Lentiviral expression of wild-type LAMA3A restores cell adhesion in airway basal cells from children with epidermolysis bullosa.

The hallmark of epidermolysis bullosa (EB) is fragile attachment of epithelia due to genetic variants in cell adhesion genes. We describe 16 EB patients treated in the ear, nose, and throat department of a tertiary pediatric hospital linked to the United Kingdom's national EB unit between 1992 and 2023. Patients suffered a high degree of morbidity and mortality from laryngotracheal stenosis. Variants in laminin subunit alpha-3 (LAMA3) were found in 10/15 patients where genotype was available. LAMA3 encodes a subunit of the laminin-332 heterotrimeric extracellular matrix protein complex and is expressed by airway epithelial basal stem cells. We investigated the benefit of restoring wild-type LAMA3 expression in primary EB patient-derived basal cell cultures. EB basal cells demonstrated weak adhesion to cell culture substrates, but could otherwise be expanded similarly to non-EB basal cells. In vitro lentiviral overexpression of LAMA3A in EB basal cells enabled them to differentiate in air-liquid interface cultures, producing cilia with normal ciliary beat frequency. Moreover, transduction restored cell adhesion to levels comparable to a non-EB donor culture. These data provide proof of concept for a combined cell and gene therapy approach to treat airway disease in LAMA3-affected EB.

Observing Dynamic Conformational Changes within the Coiled-Coil Domain of Different Laminin Isoforms Using High-Speed Atomic Force Microscopy.

Laminins are trimeric glycoproteins with important roles in cell-matrix adhesion and tissue organization. The laminin α, ß, and γ-chains have short N-terminal arms, while their C-termini are connected via a triple coiled-coil domain, giving the laminin molecule a well-characterized cross-shaped morphology as a result. The C-terminus of laminin alpha chains contains additional globular laminin G-like (LG) domains with important roles in mediating cell adhesion. Dynamic conformational changes of different laminin domains have been implicated in regulating laminin function, but so far have not been analyzed at the single-molecule level. High-speed atomic force microscopy (HS-AFM) is a unique tool for visualizing such dynamic conformational changes under physiological conditions at sub-second temporal resolution. After optimizing surface immobilization and imaging conditions, we characterized the ultrastructure of laminin-111 and laminin-332 using HS-AFM timelapse imaging. While laminin-111 features a stable S-shaped coiled-coil domain displaying little conformational rearrangement, laminin-332 coiled-coil domains undergo rapid switching between straight and bent conformations around a defined central molecular hinge. Complementing the experimental AFM data with AlphaFold-based coiled-coil structure prediction enabled us to pinpoint the position of the hinge region, as well as to identify potential molecular rearrangement processes permitting hinge flexibility. Coarse-grained molecular dynamics simulations provide further support for a spatially defined kinking mechanism in the laminin-332 coiled-coil domain. Finally, we observed the dynamic rearrangement of the C-terminal LG domains of laminin-111 and laminin-332, switching them between compact and open conformations. Thus, HS-AFM can directly visualize molecular rearrangement processes within different laminin isoforms and provide dynamic structural insight not available from other microscopy techniques.

Insights into clinical and diagnostic findings as well as treatment responses in patients with mucous membrane pemphigoid: A retrospective cohort study.

BACKGROUND: The variable clinical severity of mucous membrane pemphigoid (MMP) often leads to diagnostic and therapeutic delays. OBJECTIVE: To describe the characteristics of a large cohort of patients with MMP. METHODS: A retrospective review of clinical and diagnostic characteristics as well as treatment responses in 145 patients with MMP. RESULTS: Monosite involvement was seen in 41.4% and multisite involvement in 58.6% of the patients. The oral mucosa was affected in 86.9% of the patients, followed by the ocular mucosa (30.3%), skin (26.2%), genital mucosa (25.5%), nasal mucosa (23.4%), and pharyngeal and/or laryngeal mucosa (17.2%). Ocular disease developed during the disease course in 41.7% of patients with initially other mucosal site involvement. The malignancy rate was significantly higher in patients with autoantibodies against laminin-332 than in patients with MMP without laminin-332 autoantibodies (35.3% vs 10.9%, respectively; P = .007). Systemic immunosuppressive or immunomodulatory therapy was administered to 77.1% of the patients, mainly to patients with multisite (P < .001), ocular (P < .001), and pharyngeal and laryngeal involvement (P = .002). The remaining patients (22.9%) received topical therapy. Adverse events were frequently reported. LIMITATIONS: Retrospective design. CONCLUSION: Patients with MMP present with a heterogeneous clinical presentation, and new symptoms may develop during the disease course. Cancer screening should be considered for patients with MMP and, in particular, for those with autoantibodies against laminin-332.

