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OBJECTIVE: Restenosis is a frequent complication of angioplasty. It consists of a neointimal hyperplasia resulting from progression and migration of vascular smooth muscle cells (VSMC) into the vessel lumen. microRNA miR-223 has recently been shown to be involved in cardiovascular diseases including atherosclerosis, vascular calcification and arterial thrombosis. In this study, our aim was to assess the impact of miR-223 modulation on restenosis in a rat model of carotid artery after balloon injury. METHODS: The over and down-expression of miR-223 was induced by adenoviral vectors, containing either a pre-miR-223 sequence allowing artificial miR-223 expression or a sponge sequence, trapping the native microRNA, respectively. Restenosis was quantified on stained rat carotid sections. RESULTS: In vitro, three mRNA (Myocyte Enhancer Factor 2C (MEF2C), Ras homolog gene family, member B (RhoB) and Nuclear factor 1 A-type (NFIA)) reported as miR-223 direct targets and known to be implicated in VSMC differentiation and contractility were studied by RT-qPCR. Our findings showed that down-expression of miR-223 significantly reduced neointimal hyperplasia by 44% in carotids, and was associated with a 2-3-fold overexpression of MEF2C, RhoB and NFIA in a murine monocyte macrophage cell line, RAW 264.7 cells. CONCLUSION: Down-regulating miR-223 could be a potential therapeutic approach to prevent restenosis after angioplasty.
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Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/terapia , Estenosis Carotídea/prevención & control , Terapia Genética , MicroARNs/metabolismo , Angioplastia de Balón , Animales , Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Estenosis Carotídea/genética , Estenosis Carotídea/metabolismo , Estenosis Carotídea/patología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Masculino , Ratones , MicroARNs/genética , Neointima , Células RAW 264.7 , Ratas Wistar , Transducción de SeñalAsunto(s)
Regulación de la Expresión Génica/fisiología , Macrófagos/metabolismo , MicroARNs/metabolismo , Monocitos/metabolismo , Animales , Proteínas Portadoras/biosíntesis , Proteínas de Drosophila/biosíntesis , Genómica , Macrófagos/citología , Ratones , Monocitos/citología , Proteína-Arginina N-Metiltransferasas/biosíntesis , Células RAW 264.7 , Enzimas Ubiquitina-Conjugadoras/biosíntesisRESUMEN
[This retracts the article DOI: 10.1093/ckj/sfw060.].
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BACKGROUND: MicroRNAs (miRNAs) are innovative and informative blood-based biomarkers involved in numerous pathophysiological processes. In this study and based on our previous experimental data, we investigated miR-126, miR-143, miR-145, miR-155 and miR-223 as potential circulating biomarkers for the diagnosis and prognosis of patients with chronic kidney disease (CKD). The primary objective of this study was to assess the levels of miRNA expression at various stages of CKD. METHODS: RNA was extracted from serum, and RT-qPCR was performed for the five miRNAs and cel-miR-39 (internal control). RESULTS: Serum levels of miR-143, -145 and -223 were elevated in patients with CKD compared with healthy controls. They were further increased in chronic haemodialysis patients, but were below control levels in renal transplant recipients. In contrast, circulating levels of miR-126 and miR-155 levels, which were also elevated in CKD patients, were lower in the haemodialysis group and even lower in the transplant group. Four of the five miRNA species were correlated with estimated glomerular filtration rate, and three were correlated with circulating uraemic toxins. CONCLUSIONS: This exploratory study suggests that specific miRNAs could be biomarkers for complications of CKD, justifying further studies to link changes of miRNA levels with outcomes in CKD patients.
