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1.
Mol Med Rep ; 25(4)2022 04.
Article in English | MEDLINE | ID: covidwho-1753714

ABSTRACT

Aberrant TGF­ß/Smad7 signaling has been reported to be an important mechanism underlying the pathogenesis of ulcerative colitis. Therefore, the present study aimed to investigate the effects of a number of potential anti­colitis agents on intestinal epithelial permeability and the TGF­ß/Smad7 signaling pathway in an experimental model of colitis. A mouse model of colitis was first established before anti­TNF­α and 5­aminosalicyclic acid (5­ASA) were administered intraperitoneally and orally, respectively. Myeloperoxidase (MPO) activity, histological index (HI) of the colon and the disease activity index (DAI) scores were then detected in each mouse. Transmission electron microscopy (TEM), immunohistochemical and functional tests, including Evans blue (EB) and FITC­dextran (FD­4) staining, were used to evaluate intestinal mucosal permeability. The expression of epithelial phenotype markers E­cadherin, occludin, zona occludens (ZO­1), TGF­ß and Smad7 were measured. In addition, epithelial myosin light chain kinase (MLCK) expression and activity were measured. Anti­TNF­α and 5­ASA treatments was both found to effectively reduce the DAI score and HI, whilst decreasing colonic MPO activity, plasma levels of FD­4 and EB permeation of the intestine. Furthermore, anti­TNF­α and 5­ASA treatments decreased MLCK expression and activity, reduced the expression of Smad7 in the small intestine epithelium, but increased the expression of TGF­ß. In mice with colitis, TEM revealed partial epithelial injury in the ileum, where the number of intercellular tight junctions and the expression levels of E­cadherin, ZO­1 and occludin were decreased, all of which were alleviated by anti­TNF­α and 5­ASA treatment. In conclusion, anti­TNF­α and 5­ASA both exerted protective effects on intestinal epithelial permeability in an experimental mouse model of colitis. The underlying mechanism may be mediated at least in part by the increase in TGF­ß expression and/or the reduction in Smad7 expression, which can inhibit epithelial MLCK activity and in turn reduce mucosal permeability during the pathogenesis of ulcerative colitis.


Subject(s)
Colitis, Ulcerative/metabolism , Smad7 Protein/genetics , Smad7 Protein/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Animals , Cadherins/metabolism , Colitis, Ulcerative/chemically induced , Colon/pathology , Dextran Sulfate/toxicity , Disease Models, Animal , Female , Intestinal Mucosa/drug effects , Intestinal Mucosa/pathology , Intestinal Mucosa/ultrastructure , Male , Mesalamine/administration & dosage , Mice, Inbred C57BL , Myosin-Light-Chain Kinase/metabolism , Occludin/metabolism , Peroxidase/drug effects , Severity of Illness Index , Signal Transduction/drug effects , Tight Junctions/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Zonula Occludens-1 Protein/metabolism
2.
Cells ; 11(4)2022 02 11.
Article in English | MEDLINE | ID: covidwho-1688673

ABSTRACT

Transmembrane proteins of adherens and tight junctions are known targets for viruses and bacterial toxins. The coronavirus receptor ACE2 has been localized at the apical surface of epithelial cells, but it is not clear whether ACE2 is localized at apical Cell-Cell junctions and whether it associates with junctional proteins. Here we explored the expression and localization of ACE2 and its association with transmembrane and tight junction proteins in epithelial tissues and cultured cells by data mining, immunoblotting, immunofluorescence microscopy, and co-immunoprecipitation experiments. ACE2 mRNA is abundant in epithelial tissues, where its expression correlates with the expression of the tight junction proteins cingulin and occludin. In cultured epithelial cells ACE2 mRNA is upregulated upon differentiation and ACE2 protein is widely expressed and co-immunoprecipitates with the transmembrane proteins ADAM17 and CD9. We show by immunofluorescence microscopy that ACE2 colocalizes with ADAM17 and CD9 and the tight junction protein cingulin at apical junctions of intestinal (Caco-2), mammary (Eph4) and kidney (mCCD) epithelial cells. These observations identify ACE2, ADAM17 and CD9 as new epithelial junctional transmembrane proteins and suggest that the cytokine-enhanced endocytic internalization of junction-associated protein complexes comprising ACE2 may promote coronavirus entry.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Intercellular Junctions/metabolism , Intercellular Junctions/virology , ADAM17 Protein/metabolism , Adherens Junctions/metabolism , Angiotensin-Converting Enzyme 2/genetics , Cadherins/metabolism , Carrier Proteins/metabolism , Cell Line , Cell Membrane Permeability , Coronavirus/metabolism , Epithelial Cells/metabolism , Epithelial Cells/virology , Gene Expression/genetics , Tetraspanin 29/metabolism , Tight Junction Proteins/metabolism , Tight Junctions/metabolism , Transcriptome/genetics
3.
Biol Pharm Bull ; 44(10): 1371-1379, 2021.
Article in English | MEDLINE | ID: covidwho-1445700

