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• W2511855771 abstract "The regulation of function of endothelial cell–cell junctions is fundamental in sustaining vascular integrity. The polycistronic microRNA (miR) complexes containing miR-23a-27a-24-2, and 23b-27b-24-1 are predicted to target the majority of major endothelial junctional proteins. We focus on miR-23a and miR-23b, and investigate the functional effects of these miRs on junctions. While miR-23a and 23b only differ by 1 nucleotide (g19) outside the seed region and thus are predicted to have the same targets, they function differently with miR-23a inhibiting permeability and miR-23b inhibiting angiogenesis. Both miRs target the junctional attractive molecule (tight junction protein 2) ZO-2 and the repulsive molecule junctional adhesion molecule C (JAM-C), although the inhibition of JAM-C by miR-23a is more profound than by miR-23b. The difference in potency is attributable to differences at g19 since a mutation of the t17, the g19 binding site of miR-23b in the 3′UTR of JAM-C restores identity. We also show that the pattern of expression of miR-23a and miR-23b and their targets are different. Thus, the paralogues miR-23a and miR-23b can have profoundly different effects on endothelial cell function due at least partially to selective effects on target proteins and differences in expression patterns of the miRs. This work exposes a hitherto unappreciated complexity in therapeutically targeting miRs. The regulation of function of endothelial cell–cell junctions is fundamental in sustaining vascular integrity. The polycistronic microRNA (miR) complexes containing miR-23a-27a-24-2, and 23b-27b-24-1 are predicted to target the majority of major endothelial junctional proteins. We focus on miR-23a and miR-23b, and investigate the functional effects of these miRs on junctions. While miR-23a and 23b only differ by 1 nucleotide (g19) outside the seed region and thus are predicted to have the same targets, they function differently with miR-23a inhibiting permeability and miR-23b inhibiting angiogenesis. Both miRs target the junctional attractive molecule (tight junction protein 2) ZO-2 and the repulsive molecule junctional adhesion molecule C (JAM-C), although the inhibition of JAM-C by miR-23a is more profound than by miR-23b. The difference in potency is attributable to differences at g19 since a mutation of the t17, the g19 binding site of miR-23b in the 3′UTR of JAM-C restores identity. We also show that the pattern of expression of miR-23a and miR-23b and their targets are different. Thus, the paralogues miR-23a and miR-23b can have profoundly different effects on endothelial cell function due at least partially to selective effects on target proteins and differences in expression patterns of the miRs. This work exposes a hitherto unappreciated complexity in therapeutically targeting miRs. IntroductionThe sustenance of normal vascular permeability is one of the chief functions of endothelial cells (ECs) and is achieved mainly by the regulation of tightness of endothelial cell–cell junctions. Junctional integrity is maintained by a balance of adhesive junctional proteins, such as VE-Cadherin, ZO-1, ZO-2, and repulsive proteins such as JAM-C, SEMA-6A, and semaphorin.1Vestweber D VE-cadherin: the major endothelial adhesion molecule controlling cellular junctions and blood vessel formation.Arterioscler Thromb Vasc Biol. 2008; 28: 223-232Crossref PubMed Scopus (510) Google Scholar,2Tsukita S Katsuno T Yamazaki Y Umeda K Tamura A Tsukita S Roles of ZO-1 and ZO-2 in establishment of the belt-like adherens and tight junctions with paracellular permselective barrier function.Ann NY Acad Sci. 2009; 1165: 44-52Crossref PubMed Scopus (80) Google Scholar,3Li X Stankovic M Lee BP Aurrand-Lions M Hahn CN Lu Y et al.JAM-C induces endothelial cell permeability through its association and regulation of β3 integrins.Arterioscler Thromb Vasc Biol. 2009; 29: 1200-1206Crossref PubMed Scopus (38) Google Scholar,4Serini G Maione F Giraudo E Bussolino F Semaphorins and tumor angiogenesis.Angiogenesis. 