Laminin γ2-enriched extracellular vesicles of oral squamous cell carcinoma cells enhance in vitro lymphangiogenesis via integrin α3-dependent uptake by lymphatic endothelial cells

Oral squamous cell carcinoma (OSCC) LN1-1 cells previously showed greater capacities for lymphangiogenesis and lymph node metastasis compared to their parental OEC-M1 cells, in addition to an ability to enhance the migration and tube formation of lymphatic endothelial cells (LECs). Purified by a series of differential centrifugations and characterized using electron microscopy, dynamic light scattering and western blot, LN1-1 cell-derived extracellular vesicles (LN1-1 EVs) were shown to promote LEC migration, tube formation and uptake by LECs more effectively than did OEC-M1 cell-derived EVs (OEC-M1 EVs). Using stable isotope labeling with amino acids in cell culture/liquid chromatography–tandem mass spectrometry-based proteomic platform, the laminin-332 proteins, including laminin α3, β3 and γ2, were validated as highly expressed proteins in LN1-1 EVs. Clinically, a higher level of laminin-332 was detected in plasma EVs from OSCC patients with lymph node metastasis than in both healthy controls and OSCC patients without lymphatic metastasis, suggesting EV-borne laminin-332 as a novel and noninvasive biomarker for the detection of lymph node metastasis in OSCC. The knockdown of laminin γ2 and inhibition by anti-laminin-332 neutralizing antibodies impaired LN1-1 EV-mediated LEC migration, tube formation and uptake by LECs. Importantly, laminin γ2-deficient EVs showed a reduced ability to drain into lymph nodes in comparison with the control EVs. In addition, the laminin 332/γ2-mediated EV uptake was dependent on integrin α3 but not β1, β4 or α6. Collectively, the uptake of laminin γ2-enriched EVs by LECs enhanced in vitro lymphangiogenesis and EV-borne laminin-332 is thus a viable biomarker for OSCC.     

Laminin and collagen IV distribution and ultrastructure of the basement membrane of the gingiva of the rat incisor

The continuous growth of the rat incisor is associated with renovation of the junctional epithelium and resorption of the periodontal ligament. The circumdental papilla separates the connective tissue suffering resorption from the rest of the gingiva. Laminin and collagen IV were detected by the immunoperoxidase technique on the basement membrane of all regions of the gingival epithelium of the rat incisor, except the internal basal lamina and the internal surface of the circumdental papilla. The internal basal lamina is formed by a granular electron-dense material, without the organization of a typical basal lamina. Areas of the internal surface of the circumdental papilla, negative for laminin and collagen IV, lack the basal lamina. These data suggest that these molecules are not components of the dento-epithelial junction of the distal surface of the rat incisor. In addition, the basal lamina is absent or fragmented on the internal surface of the circumdental papilla, adjacent to the areas of the connective tissue undergoing resorption.     

Differential localization of laminin gamma and integrin beta in primary cultures of the rat gingival epithelium

OBJECTIVES: The aim of this study was to investigate the differential immunolocalization of laminin gamma(2) and integrin beta(4) in primary cultures of the rat gingival epithelium. METHODS: The gingival epithelium was obtained from Sprague-Dawley rats and was cultured in serum-free keratinocyte growth medium (DK-SFM). Western blotting analysis, immunofluorescence, confocal laser scanning microscopy (CLSM), and immuno-gold labeling for laminin gamma(2) and integrin beta(4) were employed. CLSM images for laminin and integrin were analyzed in horizontal (x-y axis) and in vertical (x-z axis) sections. RESULTS: Both laminin gamma(2) and integrin beta(4) were detected by Western blot analysis in the gingival epithelium. Immunolocalization of laminin gamma(2) was distinct in the cytoplasm to form one or two irregular rings in gingival epithelial cells. By contrast, integrin beta(4) was localized diffusely in the cytoplasm. F-actin (indicating actin filaments) was clearly discernible at the periphery of the cytoplasm to form a cellular fringe. In x-z axis images obtained by CLSM, laminin gamma(2) was recognized as large foci in the most inner portion just above the basal plasma membrane. Integrin beta(4) existed in the area where F-actin was labeled surrounding the membrane. Immuno-electron microscopy showed that 10nm colloidal gold particles indicating laminin gamma(2) were mainly localized at the extracellular portion and in the peripheral cytoplasm, whereas integrin beta(4) was distributed in the cytoplasm close to the basal plasma membrane but not in extracellular regions. CONCLUSIONS: In primary cultures of the rat gingival epithelium, both laminin gamma(2) and integrin beta(4) may be produced by the epithelium, and irregular rings of laminin gamma(2) are formed in areas where gingival cells adhere to the extracellular matrix.     

