RESUMEN
The sense of touch is conferred by the conjoint function of somatosensory neurons and skin cells. These cells meet across a gap filled by a basal lamina, an ancient structure found in metazoans. Using Caenorhabditis elegans, we investigate the composition and ultrastructure of the extracellular matrix at the epidermis and touch receptor neuron (TRN) interface. We show that membrane-matrix complexes containing laminin, nidogen, and the MEC-4 mechano-electrical transduction channel reside at this interface and are central to proper touch sensation. Interestingly, the dimensions and spacing of these complexes correspond with the discontinuous beam-like extracellular matrix structures observed in serial-section transmission electron micrographs. These complexes fail to coalesce in touch-insensitive extracellular matrix mutants and in dissociated neurons. Loss of nidogen reduces the density of mechanoreceptor complexes and the amplitude of the touch-evoked currents they carry. Thus, neuron-epithelium cell interfaces are instrumental in mechanosensory complex assembly and function. Unlike the basal lamina ensheathing the pharynx and body wall muscle, nidogen recruitment to the puncta along TRNs is not dependent upon laminin binding. MEC-4, but not laminin or nidogen, is destabilized by point mutations in the C-terminal Kunitz domain of the extracellular matrix component, MEC-1. These findings imply that somatosensory neurons secrete proteins that actively repurpose the basal lamina to generate special-purpose mechanosensory complexes responsible for vibrotactile sensing.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Mecanorreceptores , Mecanotransducción Celular , Animales , Caenorhabditis elegans/fisiología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Mecanorreceptores/metabolismo , Mecanorreceptores/fisiología , Mecanotransducción Celular/fisiología , Tacto/fisiología , Membrana Basal/metabolismo , Membrana Basal/fisiología , Matriz Extracelular/metabolismo , Laminina/metabolismo , Glicoproteínas de Membrana , Proteínas de la MembranaRESUMEN
Flow in a porous medium can be driven by the deformations of the boundaries of the porous domain. Such boundary deformations locally change the volume fraction accessible by the fluid, creating non-uniform porosity and permeability throughout the medium. In this work, we construct a deformation-driven porous medium transport model with spatially and temporally varying porosity and permeability that are dependent on the boundary deformations imposed on the medium. We use this model to study the transport of interstitial fluid along the basement membranes in the arterial walls of the brain. The basement membrane is modeled as a deforming annular porous channel with the compressible pore space filled with an incompressible, Newtonian fluid. The role of a forward propagating peristaltic heart pulse wave and a reverse smooth muscle contraction wave on the flow within the basement membranes is investigated. Our results identify combinations of wave amplitudes that can induce either forward or reverse transport along these transport pathways in the brain. The magnitude and direction of fluid transport predicted by our model can help in understanding the clearance of fluids and solutes along the Intramural Periarterial Drainage route and the pathology of cerebral amyloid angiopathy.
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Encéfalo , Líquido Extracelular , Líquido Extracelular/metabolismo , Líquido Extracelular/fisiología , Porosidad , Humanos , Encéfalo/metabolismo , Encéfalo/irrigación sanguínea , Encéfalo/fisiología , Membrana Basal/metabolismo , Membrana Basal/fisiología , Conceptos Matemáticos , Transporte Biológico/fisiología , Modelos Biológicos , Simulación por Computador , Modelos Neurológicos , Animales , PermeabilidadRESUMEN
The glomerular basement membrane (GBM) is an important component of the kidney filtration barrier. The ability to evaluate the molecular transport properties of the GBM and determining how changes in the structure, composition, and mechanical properties of the GBM regulate its size selective transport properties may provide additional insight into glomerular function. This chapter details a method for making in vitro models of the glomerular filtration barrier using animal-derived decellularized glomeruli. FITC-labelled Ficoll is used as a filtration probe to evaluate the molecular transport properties during passive diffusion and under applied pressure. These systems can serve as a platform to evaluate the molecular permeability of basement membrane systems using conditions that simulate normal or pathophysiological conditions.
