scholarly journals Surface relocation of alpha 6 beta 4 integrins and assembly of hemidesmosomes in an in vitro model of wound healing.

1991 ◽  
Vol 115 (6) ◽  
pp. 1737-1750 ◽  
Author(s):  
M A Kurpakus ◽  
V Quaranta ◽  
J C Jones
Keyword(s):  
Wound Healing ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Connective Tissue ◽  
Collagen Type ◽  
Polyclonal Antibodies ◽  
Vitro Model ◽  
Tissue Attachment ◽  
Collagen Type Vii

A transmembrane extracellular matrix receptor of the integrin family, alpha 6 beta 4, is a component of the hemidesmosome, an adhesion complex of importance in epithelial cell-connective tissue attachment (Stepp, M. A., S. Spurr-Michaud, A. Tisdale, J. Elwell, and I. K. Gipson. 1990. Proc. Natl. Acad. Sci. USA. 87:8970-8974; Jones, J. C. R., M. A. Kurpakus, H. M. Cooper, and V. Quaranta. 1991. Cell Regulation. 2:427-438). Cytosolic components of hemidesmosomes include bullous pemphigoid (BP) antigens while extracellular components include a 125-kD component of anchoring filaments (CAF) and collagen type VII-containing anchoring fibrils. We have monitored the incorporation of the alpha 6 beta 4 integrins into forming hemidesmosomes in an in vitro wound-healing explant model. In epithelial cells recently migrated from the edges of unwounded sites over bare connective tissue, alpha 6 beta 4 first appears along the entire cell surface. At this stage, these cells contain little or no cytosolic hemidesmosomal components, at least as detectable by immunofluorescence using BP autoantibodies, whereas they are already positive for laminin and CAF. At a later stage, as cells become positive for cytosolic hemidesmosome components such as BP antigens as well as collagen type VII, alpha 6 beta 4 becomes concentrated along the basal pole of the epithelial cell where it abuts the connective tissue of the explant. Polyclonal antibodies to beta 4 do not interfere with the migration of epithelial cells in the explant. However, they prevent assembly of hemidesmosomal complexes and inhibit expression of collagen type VII in cells that have migrated over wound areas. In addition, they induce disruption of established hemidesmosomes in nonmigrating cells of the unwounded area of the explant. Monoclonal antibodies to alpha 6 have a more dramatic effect, since they completely detach epithelial cells in the unwounded area of the explant. Antibodies to CAF also detach epithelial cells in unwounded areas, apparently by inducing separation between epithelium and connective tissue at the lamina lucida of the basement membrane zone. These results suggest a model whereby polarization of alpha 6 beta 4 to the basal surface of the cells, perhaps induced by a putative anchoring filament-associated ligand, triggers assembly of hemidesmosome plaques.

Analysis of wound healing in an in vitro model: early appearance of laminin and a 125 × 10(3) Mr polypeptide during adhesion complex formation

1990 ◽  
Vol 96 (4) ◽  
pp. 651-660
Author(s):  
M.A. Kurpakus ◽  
E.L. Stock ◽  
J.C. Jones
Keyword(s):  
Wound Healing ◽  
Basement Membrane ◽  
In Vitro Model ◽  
Collagen Type ◽  
Basement Membrane Zone ◽  
Lamina Densa ◽  
Vitro Model ◽  
Collagen Type Vii ◽  
Adhesion Complex

The adhesion complex, which plays an important role in cell-substratum attachment, consists of a cellular hemidesmosomal plaque, anchoring filaments, the basement membrane zone and anchoring fibrils. An analysis of the temporal sequence of assembly of the adhesion complex was undertaken in an in vitro model of epithelial cell wound healing by immunofluorescence and electron microscopy. A monoclonal antibody directed against a 125K (K = 10(3) Mr) polypeptide (mAbHD), bullous pemphigoid (BP) autoantibodies, antibodies directed against collagen type VII and laminin antibodies were used as markers for anchoring filaments, the hemidesmosome, anchoring fibrils and the laminin component of the basement membrane zone, respectively. Fluorescence labeling could be detected with mAbHD before labeling with BP autoantibodies or collagen type VII antibodies. Laminin fluorescence was detected at the same time as mAbHD. Furthermore, the 125K polypeptide and laminin were located extracellularly prior to the appearance of BP antigen and collagen type VII. The appearance of the hemidesmosomal plaque at the electron microscope level succeeded the localization of BP antigen in basal cells detected by immunofluorescence microscopy. No evidence for the coordinated appearance of BP antigen, collagen type VII and laminin was observed in this model. We discuss the possibility that the 125K protein and laminin may play roles in the initiation of complex formation. Furthermore, although basement membrane zone components were detected early in adhesion complex re-formation, formation of the lamina densa region of the basement membrane zone followed the appearance of the hemidesmosomal plaque, indicating a role for the hemidesmosomal plaque in organizing the structure of the lamina densa.

