Training-Associated Alterations In Equine Respiratory Immunity – A Multi-Omics Comparative Approach.

Abstract Background: Neutrophilic airway inflammation is highly prevalent in racehorses in training, with the term mild to moderate equine asthma (MMEA) being applied to the majority of such cases. Our proposed study is largely derived from the strong association between MMEA in racehorses and their entry into a race training program; this has led to our primary aim of measuring the effect of race training on pulmonary immune cell function. The objectives of this study are to characterise the effect of training on the local pulmonary immune system and quantify the magnitude of effect by defining (a) the gene expression of tracheal wash (TW) derived cells and (b) the protein expression of TW samples derived from Thoroughbred racehorses prior to (T0) and following commencement of race training (T1). Results: Tracheal wash samples were obtained at both time points (T0, T1) from the same Thoroughbred horses (n=16). Gene expression of TW derived cells, determined by RNAseq and analysed by DEseq2 detected 2,138 differentially expressed (DE) genes during the training period; 1,122 of these were upregulated. The proteome of TW samples was also evaluated and contained 260 DE proteins during the training period; 103 of these were upregulated and the rest downregulated. Gene and protein sets were enriched for biological processes related to acute phase response and oxidative stress, as well as to immune response and inflammation. Many genes, including ISG15, ISG20, IFI35, SOCS1 and TRIM21, were highly enriched for IFN signaling. Interestingly, pathway analysis also highlighted genes and proteins related to haemopoietic processes.Conclusions: This study demonstrated TW samples to represent a rich source of airway cells, protein and RNA to study airway immunity in the horse and highlighted the benefits of a multi-omics methodological approach to studying the dynamics of equine airway immunity. Intense training induced quantifiable alterations in both gene and protein expression of airway derived cells is consistent with deregulation of airway immunity and haemopoietic abnormalities. Respectively, these findings likely reflect the known associations between race training and both airway inflammation and bleeding, in particular offering further insight into the potential mechanisms which underpin training associated airway inflammation.

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Training associated alterations in equine respiratory immunity using a multiomics comparative approach

Scientific Reports ◽

10.1038/s41598-021-04137-3 ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Anna E. Karagianni ◽

Dominic Kurian ◽

Eugenio Cillán-Garcia ◽

Samantha L. Eaton ◽

Thomas M. Wishart ◽

...

Keyword(s):

Airway Inflammation ◽

Methodological Approach ◽

Strong Association ◽

Comparative Approach ◽

Biological Processes ◽

Period Gene ◽

Thoroughbred Racehorses ◽

Gene And Protein Expression ◽

Pulmonary Immune System ◽

Insight Into

AbstractNeutrophilic airway inflammation is highly prevalent in racehorses in training, with the term mild to moderate equine asthma (MMEA) being applied to the majority of such cases. Our proposed study is largely derived from the strong association between MMEA in racehorses and their entry into a race training program. The objectives of this study are to characterise the effect of training on the local pulmonary immune system by defining the gene and protein expression of tracheal wash (TW) derived samples from Thoroughbred racehorses prior to and following commencement of race training. Multiomics analysis detected 2138 differentially expressed genes and 260 proteins during the training period. Gene and protein sets were enriched for biological processes related to acute phase response, oxidative stress, haemopoietic processes, as well as to immune response and inflammation. This study demonstrated TW samples to represent a rich source of airway cells, protein and RNA to study airway immunity in the horse and highlighted the benefits of a multiomics methodological approach to studying the dynamics of equine airway immunity. Findings likely reflect the known associations between race-training and both airway inflammation and bleeding, offering further insight into the potential mechanisms which underpin training associated airway inflammation.

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Training Associated Alterations in Equine Respiratory Immunity Using a Multiomics Comparative Approach

10.21203/rs.3.rs-787990/v1 ◽

2021 ◽

Author(s):

Anna Eleonora Karagianni ◽

Dominic Kurian ◽

Eugenio Cillán-Garcia ◽

Samantha L. Eaton ◽

Thomas M. Wishart ◽

...