ROLE of exercise in maintaining the integrity of the neuromuscular junction

Physical activity plays an important role in preventing chronic disease in adults and the elderly. Exercise has beneficial effects on the nervous system, including at the neuromuscular junction (NMJ). Exercise causes hypertrophy of NMJs and improves recovery from peripheral nerve injuries, whereas decreased physical activity causes degenerative changes in NMJs. Recent studies have begun to elucidate molecular mechanisms underlying the beneficial effects of exercise. These mechanisms involve Bassoon, neuregulin‐1, peroxisome proliferator–activated receptor gamma coactivator 1α, insulin‐like growth factor‐1, glial cell line–derived neurotrophic factor, neurotrophin 4, Homer, and nuclear factor of activated T cells c1. For example, NMJ denervation and active zone decreases have been observed in aged NMJs, but these age‐dependent degenerative changes can be ameliorated by exercise. In this review we assess the effects of exercise on the maintenance and regeneration of NMJs and highlight recent insights into the molecular mechanisms underlying these exercise effects. Muscle Nerve 49 :315–324, 2014     

Responsiveness and Minimal Clinically Important Difference of the Motor Function Measure in Collagen VI-Related Dystrophies and Laminin Alpha2-Related Muscular Dystrophy

ObjectivesTo investigate the responsiveness of the motor function measure (MFM) and determine the minimal clinically important difference (MCID) in individuals with 2 common types of congenital muscular dystrophy (CMD).DesignObservational, prospective, single center, cohort study.SettingNational Institute of Neurological Disorders and Stroke (NINDS) of the National Institutes of Health (NIH).ParticipantsIndividuals (N=44) with collagen VI-related dystrophies (COL6-RD, n=23) and 21 individuals laminin alpha2-related muscular dystrophy (LAMA2-RD, n=21) enrolled in a 4-year longitudinal natural history study.InterventionsNot applicable.Main Outcome MeasuresResponsiveness of the MFM-32 and the Rasch-scaled MFM-25 and the MCID of the MFM-32 determined from a patient-reported anchor with 2 different methods, within-patient and between-patient.ResultsThe original MFM-32 and Rasch-scaled MFM-25 performed similarly overall in both the COL6-RD and LAMA2-RD populations, with all subscores (D1, standing and transfers; D2, axial and proximal; D3, distal) showing a significant decrease over time, except MFM D1 and D3 for LAMA2-RD. The MFM D1 subscore was the most sensitive to change for ambulant individuals, whereas the MFM D2 subscore was the most sensitive to change for nonambulant individuals. The MCID for the MFM-32 total score was calculated as 2.5 and 3.9 percentage points according to 2 different methods.ConclusionsThe MFM showed strong responsiveness in individuals with LAMA2-RD and COL6-RD. Because a floor effect was identified more prominently with the Rasch-Scaled MFM-25, the use of the original MFM-32 as a quantitative variable with the assumption of scale linearity appears to be a good compromise. When designing clinical trials in congenital muscular dystrophies, the use of MCID for MFM should be considered to determine if a given intervention effects show not only a statistically significant change but also a clinically meaningful change.     