2009; 12: 187-193Crossref PubMed Scopus (42) Google Scholar,5Zhou Q Gallagher R Ufret-Vincenty R Li X Olson EN Wang S Regulation of angiogenesis and choroidal neovascularization by members of microRNA-23≃27≃24 clusters.Proc Natl Acad Sci USA. 2011; 108: 8287-8292Crossref PubMed Scopus (292) Google Scholar The coordination of regulation of expression and signaling through these proteins is very important to allow transient changes as seen in acute inflammation and prevent chronic changes evident in almost all inflammatory and degenerative diseases. Targeting such junctional proteins is a possible approach for development of therapeutics particularly for diseases where vascular leak is central to the underlying pathology.MiRNAs (miRs) are a highly conserved class of endogenous small noncoding RNAs (20–25 nucleotides), which regulate genes at the post-transcriptional stage of expression.6Bartel DP MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (28968) Google Scholar MiRs have been shown to be dysregulated and contribute to the initiation and development of many diseases.7Negrini M Ferracin M Sabbioni S Croce CM MicroRNAs in human cancer: from research to therapy.J Cell Sci. 2007; 120: 1833-1840Crossref PubMed Scopus (206) Google Scholar,8Ventura A Jacks T MicroRNAs and cancer: short RNAs go a long way.Cell. 2009; 136: 586-591Abstract Full Text Full Text PDF PubMed Scopus (801) Google Scholar MiRs are able to inhibit the post-transcriptional expression of multiple genes or gene families involved in development or complex cellular functions,9Alvarez-Garcia I Miska EA MicroRNA functions in animal development and human disease.Development. 2005; 132: 4653-4662Crossref PubMed Scopus (1125) Google Scholar and are thus well suited to coordinate the function of cell junctions.The complexity and importance of these interactions was highlighted by our recent description of miR-27a as a potent regulator of the proadhesive protein VE-Cadherin,10Young JA Ting KK Li J Moller T Dunn L Lu Y et al.Regulation of vascular leak and recovery from ischemic injury by general and VE-cadherin-restricted miRNA antagonists of miR-27.Blood. 2013; 122: 2911-2919Crossref PubMed Scopus (49) Google Scholar while others have demonstrated miR-27 regulation of the antiadhesive proteins SEMA-6A and semaphorin.5Zhou Q Gallagher R Ufret-Vincenty R Li X Olson EN Wang S Regulation of angiogenesis and choroidal neovascularization by members of microRNA-23≃27≃24 clusters.Proc Natl Acad Sci USA. 2011; 108: 8287-8292Crossref PubMed Scopus (292) Google Scholar,11Urbich C Kaluza D Frömel T Knau A Bennewitz K Boon RA et al.MicroRNA-27a/b controls endothelial cell repulsion and angiogenesis by targeting semaphorin 6A.Blood. 2012; 119: 1607-1616Crossref PubMed Scopus (182) Google Scholar,12Dhanabal M Wu F Alvarez E McQueeney KD Jeffers M MacDougall J et al.Recombinant semaphorin 6A-1 ectodomain inhibits in vivo growth factor and tumor cell line-induced angiogenesis.Cancer Biol Ther. 2005; 4: 659-668Crossref PubMed Scopus (56) Google Scholar This contrasting function led to the development of an RNA-derived drug that specifically targets the miR-27a-VE-Cadherin interaction and has potent antipermeability properties and has suitable drug characteristics.10Young JA Ting KK Li J Moller T Dunn L Lu Y et al.Regulation of vascular leak and recovery from ischemic injury by general and VE-cadherin-restricted miRNA antagonists of miR-27.Blood. 