Keyword(s):

Airway Inflammation ◽

Methodological Approach ◽

Strong Association ◽

Comparative Approach ◽

Biological Processes ◽

Period Gene ◽

Thoroughbred Racehorses ◽

Gene And Protein Expression ◽

Pulmonary Immune System ◽

Insight Into

Abstract Neutrophilic airway inflammation is highly prevalent in racehorses in training, with the term mild to moderate equine asthma (MMEA) being applied to the majority of such cases. Our proposed study is largely derived from the strong association between MMEA in racehorses and their entry into a race training program. The objectives of this study are to characterise the effect of training on the local pulmonary immune system by defining the gene and protein expression of tracheal wash (TW) derived samples from Thoroughbred racehorses prior to and following commencement of race training. Multiomics analysis detected 2,138 differentially expressed genes and 260 proteins during the training period. Gene and protein sets were enriched for biological processes related to acute phase response, oxidative stress, haemopoietic processes, as well as to immune response and inflammation. This study demonstrated TW samples to represent a rich source of airway cells, protein and RNA to study airway immunity in the horse and highlighted the benefits of a multiomics methodological approach to studying the dynamics of equine airway immunity. Findings likely reflect the known associations between race-training and both airway inflammation and bleeding, offering further insight into the potential mechanisms which underpin training associated airway inflammation.

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The Effects of Simulated Microgravity on Macrophage Phenotype

Biomedicines ◽

10.3390/biomedicines9091205 ◽

2021 ◽

Vol 9 (9) ◽

pp. 1205

Author(s):

Christopher Ludtka ◽

Erika Moore ◽

Josephine B. Allen

Keyword(s):

Protein Expression ◽

Simulated Microgravity ◽

Prolonged Exposure ◽

Cell Function ◽

Immune Cell ◽

Expression Profiles ◽

Microgravity Environment ◽

M2 Macrophage ◽

Macrophage Function ◽

Gene And Protein Expression

The effects of spaceflight, including prolonged exposure to microgravity, can have significant effects on the immune system and human health. Altered immune cell function can lead to adverse health events, though precisely how and to what extent a microgravity environment impacts these cells remains uncertain. Macrophages, a key immune cell, effect the inflammatory response as well as tissue remodeling and repair. Specifically, macrophage function can be dictated by phenotype that can exist between spectrums of M0 macrophage: the classically activated, pro-inflammatory M1, and the alternatively activated, pro-healing M2 phenotypes. This work assesses the effects of simulated microgravity via clinorotation on M0, M1, and M2 macrophage phenotypes. We focus on phenotypic, inflammatory, and angiogenic gene and protein expression. Our results show that across all three phenotypes, microgravity results in a decrease in TNF-α expression and an increase in IL-12 and VEGF expression. IL-10 was also significantly increased in M1 and M2, but not M0 macrophages. The phenotypic cytokine expression profiles observed may be related to specific gravisensitive signal transduction pathways previously implicated in microgravity regulation of macrophage gene and protein expression. Our results highlight the far-reaching effects that simulated microgravity has on macrophage function and provides insight into macrophage phenotypic function in microgravity.

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Toll-like receptor 2 induced senescence in intervertebral disc cells of patients with back pain can be attenuated by o-vanillin

Arthritis Research & Therapy ◽

10.1186/s13075-021-02504-z ◽

2021 ◽

Vol 23 (1) ◽

Author(s):

Matthew Mannarino ◽

Hosni Cherif ◽

Li Li ◽

Kai Sheng ◽

Oded Rabau ◽

...

