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 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.

In vitro potential of human mesenchymal stem cells for corneal epithelial regeneration

2020 ◽  
Vol 15 (3) ◽  
pp. 1409-1426 ◽  
Author(s):  
Núria Nieto-Nicolau ◽  
Beatriz Martín-Antonio ◽  
Claudia Müller-Sánchez ◽  
Ricardo P Casaroli-Marano
Keyword(s):  
Stem Cells ◽  
Wound Healing ◽  
Mesenchymal Stem Cells ◽  
Epithelial Cells ◽  
Electric Resistance ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial ◽  
Epithelial Regeneration ◽  
Human Corneal Epithelial Cells

Aim: To determine the potential of mesenchymal stem cells (MSC) for corneal epithelial regeneration in vitro. Materials & methods: Bone marrow MSC (BM-MSC) and adipose tissue MSC were analyzed for corneal epithelial and mesenchymal markers, using limbal stem cells and corneal cells as controls. MSC with better potential were cultured with specific mediums for epithelial induction. Transepithelial electric resistance and wound healing assay with human corneal epithelial cells were performed. Results: BM-MSC showed better potential, increased corneal markers, and higher transepithelial electric resistance values when induced with limbal epithelial culture medium. Induced BM-MSC promoted better wound healing of human corneal epithelial cells by paracrine secretion. Conclusion: BM-MSC has potential for corneal epithelial induction in a protocol compatible with human application.

scholarly journals IL-1β mediates miR-34a promotes bovine endometritis via LGR4-NF-κB axis

2021 ◽  
Author(s):  
Xiaofei Ma ◽  
Baoyi Yin ◽  
Shuai Guo ◽  
Talha Umar ◽  
Junfeng Liu ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Bacterial Infections ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Endometrial Epithelial Cell ◽  
Tnf Α ◽  
Uterine Inflammation

Abstract Background Persistent endometritis lead by bacterial infections has lethal effects on the reproductive performance of dairy cattle, which not only compromise animal welfare but also delay or prevent pregnancy. MicroRNA (miRNA) miR-34 family plays a pivotal role in the inflammatory process; however, the precise mechanism of miR-34a in endometritis is still not thoroughly revealed. Methods In this study, we established bovine endometrial epithelial cell (BENDs) inflammation model and mouse model stimulation with Lipopolysaccharide (LPS) in vitro and in vivo. CCK-8 was used to assess cell viability. H&E was used to characterize morphology. Immunohistochemistry, immunofluorescence, qRT-PCR and western blot assays were performed to measure the mRNA or protein expression of related genes. Online database, plasmid construction and dual-luciferase Reporter gene assays were applied to predict and validate the interaction between miR-34a and its target gene LGR4, and mice were injected vaginally with local antagomir to validate the role of miR-34a in murine uterine inflammation. Results Here, we report that miR-34a suppresses LGR4 gene expression by targeting its 3'untranslated regions (3'UTR). The miR-34a was up-regulated in cow uterine tissues and bovine endometrial epithelial cell (BENDs) stimulation with LPS. It further induces the release of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α by activating the phosphorylation level of NF-κB p65. Furthermore, we also revealed that IL-1β was responsible for the upregulation of miR-34a transcription and downregulation of LGR4 in an IL-1β-dependent manner. Conclusions Taken together, our study confirmed that miR-34a is regulated by IL-1β and suppress the level of LGR4 3’UTR which in turn exacerbates the inflammatory response. Thus knockdown miR-34a might be a new indirection for treatment endometritis.

scholarly journals An In Vitro Model to Assess Early Immune Markers following Co-Exposure of Epithelial Cells to Carbon Black (nano)Particles in the Presence of S. Aureus: Differential Induction of Cytokines.

2021 ◽  
Author(s):  
Scott M Brown ◽  
Stephen J Evans ◽  
Michael J Burgum ◽  
Llinos G Harris ◽  
Rowena E Jenkins ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Carbon Black ◽  
Human Skin ◽  
Lung Epithelial Cells ◽  
Nano Particles ◽  
Ageing Population ◽  
Dependent Manner ◽  
Lung Epithelial