2013; 122: 2911-2919Crossref PubMed Scopus (49) Google ScholarMiR-27 is part of a poly-cistronic group of cotranscribed miRs, including 23 and 24. There are two copies of this miR cistron in the genome, miR-23a-27a-24 being intergenic with its own promoter on chromosome 19 and miR-23b-27b-24, being intronic on chromosome 9. This organization has been highly preserved in vertebrates and also in fish, albeit less closely clustered.13Liang T Yu J Liu C Guo L An exploration of evolution, maturation, expression and function relationships in mir-23 ∼ 27 ∼ 24 cluster.PLoS One. 2014; 9: e106223Crossref PubMed Scopus (19) Google Scholar,14Wienholds E Kloosterman WP Miska E Alvarez-Saavedra E Berezikov E de Bruijn E et al.MicroRNA expression in zebrafish embryonic development.Science. 2005; 309: 310-311Crossref PubMed Scopus (1322) Google Scholar Members of the cluster have been described to have functions in several organ/developmental systems, including in cancer,15Acunzo M Romano G Palmieri D Laganá A Garofalo M Balatti V et al.Cross-talk between MET and EGFR in non-small cell lung cancer involves miR-27a and Sprouty2.Proc Natl Acad Sci USA. 2013; 110: 8573-8578Crossref PubMed Scopus (97) Google Scholar the central nervous system 16Lin ST Huang Y Zhang L Heng MY Ptácek LJ Fu YH MicroRNA-23a promotes myelination in the central nervous system.Proc Natl Acad Sci USA. 2013; 110: 17468-17473Crossref PubMed Scopus (80) Google Scholar and vascular organs.17Chhabra R Adlakha YK Hariharan M Scaria V Saini N Upregulation of miR-23a-27a-24-2 cluster induces caspase-dependent and -independent apoptosis in human embryonic kidney cells.PLoS One. 2009; 4: e5848Crossref PubMed Scopus (122) Google Scholar,18Chhabra R Dubey R Saini N Cooperative and individualistic functions of the microRNAs in the miR-23a≃27a≃24-2 cluster and its implication in human diseases.Mol Cancer. 2010; 9: 232Crossref PubMed Scopus (257) Google Scholar Most recently the cluster has been shown to regulate T cell differentiation.19Cho S Wu CJ Yasuda T Cruz LO Khan AA Lin LL et al.miR-23∼27∼24 clusters control effector T cell differentiation and function.J Exp Med. 2016; 213: 235-249Crossref PubMed Scopus (95) Google Scholar In the vascular system there has been a linkage primarily to angiogenesis, cardiac development and apoptosis.5Zhou Q Gallagher R Ufret-Vincenty R Li X Olson EN Wang S Regulation of angiogenesis and choroidal neovascularization by members of microRNA-23≃27≃24 clusters.Proc Natl Acad Sci USA. 2011; 108: 8287-8292Crossref PubMed Scopus (292) Google Scholar,11Urbich C Kaluza D Frömel T Knau A Bennewitz K Boon RA et al.MicroRNA-27a/b controls endothelial cell repulsion and angiogenesis by targeting semaphorin 6A.Blood. 2012; 119: 1607-1616Crossref PubMed Scopus (182) Google Scholar,20Chinchilla A Lozano E Daimi H Esteban FJ Crist C Aranega AE et al.MicroRNA profiling during mouse ventricular maturation: a role for miR-27 modulating Mef2c expression.Cardiovasc Res. 2011; 89: 98-108Crossref PubMed Scopus (83) Google Scholar,21Ruan W Xu JM Li SB Yuan LQ Dai RP Effects of down-regulation of microRNA-23a on TNF-α-induced endothelial cell apoptosis through caspase-dependent pathways.Cardiovasc Res. 2012; 93: 623-632Crossref PubMed Scopus (61) Google Scholar,22Biyashev D Veliceasa D Topczewski J Topczewska JM Mizgirev I Vinokour E et al.miR-27b controls venous specification and tip cell fate.Blood. 