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OBJECTIVE: Embolization of carotid stenotic plaques is the direct cause of stroke in nearly 20% of cases. Genetic mechanisms and especially the roles played by microRNAs in the regulation of plaque destabilization and rupture are mostly unknown. The aim of this pilot study was to compare the expression of seven microRNAs allegedly involved in plaque growth and instability (miR-100, 125a, 127, 133a, 145, 155, and 221), between symptomatic and asymptomatic human carotid plaques. METHODS: Thirty patients undergoing carotid endarterectomy in our department were prospectively included. Carotid plaques were subdivided into symptomatic (n = 15) and asymptomatic (n = 15) according to the presence or absence of stroke. After isolation of total RNA from atherosclerotic plaques, microRNAs were quantified by real-time polymerase chain reaction. RESULTS: The two groups of patients were comparable in terms of age, gender, risk factors for cerebral ischemia, medication, and stenosis severity. All seven microRNAs were quantified in extracted carotid plaques. miR-100, miR-125a, miR-127, miR-133a, miR-145, and miR-221 were significantly overexpressed in symptomatic vs asymptomatic plaques. miR-125a expression was significantly inversely correlated with the circulating level of low-density lipoprotein cholesterol in the symptomatic group. CONCLUSIONS: This pilot study evaluated the expression of seven selected miRNAs in human carotid plaques from a small group of patients and suggested a potential regulatory role for these miRNAs in evolution of the plaque towards growth, instability and rupture. Studies based on larger sample sizes are required to determine the potential use of miR-100, miR-125a, miR-127, miR-133a, miR-145, and miR-221 as biomarkers or therapeutic targets for stroke.
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Enfermedades de las Arterias Carótidas/genética , Arteria Carótida Interna/química , Embolia/genética , Ataque Isquémico Transitorio/genética , MicroARNs/genética , Placa Aterosclerótica , Accidente Cerebrovascular/genética , Anciano , Biomarcadores/sangre , Enfermedades de las Arterias Carótidas/complicaciones , Enfermedades de las Arterias Carótidas/diagnóstico , Enfermedades de las Arterias Carótidas/cirugía , Arteria Carótida Interna/diagnóstico por imagen , Arteria Carótida Interna/cirugía , Creatinina/sangre , Embolia/diagnóstico , Endarterectomía Carotidea , Femenino , Francia , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Ataque Isquémico Transitorio/diagnóstico , Lípidos/sangre , Masculino , Persona de Mediana Edad , Fenotipo , Proyectos Piloto , Pronóstico , Estudios Prospectivos , Reacción en Cadena en Tiempo Real de la Polimerasa , Rotura Espontánea , Accidente Cerebrovascular/diagnóstico , Tomografía Computarizada por Rayos X , Ultrasonografía DopplerRESUMEN
Chronic kidney disease-mineral and bone disorder (CKD-MBD) is a common complication of CKD, and uremic toxins have been shown to be instrumental in this process. We have previously shown that miR-223 is increased in smooth muscle cells subjected to the uremic toxin inorganic phosphate (Pi). In the present study we investigated the influence of this miRNA in osteoclastogenesis in order to elucidate its role in the course of CKD-MBD. RT-qPCR demonstrated that high Pi concentration decreased miR-223 expression in differentiated RAW 264.7 cells. Up- and down-regulation of miR-223 was performed using specific pre-miR and anti-miR-223. Differentiation of monocyte/macrophage precursors was assessed by using RAW 264.7 cells and peripheral blood mononuclear cells (PBMC). TRAP activity and bone resorption were used to measure osteoclast activity. Pi induced a marked decrease in osteoclastogenesis in RAW cells and miR-223 levels were concomitantly decreased. Anti-miR-223 treatment inhibited osteoclastogenesis in the same way as Pi. In contrast, overexpression of miR-223 triggered differentiation, as reflected by TRAP activity. We showed that miR-223 affected the expression of its target genes NFIA and RhoB, but also osteoclast marker genes and the Akt signalling pathway, which induces osteoclastogenesis. These results were confirmed by measuring bone resorption activity of human PBMC differentiated into osteoclasts. We thus demonstrate a role of miR-223 in osteoclast differentiation, with rational grounds to use deregulation of this miRNA to selectively increase osteoclast-like activity in calcified vessels of CKD-MBD. This approach could alleviate vascular calcification without altering bone structure.
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Morphology and changes in gene expression of vascular endothelium are mainly due to shear stress and inflammation. Cell phenotype modulation has been clearly demonstrated to be controlled by small noncoding micro-RNAs (miRNAs). This study focused on the effect of laminar shear stress (LSS) on human endothelial cells (HUVECs), with an emphasis on the role of miRNA-126 (miR-126). Exposure of HUVECs in vitro to LSS modified the shape of HUVECs and concomitantly regulated the expression of miR-126, vascular cell adhesion molecule 1 (VCAM-1), and syndecan-4 (SDC-4). A significant upregulation of miR-126 during long-term exposure to flow was shown. Interestingly, LSS enhanced SDC-4 expression on the HUVEC membranes. Overexpression of miR-126 in HUVECs decreased the levels of targets stromal cell-derived factor-1 SDF-1/CXCL12 and VCAM-1 but increased the expression of RGS16, CXCR4, and SDC-4. No significant difference in terms of cell proliferation and apoptosis was observed between scramble, anti-miR-126, and pre-miR-126 transfected HUVECs. In Apo-E KO/CKD mice aortas expressing a high level of miR-126, SDC-4 was concomitantly increased. In conclusion, our results suggest that miR-126 (i) is overexpressed by long-term LSS, (ii) has a role in up- and downregulation of genes involved in atherosclerosis, and (iii) affects SDC-4 expression.