Keyword(s):

Gene Expression ◽

Back Pain ◽

Protein Expression ◽

Disc Degeneration ◽

Cell Senescence ◽

Enzyme Linked Immunosorbent Assay ◽

Matrix Synthesis ◽

Gene And Protein Expression ◽

Pain Patients ◽

In Cells

Abstract Background There is an increased level of senescent cells and toll-like teceptor-1, -2, -4, and -6 (TLR) expression in degenerating intervertebral discs (IVDs) from back pain patients. However, it is currently not known if the increase in expression of TLRs is related to the senescent cells or if it is a more general increase on all cells. It is also not known if TLR activation in IVD cells will induce cell senescence. Methods Cells from non-degenerate human IVD were obtained from spine donors and cells from degenerate IVDs came from patients undergoing surgery for low back pain. Gene expression of TLR-1,2,4,6, senescence and senescence-associated secretory phenotype (SASP) markers was evaluated by RT-qPCR in isolated cells. Matrix synthesis was verified with safranin-O staining and Dimethyl-Methylene Blue Assay (DMMB) confirmed proteoglycan content. Protein expression of p16INK4a, SASP factors, and TLR-2 was evaluated by immunocytochemistry (ICC) and/or by enzyme-linked immunosorbent assay (ELISA). Results An increase in senescent cells was found following 48-h induction with a TLR-2/6 agonist in cells from both non-degenerate and degenerating human IVDs. Higher levels of SASP factors, TLR-2 gene expression, and protein expression were found following 48-h induction with TLR-2/6 agonist. Treatment with o-vanillin reduced the number of senescent cells, and increased matrix synthesis in IVD cells from back pain patients. Treatment with o-vanillin after induction with TLR-2/6 agonist reduced gene and protein expression of SASP factors and TLR-2. Co-localized staining of p16INK4a and TLR-2 demonstrated that senescent cells have a high TLR-2 expression. Conclusions Taken together our data demonstrate that activation of TLR-2/6 induce senescence and increase TLR-2 and SASP expression in cells from non-degenerate IVDs of organ donors without degeneration and back pain and in cells from degenerating human IVD of patients with disc degeneration and back pain. The senescent cells showed high TLR-2 expression suggesting a link between TLR activation and cell senescence in human IVD cells. The reduction in senescence, SASP, and TLR-2 expression suggest o-vanillin as a potential disease-modifying drug for patients with disc degeneration and back pain.

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83 Spatially resolved transcriptomic and proteomic investigation of breast cancer and its immune microenvironment

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.083 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A91-A91

Author(s):

Jennifer Chew ◽

Cedric Uytingco ◽

Rapolas Spalinskas ◽

Yifeng Yin ◽

Joe Shuga ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Tumor Microenvironment ◽

Protein Expression ◽

Protein Detection ◽

Response To Therapy ◽

Pathological Examination ◽

Multi Drug Resistance ◽

Spatially Resolved ◽

Gene And Protein Expression

BackgroundThe tumor microenvironment (TME) is composed of highly heterogeneous extracellular structures and cell types such as endothelial cells, immune cells, and fibroblasts that dynamically influence and communicate with each other. The constant interaction between a tumor and its microenvironment plays a critical role in cancer development and progression and can significantly affect a tumor’s response to therapy and capacity for multi-drug resistance. High resolution analyses of gene and protein expression with spatial context can provide deeper insights into the interactions between tumor cells and surrounding cells within the TME, where a better understanding of the underlying biology can improve treatment efficacy and patient outcomes. Here, we demonstrated the ability to perform streamlined multi-omic tumor analyses by utilizing the 10X Genomics Visium Spatial Gene Expression Solution for FFPE with multiplex protein enablement. This technique simultaneously assesses gene and protein expression to elucidate the immunological profile and microenvironment of different breast cancer samples in conjunction with standard pathological methods.MethodsSerial (5 µm) sections of FFPE human breast cancer samples were placed on Visium Gene Expression (GEX) slides. The Visium GEX slides incorporate ~5,000 molecularly barcoded, spatially encoded capture spots onto which tissue sections are placed, stained, and imaged. Following incubation with a human whole transcriptome, probe-based RNA panel and an immuno-oncology oligo-tagged antibody panel, developed with Abcam conjugated antibodies, the tissues are permeabilized and the representative probes are captured. Paired GEX and protein libraries are generated for each section and then sequenced on an Illumina NovaSeq at a depth of ~50,000 reads per spot. Resulting reads from both libraries are aligned and overlaid with H&E-stained tissue images, enabling analysis of both mRNA and protein expression. Additional analyses and data visualizations were performed on the Loupe Browser v4.1 desktop software.ConclusionsSpatial transcriptomics technology complements pathological examination by combining histological assessment with the throughput and deep biological insight of highly-multiplexed protein detection and RNA-seq. Taken together, our work demonstrated that Visium Spatial technology provides a spatially-resolved, multi-analyte view of the tumor microenvironment, where a greater understanding of cellular behavior in and around tumors can help drive discovery of new biomarkers and therapeutic targets.