2012; 119: 2679-2687Crossref PubMed Scopus (103) Google Scholar However bioinformatic analysis of the potential targets of these miRs finds a concentration of molecules expressed in adherens and tight junctions,13Liang T Yu J Liu C Guo L An exploration of evolution, maturation, expression and function relationships in mir-23 ∼ 27 ∼ 24 cluster.PLoS One. 2014; 9: e106223Crossref PubMed Scopus (19) Google Scholar and in particular, we note representation of almost all known members of endothelial cell–cell junctions, giving rise to the possibility that endothelial junctional regulation is a chief function of these coevolved set of miRs.Mature sequences of miR-23a and miR-23b have identical seed sequences and only differ by one nucleotide in the nonseed region. As a result, in silico target analysis (Targetscan, DIANA lab, and www.microRNA.org) has suggested they share the same putative target genes and thus are likely to have similar biological functions. Despite reports showing distinct function between miRs with the same seed sequence, few studies have focused on the possible difference in the biological effects or the targets of these miRs.23Lovat F Fassan M Gasparini P Rizzotto L Cascione L Pizzi M et al.miR-15b/16-2 deletion promotes B-cell malignancies.Proc Natl Acad Sci USA. 2015; 112: 11636-11641Crossref PubMed Scopus (83) Google Scholar,24Zou J Li WQ Li Q Li XQ Zhang JT Liu GQ et al.Two functional microRNA-126s repress a novel target gene p21-activated kinase 1 to regulate vascular integrity in zebrafish.Circ Res. 2011; 108: 201-209Crossref PubMed Scopus (62) Google Scholar,25Lin R Chen L Chen G Hu C Jiang S Sevilla J et al.Targeting miR-23a in CD8+ cytotoxic T lymphocytes prevents tumor-dependent immunosuppression.J Clin Invest. 2014; 124: 5352-5367Crossref PubMed Scopus (89) Google Scholar By using both gain- and loss-of-function approaches, show that in ECs, miR-23a and miR-23b do not behave in the expected identical manner. Overexpression of miR-23a inhibits EC permeability whereas miR-23b increases permeability. Moreover, miR-23b but not miR-23a overexpression regulates angiogenesis. Importantly, such differences are also seen in vivo. Further, we demonstrate that these biological differences likely are attributed to the differential regulation of miR-23a and miR-23b on EC junctional molecules in either a direct or indirect manner. Together our findings provide substantial evidence for the evolution of a vascular permeability miR operon, and demonstrate the differing potent effects of miR-23s on their target genes and diverse role on EC function. The findings have both physiological and clinical relevance at a time where miRNAs and their inhibitors are entering the clinic.ResultsThe miR-23-27-24 cistron targets endothelial junctional proteinsWe examined the predicted targets of the individual members of the miR-23-27-24 cistron. 2,517 genes are predicted (by TargetScan 6.2) to be targets of at least one member of miR-23-27-24 cluster. A gene set enrichment analysis (GSEA) revealed that many of the predicted targets (see Supplementary Table S1) are involved in the adherens junctions pathway (P = 9.67 e−18) with a highly significant False Discovery Rate q-values of 8.01 e−16.All of the 17 major junctional and 18 downstream signaling or junctional regulating proteins (Figure 1a) known to play important roles in regulating EC junctions,3Li X Stankovic M Lee BP Aurrand-Lions M Hahn CN Lu Y et al.JAM-C induces endothelial cell permeability through its association and regulation of β3 integrins.Arterioscler Thromb Vasc Biol. 2009; 29: 1200-1206Crossref PubMed Scopus (38) Google Scholar,5Zhou Q Gallagher R Ufret-Vincenty R Li X Olson EN Wang S Regulation of angiogenesis and choroidal neovascularization by members of microRNA-23≃27≃24 clusters.Proc Natl Acad Sci USA. 2011; 108: 8287-8292Crossref PubMed Scopus (292) Google Scholar,26Dejana E Tournier-Lasserve E Weinstein BM The control of vascular integrity by endothelial cell junctions: molecular basis and pathological implications.Dev Cell. 2009; 16: 209-221Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar,27Dejana