In vitro model of “wound healing” analyzed by laser scanning cytometry: Accelerated healing of epithelial cell monolayers in the presence of hyaluronate

Cytometry ◽  
2003 ◽  
Vol 53A (1) ◽  
pp. 1-8 ◽  
Author(s):  
Asifa S. Haider ◽  
Jerzy Grabarek ◽  
Ben Eng ◽  
Paulina Pedraza ◽  
Nicholas R. Ferreri ◽  
...  
Keyword(s):  
Wound Healing ◽  
Epithelial Cell ◽  
Laser Scanning ◽  
In Vitro Model ◽  
Cell Monolayers ◽  
Laser Scanning Cytometry ◽  
Epithelial Cell Monolayers ◽  
Vitro Model

M2 Macrophage-Derived Exosomal miR-590-3p Attenuates DSS-Induced Mucosal Damage and Promotes Epithelial Repair via the LATS1/YAP/ β-Catenin Signalling Axis

2020 ◽  
Author(s):  
Feihong Deng ◽  
Jin Yan ◽  
Jiaxi Lu ◽  
Min Luo ◽  
Pianpian Xia ◽  
...  
Keyword(s):  
Wound Healing ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Mucosal Damage ◽  
Luciferase Reporter ◽  
M2 Macrophage ◽  
Dependent Manner ◽  
M2 Macrophages ◽  
Epithelial Regeneration

Abstract Background and Aims M2 phenotype macrophages are involved in the resolution of inflammation and intestinal repair. Exosomes are emerging as important mediators of intercellular communication in the mucosal microenvironment. Methods M2 macrophages were transfected with or without miR-590-3p. Exosomes derived from M2 macrophages were isolated and identified. Proliferation and wound healing were tested in vitro and compared between groups. The mechanism involving LATS1, and activation of YAP and β-catenin signalling was investigated by using plasmid transfection, western blotting, immunofluorescence and luciferase reporter assays. The effect of exosomes in vivo was detected in dextran saline sulphate [DSS]-induced murine colitis. Results First, we demonstrated that M2 macrophages promoted colonic epithelial cell proliferation in an exosome-dependent manner. Epithelial YAP mediated the effect of M2 macrophage-derived exosomes [M2-exos] in epithelial proliferation. Moreover, miR-590-3p, which was significantly enriched in M2-exos, could be transferred from macrophages into epithelial cells, resulting in the enhanced proliferation and wound healing of epithelial cells. Mechanistically, miR-590-3p suppressed the expression of LATS1 by binding to its coding sequence and subsequently activated the YAP/β-catenin-modulated transcription process to improve epithelial cell wound-healing ability. miR-590-3p also inhibited the induction of pro-inflammatory cytokines, including tumour necrosis factor-α, interleukin-1β [IL-1β] and IL-6. More importantly, repression of miR-590-3p in M2-exos resulted in more severe mucosal damage and impaired colon repair of mice compared with those in M2-exo-treated mice after DSS-induced colitis. Conclusion M2 macrophage-derived exosomal miR-590-3p reduces inflammatory signals and promotes epithelial regeneration by targeting LATS1 and subsequently activating YAP/β-catenin-regulated transcription, which could offer a new opportunity for clinical therapy for ulcerative colitis.

scholarly journals G-protein-coupled receptor kinase-2 is a critical regulator of TNFα signaling in colon epithelial cells

2017 ◽  
Vol 474 (14) ◽  
pp. 2301-2313 ◽  
Author(s):  
Michael D. Steury ◽  
Peter C. Lucas ◽  
Laura R. McCabe ◽  
Narayanan Parameswaran
Keyword(s):  
Wound Healing ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
G Protein ◽  
Critical Role ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