Asunto(s)
Barrera de Filtración Glomerular , Glomérulos Renales , Animales , Membrana Basal/fisiología , Tasa de Filtración Glomerular , PermeabilidadRESUMEN
BACKGROUND: We newly developed a muscle graft that employs a doxorubicin pretreatment technique. The aims of this study were to reveal the biological and morphological features of the muscle tissue in the second week (Study I), to reveal the regeneration outcomes of functional and kinematic assessments of longer-term follow-up (16 weeks, Study II), and to make assessments of the muscle graft with doxorubicin pretreatment in the critical-sized nerve defect model (20 mm, Study III). METHODS: A total of 26 adult rats were used in this study. Doxorubicin treatment was accomplished by immersion in a doxorubicin solution for 10 minutes followed by a rinsing procedure. The rats were divided into three groups: the muscle graft with and without doxorubicin pretreatment (M-graft-w-Dox and M-graft-w/o-Dox) groups and the autologous nerve graft (N-graft) group. Assays of apoptosis, immunofluorescent histochemistry including CD68 (macrophage marker), scanning electron microscopy (SEM), morphometrical studies of the regenerated axons, nerve conduction studies, and kinematic studies were performed. RESULTS: The M-graft-w-Dox group contained significantly larger numbers of apoptotic cells and CD68-positive cells. SEM revealed the existence of the basal lamina, so called "empty tubes," in the M-graft-w-Dox group. Study II showed contentious maturation of the regenerated axons, especially in the compound muscle action potentials. Study III showed that even at 20 mm, the M-graft-w-Dox group promoted axonal regeneration and functional regeneration. CONCLUSION: The M-graft-w-Dox group showed superior regeneration results, and this easy and short-term procedure can expand the muscle graft clinical indication for the treatment of peripheral nerve defects.
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Regeneración Nerviosa , Nervio Ciático , Ratas , Animales , Regeneración Nerviosa/fisiología , Nervio Ciático/cirugía , Nervio Ciático/fisiología , Músculos , Axones/fisiología , Membrana Basal/fisiología , Membrana Basal/trasplante , Doxorrubicina/farmacologíaRESUMEN
The kidney tubule consists of a single layer of epithelial cells supported by the tubular basement membrane (TBM), a thin layer of specialized extracellular matrix (ECM). The mechanical properties of the ECM are important for regulating a wide range of cell functions including proliferation, differentiation and cell survival. Increased ECM stiffness plays a role in promoting multiple pathological conditions including cancer, fibrosis and heart disease. How changes in TBM mechanics regulate tubular epithelial cell behavior is not fully understood. Here we introduce a cell culture system that utilizes in vivo-derived TBM to investigate cell-matrix interactions in kidney proximal tubule cells. Basement membrane mechanics was controlled using genipin, a biocompatibility crosslinker. Genipin modification resulted in a dose-dependent increase in matrix stiffness. Crosslinking had a marginal but statistically significant impact on the diffusive molecular transport properties of the TBM, likely due to a reduction in pore size. Both native and genipin-modified TBM substrates supported tubular epithelial cell growth. Cells were able to attach and proliferate to form confluent monolayers. Tubular epithelial cells polarized and assembled organized cell-cell junctions. Genipin modification had minimal impact on cell viability and proliferation. Genipin stiffened TBM increased gene expression of pro-fibrotic cytokines and altered gene expression for N-cadherin, a proximal tubular epithelial specific cell-cell junction marker. This work introduces a new cell culture model for cell-basement membrane mechanobiology studies that utilizes in vivo-derived basement membrane. We also demonstrate that TBM stiffening affects tubular epithelial cell function through altered gene expression of cell-specific differentiation markers and induced increased expression of pro-fibrotic growth factors.
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Iridoides , Túbulos Renales , Túbulos Renales/metabolismo , Túbulos Renales/patología , Iridoides/farmacología , Iridoides/metabolismo , Membrana Basal/fisiología , Células Epiteliales , Túbulos Renales ProximalesRESUMEN
We engineered an ultrasensitive reporter of p16INK4a, a biomarker of cellular senescence. Our reporter detected p16INK4a-expressing fibroblasts with certain senescent characteristics that appeared shortly after birth in the basement membrane adjacent to epithelial stem cells in the lung. Furthermore, these p16INK4a+ fibroblasts had enhanced capacity to sense tissue inflammation and respond through their increased secretory capacity to promote epithelial regeneration. In addition, p16INK4a expression was required in fibroblasts to enhance epithelial regeneration. This study highlights a role for p16INK4a+ fibroblasts as tissue-resident sentinels in the stem cell niche that monitor barrier integrity and rapidly respond to inflammation to promote tissue regeneration.