E Endothelial cell-cell junctions: happy together.Nat Rev Mol Cell Biol. 2004; 5: 261-270Crossref PubMed Scopus (885) Google Scholar,28Nagashima Y Oosako T Takase Y Ejiri A Watanabe O Kobayashi H et al.Observation of beat oscillation generation by coupled waves associated with parametric decay during radio frequency wave heating of a spherical tokamak plasma.Phys Rev Lett. 2010; 104: 245002Crossref PubMed Scopus (6) Google Scholar,29Nie L Guo X Esmailzadeh L Zhang J Asadi A Collinge M et al.Transmembrane protein ESDN promotes endothelial VEGF signaling and regulates angiogenesis.J Clin Invest. 2013; 123: 5082-5097Crossref PubMed Scopus (40) Google Scholar,30Singleton PA Dudek SM Chiang ET Garcia JG Regulation of sphingosine 1-phosphate-induced endothelial cytoskeletal rearrangement and barrier enhancement by S1P1 receptor, PI3 kinase, Tiam1/Rac1, and alpha-actinin.FASEB J. 2005; 19: 1646-1656Crossref PubMed Scopus (244) Google Scholar,31Bratt A Birot O Sinha I Veitonmäki N Aase K Ernkvist M et al.Angiomotin regulates endothelial cell-cell junctions and cell motility.J Biol Chem. 2005; 280: 34859-34869Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar,32Lee HJ Cho CH Hwang SJ Choi HH Kim KT Ahn SY et al.Biological characterization of angiopoietin-3 and angiopoietin-4.FASEB J. 2004; 18: 1200-1208Crossref PubMed Scopus (132) Google Scholar,33Broermann A Winderlich M Block H Frye M Rossaint J Zarbock A et al.Dissociation of VE-PTP from VE-cadherin is required for leukocyte extravasation and for VEGF-induced vascular permeability in vivo..J Exp Med. 2011; 208: 2393-2401Crossref PubMed Scopus (142) Google Scholar are potential targets of at least one member of miR-23-27-24 clusters. Thus, it is highly likely that the whole cistron has a major role in governing endothelial function.Of the predicted EC junctional targets of miR-23-27-24, three (VE-cadherin, SEMA6A, Sprouty2) have been confirmed by our group10Young JA Ting KK Li J Moller T Dunn L Lu Y et al.Regulation of vascular leak and recovery from ischemic injury by general and VE-cadherin-restricted miRNA antagonists of miR-27.Blood. 2013; 122: 2911-2919Crossref PubMed Scopus (49) Google Scholar and by others.24Zou J Li WQ Li Q Li XQ Zhang JT Liu GQ et al.Two functional microRNA-126s repress a novel target gene p21-activated kinase 1 to regulate vascular integrity in zebrafish.Circ Res. 2011; 108: 201-209Crossref PubMed Scopus (62) Google Scholar Here we focus on targets of miR-23 namely the expression of five of the most conserved (ZO-1, ZO-2, JAM-C, VE-PTP, and CCM2). Human umbilical vein endothelial cells (HUVEC) (Figure 1b) or human cerebral microvascular endothelial cell line (hCMEC/D3) (see Supplementary Figure S1a, b) transfected with either locked nucleic acid (LNA) to miR-23 or mimics of miR-23a or miR-23b. Mimics to miR-23a and miR-23b were able to specifically increase miR-23a and miR-23b expression respectively (see Supplementary Figure S2a), whereas with the LNA, both miR-23a and miR-23b was reduced to a similar level (see Supplementary Figure S2b), The manipulation of endogeneous miR-23a/b led to appropriate up or down regulation of all five genes, as measured by polymerase chain reaction (PCR). Therefore there are at least eight major EC junctional molecule targeted, either directly or indirectly, by the miR-23-27-24 cistron5Zhou Q Gallagher R Ufret-Vincenty R Li X Olson EN Wang S Regulation of angiogenesis and choroidal neovascularization by members of microRNA-23≃27≃24 clusters.Proc Natl Acad Sci USA. 2011; 108: 8287-8292Crossref PubMed Scopus (292) Google Scholar,10Young JA Ting KK Li J Moller T Dunn L Lu Y et al.Regulation of vascular leak and recovery from ischemic injury by general and VE-cadherin-restricted miRNA antagonists of miR-27.Blood. 