Laminin 332 in cancer: When the extracellular matrix turns signals from cell anchorage to cell movement.

Laminin 332 is crucial in the biology of epithelia. This large extracellular matrix protein consists of the heterotrimeric assembly of three subunits - α3, ß3, and γ2 - and its multifunctionality relies on a number of extracellular proteolytic processing events. Laminin 332 is central to normal epithelium homeostasis by sustaining cell adhesion, polarity, proliferation, and differentiation. It also supports a major function in epithelial tissue formation, repair, and regeneration by buttressing cell migration and survival and basement membrane assembly. Interest in this protein increased after the discovery that its expression is perturbed in tumor cells, cancer-associated fibroblasts, and the tumor microenvironment. This review summarizes current knowledge regarding the established involvement of the laminin 332 γ2 chain in tumor invasiveness and discusses the role of its α3 and ß3 subunits.

Autoantibody Detection for Diagnosis in Direct Immunofluorescence-Negative Mucous Membrane Pemphigoid: Ocular and Other Sites Compared.

PURPOSE: To assess whether a panel of serum pemphigoid autoantibody tests could be used to confirm an immunopathologic diagnosis of mucous membrane pemphigoid (MMP) in direct immunofluorescent negative (DIF-) MMP patients. DESIGN: Prospective cross-sectional study. PARTICIPANTS: Seventy-six patients with multisite MMP with 45 matched control participants. METHODS: Enzyme-linked immunosorbent assays (ELISAs) for BP180 and BP230 (MBL International), immunoglobulin A (IgA) A and immunoglobulin G indirect immunofluorescence (IIF) on human salt-split skin and the keratinocyte footprint assay for anti-laminin 332 antibodies. MAIN OUTCOME MEASURES: Sensitivity and specificity of autoantibody detection and significant differences for individual tests and test combinations for MMP involving different sites. RESULTS: All DIF- patients (24/73 [31.8%]) had either ocular-only disease or ocular involvement in multisite disease. Serum pemphigoid autoantibodies were detected in 29 of 76 MMP patients (38.2%) compared with 3 of 45 control participants (6.7%). Autoantibody reactivity detected by any 1 or more of the tests was present in 6 of 24 DIF- patients (25%) compared with 22 of 49 DIF positive (DIF+) patients (44.9%). Ocular-only MMP serum reactivity was not significantly different for any test or test combination compared with control participants, whereas DIF- multisite ocular MMP differed for 1 ELISA and 3 of 7 test combinations. By contrast, for DIF+ nonocular MMP patients, all the individual tests, apart from IgA IIF, and all test combinations were significantly different compared with those for control participants. For the entire MMP cohort, the sensitivity of all individual tests was low, having a maximum of 21.05% for BP180 reactivity but increasing to 38.16% for an optimal test combination. Disease activity was associated strongly with positive serologic findings. CONCLUSIONS: Pemphigoid serum autoantibody tests did not provide immunopathologic evidence of MMP in ocular-only MMP patients but showed limited value in DIF- multisite ocular MMP patients. The requirement for immunopathologic confirmation of MMP by autoantibody detection is inappropriate for DIF- ocular-only MMP patients, resulting in missed diagnoses, delayed therapy, and poor outcomes. Alternative diagnostic criteria for ocular-only MMP are required to exclude the other causes of scarring conjunctivitis until more sensitive and specific immunopathologic tests become available.

Interaction between prostate cancer cells and prostate fibroblasts promotes accumulation and proteolytic processing of basement membrane proteins.