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Angiopoietin 1 and 2 gene and protein expression is differentially regulated in acute anti-Thy1.1 glomerulonephritis

AJP Renal Physiology ◽

10.1152/ajprenal.00320.2007 ◽

2008 ◽

Vol 294 (5) ◽

pp. F1174-F1184 ◽

Cited By ~ 12

Author(s):

Valentina Câmpean ◽

Britta Karpe ◽

Christian Haas ◽

Akram Atalla ◽

Harm Peters ◽

...

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Glomerular Injury ◽

Double Immunofluorescence ◽

Real Time Quantitative Pcr ◽

Glomerular Capillary ◽

Gene And Protein Expression ◽

Nonradioactive In Situ Hybridization ◽

Angiopoietin 1

Capillary neoformation is important in repair of glomerular injury of various origins. VEGF was shown to be crucial for glomerular capillary repair in glomerulonephritis (GN). We reasoned that other angiogenic factors are likewise involved in glomerular capillary remodeling and found angiopoietin 1 and -2 (ANG1 and ANG2) mRNA to be upregulated in cDNA microarrays of microdissected glomeruli of anti-Thy1.1 GN of the rat. We then studied glomerular in situ gene and protein expression of ANG1 and ANG2 and their receptor Tie-2 in the course of anti-Thy1.1 GN, which was induced by injection of OX-7 antibody. Animals were perfusion fixed at days 6 and 12 after GN induction and compared with nonnephritic controls receiving PBS. Capillary damage and repair were quantitatively analyzed using stereological techniques. Gene and protein expression of ANG1 and ANG2 and their receptor Tie-2 was analyzed using real-time quantitative PCR from microdissected glomeruli, nonradioactive in situ hybridization, double immunofluorescence, and Western blot analysis. Glomerular capillarization assessed as length density was significantly lower at day 6 of anti-Thy1.1 GN than in controls; it was back to normal values at day 12. ANG1 and ANG2 gene expression was markedly upregulated at day 6 of the disease compared with controls. Protein expression of ANG1 and ANG2 was confined to podocytes and that of Tie-2 to endothelial cells. At day 12 of anti-Thy1.1 GN when capillary restoration was nearly completed, ANG1 and ANG2 gene expression returned to basal levels, whereas Tie-2 expression was still high. With the use of a combined molecular and in situ approach, the spatial and temporal gene and protein expression of the angiopoietins and their receptor was analyzed in anti-Thy1.1 GN. The results indicate that glomerular expression of ANG1 and ANG2 and Tie-2 is differentially regulated and may contribute to healing and endothelial cell stabilization in experimental GN.

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Comparison of the Effects of Glibenclamide on Metabolic Parameters, GLUT1 Expression, and Liver Injury in Rats With Severe and Mild Streptozotocin-Induced Diabetes Mellitus

Medicina ◽

10.3390/medicina48100078 ◽

2012 ◽

Vol 48 (10) ◽

pp. 78 ◽

Cited By ~ 5

Author(s):

Jelizaveta Sokolovska ◽

Sergejs Isajevs ◽

Olga Sugoka ◽

Jelena Sharipova ◽

Natalia Paramonova ◽

...