Asunto(s)
Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Células Epiteliales , Fibroblastos , Genes Reporteros , Pulmón , Regeneración , Nicho de Células Madre , Humanos , Membrana Basal/citología , Membrana Basal/fisiología , Biomarcadores/metabolismo , Senescencia Celular/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Fibroblastos/metabolismo , Inflamación/metabolismo , Pulmón/patología , Pulmón/fisiología , Células Epiteliales/fisiología , Nicho de Células Madre/fisiologíaRESUMEN
During animal embryogenesis, homeostasis and disease, tissues push and pull on their surroundings to move forward. Although the force-generating machinery is known, it is unknown how tissues exert physical stresses on their substrate to generate motion in vivo. Here, we identify the force transmission machinery, the substrate and the stresses that a tissue, the zebrafish posterior lateral line primordium, generates during its migration. We find that the primordium couples actin flow through integrins to the basement membrane for forward movement. Talin- and integrin-mediated coupling is required for efficient migration, and its loss is partially compensated for by increased actin flow. Using Embryogram, an approach to measure stresses in vivo, we show that the rear of the primordium exerts higher stresses than the front, which suggests that this tissue pushes itself forward with its back. This unexpected strategy probably also underlies the motion of other tissues in animals.
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Membrana Basal/fisiología , Quimiotaxis , Embrión no Mamífero/fisiología , Mecanotransducción Celular , Actinas/metabolismo , Animales , Animales Modificados Genéticamente , Membrana Basal/metabolismo , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Integrinas/genética , Integrinas/metabolismo , Morfogénesis , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Estrés Mecánico , Talina/genética , Talina/metabolismo , Factores de Tiempo , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The epidermal basement membrane deteriorates with aging. We previously reported that basement membrane reconstruction not only serves to maintain epidermal stem/progenitor cells in the epidermis, but also increases collagen fibrils in the papillary dermis. Here, we investigated the mechanism of the latter action. Collagen fibrils in the papillary dermis were increased in organotypic human skin culture treated with matrix metalloproteinase and heparinase inhibitors. The expression levels of COL5A1 and COL1A1 genes (encoding collagen type V α 1 chain and collagen type I α 1 chain, respectively) were increased in fibroblasts cultured with conditioned medium from a skin equivalent model cultured with the inhibitors and in keratinocytes cultured on laminin-511 E8 fragment-coated plates. We then examined cytokine expression, and found that the inhibitors increased the expression of PDGF-BB (platelet-derived growth factor consisting of two B subunits) in epidermis. Expression of COL5A1 and COL1A1 genes was increased in cultured fibroblasts stimulated with PDGF-BB. Further, the bifunctional inhibitor hydroxyethyl imidazolidinone (HEI) increased skin elasticity and the thickness of the papillary dermis in the skin equivalent. Taken together, our data suggests that reconstructing the basement membrane promotes secretion of PDGF-BB by epidermal keratinocytes, leading to increased collagen expression at the papillary dermis.
Asunto(s)
Membrana Basal/fisiología , Epidermis/fisiología , Colágenos Asociados a Fibrillas/fisiología , Fibroblastos/metabolismo , Fibroblastos/fisiología , Regeneración/fisiología , Envejecimiento de la Piel/patología , Envejecimiento de la Piel/fisiología , Membrana Basal/metabolismo , Becaplermina/genética , Becaplermina/metabolismo , Células Cultivadas , Cadena alfa 1 del Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Colágeno Tipo V/genética , Colágeno Tipo V/metabolismo , Células Epidérmicas/metabolismo , Epidermis/metabolismo , Epidermis/patología , Colágenos Asociados a Fibrillas/genética , Colágenos Asociados a Fibrillas/metabolismo , Expresión Génica , Humanos , Queratinocitos/metabolismo , Metaloproteinasas de la Matriz/farmacología , Regeneración/genéticaRESUMEN
Basement membranes (BMs) are complex macromolecular networks underlying all continuous layers of cells. Essential components include collagen IV and laminins, which are affected by human genetic variants leading to a range of debilitating conditions including kidney, muscle, and cerebrovascular phenotypes. We investigated the dynamics of BM assembly in human pluripotent stem cell-derived kidney organoids. We resolved their global BM composition and discovered a conserved temporal sequence in BM assembly that paralleled mammalian fetal kidneys. We identified the emergence of key BM isoforms, which were altered by a pathogenic variant in COL4A5. Integrating organoid, fetal, and adult kidney proteomes, we found dynamic regulation of BM composition through development to adulthood, and with single-cell transcriptomic analysis we mapped the cellular origins of BM components. Overall, we define the complex and dynamic nature of kidney organoid BM assembly and provide a platform for understanding its wider relevance in human development and disease.