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MicroARNs/metabolismo , Estrés Mecánico , Remodelación Vascular/genética , Actinas/metabolismo , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/metabolismo , Apoptosis/genética , Proliferación Celular/genética , Forma de la Célula , Quimiocina CXCL12/metabolismo , Femenino , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , Modelos Biológicos , Proteínas RGS/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores CXCR4/metabolismo , Sindecano-1/metabolismo , Sindecano-4/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Chronic kidney disease (CKD) is associated with vascular calcifications and atherosclerosis. There is a need for novel predictors to allow earlier diagnosis of these disorders, predict disease progression, and improve assessment of treatment response. We focused on microRNAs since they are implicated in a variety of cellular functions in cardiovascular pathology. We examined changes of microRNA expression in aortas of CKD and non-CKD wild type mice and apolipoprotein E knock-out mice, respectively. Both vascular smooth muscle-specific miR-143 and miR-145 expressions were decreased in states of atherosclerosis and/or CKD or both, and the expression level of protein target Myocardin was increased. The inflammatory miR-223 was increased in more advanced stages of CKD, and specific protein targets NFI-A and GLUT-4 were dramatically decreased. Expression of miR-126 was markedly increased and expression of protein targets VCAM-1 and SDF-1 was altered during the course of CKD. The drug sevelamer, commonly used in CKD, corrected partially these changes in microRNA expression, suggesting a direct link between the observed microRNA alterations and uremic vascular toxicity. Finally, miR-126, -143 and -223 expression levels were deregulated in murine serum during the course of experimental CKD. In conclusion, these miRNAs could have role(s) in CKD vascular remodeling and may therefore represent useful targets to prevent or treat complications of CKD.
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Aorta/metabolismo , Aterosclerosis/genética , MicroARNs/genética , Insuficiencia Renal Crónica/genética , Animales , Aorta/efectos de los fármacos , Aorta/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/complicaciones , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/patología , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Factores de Transcripción NFI/genética , Factores de Transcripción NFI/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poliaminas/farmacología , Insuficiencia Renal Crónica/complicaciones , Insuficiencia Renal Crónica/tratamiento farmacológico , Insuficiencia Renal Crónica/patología , Sevelamer , Transactivadores/genética , Transactivadores/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
BACKGROUND: An elevated serum inorganic phosphate (Pi) level is a major risk factor for kidney disease and downstream vascular complications. We focused on the effect of Pi levels on human aortic vascular smooth muscle cells (VSMCs), with an emphasis on the role of microRNAs (miRNAs). METHODOLOGY/PRINCIPAL FINDINGS: Exposure of human primary VSMCs in vitro to pathological levels of Pi increased calcification, migration rate and concomitantly reduced cell proliferation and the amount of the actin cytoskeleton. These changes were evidenced by significant downregulation of miRNA-143 (miR-143) and miR-145 and concomitant upregulation of their targets and key markers in synthetic VSMCs, such as Krüppel-like factors-4 and -5 and versican. Interestingly, we also found that miR-223 (a marker of muscle damage and a key factor in osteoclast differentiation) is expressed in VSMCs and is significantly upregulated in Pi-treated cells. Over-expressing miR-223 in VSMCs increased proliferation and markedly enhanced VSMC migration. Additionally, we found that the expression of two of the known miR-223 targets, Mef2c and RhoB, was highly reduced in Pi treated as well as miR-223 over-expressing VSMCs. To complement these in vitro findings, we also observed significant downregulation of miR-143 and miR-145 and upregulation of miR-223 in aorta samples collected from ApoE knock-out mice, which display vascular calcification. CONCLUSIONS/SIGNIFICANCE: Our results suggest that (i) high levels of Pi increase VSMC migration and calcification, (ii) altered expression levels of miR-223 could play a part in this process and (iii) miR-223 is a potential new biomarker of VSMC damage.