research on the role of elastin in melanocyte functions has greatly increased even in human skin, whereas no full review article on this subject has been presented so far. Thus, the author tried to present a complete review article on this subject in animals and humans. Methods: The author searched the literature on elastin and melanocytes in research databases. Results: Elastin fibers can affect the normal functions of melanoblasts/melanocytes in animals and humans. Moreover, elastin fibers influence melanocyte functions in abnormal skin. In vitiliginous skin, where melanin and melanoblasts/melanocytes are completely lost, elastin fibers are dramatically reduced. However, elastin fibers are completely restored or even increased in the dermis of repigmented skin after phototherapy and/or skin transplantation. Moreover, elastin fibers penetrate the basement membrane of repigmented skin, suggesting a direct contact between dermal elastin fibers and epidermal melanoblasts/melanocytes. Conclusions: Elastin/elastin fibers may control the functions of mammalian melanocytes in normal and abnormal skin.

283 Optimising specificity and applicability of base editing for COL7A1 mutations in recessive dystrophic epidermolysis bullosa

Base and prime editors are novel CRISPR variants that reduce the risk of off-target effects by avoiding double-stranded DNA cleavage, bypassing the need for homology-directed repair. As such, these tools offer great therapeutic potential for inherited skin diseases such as recessive dystrophic epidermolysis bullosa (RDEB), a severe blistering skin disorder caused by bi-allelic loss-of-function mutations in COL7A1, which encodes type VII collagen (C7) the major component of anchoring fibrils in skin basement membrane.

292 Heterozygous pathogenic variants affecting the the highly conserved VWFA domain of integrin-β4 cause isolated nail dystrophy

Pathogenic variants in ITGB4 cause various forms of autosomal recessive epidermolysis bullosa (EB). ITGB4 encodes integrin-β4, which plays a critical role in the maintenance of the skin basement membrane integrity. Here, we aimed to delineate the genetic basis of dominant isolated nail dystrophy in 2 families. We studied a total of five affected individuals with nail dystrophy and no other cutaneous or extra-cutaneous findings. Whole exome sequencing revealed a different heterozygous missense variant in ITGB4 gene in each of the 2 families.

184 The role of the Laminin 332 in cholesterol transport in dry and Aged skin

The skin provides a barrier function mediated by a combination of cornified epithelial cells, cell-cell adhesions and epidermal lipids. The barrier function is compromised in old age which is exemplified by features such as improper lipid metabolism and flattened dermal-epidermal junction. Laminin 332 is a skin basement membrane glycoprotein which was shown to be decreased in aged skin. We have previously demonstrated that the loss of laminin 332 leads to an upregulation of cholesterol biosynthesis genes and alters the skin’s lipid profile. We identified that this change is due to an impaired intracellular cholesterol transport. To understand the mechanism around cholesterol transport we have identified a panel of proteins of interest for further investigation. We investigated the role that laminin 332, actin and microtubule disruption have on both subcellular location of our proteins of interest and cholesterol transport. We have demonstrated that NPC2, Myo5b and e-cadherin are decreased in laminin α3 knockdown nTERT keratinocytes compared to shC controls. Interestingly, NPC1, a binding partner for NPC2, was shown to be increased in the laminin α3 knockdown nTERT keratinocytes. Normal levels and localisation of each protein can be restored by the addition of recombinant laminin 332. We have also demonstrated that in keratinocytes free cholesterol localises to Rab11a-positive vesicles which is a binding partner for Myo5b. By using actin- and microtubule-disrupting drugs, we have shown that it is the loss of actin that’s driving the observed phenotype. These findings suggest that the disrupted cholesterol transport and impaired barrier function in laminin α3 knockdown is due to lower levels of Myo5b and e-cadherin resulting from a disorganised actin cytoskeleton with a concomitant weakening of cell-cell contacts.

Investigation of comorbid autoimmune diseases in women with autoimmune bullous diseases: An interplay of autoimmunity and practical implications.

Autoimmune bullous diseases are a group of skin disorders resulting from an autoimmune reaction against intercellular adhesion molecules or components of the basement membrane of skin and mucosa. Autoimmune disorders often occur in patients with a history of another autoimmune disease and most autoimmune diseases have a striking female predominance. In this review, we aim to analyze the different associations of autoimmune bullous diseases with other autoimmune diseases and highlight the distinctiveness of the female gender in these associations.

Melanin accumulation in dermal stem cells deteriorates their exosome-mediated skin basement membrane construction in solar lentigo.