2013; 122: 2911-2919Crossref PubMed Scopus (49) Google Scholar,11Urbich C Kaluza D Frömel T Knau A Bennewitz K Boon RA et al.MicroRNA-27a/b controls endothelial cell repulsion and angiogenesis by targeting semaphorin 6A.Blood. 2012; 119: 1607-1616Crossref PubMed Scopus (182) Google Scholar,22Biyashev D Veliceasa D Topczewski J Topczewska JM Mizgirev I Vinokour E et al.miR-27b controls venous specification and tip cell fate.Blood. 2012; 119: 2679-2687Crossref PubMed Scopus (103) Google Scholar,29Nie L Guo X Esmailzadeh L Zhang J Asadi A Collinge M et al.Transmembrane protein ESDN promotes endothelial VEGF signaling and regulates angiogenesis.J Clin Invest. 2013; 123: 5082-5097Crossref PubMed Scopus (40) Google Scholar,34Ferreira Tojais N Peghaire C Franzl N Larrieu-Lahargue F Jaspard B Reynaud A et al.Frizzled7 controls vascular permeability through the Wnt-canonical pathway and cross-talk with endothelial cell junction complexes.Cardiovasc Res. 2014; 103: 291-303Crossref PubMed Scopus (24) Google Scholar(bold in Figure 1a).Taken together, these data indicate that miR-23-27-24 targets are specifically highly enriched in EC tight and adherens junctions. We thus propose that the miR-23-27-24 cluster serves as a vascular cell–cell junctional operon. It should be noted that the miRs target both attractive and repulsive proteins, thus there is likely to be a fine tuning in the release of these miRs or access to their respective targets to coordinate the opening and closing of cell–cell junctions.Expression patterns of miR-23a and miR-23b in angiogenesisThe miR-23-27-24 cluster was originally identified using microarray analysis (GSE50437) and found to be regulated in ECs during in vitro 3-dimensional (3D) collagen angiogenesis assay. Both miR-23a and miR-23b showed a rapid and sustained downregulation in their expression (Figure 2a,b). This microarray data for miR23 was validated in the 3D collagen angiogenesis assay by quantitative PCR (Figure 2c). The pattern is similar to that we have previously observed with miR-27a.10Young JA Ting KK Li J Moller T Dunn L Lu Y et al.Regulation of vascular leak and recovery from ischemic injury by general and VE-cadherin-restricted miRNA antagonists of miR-27.Blood. 2013; 122: 2911-2919Crossref PubMed Scopus (49) Google Scholar Since the earliest events in in vitro angiogenesis are cell movement and alignment, the rapid decrease in miR maybe associated with loss of cell–cell interactions or cell migration. To test this we measured miR expression in migrating ECs, in a modified “scratch assay”, from a wounded edge of a confluent monolayer (Figure 2d). Both miR-23a and miR-23b were rapidly down-regulated within 30 minutes of wounding and also at times that reflect the migration stage. Normal levels of expression of both miRs are achieved by 6 hours and times thereafter (Figures 2e,f). These data indicate that miR-23a and miR-23b were both down-regulated in the early stage of angiogenesis, times associated with loss of cell–cell interactions and the induction of migration.Figure 2Expression pattern of miR-23a and miR-23b during angiogenesis. (a) Expression of miR-23a and (b) miR-23b in EC during in vitro 3D collagen angiogenesis assay measured by microarray. (c) Confirmation of microarray by real-time PCR. C = nonangiogenic, 3D = in vitro 3D collagen angiogenesis assay. mean ± SEM; n = 2. (d) Morphology of EC either before wounding (0 hour) and 0.5 and 24 hours after wounding. Representative of three experiments. Scale bar = 100 μm. Quantification of miR-23a (e) and miR-23b(f) after scratch (mean ± SEM; n = 3). Data is compared with the levels in the confluent cells. Expression levels were measured by qRT-PCR with results of microRNA normalized to U48. *P < 0.05; ***P < 0.001; ****P < 0.0001 by unpaired two-tailed t-test. qRT-PCR, quantitative reverse transcriptase polymerase chain reaction.View Large Image Figure ViewerDownload Hi-res image Download (PPT)MiR-23a and miR-23b differentially regulate angiogenesisTo test whether the down-regulation of miR-23 family is essential to the angiogenic process, we manipulated the levels of the miRs and investigated changes in the early phases of an angiogenesis assay.The knockdown of miR-23a and miR-23b by LNA-23 significantly enhanced tube formation on Matrigel, as quantified by tube length, tube number and number of branching points (Figure 3a). These results indicate that down-regulation of miR-23 promotes angiogenesis in vitro. Next, we investigated the role of each of the individual miRs in angiogenesis. Since LNAs against the individual members could not be designed to achieve specificity, we used mimics of each of these transfected into ECs. Cells were seeded onto Matrigel 48 hours after transfection. Different cell concentrations were used in order to see enhancement or inhibition of tube formation (See “Materials and Methods”). Overexpression of miR-23b, but not miR-23a significantly impaired the formation of capillary-like structures in ECs causing a decrease of total tube length, total tube numbers, and total branching points (Figure 3b). This was interesting as miR-23a and miR-23b are predicted to have the same targets due to their identical seed sequence and were similarly downregulated in angiogenesis (Figure 2a,b). The difference in the regulation of angiogenesis between miR-23a and miR-23b also occurs in vivo. Matrigel plugs containing either miR23a, miR-23b or control miR mimic were injected into mice and the neovascularization quantified by total vessel area and average vessel size, as determined by immunohistochemical analyses of CD31 expression (Figure 3c). MiR-23b containing plugs significantly inhibited the angiogenic response whereas the miR-23a mimic treated mice did not show any significant change compared with control. Taken together, these data indicate that miR-23b but not miR-23a regulates angiogenesis.Figure 3Mir-23b but not and miR-23a regulates EC tube formation. (a) LNA-23 increased HUVEC tubule formation on Matrigel. (b) Over-expression of miR-23b but not miR-23a inhibits tubule formation on Matrigel. Cells were monitored and photos taken every hour, for 7 hours. Representative images all taken at the same time point for each experiment are shown with a scale bar = 100 μm. Images analysis and quantification were performed by Wimasis software (Wimasis GmbH). Graphs of tube length, number and branching points show the mean ± SEM. *P < 0.05; **P < 0.005; ****P < 0.0001 by unpaired two-tailed t-test. n = 3 independent EC lines. (c) Angiogenic effects of miR-23a and miR-23b in mice. Mice received subcutaneous injection of 0.5 ml of a Matrigel mixture containing FGF-2, Heparin, and mimics. The animals were killed and gels dissected 2 weeks later. 5 μm sections were stained for CD31 (n = 3–4 for each plug) and a representative image is shown. Pixel histogram quantitation of percentages of vessel area (CD31 positive staining) and average vessel size with miR-23a, miR-23b or control mimic is shown. Each group contained 3–4 animals. Scale bar = 200 μm, *P < 0.05, **P < 0.01. NS = not significant. LNA, locked nucleic acid; HUVEC, human umbilical vein endothelial cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)MiR-23a and miR-23b regulate vascular permeability differentlyChanges in vascular permeability are associated with pathological angiogenesis.35Dvorak HF Brown LF Detmar M Dvorak AM Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis.Am J Pathol. 