BACKGROUND: Tumor microenvironment or stroma has the potency to regulate the behavior of malignant cells. Fibroblast-like cells are abundant in tumor stroma and they are also responsible for the synthesis of many extracellular matrix components. Fibroblast-cancer cell interplay can modify the functions of both cell types. METHODS: We applied mass spectrometry and proteomics to unveil the matrisome in 3D spheroids formed by DU145 prostate cancer cells, PC3 prostate cancer cells, or prostate-derived fibroblasts. Similarly, DU145/fibroblast and PC3/fibroblast coculture spheroids were also analyzed. Western blot analysis and immunofluorescence were used to confirm the presence of specific proteins in spheroids. Cancer dissemination was studied by utilizing "out of spheroids" migration and invasion assays. RESULTS: In the spheroid model cancer cell-fibroblast interplay caused remarkable changes in the extracellular matrix and accelerated the invasion of DU145 cells. Fibroblasts produced structural matrix proteins, growth factors, and matrix metalloproteinases. In cancer cell/fibroblast cocultures basement membrane components, including laminins (α3, α5, ß2, and ß3), heparan sulfate proteoglycan (HSPG2 gene product), and collagen XVIII accumulated in a prominent manner when compared with spheroids that contained fibroblasts or cancer cells only. Furthermore, collagen XVIII was intensively processed to different endostatin-containing isoforms by cancer cell-derived cathepsin L. CONCLUSIONS: Fibroblasts can promote carcinoma cell dissemination by several different mechanisms. Extracellular matrix and basement membrane proteins provide attachment sites for cell locomotion promoting adhesion receptors. Growth factors and metalloproteinases are known to accelerate cell invasion. In addition, cancer cell-fibroblast interplay generates biologically active fragments of basement membrane proteins, such as endostatin.

Mechanism of anchorage-independency and tumor formation of cancer cells: possible involvement of cell membrane-bound laminin-332.

Cancer cells are characterized by anchorage-independency and tumor formation. Involvement of laminin-332 in the pathogenesis of cancer has also been reported. I present a theory that can explain these characteristics together. Proliferating keratinocytes in wound healing produce and deposit laminin-332, which is shown in the provisional basement membrane of a wound. In association with wound closure, expression of LG4/5 domain on the α3 chain of laminin-332 disappears, implicating cleavage of LG4/5 domain. LG4/5 domain expression indicates that laminin-332 prior to the cleavage is bound to the cell membrane, because LG4/5 domain is a cell binding site. In this binding, heparan sulfate proteoglycan on the cell surface seems to be the acceptor for LG4/5 domain. I named this laminin "cell membrane-bound laminin-332" (ML332). ML332 would then bind to integrin α3ß1 via LG1-3 domain, the integrin binding site, and activate FAK and the following Ras/MAPK pathway. Therefore, ML332 eliminates the need for proliferating keratinocytes to bind to processed laminin-332 secreted and deposited into the basement membrane for their proliferation (anchorage-independency). This may hold true of every proliferating epithelial cell, either benign or malignant. Whereas wound closure deprives keratinocytes of anchorage-independency, such events do not occur in cancer cells, and cancer cells maintain anchorage-independency. In the basement membrane formation by epithelial cells, short arms of laminin-332 anchored to the cell membrane bind each other and generate a meshwork polymer. This is the three-arm interaction model. In a similar manner, short-arm interactions between adjacent cancer cells may occur and induce tumor formation.

The impact of surface alteration on epithelial tissue attachment after the mechanical cleaning of titanium or zirconia surface.

OBJECTIVE: Mechanical plaque removal may alter the surface morphology of the gingival penetration part of the implant. We applied an air-powered abrasive system (AP), titanium curette (TC), stainless curette (SC), ultrasound scaler (US), and titanium brush (TB) which are commonly used to remove plaque, to titanium or zirconia and the changes in surface morphology and the epithelial attach against substrata. MATERIALS AND METHODS: (a) The morphological changes of titanium and zirconia after mechanical cleaning were assessed by scanning electron microscopy and a roughness analyser. (b) Oral epithelial cells of rats were inoculated on the surface of the materials after mechanical cleaning, and the adherence of epithelial cells was observed. (c) The maxillary first molars were extracted from the rats and replaced by experimental titanium or zirconia implants. The length of the immunoreactive laminin-332 band was observed at the implant-peri-implant epithelium interface. RESULTS: (a) The surface roughness increased in experimental groups except the AP group. (b) Among the experimental groups, the AP group showed the highest number of attached cells. (c) The length of the immunoreactive laminin-332 band was longer in the control group than those in all five experimental groups. Among the experimental groups, the AP group showed the longest band. CONCLUSION: All mechanical cleaning methods increased the surface roughness of the materials except AP. AP did not cause distinct implant surface alterations. Surface alteration caused by mechanical cleaning may evoke inferior for epithelial attachment and reduce resistance against foreign infiltration.