Abstract Human exposure to carbon black (CB) is inevitable due to its widespread applications in the medical, industrial and consumer sectors. With an ageing population, it is imperative that the effects of (nano)particle exposure in individuals with compromised immunity or infection are considered. Since barrier immunity provides the first line of defence against CB and the human skin and lung pathogen, Staphylococcus aureus, this work focuses on studying the impact of CB exposure upon compromised immunity during infection on human skin and lung epithelial cells in vitro. The principal aim of the work was to develop an epithelial cell model to characterise (co-)exposure to CB and S. aureus. The work used two human epithelial cell lines, HaCaT (skin) and A549 (lung), ELISA technology to assess the (pro-)inflammatory response, aseptic microbiology techniques to grow S. aureus and a Zetasizer, EDX spectroscopy, and both scanning and transmission electron microscopy (SEM and TEM) to characterise the CB under the conditions used in the study. Physicochemical characterisation of CB confirmed its shape, dramatic polydispersity and potential to aggregate. CB significantly inhibited S. aureus growth, but in a biological media dependent manner. CB did not induce cytokines or antimicrobial peptides from skin and lung epithelial cells, when given alone. In contrast, S. aureus induced a robust interleukin (IL)-8 response in both skin and lung epithelial cells. IL-6 and human beta defensin (hβD)-2 could only be detected when cells were stimulated with S. aureus. However, co-exposure to CB (100µg/ml) and S. aureus resulted in significant inhibition of IL-8 (compared to S. aureus only induced levels). Furthermore, the same co-exposure induced significantly more hβD-2 (compared to S. aureus alone). The ability to detect pathogen responses to particle, in addition to epithelial responses to particle and pathogen is an advance on assessing cell responses under ‘healthy’ conditions and supports the need for developing exposure models under stressed or immunosuppressed conditions. This model will be useful for studying mechanisms of exposure in at-risk groups, including factory workers, the elderly and immunocompromised. Advanced models, that better represent human pathophysiology are essential for understanding cellular mechanisms of toxicity in the 21st Century.

CXCL9 Contributes To Chemotherapy-Induced Acute Intestinal Damage Through Proliferative Inhibition Of Epithelial Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5563-5563
Author(s):  
Huili Lu ◽  
Hongyu Liu ◽  
Jiaqing Shen ◽  
Shunyan Weng ◽  
Lan Qian ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Conflicts Of Interest ◽  
Intestinal Damage ◽  
Dependent Manner ◽  
Epithelial Regeneration ◽  
Proliferative Effect ◽  
Ribosomal S6 Kinase ◽  
Mcf10a Cells

Abstract Apart from angiostasis and chemoattraction, CXCL9 can derange hematopoiesis by its influence on mesenchymal stroma cells. The receptor of CXCL9, CXCR3, is abundantly expressed intracellularly in epithelial cells, even though it is rarely present on the surface of these cells. Here, we hypothesized that CXCL9 influences the proliferative and degenerative activity of epithelial cells in vitro and in vivo. CXCL9 inhibited the proliferation of MCF10A cells in a dose-dependent manner. In vivo, rhCXCL9 caused an intestinal weight loss of 30% in normal mice (n=6, 0.59±0.05 g of rhCXCL9 treated mice versus 0.83±0.06 g controls, P = 0.0007 determined by 2-tailed student’s t-test). Intestinal epithelial cells of 5-Fluorouracil (5-FU) treated mice developed a 2.55 fold higher level of cxcl9 expression, indicating that CXCL9 may participate in a chemotherapy-induced damage of the intestinal epithelium. Neutralization of the up-regulated endogenous CXCL9 by anti-CXCL9 monoclonal antibodies accelerated epithelial regeneration determined by villi length (317.5±19.9 μm versus no-antibody control 283.7±17.1 μm, P < 0.001) and crypt depth (78.0±8 μm versus control 67.9±10.9 μm, P = 0.326). CXCL9 function was highly associated with p70 ribosomal S6 kinase (p70S6K) activation (50.0±2.2 MFI versus control 27.9±1.4 MFI, P = 0.007), which was reversed by anti-CXCR3 (31.4±5.7 MFI, n=4). CXCL9 downstreamingly stimulated TGF-β secretion of epithelial cells through the mTOR/p70S6K pathway (66.3±17.1 pg/mL versus on-treated control 39.8±12.2 pg/mL, P <0.05), which was reversed by anti-CXCR3 (46.8±21.6 pg/mL, n=8). That explains the anti-proliferative effect of CXCL9 on these cells. Our results strongly suggest that anti-CXCL9 may help to mitigate a chemotherapy-induced intestinal damage. The work was supported by the National Science Foundation China (81273576, 30801419, 30901873), and the German Academic Exchange Service (A/09/90104). Disclosures: No relevant conflicts of interest to declare.

Protein kinase D2 mediates lysophosphatidic acid-induced interleukin 8 production in nontransformed human colonic epithelial cells through NF-κB

2007 ◽  
Vol 292 (2) ◽  
pp. C767-C777 ◽  
Author(s):  
Terence T. Chiu ◽  
Wai Yin Leung ◽  
Mary Pat Moyer ◽  
Robert M. Strieter ◽  
Enrique Rozengurt
Keyword(s):  
Epithelial Cells ◽  
Lysophosphatidic Acid ◽  
Interleukin 8 ◽  
Kinase Assay ◽  
Luciferase Reporter ◽  
Early Event ◽  
Potential Contribution ◽  
Dependent Manner ◽  
Small Interfering Rnas