Keyword(s):

Gene Expression ◽

Diabetes Mellitus ◽

Protein Expression ◽

Diabetic Rats ◽

Metabolic Parameters ◽

Gene And Protein Expression ◽

Glut1 Expression ◽

Mild Diabetes ◽

Streptozotocin Induced Diabetes ◽

Glut1 Gene

Background and Objective. Glucose transport via GLUT1 protein could be one of additional mechanisms of the antidiabetic action of sulfonylureas. Here, the GLUT1 gene and the protein expression was studied in rats in the course of severe and mild streptozotocin-induced diabetes mellitus and under glibenclamide treatment. Material and Methods. Severe and mild diabetes mellitus was induced using different streptozotocin doses and standard or high fat chow. Rats were treated with glibenclamide (2 mg/kg daily, per os for 6 weeks). The therapeutic effect of glibenclamide was monitored by measuring several metabolic parameters. The GLUT1 mRNA and the protein expression in the kidneys, heart, and liver was studied by means of real-time R T-PCR and immunohistochemistry. Results. The glibenclamide treatment decreased the blood glucose concentration and increased the insulin level in both models of severe and mild diabetes mellitus. Severe diabetes mellitus provoked an increase in both GLUT1 gene and protein expression in the kidneys and the heart, which was nearly normalized by glibenclamide. In the kidneys of mildly diabetic rats, an increase in the GLUT1 gene expression was neither confirmed on the protein level nor influenced by the glibenclamide treatment. In the liver of severely diabetic rats, the heart and the liver of mildly diabetic rats, the GLUT1 gene and the protein expression was changed independently of each other, which might be explained by abortive transcription, and pre- and posttranslational modifications of gene expression. Conclusions. The GLUT1 expression was found to be affected by the glucose and insulin levels and can be modulated by glibenclamide in severely and mildly diabetic rats. Glibenclamide can prevent the liver damage caused by severe hyperglycemia.

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Preconditioning of mesenchymal stromal cells with low-intensity ultrasound: influence on chondrogenesis and directed SOX9 signaling pathways

Stem Cell Research & Therapy ◽

10.1186/s13287-019-1532-2 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Neety Sahu ◽