Asunto(s)
Membrana Basal/patología , Membrana Basal/fisiología , Enfermedades Renales/patología , Riñón/fisiología , Organoides/fisiología , Animales , Biopsia , Técnicas de Cultivo Tridimensional de Células/métodos , Línea Celular , Preescolar , Colágeno Tipo IV/genética , Proteínas de la Matriz Extracelular/genética , Femenino , Humanos , Riñón/patología , Enfermedades Renales/genética , Masculino , Ratones , Células Madre Pluripotentes/fisiología , Proteómica/métodosRESUMEN
PURPOSE: To investigate the interrelationship among the outer retinal layers after macular hole surgery and elucidate the restoration process. METHODS: This retrospective observational study included 50 eyes of 47 consecutive patients with closed macular holes in the first vitrectomy. Optical coherence tomography was obtained before surgery; at 1, 3, and 6 months postsurgery; and at the last visit. The complete continuous layer rate and mean defect length were evaluated for the outer nuclear layer (ONL), external limiting membrane (ELM), and ellipsoid zone (EZ). RESULTS: At all postoperative visits, the complete continuous layer rate was in the descending order of ELM, ONL, and EZ and the mean defect length was in the ascending order of ELM, ONL, and EZ. External limiting membrane was necessary for ONL restoration. External limiting membrane and ONL were necessary for EZ restoration. Hyperreflective protrusions were observed from the area lacking ELM into the subretinal space after surgery. Ellipsoid zone was not formed in coexistence with the hyperreflective protrusions. Intermediate reflective protrusions appeared under the ONL plus ELM after surgery and were eventually replaced by EZ. CONCLUSION: Restoration of the outer retinal layers after surgical macular hole closure occurs in the order of ELM, ONL, and EZ.
Asunto(s)
Membrana Basal/fisiología , Endotaponamiento , Neuronas Retinianas/fisiología , Perforaciones de la Retina/cirugía , Epitelio Pigmentado de la Retina/fisiología , Vitrectomía , Anciano , Membrana Basal/diagnóstico por imagen , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Perforaciones de la Retina/diagnóstico por imagen , Perforaciones de la Retina/fisiopatología , Epitelio Pigmentado de la Retina/diagnóstico por imagen , Estudios Retrospectivos , Hexafluoruro de Azufre/administración & dosificación , Tomografía de Coherencia Óptica , Agudeza Visual/fisiologíaRESUMEN
Collagen α3 (IV) chains are one of the major constituent components of the basement membrane in the mammalian testis. Studies have shown that biologically active fragments, such as noncollagenase domain (NC1)-peptide, can be released from the C-terminal region of collagen α3 (IV) chains, possibly through the proteolytic action of metalloproteinase 9 (MMP9). NC1-peptide was shown to promote blood-testis barrier (BTB) remodeling and fully developed spermatid (e.g., sperm) release from the seminiferous epithelium because this bioactive peptide was capable of perturbing the organization of both actin- and microtubule (MT)-based cytoskeletons at the Sertoli cell-cell and also Sertoli-spermatid interface, the ultrastructure known as the basal ectoplasmic specialization (ES) and apical ES, respectively. More importantly, recent studies have shown that this NC1-peptide-induced effects on cytoskeletal organization in the testis are mediated through an activation of mammalian target of rapamycin complex 1/ribosomal protein S6/transforming retrovirus Akt1/2 protein (mTORC1/rpS6/Akt1/2) signaling cascade, involving an activation of cell division control protein 42 homolog (Cdc42) GTPase, but not Ras homolog family member A GTPase (RhoA), and the participation of end-binding protein 1 (EB1), a microtubule plus (+) end tracking protein (+TIP), downstream. Herein, we critically evaluate these findings, providing a critical discussion by which the basement membrane modulates spermatogenesis through one of its locally generated regulatory peptides in the testis.