ABSTRACT

The vascular permeability of the endothelium is finely controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions. In the majority of normal adult tissues, endothelial cells in blood vessels maintain vascular permeability at a relatively low level, while in response to inflammation, they limit vascular barrier function to induce plasma leakage and extravasation of immune cells as a defense mechanism. Thus, the dynamic but also simultaneously tight regulation of vascular permeability by endothelial cells is responsible for maintaining homeostasis and, as such, impairments of its underlying mechanisms result in hyperpermeability, leading to the development and progression of various diseases including coronavirus disease 2019 (COVID-19), a newly emerging infectious disease. Recently, increasing numbers of studies have been unveiling the important role of Rap1, a small guanosine 5'-triphosphatase (GTPase) belonging to the Ras superfamily, in the regulation of vascular permeability. Rap1 enhances VE-cadherin-mediated endothelial cell-cell junctions to potentiate vascular barrier functions via dynamic reorganization of the actin cytoskeleton. Importantly, Rap1 signaling activation reportedly improves vascular barrier function in animal models of various diseases associated with vascular hyperpermeability, suggesting that Rap1 might be an ideal target for drugs intended to prevent vascular barrier dysfunction. Here, we describe recent progress in understanding the mechanisms by which Rap1 potentiates VE-cadherin-mediated endothelial cell-cell adhesions and vascular barrier function. We also discuss how alterations in Rap1 signaling are related to vascular barrier dysfunction in diseases such as acute pulmonary injury and malignancies. In addition, we examine the possibility of Rap1 signaling as a target of drugs for treating diseases associated with vascular hyperpermeability.


Subject(s)
Antigens, CD/metabolism , Cadherins/metabolism , Capillary Permeability , Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Intercellular Junctions/metabolism , rap1 GTP-Binding Proteins/metabolism , Animals , Humans
5.
Cells ; 10(7)2021 07 02.
Article in English | MEDLINE | ID: covidwho-1323125

ABSTRACT

The open carrier system (OC) is used for vitrification due to its high efficiency in preserving female fertility, but concerns remain that it bears possible risks of cross-contamination. Closed carrier systems (CC) could be an alternative to the OC to increase safety. However, the viability and developmental competence of vitrified/warmed (VW) oocytes using the CC were significantly lower than with OC. We aimed to improve the efficiency of the CC. Metaphase II oocytes were collected from mice after superovulation and subjected to in vitro fertilization after vitrification/warming. Increasing the cooling/warming rate and exposure time to cryoprotectants as key parameters for the CC effectively improved the survival rate and developmental competence of VW oocytes. When all the conditions that improved the outcomes were applied to the conventional CC, hereafter named the modified vitrification/warming procedure using CC (mVW-CC), the viability and developmental competence of VW oocytes were significantly improved as compared to those of VW oocytes in the CC. Furthermore, mVW-CC increased the spindle normality of VW oocytes, as well as the cell number of blastocysts developed from VW oocytes. Collectively, our mVW-CC optimized for mouse oocytes can be utilized for humans without concerns regarding possible cross-contamination during vitrification in the future.