The signaling pathways mediating lysophosphatidic acid (LPA)-stimulated PKD2 activation and the potential contribution of PKD2 in regulating LPA-induced interleukin 8 (IL-8) secretion in nontransformed, human colonic epithelial NCM460 cells were examined. Treatment of serum-deprived NCM460 cells with LPA led to a rapid and striking activation of PKD2, as measured by in vitro kinase assay and phosphorylation at the activation loop (Ser706/710) and autophosphorylation site (Ser876). PKD2 activation induced by LPA was abrogated by preincubation with selective PKC inhibitors GF-I and Ro-31-8220 in a dose-dependent manner. These inhibitors did not have any direct inhibitory effect on PKD2 activity. LPA induced a striking increase in IL-8 production and stimulated NF-κB activation, as measured by NF-κB-DNA binding, NF-κB-driven luciferase reporter activity, and IκBα phosphorylation. PKD2 gene silencing utilizing small interfering RNAs targeting distinct PKD2 sequences dramatically reduced LPA-stimulated NF-κB promoter activity and IL-8 production. PKD2 activation is a novel early event in the biological action of LPA and mediates LPA-stimulated IL-8 secretion in NCM460 cells through a NF-κB-dependent pathway. Our results demonstrate, for the first time, the involvement of a member of the PKD family in the production of IL-8, a potent proinflammatory chemokine, by epithelial cells.

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.

scholarly journals Green Synthesis of CeO2 Nanoparticles from the Abelmoschus esculentus Extract: Evaluation of Antioxidant, Anticancer, Antibacterial, and Wound-Healing Activities

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4659
Author(s):  
Hafiz Ejaz Ahmed ◽  
Yasir Iqbal ◽  
Muhammad Hammad Aziz ◽  
Muhammad Atif ◽  
Zahida Batool ◽  
...  
Keyword(s):  
Wound Healing ◽  
Metal Oxide Nanoparticles ◽  
In Vitro Cytotoxicity ◽  
Ceo2 Nanoparticles ◽  
Abelmoschus Esculentus ◽  
Oxide Nanoparticles ◽  
Dependent Manner ◽  
Hydrogel Membrane ◽  
Cancerous Cells

Metal oxide nanoparticles synthesized by the biological method represent the most recent research in nanotechnology. This study reports the rapid and ecofriendly approach for the synthesis of CeO2 nanoparticles mediated using the Abelmoschus esculentus extract. The medicinal plant extract acts as both a reducing and stabilizing agent. The characterization of CeO2 NPs was performed by scanning electron microscopy (SEM), X-ray diffraction (XRD), ultraviolet-visible spectroscopy (UV-Vis), and Fourier transform infrared spectroscopy (FTIR). The in vitro cytotoxicity of green synthesized CeO2 was assessed against cervical cancerous cells (HeLa). The exposure of CeO2 to HeLa cells at 10–125 µg/mL caused a loss in cellular viability against cervical cancerous cells in a dose-dependent manner. The antibacterial activity of the CeO2 was assessed against S. aureus and K. pneumonia. A significant improvement in wound-healing progression was observed when cerium oxide nanoparticles were incorporated into the chitosan hydrogel membrane as a wound dressing.

scholarly journals Blockade of LINC01605-enriched exosome generation in M2 macrophages impairs M2 macrophage-induced proliferation, migration, and invasion of human dermal fibroblasts

2021 ◽  
Vol 35 ◽  
pp. 205873842110167
Author(s):  
Zhensen Zhu ◽  
Bo Chen ◽  
Liang Peng ◽  
Songying Gao ◽  
Jingdong Guo ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Dermal Fibroblast ◽  
Dermal Fibroblasts ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
M2 Macrophage ◽  
M2 Macrophages ◽  
Luciferase Reporter Gene ◽  
Gene Assay

Activated M2 macrophages are involved in hypertrophic scar (HS) formation via manipulating the differentiation of fibroblasts to myofibroblasts having the proliferative capacity and biological function. However, the function of exosomes derived from M2 macrophages in HS formation is unclear. Thus, this study aims to investigate the role of exosomes derived by M2 in the formation of HS. To understand the effect of exosomes derived from M2 macrophages on formation of HS, M2 macrophages were co-cultured with human dermal fibroblast (HDF) cells. Cell Counting Kit-8 assay was performed to evaluate HDF proliferation. To evaluate the migration and invasion of HDFs, wound-healing and transwell invasion assays were performed, respectively. To investigate the interaction between LINC01605 and miR-493-3p, a dual-luciferase reporter gene assay was adopted; consequently, an interaction between miR-493-3p and AKT1 was detected. Our results demonstrated that exosomes derived from M2 macrophages promoted the proliferation, migration, and invasion of HDFs. Additionally, we found that long noncoding RNA LINC01605, enriched in exosomes derived from M2 macrophages, promoted fibrosis of HDFs and that GW4869, an inhibitor of exosomes, could revert this effect. Mechanistically, LINC01605 promoted fibrosis of HDFs by directly inhibiting the secretion of miR-493-3p, and miR-493-3p down-regulated the expression of AKT1. Exosomes derived from M2 macrophages promote the proliferation and migration of HDFs by transmitting LINC01605, which may activate the AKT signaling pathway by sponging miR-493-3p. Our results provide a novel approach and basis for further investigation of the function of M2 macrophages in HS formation.

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.

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