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Membrana Basal/metabolismo , Barrera Hematotesticular/metabolismo , Colágeno Tipo IV/metabolismo , Fragmentos de Péptidos/metabolismo , Epitelio Seminífero/metabolismo , Espermatogénesis/fisiología , Citoesqueleto de Actina , Animales , Membrana Basal/fisiología , Barrera Hematotesticular/fisiología , Comunicación Celular , Colágeno Tipo IV/fisiología , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos , Fragmentos de Péptidos/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína S6 Ribosómica/metabolismo , Epitelio Seminífero/fisiología , Células de Sertoli/metabolismo , Células de Sertoli/fisiología , Transducción de Señal , Espermátides/metabolismo , Espermátides/fisiología , Testículo , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Follicular thyroid tissue originates from progenitors derived from a midline endodermal primordium. Current understanding infers that folliculogenesis in the embryonic thyroid designates the latest morphogenetic event taking place after the final anatomical shape and position of the gland is established. However, this concept does not consider the fact that the thyroid isthmus develops chronologically before the lobes and also contains all progenitors required for lobulation. To elucidate whether cells committed to a thyroid fate might be triggered to differentiate asynchronously related to maturation and developmental stage, mouse embryonic thyroid tissues from E12.5-17.5 were subjected to immunofluorescent labeling of biomarkers (progenitors: NKX2-1; differentiation: thyroglobulin/TG); folliculogenesis: E-cadherin/CDH1; luminogenesis: mucin 1/MUC1; apical polarity: pericentrin/PCNT; basement membrane: laminin; growth: Ki67), quantitative RT-PCR analysis (Nkx2.1, Tg, Muc1) and transmission electron microscopy. Tg expression was detectable as early as E12.5 and gradually increased >1000-fold until E17.5. Muc1 and Nkx2.1 transcript levels increased in the same time interval. Prior to lobulation (E12.5-13.5), MUC1 and TG distinguished pre-follicular from progenitor cells in the developing isthmus characterized by intense cell proliferation. Luminogenesis comprised redistribution of MUC1+ vesicles or vacuoles, transiently associated with PCNT, to the apical cytoplasm and the subsequent formation of MUC1+ nascent lumens. Apical polarization of pre-follicular cells and lumen initiation involved submembraneous vesicular traffic, reorganization of adherens junctions and ciliogenesis. MUC1 did not co-localize with TG until a lumen with a MUC1+ apical membrane was established. MUC1 delineated the lumen of all newly formed follicles encountered in the developing lobes at E15.5-17.5. Folliculogenesis started before establishment of a complete follicular basal lamina. These observations indicate that embryonic thyroid differentiation is an asynchronous process consistent with the idea that progenitors attaining a stationary position in the connecting isthmus portion undergo apical polarization and generate follicles already at a primordial stage of thyroid development, i.e. foregoing growth of the lobes. Although the thyroid isthmus eventually comprises minute amounts of the total thyroid volume and contributes little to the overall hormone production, it is of principal interest that local cues related to the residence status of cells - independently of a prevailing high multiplication rate - govern the thyroid differentiation program.