Subject(s)
Blastocyst/cytology , Cryopreservation/methods , Fertilization in Vitro/methods , Oocytes/cytology , Vitrification , Animals , Biomarkers/metabolism , Blastocyst/metabolism , CDX2 Transcription Factor/genetics , CDX2 Transcription Factor/metabolism , Cadherins/genetics , Cadherins/metabolism , Cell Survival/drug effects , Cells, Cultured , Cryoprotective Agents/pharmacology , Dimethyl Sulfoxide/pharmacology , Ethylene Glycol/pharmacology , Female , Gene Expression , Male , Metaphase , Mice , Oocytes/drug effects , Oocytes/metabolism , Spermatozoa/physiology , Sucrose/pharmacology
6.
PLoS One ; 16(6): e0253347, 2021.
Article in English | MEDLINE | ID: covidwho-1280628

ABSTRACT

The unprecedented global COVID-19 pandemic has prompted a desperate international effort to accelerate the development of anti-viral candidates. For unknown reasons, COVID-19 infections are associated with adverse cardiovascular complications, implicating that vascular endothelial cells are essential in viral propagation. The etiological pathogen, SARS-CoV-2, has a higher reproductive number and infection rate than its predecessors, indicating it possesses novel characteristics that infers enhanced transmissibility. A unique K403R spike protein substitution encodes an Arg-Gly-Asp (RGD) motif, introducing a potential role for RGD-binding host integrins. Integrin αVß3 is widely expressed across the host, particularly in the endothelium, which acts as the final barrier before microbial entry into the bloodstream. This mutagenesis creates an additional binding site, which may be sufficient to increase SARS-CoV-2 pathogenicity. Here, we investigate how SARS-CoV-2 passes from the epithelium to endothelium, the effects of αVß3 antagonist, Cilengitide, on viral adhesion, vasculature permeability and leakage, and also report on a simulated interaction between the viral and host protein in-silico.


Subject(s)
Endothelium, Vascular/virology , Integrin alphaVbeta3/metabolism , SARS-CoV-2/pathogenicity , Snake Venoms/pharmacology , Antigens, CD/metabolism , Binding Sites , COVID-19/metabolism , COVID-19/physiopathology , Caco-2 Cells , Cadherins/metabolism , Computer Simulation , Endothelium, Vascular/cytology , Endothelium, Vascular/physiopathology , Host-Pathogen Interactions/drug effects , Humans , Integrin alphaVbeta3/chemistry , Models, Molecular , Mutation , Permeability , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
8.
Virology ; 552: 43-51, 2021 01 02.
Article in English | MEDLINE | ID: covidwho-843443

ABSTRACT

This study focused on intestinal restitution including phenotype switching of absorptive enterocytes and the abundance of different enterocyte subtypes in weaned pigs after porcine epidemic diarrhea virus (PEDV) infection. At 10 days post-PEDV-inoculation, the ratio of villus height to crypt depth in both jejunum and ileum had restored, and the PEDV antigen was not detectable. However, enterocytes at the villus tips revealed epithelial-mesenchymal transition (EMT) in the jejunum in which E-cadherin expression decreased while expression of N-cadherin, vimentin, and Snail increased. Additionally, there was reduced expression of actin in microvilli and Zonula occludens-1 (ZO-1) in tight junctions. Moreover, the protein concentration of transforming growth factor ß1 (TGFß1), which mediates EMT and cytoskeleton alteration, was increased. We also found a decreased number of Peyer's patch M cells in the ileum. These results reveal incomplete restitution of enterocytes in the jejunum and potentially impaired immune surveillance in the ileum after PEDV infection.


Subject(s)
Coronavirus Infections/veterinary , Enterocytes/pathology , Epithelial-Mesenchymal Transition , Gastroenteritis, Transmissible, of Swine/pathology , Peyer's Patches/pathology , Porcine epidemic diarrhea virus/pathogenicity , Animals , Cadherins/metabolism , Coronavirus Infections/immunology , Coronavirus Infections/pathology , Coronavirus Infections/virology , Gastroenteritis, Transmissible, of Swine/immunology , Gastroenteritis, Transmissible, of Swine/virology , Ileum/immunology , Ileum/pathology , Intestinal Mucosa/pathology , Jejunum/immunology , Jejunum/pathology , Microvilli/pathology , Swine , Tight Junctions/pathology , Transforming Growth Factor beta1/metabolism , Weaning
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