G-protein-coupled receptor kinase-2 (GRK2) belongs to the GRK family of serine/threonine protein kinases critical in the regulation of G-protein-coupled receptors. Apart from this canonical role, GRK2 is also involved in several signaling pathways via distinct intracellular interactomes. In the present study, we examined the role of GRK2 in TNFα signaling in colon epithelial cell–biological processes including wound healing, proliferation, apoptosis, and gene expression. Knockdown of GRK2 in the SW480 human colonic cells significantly enhanced TNFα-induced epithelial cell wound healing without any effect on apoptosis/proliferation. Consistent with wound-healing effects, GRK2 knockdown augmented TNFα-induced matrix metalloproteinases (MMPs) 7 and 9, as well as urokinase plasminogen activator (uPA; factors involved in cell migration and wound healing). To assess the mechanism by which GRK2 affects these physiological processes, we examined the role of GRK2 in TNFα-induced MAPK and NF-κB pathways. Our results demonstrate that while GRK2 knockdown inhibited TNFα-induced IκBα phosphorylation, activation of ERK was significantly enhanced in GRK2 knockdown cells. Our results further demonstrate that GRK2 inhibits TNFα-induced ERK activation by inhibiting generation of reactive oxygen species (ROS). Together, these data suggest that GRK2 plays a critical role in TNFα-induced wound healing by modulating MMP7 and 9 and uPA levels via the ROS–ERK pathway. Consistent with in vitro findings, GRK2 heterozygous mice exhibited enhanced intestinal wound healing. Together, our results identify a novel role for GRK2 in TNFα signaling in intestinal epithelial cells.

An in vitro model for investigation of vitamin A effects on wound healing

2021 ◽  
pp. 1-6
Author(s):  
Hoda Keshmiri Neghab ◽  
Mohammad Hasan Soheilifar ◽  
Gholamreza Esmaeeli Djavid
Keyword(s):  
Wound Healing ◽  
Endothelial Cell ◽  
Vitamin A ◽  
In Vitro Model ◽  
Cellular Proliferation ◽  
Endothelial Cell Migration ◽  
Normal Fibroblast ◽  
Anti Inflammatory ◽  
Vitro Model

Abstract. Wound healing consists of a series of highly orderly overlapping processes characterized by hemostasis, inflammation, proliferation, and remodeling. Prolongation or interruption in each phase can lead to delayed wound healing or a non-healing chronic wound. Vitamin A is a crucial nutrient that is most beneficial for the health of the skin. The present study was undertaken to determine the effect of vitamin A on regeneration, angiogenesis, and inflammation characteristics in an in vitro model system during wound healing. For this purpose, mouse skin normal fibroblast (L929), human umbilical vein endothelial cell (HUVEC), and monocyte/macrophage-like cell line (RAW 264.7) were considered to evaluate proliferation, angiogenesis, and anti-inflammatory responses, respectively. Vitamin A (0.1–5 μM) increased cellular proliferation of L929 and HUVEC (p < 0.05). Similarly, it stimulated angiogenesis by promoting endothelial cell migration up to approximately 4 fold and interestingly tube formation up to 8.5 fold (p < 0.01). Furthermore, vitamin A treatment was shown to decrease the level of nitric oxide production in a dose-dependent effect (p < 0.05), exhibiting the anti-inflammatory property of vitamin A in accelerating wound healing. These results may reveal the therapeutic potential of vitamin A in diabetic wound healing by stimulating regeneration, angiogenesis, and anti-inflammation responses.

Transforming growth factor-alpha enhances alveolar epithelial cell repair in a new in vitro model

1994 ◽  
Vol 267 (6) ◽  
pp. L728-L738 ◽  
Author(s):  
F. Kheradmand ◽  
H. G. Folkesson ◽  
L. Shum ◽  
R. Derynk ◽  
R. Pytela ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epithelial Cell ◽  
Wound Repair ◽  
Wound Closure ◽  
Transforming Growth Factor ◽  
Alveolar Epithelial Cell ◽  
Alveolar Epithelial ◽  
Factor Alpha ◽  
Vitro Model

Alveolar epithelial type II cells are essential for regenerating an intact alveolar barrier after destruction of type I cells in vivo. The first objective of these experimental studies was to develop an in vitro model to quantify alveolar epithelial cell wound repair. The second objective was to investigate mechanisms of alveolar epithelial cell wound healing by studying the effects of serum and transforming growth factor-alpha (TGF-alpha) on wound closure. Primary cultures of rat alveolar type II cells were prepared by standard methods and grown to form confluent monolayers in 48 h. Then a wound was made by denuding an area (mean initial area of 2.1 +/- 0.6 mm2) of the monolayer. Re-epithelialization of the denuded area over time in the presence or absence of serum was measured using quantitative measurements from time-lapse video microscopy. The half time of wound healing was significantly enhanced in the presence of serum compared with serum-free conditions (2.4 +/- 0.2 vs. 17.4 +/- 0.8 h, P < 0.001). We then tested the hypothesis that TGF-alpha is an important growth factor for stimulating wound repair of alveolar epithelial cells. Exogenous addition of TGF-alpha in serum-free medium resulted in a significantly more rapid wound closure, and, furthermore, the addition of a monoclonal antibody to TGF-alpha in the presence of serum significantly decreased fourfold the rate of wound closure. Measurement of internuclear cell distance confirmed that both cell motility and cell spreading were responsible for closure of the wound. These data demonstrate that 1) the mechanisms of alveolar cell repair can be studied in vitro and that 2) TGF-alpha is a potent growth factor that enhances in vitro alveolar epithelial cell wound closure.