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Membrana Basal/fisiología , Diferenciación Celular/fisiología , Desarrollo Embrionario/fisiología , Células Epiteliales Tiroideas/fisiología , Glándula Tiroides/fisiología , Animales , Membrana Basal/metabolismo , Biomarcadores/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Células Madre/metabolismo , Células Madre/fisiología , Células Epiteliales Tiroideas/metabolismo , Glándula Tiroides/metabolismoRESUMEN
PURPOSE: To report the anatomic and functional outcomes of autologous retinal transplantation (ART). DESIGN: Multicenter, retrospective, interventional, consecutive case series. PARTICIPANTS: One hundred thirty eyes of 130 patients undergoing ART for the repair of primary and refractory macular holes (MHs), as well as combined MH-rhegmatogenous retinal detachment (MH-RRD), between January 2017 and December 2019. METHODS: All patients underwent pars plana vitrectomy and ART, with surgeon modification of intraoperative variables. A large array of preoperative, intraoperative, and postoperative data was collected. Two masked reviewers graded OCT images. Multivariate statistical analysis and subgroup analysis were performed. MAIN OUTCOME MEASURES: Macular hole closure rate, visual acuity (VA), external limiting membrane and ellipsoid zone (EZ) band integrity, and alignment of neurosensory layers (ANL) on OCT. RESULTS: One hundred thirty ART surgeries were performed by 33 vitreoretinal surgeons worldwide. Patient demographics were: mean age of 63 ± 6.3 years, 58% female, 41% White, 23% Black, 19% Asian, and 17% Latino. Preoperative VA was 1.37 ± 0.12 logarithm of the minimum angle of resolution (logMAR; Snellen equivalent, approximately 20/500), which improved significantly to 1.05 ± 0.09 logMAR (Snellen equivalent, approximately 20/225; P < 0.001) after surgery (mean follow-up, 8.6 ± 0.8 months). Autologous retinal transplantation was performed for primary MH repair in 27% of patients (n = 35), for refractory MH in 58% of patients (n = 76; mean number of previous surgeries, 1.6 ± 0.2), and for MH-RRD in 15% of patients (n = 19). Mean maximum MH diameter was 1470 ± 160 µm, mean minimum diameter was 840 ± 94 µm, and mean axial length was 24.6 ± 3.2 mm. Overall, 89% of MHs closed (78.5% complete; 10% small eccentric defect), with a 95% closure rate in MH-RRD (68.4% complete; 26.3% small eccentric defect). Visual acuity improved by at least 3 lines in 43% of eyes and by at least 5 lines in 29% of eyes. Reconstitution of the EZ (P = 0.02) and ANL (P = 0.01) on OCT were associated with better final VA. Five cases of ART graft dislocation (3.8%), 5 cases of postoperative retinal detachment (3.8%), and 1 case of endophthalmitis (0.77%) occurred. CONCLUSIONS: In this global experience, patients undergoing ART for large primary and refractory MHs and MH-RRDs achieved good anatomic and functional outcomes, with low complication rates despite complex surgical pathologic features.
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Retina/trasplante , Desprendimiento de Retina/cirugía , Perforaciones de la Retina/cirugía , Anciano , Membrana Basal/fisiología , Femenino , Estudios de Seguimiento , Salud Global , Humanos , Masculino , Persona de Mediana Edad , Desprendimiento de Retina/diagnóstico , Desprendimiento de Retina/fisiopatología , Perforaciones de la Retina/diagnóstico , Perforaciones de la Retina/fisiopatología , Estudios Retrospectivos , Tomografía de Coherencia Óptica , Trasplante Autólogo , Resultado del Tratamiento , Agudeza Visual/fisiología , VitrectomíaRESUMEN
The purpose of this study was to investigate the expression and localization of transforming growth factor (TGF) ß1 and TGFß2 in rabbit corneas that healed with and without stromal fibrosis, and to further study defective perlecan incorporation in the epithelial basement membrane (EBM) in corneas with scarring fibrosis. A total of 120 female rabbits had no surgery, -4.5D PRK, or -9D PRK. Immunohistochemistry (IHC) was performed at time points from unwounded to eight weeks after surgery, with four corneas at each time point in each group. Multiplex IHC was performed for TGFß1 or TGFß2, with Image-J quantitation, and keratocan, vimentin, alpha-smooth muscle actin (SMA), perlecan, laminin-alpha 5, nidogen-1 or CD11b. Corneas at the four-week peak for myofibroblast and fibrosis development were evaluated using Imaris 3D analysis. Delayed