miRNA-27a-3p and miRNA-222-3p as Novel Modulators of Phosphodiesterase 3a (PDE3A) in Cerebral Microvascular Endothelial Cells
Endothelial dysfunction is a key element in cerebral small vessel disease (CSVD), which may cause stroke and cognitive decline. Cyclic nucleotide signaling modulates endothelial function. The cyclic adenosine monophosphate-degrading enzyme phosphodiesterase 3 (PDE3) is an important treatment target which may be modulated by microRNAs (miRNAs) important for regulating gene expression. We aimed to identify PDE3-targeting miRNAs to highlight potential therapeutic targets for endothelial dysfunction and CSVD. PDE3-targeting miRNAs were identified by in silico analysis (TargetScan, miRWalk, miRanda, and RNA22). The identified miRNAs were ranked on the basis of TargetScan context scores and their expression (log2 read counts) in a human brain endothelial cell line (hCMEC/D3) described recently. miRNAs were subjected to co-expression meta-analysis (CoMeTa) to create miRNA clusters. The pathways targeted by the miRNAs were assigned functional annotations via the KEGG pathway and COOL. hCMEC/D3 cells were transfected with miRNA mimics miR-27a-3p and miR-222-3p, and the effect on PDE3A protein expression was analyzed by Western blotting. Only PDE3A is expressed in hCMEC/D3 cells. The in silico prediction identified 67 PDE3A-related miRNAs, of which 49 were expressed in hCMEC/D3 cells. Further analysis of the top two miRNA clusters (miR-221/miR-222 and miR-27a/miR-27b/miR-128) indicated a potential link to pathways relevant to cerebral and vascular integrity and repair. hCMEC/D3 cells transfected with miR-27a-3p and miR-222-3p mimics had reduced relative expression of PDE3A protein. PDE3A-related miRNAs miR-221/miR-222 and miR-27a/miR-27b/miR-128 are potentially linked to pathways essential for immune regulation as well as cerebral and vascular integrity/function. Furthermore, relative PDE3A protein expression was reduced by miR27a-3p and miR-222-3p.
KeywordsSmall vessel disease microRNA PDE3 Stroke Endothelial cells
Study concept and design: CK, AHM, SY, SK, and FP; acquisition of data: SK, AHM and SY; analysis and interpretation of data: SY, SK, CK, FP and BB; drafting of the manuscript: SY; critical revision of the manuscript for important intellectual content: SY, SK, CK, FP, AHM, and BB; approval of final manuscript: CK, SY, SK, FP, BB and AHM; obtained funding: SY and CK; study supervision, CK, FP and BB.
This work was funded by the Herlev Research Council. S.Y. is supported by the Department of Neurology, Herlev University Hospital. Running costs are supported by the Aase and Ejnar Danielsens Foundation, the Fonden for Lægevidenskabens Fremme, the Novo Nordic Foundation, and Direktør Jacob Madsen og Hustru Olga Madsens Fond.
Compliance with Ethical Standards
Conflicts of Interest
The authors declare that they have no conflict of interest.
- 5.de Donato G, Setacci F, Galzerano G, Mele M, Ruzzi U, Setacci C (2016) The use of cilostazol in patients with peripheral arterial disease: results of a national physician survey. J Cardiovasc Surg 57(3):457–465Google Scholar
- 8.Fukuhara S, Sakurai A, Sano H, Yamagishi A, Somekawa S, Takakura N, Saito Y, Kangawa K et al (2005) Cyclic AMP potentiates vascular endothelial cadherin-mediated cell-cell contact to enhance endothelial barrier function through an Epac-Rap1 signaling pathway. Mol Cell Biol 25(1):136–146PubMedPubMedCentralCrossRefGoogle Scholar
- 19.Keravis T, Lugnier C (2012) Cyclic nucleotide phosphodiesterase (PDE) isozymes as targets of the intracellular signalling network: benefits of PDE inhibitors in various diseases and perspectives for future therapeutic developments. Br J Pharmacol 165(5):1288–1305PubMedPubMedCentralCrossRefGoogle Scholar
- 23.Harndahl L, Wierup N, Enerback S, Mulder H, Manganiello VC, Sundler F, Degerman E, Ahren B et al (2004) Beta-cell-targeted overexpression of phosphodiesterase 3B in mice causes impaired insulin secretion, glucose intolerance, and deranged islet morphology. J Biol Chem 279(15):15214–15222PubMedCrossRefGoogle Scholar
- 24.Kwon SU, Cho YJ, Koo JS, Bae HJ, Lee YS, Hong KS, Lee JH, Kim JS (2005) Cilostazol prevents the progression of the symptomatic intracranial arterial stenosis: the multicenter double-blind placebo-controlled trial of cilostazol in symptomatic intracranial arterial stenosis. Stroke 36:782–786PubMedCrossRefGoogle Scholar
- 27.Hori M (2007) The phosphodiesterase 4D gene for early onset ischemic stroke among normotensive patients. Stroke 5(2):436–438Google Scholar
- 45.Nielsen LB, Wang C, Sorensen K, Bang-Berthelsen CH, Hansen L, Andersen ML, Hougaard P, Juul A et al (2012) Circulating levels of microRNA from children with newly diagnosed type 1 diabetes and healthy controls: evidence that miR-25 associates to residual beta-cell function and glycaemic control during disease progression. Exp Diabetes Res 2012:896362PubMedPubMedCentralGoogle Scholar
- 48.Urabe T (2013) Cilostazol strengthens barrier integrity in brain endothelial cells. Neurosci Res 33(2):291–307Google Scholar
- 50.Uchiyama S (2009) Stroke prevention by cilostazol in patients with atherothrombosis: meta-analysis of placebo-controlled randomized trials. J Stroke Cerebrovasc Dis 18(6):482–90Google Scholar
- 59.Sabirzhanov B, Zhao Z, Stoica BA, Loane DJ, Wu J, Borroto C, Dorsey SG, Faden AI (2014) Downregulation of miR-23a and miR-27a following experimental traumatic brain injury induces neuronal cell death through activation of proapoptotic Bcl-2 proteins. J Neurosci 34(30):10055–10071PubMedPubMedCentralCrossRefGoogle Scholar
- 61.Regev K, Paul A, Healy B, von Glenn F, Diaz-Cruz C, Gholipour T, Mazzola MA, Raheja R et al (2016) Comprehensive evaluation of serum microRNAs as biomarkers in multiple sclerosis. Neurol Neurophysiol Neurosci 3(5):e267Google Scholar
- 66.Malik R, Dichgans M, A.F. Consortium, H. Cohorts for, C. Aging Research in Genomic Epidemiology, C. International Genomics of Blood Pressure, I. Consortium, Starnet, G. BioBank Japan Cooperative Hospital, C. Consortium, E.-C. Consortium, E.P.-I. Consortium, C. International Stroke Genetics, M. Consortium, C.C. Neurology Working Group of the, N.S.G. Network, U.K.Y.L.D. Study, M. Consortium, M. Consortium (2018) Multiancestry genome-wide association study of 520,000 subjects identifies 32 loci associated with stroke and stroke subtypes. Nat Genet 50(4):524–537PubMedPubMedCentralCrossRefGoogle Scholar