1995; 146: 1029-1039PubMed Google Scholar,36Dvorak HF Detmar M Claffey KP Nagy JA van de Water L Senger DR Vascular permeability factor/vascular endothelial growth factor: an important mediator of angiogenesis in malignancy and inflammation.Int Arch Allergy Immunol. 1995; 107: 233-235Crossref PubMed Scopus (318) Google Scholar We investigated whether miR-23a and miR-23b impacted on permeability. Since EC junctions are directly involved in regulating vascular permeability, we first examined the EC junction pattern, as shown by VE-cadherin staining, after miR-23 mimics treatment. In a loosely packed HUVEC monolayer, the control cells showed open zipper–like staining for VE-cadherin, whereas miR-23a mimic treated cells showed a smoother and continuous VE-cadherin staining at both 1.5 nmol/l and 15 nmol/l, indicating tightly apposed junctions (Figure 4a). In contrast, more gaps and wider zipper-like staining for VE-cadherin between cells were observed in miR-23b mimic treated cells particularly in the cells that received 15 nmol/l of 23b mimic (Figure 4a, white arrows). These data indicate miR-23a and miR-23b may have opposite roles in the regulation of cellular junctions and hence permeability, where miR-23a may inhibit permeability while miR-23b may promote permeability.Figure 4MiR-23a inhibits and miR-23b enhances EC permeability. Cells were stimulated with 0.1–0.2 U/ml of thrombin, 10 µmol/l of histamine or 50 ng/ml of human VEGF165 for 30 minutes as indicated for in vitro experiments. (a) 48 hours after 1.5 or 15 nmol/l of miR-23a or miR-23b mimic treatment, HUVEC were stained for VE-cadherin (green). White arrows = examples of changed adherens junctions (VE-cadherin-staining). (b,d) Permeability measured in control or miR-23a (b) or miR-23b (d)-mimic–transfected cells without (N) or with thrombin stimulation (T). n = 4 experiments. (c,e) Permeability measured at basal level in control or cells transfected with 1.5 or 15 nmol/l miR-23a-mimic (c) or miR-23b (e). n = 3 experiments, where values are normalized to control (C). (f,g) Permeability was measured without (N) or with thrombin (T, n = 6 experiments), histamine (H, n = 4 experiments) or VEGF (V, n = 5 experiments) stimulation in control or miR-23 LNA transfected EC. (h) The Miles assay was performed with 4 µg of control mimic or miR-23a or miR-23b mimic injected intradermally into the back of the mice. 24 hours later, 200 μl 0.5% Evan's Blue was injected intravenously. After 30 minutes, 10 ng VEGF (V) or phosphate-buffered saline (N) was given into the same site as the mimic. Mice were sacrificed 30 minutes later and the dye was extracted from skin samples and quantified; # mice are: N+C, n = 10; N+23a, n = 6; N+23b, n = 5; V+C, n = 12, V+23a, n = 11; V+23b, n = 6. Representative photos of lesions are given. (i) The Miles assay was performed with 4 µg of control LNA, LNA-23 or LNA-27 without (N) or with VEGF stimulation (V) with set up given as in f above. # mice are: N+C, n = 16; N+23, n = 16; N+27, n = 8; V+C, n = 16, V+23, n = 16; V+27, n = 8. All graph represents mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001 by unp" @default.
• W2511855771 created "2016-09-16" @default.
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• W2511855771 creator A5039810326 @default.
• W2511855771 creator A5043141098 @default.
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• W2511855771 date "2016-01-01" @default.
• W2511855771 modified "2023-10-16" @default.
• W2511855771 title "The Poly-cistronic miR-23-27-24 Complexes Target Endothelial Cell Junctions: Differential Functional and Molecular Effects of miR-23a and miR-23b" @default.
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• W2511855771 doi "https://doi.org/10.1038/mtna.2016.62" @default.
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