scholarly journals c-Src inactivation reduces renal epithelial cell-matrix adhesion, proliferation, and cyst formation

2011 ◽  
Vol 301 (2) ◽  
pp. C522-C529 ◽  
Author(s):  
Justine Elliott ◽  
Nadezhda N. Zheleznova ◽  
Patricia D. Wilson
Keyword(s):  
Cell Proliferation ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Collecting Duct ◽  
Specific Inhibitor ◽  
Cell Phenotype ◽  
Epithelial Cell Proliferation ◽  
Matrix Adhesion ◽  
Cyst Lining

c-Src is a non-receptor tyrosine kinase whose activity is induced by phosphorylation at Y418 and translocation from the cytoplasm to the cell membrane. Increased activity of c-Src has been associated with cell proliferation, matrix adhesion, motility, and apoptosis in tumors. Immunohistochemistry suggested that activated (pY418)-Src activity is increased in cyst-lining autosomal dominant polycystic kidney disease (ADPKD) epithelial cells in human and mouse ADPKD. Western blot analysis showed that SKI-606 (Wyeth) is a specific inhibitor of pY418-Src without demonstrable effects on epidermal growth factor receptor or ErbB2 activity in renal epithelia. In vitro studies on mouse inner medullary collecting duct (mIMCD) cells and human ADPKD cyst-lining epithelial cells showed that SKI-606 inhibited epithelial cell proliferation over a 24-h time frame. In addition, SKI-606 treatment caused a striking statistically significant decrease in adhesion of mIMCD and human ADPKD to extracellular collagen matrix. Retained viability of unattached cells was consistent with a primary effect on epithelial cell anchorage dependence mediated by the loss of extracellular matrix (ECM)-attachment due to α2β1-integrin function. SKI-606-mediated attenuation of the human ADPKD hyperproliferative and hyper-ECM-adhesive epithelial cell phenotype in vitro was paralleled by retardation of the renal cystic phenotype of Pkd1 orthologous ADPKD heterozygous mice in vivo. This suggests that SKI-606 has dual effects on cystic epithelial cell proliferation and ECM adhesion and may have therapeutic potential for ADPKD patients.

scholarly journals A Dual Role for Oncostatin M Signaling in the Differentiation and Death of Mammary Epithelial Cells in Vivo

2008 ◽  
Vol 22 (12) ◽  
pp. 2677-2688 ◽  
Author(s):  
Paul G. Tiffen ◽  
Nader Omidvar ◽  
Nuria Marquez-Almuina ◽  
Dawn Croston ◽  
Christine J. Watson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Oncostatin M ◽  
Specific Gene ◽  
Mammary Involution

Abstract Recent studies in breast cancer cell lines have shown that oncostatin M (OSM) not only inhibits proliferation but also promotes cell detachment and enhances cell motility. In this study, we have looked at the role of OSM signaling in nontransformed mouse mammary epithelial cells in vitro using the KIM-2 mammary epithelial cell line and in vivo using OSM receptor (OSMR)-deficient mice. OSM and its receptor were up-regulated approximately 2 d after the onset of postlactational mammary regression, in response to leukemia inhibitory factor (LIF)-induced signal transducer and activator of transcription-3 (STAT3). This resulted in sustained STAT3 activity, increased epithelial apoptosis, and enhanced clearance of epithelial structures during the remodeling phase of mammary involution. Concurrently, OSM signaling precipitated the dephosphorylation of STAT5 and repressed expression of the milk protein genes β-casein and whey acidic protein (WAP). Similarly, during pregnancy, OSM signaling suppressed β-casein and WAP gene expression. In vitro, OSM but not LIF persistently down-regulated phosphorylated (p)-STAT5, even in the continued presence of prolactin. OSM also promoted the expression of metalloproteinases MMP3, MMP12, and MMP14, which, in vitro, were responsible for OSM-specific apoptosis. Thus, the sequential activation of IL-6-related cytokines during mammary involution culminates in an OSM-dependent repression of epithelial-specific gene expression and the potentiation of epithelial cell extinction mediated, at least in part, by the reciprocal regulation of p-STAT5 and p-STAT3.

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