The intercellular expression of type-XVII collagen, laminin-332, and integrin-ß1 promote contact following during the collective invasion of a cancer cell population.

Cancer cells can invade as a population in various cancer tissues. This phenomenon is called collective invasion, which is associated with the metastatic potential and prognosis of cancer patients. The collectiveness of cancer cells is necessary for collective invasion. However, the mechanism underlying the generation of collectiveness by cancer cells is not well known. In this study, the phenomenon of contact following, where neighboring cells move in the same direction via intercellular adhesion, was investigated. An experimental system was created to observe the two-dimensional invasion using a collagen gel overlay to study contact following in collective invasion. The role of integrin-ß1, one of the major extracellular matrix (ECM) receptors, in contact following was examined through the experimental system. Integrin-ß1 was localized to the intercellular site in squamous carcinoma cells. Moreover, the intercellular adhesion and contact following were suppressed by treatment of an integrin-ß1 inhibitory antibody. ECM proteins such as laminin-332 and type-XVII collagen were also localized to the intercellular site and critical for contact following. Collectively, it was demonstrated that the activity of integrin-ß1 and expression of ECM proteins in the intercellular site promote contact following in the collective invasion of a cancer cell population.

Epidermolysis Bullosa-Associated Squamous Cell Carcinoma: From Pathogenesis to Therapeutic Perspectives.

Epidermolysis bullosa (EB) is a heterogeneous group of inherited skin disorders determined by mutations in genes encoding for structural components of the cutaneous basement membrane zone. Disease hallmarks are skin fragility and unremitting blistering. The most disabling EB (sub)types show defective wound healing, fibrosis and inflammation at lesional skin. These features expose patients to serious disease complications, including the development of cutaneous squamous cell carcinomas (SCCs). Almost all subjects affected with the severe recessive dystrophic EB (RDEB) subtype suffer from early and extremely aggressive SCCs (RDEB-SCC), which represent the first cause of death in these patients. The genetic determinants of RDEB-SCC do not exhaustively explain its unique behavior as compared to low-risk, ultraviolet-induced SCCs in the general population. On the other hand, a growing body of evidence points to the key role of tumor microenvironment in initiation, progression and spreading of RDEB-SCC, as well as of other, less-investigated, EB-related SCCs (EB-SCCs). Here, we discuss the recent advances in understanding the complex series of molecular events (i.e., fibrotic, inflammatory, and immune processes) contributing to SCC development in EB patients, cross-compare tumor features in the different EB subtypes and report the most promising therapeutic approaches to counteract or delay EB-SCCs.

[Epithelial to Mesenchymal Transition Marker in 2D and 3D Colon Cancer Cell Cultures in the Presence of Laminin 332 and 411].

Received August 28, 2018; revised October 10, 2018; accepted November 6, 2018 The loss of apical-basal cell polarity is a necessary stage of the epithelial-mesenchymal transition (EMT). Polarized epithelial cells interact with the basement membrane (BM) and, in particular, with laminins, the major components of BM. Here, we examined the effect of the transition of colon cancer cells from 2D polarized state to non-polarized 3D state on the expression of EMT associated genes, as well as the role of laminins 332 and 411 (LM-332 and LM-411) in this process. The three studied cell lines, HT-29, HCT-116 and RKO, were found to have different sensitivity to cultivation conditions (2D to 3D changes) and to addition of laminins. One of the possible reasons for this maybe a difference in the initial 2D state of the cells. In particular, it was shown that the cell lines were at different EMT stages. HT-29 exhibited more epithelial expression profile, RKO was more mesenchymal, and HCT-116 was in an intermediate state. The most laminin-sensitive cell line was HCT-116. The magnitude and the specificity of cell response to LM-332 and LM-411 depended on the expression pattern of laminins' receptors. EMT gene expression profile was not substantially changed neither during the transition from 2D to 3D state, nor the presence of laminins' isoforms. However, we detected changes in expression of SNAI1 and ZEB1 genes encoding transcription factors that control the EMT process. Notably, in all three studied cell lines, the expression of SNAI1 was enhanced in response to laminin treatment.