regeneration of both an apical epithelial growth factor barrier and EBM barrier function, including defective EBM perlecan incorporation, was greater in high injury -9D PRK corneas compared to -4.5D PRK corneas without fibrosis. Defective apical epithelial growth factor barrier and EBM allowed epithelial and tear TGFß1 and tear TGFß2 to enter the corneal stroma to drive myofibroblast generation in the anterior stroma from vimentin-positive corneal fibroblasts, and likely fibrocytes. Vimentin-positive cells and unidentified vimentin-negative, CD11b-negative cells also produce TGFß1 and/or TGFß2 in the stroma in some corneas. TGFß1 and TGFß2 were at higher levels in the anterior stroma in the weeks preceding myofibroblast development in the -9D group. All -9D corneas (beginning two to three weeks after surgery), and four -4.5D PRK corneas developed significant SMA + myofibroblasts and stromal fibrosis. Both the apical epithelial growth factor barrier and/or EBM barrier functions tended to regenerate weeks earlier in -4.5D PRK corneas without fibrosis, compared to -4.5D or -9D PRK corneas with fibrosis. SMA-positive myofibroblasts were markedly reduced in most corneas by eight weeks after surgery. The apical epithelial growth factor barrier and EBM barrier limit TGFß1 and TGFß2 entry into the corneal stroma to modulate corneal fibroblast and myofibroblast development associated with scarring stromal fibrosis. Delayed regeneration of these barriers in corneas with more severe injuries promotes myofibroblast development, prolongs myofibroblast viability and triggers stromal scarring fibrosis.
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Membrana Basal/fisiología , Córnea/metabolismo , Sustancia Propia/patología , Epitelio Corneal/fisiología , Regeneración/fisiología , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta2/metabolismo , Animales , Opacidad de la Córnea/metabolismo , Opacidad de la Córnea/patología , Sustancia Propia/metabolismo , Femenino , Fibrosis/metabolismo , Fibrosis/patología , Proteínas de la Membrana/metabolismo , Microscopía Confocal , ConejosRESUMEN
The present study shows chronic adjustments in the myotendinous junction (MTJ) in response to different ladder-based resistance training (LRT) protocols. Thirty adult male Wistar rats were divided into groups: sedentary (S), calisthenics (LRT without additional load [C]), and resistance-trained (LRT with extra weight [R]). We demonstrated longer lengths of sarcoplasmatic invaginations in the trained groups; however, evaginations were seen mainly in group R. We showed a greater thickness of sarcoplasmatic invaginations in groups C and R, in addition to greater evaginations in R. We also observed thinner basal lamina in trained groups. The support collagen layer (SCL) adjacent to the MTJ and the diameters of the transverse fibrils were larger in R. We also discovered a niche of telocytes in the MTJ with electron micrographs of the plantar muscle and with immunostaining with CD34+ in the gastrocnemius muscle near the blood vessels and pericytes. We concluded that the continuous adjustments in the MTJ ultrastructure were the result of tissue plasticity induced by LRT, which is causally related to muscle hypertrophy and, consequently, to the remodeling of the contact interface. Also, we reveal the existence of a collagen layer adjacent to MTJ and discover a new micro anatomic location of telocytes.
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Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/fisiología , Condicionamiento Físico Animal/fisiología , Entrenamiento de Fuerza/métodos , Retículo Sarcoplasmático/fisiología , Telocitos/fisiología , Adaptación Fisiológica/fisiología , Uniones Adherentes/fisiología , Animales , Membrana Basal/fisiología , Adhesión Celular , Movimiento Celular/fisiología , Uniones Célula-Matriz/fisiología , Colágeno/metabolismo , Masculino , Ratas , Ratas Wistar , Conducta SedentariaRESUMEN
Three-dimensional (3D) cultures are better able to reflect the tumor microenvironment than two-dimensional (2D) monolayer cultures by facilitating cell-cell interactions in the appropriate spatial dimensions. Here I describe the isolation and co-culture of immune cells with tumor cell lines in a three-dimensional system facilitated by a basement membrane extract. This allows for further downstream applications to analyze interactions between these cell types.