Gaurav Budhiraja ◽

Anuradha Subramanian

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Signaling Pathways ◽

Mesenchymal Stromal Cells ◽

Nuclear Localization ◽

Stromal Cells ◽

Actin Reorganization ◽

Low Intensity ◽

Gene And Protein Expression ◽

Low Intensity Ultrasound

Abstract Background Continuous low-intensity ultrasound (cLIUS) facilitates the chondrogenic differentiation of human mesenchymal stromal cells (MSCs) in the absence of exogenously added transforming growth factor-beta (TGFβ) by upregulating the expression of transcription factor SOX9, a master regulator of chondrogenesis. The present study evaluated the molecular events associated with the signaling pathways impacting SOX9 gene and protein expression under cLIUS. Methods Human bone marrow-derived MSCs were exposed to cLIUS stimulation at 14 kPa (5 MHz, 2.5 Vpp) for 5 min. The gene and protein expression of SOX9 was evaluated. The specificity of SOX9 upregulation under cLIUS was determined by treating the MSCs with small molecule inhibitors of select signaling molecules, followed by cLIUS treatment. Signaling events regulating SOX9 expression under cLIUS were analyzed by gene expression, immunofluorescence staining, and western blotting. Results cLIUS upregulated the gene expression of SOX9 and enhanced the nuclear localization of SOX9 protein when compared to non-cLIUS-stimulated control. cLIUS was noted to enhance the phosphorylation of the signaling molecule ERK1/2. Inhibition of MEK/ERK1/2 by PD98059 resulted in the effective abrogation of cLIUS-induced SOX9 expression, indicating that cLIUS-induced SOX9 upregulation was dependent on the phosphorylation of ERK1/2. Inhibition of integrin and TRPV4, the upstream cell-surface effectors of ERK1/2, did not inhibit the phosphorylation of ERK1/2 and therefore did not abrogate cLIUS-induced SOX9 expression, thereby suggesting the involvement of other mechanoreceptors. Consequently, the effect of cLIUS on the actin cytoskeleton, a mechanosensitive receptor regulating SOX9, was evaluated. Diffused and disrupted actin fibers observed in MSCs under cLIUS closely resembled actin disruption by treatment with cytoskeletal drug Y27632, which is known to increase the gene expression of SOX9. The upregulation of SOX9 under cLIUS was, therefore, related to cLIUS-induced actin reorganization. SOX9 upregulation induced by actin reorganization was also found to be dependent on the phosphorylation of ERK1/2. Conclusions Collectively, preconditioning of MSCs by cLIUS resulted in the nuclear localization of SOX9, phosphorylation of ERK1/2 and disruption of actin filaments, and the expression of SOX9 was dependent on the phosphorylation of ERK1/2 under cLIUS.

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P4460microRNA-17-5p and microRNA-20a-5p downregulation by atorvastatin in Hepg2 cells: a new mechanism involved in the lipid-lowering therapy

European Heart Journal ◽

10.1093/eurheartj/ehz745.0857 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

K Saavedra ◽

K Leal ◽

D Zapata ◽

S Sagardia ◽

F Ortega ◽

...

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Hepg2 Cells ◽

Lipid Lowering ◽

Lipid Lowering Therapy ◽

Ldlr Gene ◽

Variable Effect ◽

Incidence And Mortality ◽

Gene And Protein Expression ◽

In Silico Studies

Abstract Background Hypercholesterolemia increases the risk of coronary artery disease and its pharmacological treatment has demonstrated, besides reducing cholesterol levels, a decrease in the incidence and mortality from coronary events. The treatment of hypercholesterolemia is mainly driven by using statins. However, the response to pharmacological therapy shows high inter-individual variability, resulting in a variable effect in both lipid lowering and risk reduction. Thus, a better understanding of lipid-lowering mechanism and response variability at molecular level is required. Previously, we demonstrated a deregulation of microRNA (miR) profile in HepG2 cells after atorvastatin treatment, including the downregulation of miR-17-5p and miR-20a-5p which potentially targets the LDL receptor gene (LDLR), suggesting that it might be involved in allowing the LDLR overexpression on the surface of hepatic cells to subsequently capture circulating LDL and to reach the expected lipid-lowering effect. Purpose To determine the role of miR-17-5p and miR-20a-5p on the regulation of LDLR gene expression in HepG2 cells. Methods Cells HepG2 were treated with atorvastatin 10μM for 24 hours. RNA extraction and enrichment of smallRNAs were performed. The gene expression of miR-17-5p, miR-20a-5p, miR-24-3p, miR-93-5p, miR-106a-5p and LDLR were evaluated. To evaluate the effect of miR-17-5p and miR-20a-5p on LDLR gene expression, both miRs were overexpressed or repressed by transfection of mimics or inhibitors respectively into HepG2 cells for 24, 48 and 72 Hours. The gene expression of LDLR was quantified by real time PCR using RPL27 gene as reference gene. The protein expression of LDLR and beta actin were evaluated using western blot and quantified using the ImageJ software. Results Our data showed that atorvastatin significantly repressed the expression of miR-17-5p (P<0.0001) and miR-20a-5p (P=0.0456) in HepG2 cells. In silico studies showed that miR-17-5p interact with the 3'-UTR region of the LDLR. Consistently, when miR-17-5p or miR-20a-5p were overexpressed by using mimics, we observed that gene and protein expression of LDLR decreased significantly (P<0.0001 and P<0.05 respectively). Consistently, when miR-17-5p or miR-20a-5p were repressed by the use inhibitors, we observed that the gene and protein expression of LDLR increases significantly (P<0.005). Conclusions In conclusion, we demonstrate that atorvastatin induces a significant down-regulation of the miR-17-5p and miR-20a-5p in HepG2 cells. The overexpression or repression regulate the gene and protein expression of LDLR. Acknowledgement/Funding FONDECYT N°3160567, FONDECYT N°1171765 and DIUFRO DI19-0094