Effects of Laminins 332 and 411 on the Epithelial-Mesenchymal Status of Colorectal Cancer Cells.

The effects of laminins 332 and 411 (LM-332 and LM-411) on the epithelial-mesenchymal transformation of colorectal cancer cells (lines HT-29, HCT-116, and RKO) with different metastatic potential were studied. Culturing of RKO cells on both laminins was associated with modification of the cell shape, which became more spindle-like or stellate, and with higher expression of EMT-associated transcription factors SNAI1 and ZEB1. In addition, culturing on LM-332 led to a decrease in the expression of laminin α5 chain (LAMA5), while culturing on LM-411 led to an increase in the expression of a cell-cell junction component (DSP). Culturing of HT-29 cells on LM-332 was associated with the formation of more close contacts between the cells and by a higher expression of epithelial markers (CDH1 and DSP genes) and a decrease in SNAI1 expression. Culturing of HCT-116 cells on both laminins led to a decrease in FN1 expression, on LM-332 - to an increase in laminin α4 chain (LAMA4) expression, and on LM-411 - to a lesser expression of LAMA4 and transcription factors SNAI2 and ZEB1. These data indicated that colorectal cancer cell adhesion to laminins contributed to the probability of epithelial-mesenchymal transformation of cells. The direction of this transformation seemed to depend on the initial characteristics of the cells.

LAMC2 is a predictive marker for the malignant progression of leukoplakia.

BACKGROUND: LAMC2 plays an important role in cancer invasion. The aim of this study was to (i) compare the immunoexpression of LAMC2 in different stages of oral squamous cell carcinoma (OSCC), early and advanced, and (ii) to evaluate LAMC2 as a marker of malignant transformation in leukoplakia. MATERIALS AND METHODS: The expression of LAMC2 was examined by immunohistochemistry in 50 surgical specimens of advanced OSCC assembled as tissue microarrays, and by cDNA microarray in 43 surgical specimens of advanced OSCC. LAMC2 expression was further examined in 39 surgical specimens of early OSCC and in 93 incisional biopsy specimens of leukoplakia of the tongue, which exhibited epithelial dysplasia. The relationship of LAMC2 expression score with clinico-pathological characteristics was analyzed. RESULTS: LAMC2 was remarkably upregulated in OSCC at the cancer-stroma interface. The grade of LAMC2 expression was significantly associated with the pattern and depth of invasion of OSCC. Foci of LAMC2-positive cells were observed in some cases of leukoplakia. The number and size of LAMC2-positive foci were significantly associated with the grade of dysplasia. The presence of LAMC2-positive foci was a significant predictive factor for the malignant progression of leukoplakia. LAMC2-positive leukoplakia had an approximately 11-fold increased risk of malignancy compared with LAMC2-negative leukoplakia. CONCLUSIONS: The results of this study highlight the value of LAMC2 as a marker of cancer invasion. LAMC2-positive foci in leukoplakia suggest an imminent risk of cancer. LAMC2 immunostaining is expected to contribute to a more precise assessment of the malignancy of leukoplakia.

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.

Ultrastructural and immunocytochemical characterization of ameloblast-enamel adhesion at maturation stage in amelogenesis in Macaca fuscata tooth germ.

Maturation-stage ameloblasts are firmly bound to the tooth enamel by a basal lamina-like structure. The mechanism underlying this adhesion, however, remains to be fully clarified. The goal of this study was to investigate the mechanism underlying adhesion between the basal lamina-like structure and the enamel in monkey tooth germ. High-resolution immunogold labeling was performed to localize amelotin and laminin 332 at the interface between ameloblasts and tooth enamel. Minute, electron-dense strands were observed on the enamel side of the lamina densa, extending into the degrading enamel matrix to produce a well-developed fibrous layer (lamina fibroreticularis). In un-demineralized tissue sections, mineral crystals smaller than those in the bulk of the enamel were observed adhering to these strands where they protruded into the surface enamel. Immunogold particles reactive for amelotin were preferentially localized on these strands in the fibrous layer. On the other hand, those for laminin 332 were localized solely in the lamina densa; none were observed in the fibrous layer. These results suggest that the fibrous layer of the basal lamina-like structure is partly composed of amelotin molecules, and that these molecules facilitate ameloblast-enamel adhesion by promoting mineralization of the fibrous layer during the maturation stage of amelogenesis.