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Comunicación Celular/fisiología , Técnicas de Cocultivo/métodos , Linfocitos/fisiología , Neoplasias/patología , Microambiente Tumoral/fisiología , Membrana Basal/fisiología , Línea Celular Tumoral , HumanosRESUMEN
Skeletal muscle regeneration implies the coordination of myogenesis with the recruitment of myeloid cells and extracellular matrix (ECM) remodelling. Currently, there are no specific biomarkers to diagnose the severity and prognosis of muscle lesions. In order to investigate the gene expression profile of extracellular matrix and adhesion molecules, as premises of homo- or heterocellular cooperation and milestones for skeletal muscle regeneration, we performed a gene expression analysis for genes involved in cellular cooperation, migration and ECM remodelling in a mouse model of acute crush injury. The results obtained at two early time-points post-injury were compared to a GSE5413 data set from two other trauma models. Third day post-injury, when inflammatory cells invaded, genes associated with cell-matrix interactions and migration were up-regulated. After day 5, as myoblast migration and differentiation started, genes for basement membrane constituents were found down-regulated, whereas genes for ECM molecules, macrophage, myoblast adhesion, and migration receptors were up-regulated. However, the profile and the induction time varied according to the experimental model, with only few genes being constantly up-regulated. Gene up-regulation was higher, delayed and more diverse following more severe trauma. Moreover, one of the most up-regulated genes was periostin, suggestive for severe muscle damage and unfavourable architecture restoration.
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Proteínas de la Matriz Extracelular/genética , Matriz Extracelular/genética , Músculo Esquelético/fisiología , Regeneración/genética , Transcriptoma/genética , Animales , Membrana Basal/fisiología , Diferenciación Celular/genética , Movimiento Celular/genética , Regulación hacia Abajo/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Mioblastos/fisiología , Regulación hacia Arriba/genéticaRESUMEN
PURPOSE OF REVIEW: The goal of this review is to present recent models of the filtration barrier that may suggest mechanism-based treatments for proteinuric renal disease. The vast majority of renal failure occurs in diseases of glomerular proteinuria. The physiology of the filtration barrier remains incompletely understood, preventing invention of mechanism-based therapies. Research is currently dominated by molecular biology approaches to the kidney instead of engineering-based filtration and transport models. RECENT FINDINGS: Reexamination of two older paradigms (basement membrane and slit diaphragm) and critical analysis of newer models may provide mechanistic insight to guide further research. We expand on our theory of podocyte-basement membrane mechanical interactions and speculate on mechanisms of action of the leading treatment for proteinuria, angiotensin blockade. SUMMARY: Treatment of proteinuria remains largely empiric and based on inhibition of the renin-angiotensin-aldosterone system, with additional benefit from statins and vitamin D. Improved definition of transport phenomena in the capillary wall may suggest rational design of new interventions.
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Barrera de Filtración Glomerular , Animales , Membrana Basal/fisiología , Barrera de Filtración Glomerular/fisiología , Tasa de Filtración Glomerular , Humanos , Podocitos/fisiología , Proteinuria/tratamiento farmacológico , Sistema Renina-Angiotensina/efectos de los fármacos , Sistema Renina-Angiotensina/fisiologíaRESUMEN
Islet transplantation in man is limited by multiple factors including islet availability, islet cell damage caused by collagenase during isolation, maintenance of islet function between isolation and transplantation, and allograft rejection. In this study, we describe a new approach for preparing islets that enhances islet function in vitro and reduces immunogenicity. The approach involves culture on native decellularized 3D bone marrow-derived extracellular matrix (3D-ECM), which contains many of the matrix components present in pancreas, prior to islet transplantation. Compared to islets cultured on tissue culture plastic (TCP), islets cultured on 3D-ECM exhibited greater attachment, higher survival rate, increased insulin content, and enhanced glucose-stimulated insulin secretion. In addition, culture of islets on 3D-ECM promoted recovery of vascular endothelial cells within the islets and restored basement membrane-related proteins (eg, fibronectin and collagen type VI). More interestingly, culture on 3D-ECM also selectively decontaminated islets of "passenger" cells (co-isolated with the islets) and restored basement membrane-associated type VI collagen, which were associated with an attenuation in islet immunogenicity. These results demonstrate that this novel approach has promise for overcoming two major issues in human islet transplantation: (a) poor yield of islets from donated pancreas tissue and (b) the need for life-long immunosuppression.