Solar lentigo (SL) is a hyperpigmented macule that occurs in sun-exposed areas and is characterized by the accumulation of melanin pigment in the epidermis. On the contrary, melanin-incorporated macrophages have also been identified in the dermis, which is thought to be caused by melanin transfer due to disruption of the basement membrane, but the detailed mechanism remains unclear. In this study, we analysed SL lesions by pathological methods and examined the mechanism of melanin accumulation in the dermis using cultured skin models in vitro. First, we observed a significant decrease in type IV collagen (COL4), a major component of the basement membrane, in SL lesions. The basement membrane is known to be formed by the interaction of keratinocytes and dermal cells. Therefore, we constructed skin models containing fibroblasts or dermal stem cells and examined their effects on basement membrane formation. The results showed a markedly enhanced production of COL4 mediated by dermal stem cell-derived exosomes. The analysis of melanin localization in the SL dermis revealed that CD163-positive macrophages and CD271-positive dermal stem cells both took up melanin pigment. Exosomes of dermal stem cells incorporating melanosomes were less effective in promoting COL4 expression. These findings suggest that while the promotion of COL4 production in keratinocytes by dermal stem cell-derived exosomes is important for maintaining basement membrane homeostasis, this mechanism is disrupted in SL lesions, leading to chronic melanin accumulation in the dermis.

Protein Ligand Nanopattern Size Selects for Cellular Adhesion via Hemidesmosomes over Focal Adhesions.

Hemidesmosomes (HDs) are multiprotein complexes that firmly anchor epidermal cells to the basement membrane of skin through the interconnection of the cytoplasmic intermediate filaments with extracellular laminin 332 (Ln332). Considerably less attention has been paid to HDs compared to focal complexes/focal adhesions (FC/FAs) in mechanistic single-cell structures due to the lack of suitable in vitro model systems. Here nanopatterns of Ln332 (100-1000nm) are created to direct and study the formation of HD in adherent HaCaT cells. It is observed that HaCaT cells at Ln 332 nanopatterns adhere via hemidesmosomes, in stark contrast to cells at homogeneous Ln332 surfaces that adhere via FC/FAs. Clustering of α6 integrin is observed at nanopatterned Ln332 of 300nm patches and larger. Cells at 500nm diameter patterns show strong colocalization of α6 integrin with ColXVII or pan-cytokeratin compared to 300nm/1000nm indicating a threshold for HD initiation >100nm but a pattern size selection for maturation of HDs. It is demonstrated that the pattern of Ln332 can determine the cellular selection of adhesion types with a size-dependent initiation and maturation of HDs. The protein nanopatterning approach that is presented provides a new in vitro route to study the role of HDs in cell signaling and function.

Dermatologic Manifestations of Systemic Diseases in Childhood

Dermatologic Manifestations of Systemic Diseases in Childhood

Involvement of COL5A2 and TGF-β1 in pathological scarring.

Dysregulation in the cutaneous wound-healing process is a consequence of alterations in the efficiency and activity of the various components involved in the healing process. This dysregulation may result in various clinical appearances of a lesion, such as skin ulcers, keloids, hypertrophic and atrophic scars. The collagen type V alpha 2 (COL5A2) gene provides a template for a component of type V collagen, found primarily within the skin basement membrane. Transforming growth factor (TGF)-β is involved in inflammation, angiogenesis, proliferation of fibroblasts, collagen synthesis and extracellular matrix remodeling. Hypertrophic scar fibroblasts possess a disrupted expression pattern of the TGF-β signaling compared to normal healing, while an increased TGF-β signaling reduces the epidermal proliferation rate, triggering atrophic scarring. In the present study, 71 female patients who had undergone planned Caesarean section, without postoperative complications, were examined. These patients were clinically and molecularly evaluated after developing scars in order to determine the role of TGF-β1 (rs201700967 and rs200230083) and COL5A2 (rs369072636) in pathological scarring. Clinical scar evaluation was carried out using SCAR and POSAS scales and genotyping was performed by RT-PCR. No statistical differences were found between the subgroups regarding the genotype and the pathological scarring, since all the patients included were wild-type allele carriers. Further investigations and a more representative study group may highlight the involvement of COL5A2 and TGF-β1 single nucleotide variants in pathological scarring.

Mapping the molecular and structural specialization of the skin basement membrane for inter-tissue interactions

Inter-tissue interaction is fundamental to multicellularity. Although the basement membrane (BM) is located at tissue interfaces, its mode of action in inter-tissue interactions remains poorly understood, mainly because the molecular and structural details of the BM at distinct inter-tissue interfaces remain unclear. By combining quantitative transcriptomics and immunohistochemistry, we systematically identify the cellular origin, molecular identity and tissue distribution of extracellular matrix molecules in mouse hair follicles, and reveal that BM composition and architecture are exquisitely specialized for distinct inter-tissue interactions, including epithelial–fibroblast, epithelial–muscle and epithelial–nerve interactions. The epithelial–fibroblast interface, namely, hair germ–dermal papilla interface, makes asymmetrically organized side-specific heterogeneity in the BM, defined by the newly characterized interface, hook and mesh BMs. One component of these BMs, laminin α5, is required for hair cycle regulation and hair germ–dermal papilla anchoring. Our study highlights the significance of BM heterogeneity in distinct inter-tissue interactions.

Gestational pemphigoid

Pemphigoid gestationis is a rare, autoimmune blistering dermatosis. It is unique in that it is diagnosed primarily in association with pregnancy. Autoantibodies against placental BP180 (also known as BPAG2 or collagen XVII) cause damage to the skin basement membrane, resulting in severe itching and blistering rash over the body and the extremities.