Ultrastructural Characterization of Mesenchymal and Epithelial Cells Co-Cultured from Human Dental Root Apical Explants

Previous studies have shown the role of cell-cell and cell-matrix interactions in the differentiation of the specific secretory cells of the tooth. In order to elucidate the mechanisms implicated in root dentin formation, we developed a co-culture system of human pulpal mesenchymal and epithelial root sheath cells. Root tips of premolars were cultured in Eagle's basal Medium supplemented with fetal calf serum, ascorbic acid, antibiotics and, for some of them, with sodium β-glycerophosphate. After 60 days of culture, cells were prepared for light and electron microscopy. Three main cell types were observed: (1) polygonal mesenchymal cells showing a functional polarity and producing a dense network of tactoid collagenous fibers. The latter had a specific circular organization that delimited small lacunae around the cells and mineralized in the presence of β-glycerophosphate; (2) spindle-shaped mesenchymal cells mainly localized inside epithelial-mesenchymal knots and synthesizing an abundant collagenous matrix; and (3) epithelial cells lying on the plastic culture dish, on the dense collagenous matrix, or on spindle-shaped cells. Epithelial cells deposited a structured basement membrane when they were lying on the plastic culture dish or on spindle-shaped cells. On the contrary, no basement membrane was found when epithelial cells were overlying the dense collagenous network. Immunoelectron microscopic analysis of type IV collagen and laminin indicated that these two specific basement membrane components were produced by all cell types. These results show that the co-culture system should be valuable for (1) studying the in vitro formation of human dental root hard tissues, (2) characterizing cell-cell and cell-matrix interactions implicated in dental basement membrane production, and (3) isolating populations of cells implicated in dental root formation.     

Protease Digestion Shortens Procedure for the Demonstration of Laminin

Abstract

Protease type XIV was found to be a suitable substitute for pepsin digestion in the immunohistochemical demonstration of laminin. The method enables pathologists to distinguish vascular spaces from artifactual tissue spaces, which has prognostic value in the evaluation of tumors. The authors speculate that protease type XIV has multiple action sites that uncover antigenic binding sites more quickly than pepsin does. (The J Histotechnol 10: 185, 1987).     

Review: Lutheran/B-CAM: A Laminin Receptor on Red Blood Cells and in Various Tissues

The Lutheran blood group glycoprotein (Lu), also known as basal cell adhesion molecule (B-CAM), is a transmembrane receptor with five immunoglobulin-like domains in its extracellular region; it is therefore classified as a member of the immunoglobulin (Ig) gene family. Lu/B-CAM is observed not only on red blood cells, but also on a subset of muscle and epithelial cells in various tissues. Recently, several groups have reported that Lu/B-CAM is a novel receptor for laminin α5. The laminin α5 chain is a component of the laminin-511 (α5β1γ1), -521 (α5β2γ1), and -523 (α5β2γ3) heterotrimers and is expressed throughout the mammalian body. We also have shown that Lu/B-CAM is co-localized with laminin α5 in various tissues. Although the biological role of Lu/B-CAM remains unclear, the specific binding of Lu/B-CAM to laminin α5 suggests that it plays an important role in developmental and physiological processes. It also is necessary to investigate further the interaction between Lu/B-CAM and laminin α5 in pathological processes, including sickle cell disease and cancer.     

Normal mammary epithelial cells promote carcinoma basement membrane invasion by inducing microtubule-rich protrusions

Recent work suggests that the dissemination of tumor cells may occur in parallel with, and even preceed, tumor growth. The mechanism for this early invasion is largely unknown. Here, we find that mammary epithelial cells (MECs) induce neighboring breast carcinoma cells (BCCs) to cross the basement membrane by secreting soluble laminin. Laminin continuously produced by MECs induce long membrane cellular protrusions in BCCs that promote their contractility and invasion into the surrounding matrix. These protrusions depend on microtubule bundles assembled de novo through laminin-integrin β1 signaling. These results describe how non-cancerous MECs can actively participate in the invasive process of BCCs.