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Compromised barrier integrity of human feto-placental vessels from gestational diabetic pregnancies is related to downregulation of occludin expression

Diabetologia ◽

10.1007/s00125-020-05290-6 ◽

2020 ◽

Vol 64 (1) ◽

pp. 195-210

Author(s):

Stephany Daniela Villota ◽

Maria Toledo-Rodriguez ◽

Lopa Leach

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Splice Variants ◽

Fold Increase ◽

Lifestyle Changes ◽

Protein Isoforms ◽

Lower Percentage ◽

Systematic Sampling ◽

Regulation Of Expression ◽

Gene And Protein Expression

Abstract Aims/hypothesis Reduced occupancy of junctional occludin is a feature of human placental vessels in the diabetic milieu. However, the functional consequence of this and whether this loss is due to differential expression of occludin splice variants is not known. Our study aimed to investigate the effects of gestational diabetes mellitus (GDM), and its treatment, on endothelial junctional integrity, gene and protein expression of occludin splice variants, and potential regulation of expression by microRNAs (miRNAs). Methods Term placentas were obtained from normal pregnancies (n = 21), and pregnancies complicated by GDM where glucose levels were controlled by diet (n = 11) or metformin (n = 6). Gene and microRNA (miRNA) expression were determined by quantitative real-time PCR; protein expression by immunoblotting; endothelial junctional occupancy by fluorescence microscopy and systematic sampling; and paracellular leakage by perfusion of placental microvascular beds with 76 Mr dextran. Transfection studies of miRNAs that target OCLN were performed in HUVECs, and the trans-endothelial electrical resistance and tracer permeability of the HUVECs were measured. Results All three predicted OCLN gene splice variants and two occludin protein isoforms were found in human placental samples. In placental samples from diet-controlled GDM (d-GDM) pregnancies we found a lower percentage of conduit vessels showing occludin immunoreactivity (12%, p < 0.01), decreased levels of the fully functional occludin isoform-A protein (29%), and differential gene expression of OCLN variant 2 (33% decrease), variant 3 (3.3-fold increase). These changes were not seen in samples from the group with metformin-controlled GDM. In d-GDM placentas, increased numbers of conduit microvessels demonstrated extravasation of 76 Mr dextran (2.0-fold). In d-GDM expression of one of the five potential miRNAs targeting OCLN, miR-181a-5p, expression was 2.1-fold that in normal pregnancies. Experimental overexpression of miR-181a-5p in HUVECs from normal pregnancies resulted in a highly significant downregulation of OCLN variant 1 (69%) and variant 2 (46%) gene expression, with decreased trans-endothelial resistance (78%) and increase in tracer permeability (1.3-fold). Conclusions/interpretation Downregulation of expression of OCLN variant 2 and the fully functional occludin isoform-A protein are a feature of placentas in d-GDM pregnancies. These may be behind the loss of junctional occludin and the increased extravasation of exogenous dextran observed. miR-181a-5p was in part responsible for the downregulation of occludin in placentas from d-GDM pregnancies. Induced overexpression of miR-181a-5p compromised the integrity of the endothelial barrier. Our data suggest that, despite good glucose control, the adoption of lifestyle changes alone during a GDM pregnancy may not be enough to prevent an alteration in the expression of occludin and the subsequent functional consequences in placentas and impaired vascular barrier function in offspring.

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