RESUMEN
BACKGROUND: eNOS (endothelial nitric oxide synthase) is an endothelial cell (EC)-specific gene predominantly expressed in medium- to large-sized arteries where ECs experience atheroprotective laminar flow with high shear stress. Disturbed flow with lower average shear stress decreases eNOS transcription, which leads to the development of atherosclerosis, especially at bifurcations and curvatures of arteries. This prototypic arterial EC gene contains 2 distinct flow-responsive cis-DNA elements in the promoter, the shear stress response element (SSRE) and the KLF (Krüppel-like factor) element. Previous in vitro studies suggested their positive regulatory functions on flow-induced transcription of EC genes including eNOS. However, the in vivo function of these cis-DNA elements remains unknown. METHODS: Insertional transgenic mice with a mutation at each flow-responsive cis-DNA element were generated using a murine eNOS promoter-ß-galactosidase reporter by linker-scanning mutagenesis and compared with episomal-based mutations in vitro. DNA methylation at the eNOS proximal promoter in mouse ECs was assessed by bisulfite sequencing or pyrosequencing. RESULTS: Wild type mice with a functional eNOS promoter-reporter transgene exhibited reduced endothelial reporter expression in the atheroprone regions of disturbed flow (n=5). It is surprising that the SSRE mutation abrogated reporter expression in ECs and was associated with aberrant hypermethylation at the eNOS proximal promoter (n=7). Reporter gene silencing was independent of transgene copy number and integration position, indicating that the SSRE is a critical cis-element necessary for eNOS transcription in vivo. The KLF mutation demonstrated an integration site-specific decrease in eNOS transcription, again with marked promoter methylation (n=8), suggesting that the SSRE alone is not sufficient for eNOS transcription in vivo. In wild type mice, the native eNOS promoter was significantly hypermethylated in ECs from the atheroprone regions where eNOS expression was markedly repressed by chronic disturbed flow, demonstrating that eNOS expression is regulated by flow-dependent DNA methylation that is region-specific in the arterial endothelium in vivo. CONCLUSIONS: We report, for the first time, that the SSRE and KLF elements are critical flow sensors necessary for a transcriptionally permissive, hypomethylated eNOS promoter in ECs under chronic shear stress in vivo. Moreover, eNOS expression is regulated by flow-dependent epigenetic mechanisms, which offers novel mechanistic insight on eNOS gene regulation in atherogenesis.
Asunto(s)
Regulación de la Expresión Génica , Óxido Nítrico Sintasa de Tipo III/genética , Secuencias Reguladoras de Ácidos Nucleicos , Elementos de Respuesta , Animales , Biomarcadores , Velocidad del Flujo Sanguíneo , Cromatina/genética , Cromatina/metabolismo , Metilación de ADN , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Células Endoteliales/metabolismo , Epigénesis Genética , Dosificación de Gen , Silenciador del Gen , Genes Reporteros , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Transgénicos , Mutación , Óxido Nítrico Sintasa de Tipo III/metabolismo , Regiones Promotoras Genéticas , Activación TranscripcionalRESUMEN
BACKGROUND: MicroRNA are essential posttranscriptional modulators of gene expression implicated in various chronic diseases. Because microRNA-145 is highly expressed in vascular smooth muscle cells (VSMC) and regulates VSMC fate and plasticity, we hypothesized that it may be a novel regulator of atherosclerosis and plaque stability. METHODS AND RESULTS: Apolipoprotein E knockout mice (ApoE(-/-)) mice were treated with either a microRNA-145 lentivirus under the control of the smooth muscle cell (SMC)-specific promoter SM22α or a SM22α control lentivirus before commencing the Western diet for 12 weeks. The SMC-targeted microRNA-145 treatment markedly reduced plaque size in aortic sinuses, ascending aortas, and brachiocephalic arteries. It also significantly increased fibrous cap area, reduced necrotic core area, and increased plaque collagen content. Cellular plaque composition analyses revealed significantly less macrophages in ApoE(-/-) mice treated with the SMC-specific microRNA-145. These mice also demonstrated marked increases in calponin levels and α-smooth muscle actin-positive SMC areas in their atherosclerotic lesions. Furthermore, lentiviral delivery of microRNA-145 resulted in reduced KLF4 and elevated myocardin expression in aortas from ApoE(-/-) mice, consistent with an effect of microRNA-145 to promote a contractile phenotype in VSMC. CONCLUSIONS: VSMC-specific overexpression of microRNA-145 is a novel in vivo therapeutic target to limit atherosclerotic plaque morphology and cellular composition, shifting the balance toward plaque stability vs plaque rupture.
Asunto(s)
Aterosclerosis/prevención & control , Terapia Genética , Vectores Genéticos/uso terapéutico , MicroARNs/fisiología , Actinas/genética , Animales , Aorta/citología , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/prevención & control , Apolipoproteínas E/deficiencia , Aterosclerosis/genética , Aterosclerosis/patología , Arterias Carótidas/metabolismo , Células Cultivadas , Dieta Aterogénica , Genes Reporteros , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/biosíntesis , Factores de Transcripción de Tipo Kruppel/genética , Lentivirus/genética , Lípidos/sangre , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/fisiología , Transducción GenéticaRESUMEN
Hemolytic uremic syndrome (HUS) is a potentially life-threatening condition. It often occurs after gastrointestinal infection with E. coli O157:H7, which produces Shiga toxins (Stx) that cause hemolytic anemia, thrombocytopenia, and renal injury. Stx-mediated changes in endothelial phenotype have been linked to the pathogenesis of HUS. Here we report our studies investigating Stx-induced changes in gene expression and their contribution to the pathogenesis of HUS. Stx function by inactivating host ribosomes but can also alter gene expression at concentrations that minimally affect global protein synthesis. Gene expression profiling of human microvascular endothelium treated with Stx implicated a role for activation of CXCR4 and CXCR7 by their shared cognate chemokine ligand (stromal cell-derived factor-1 [SDF-1]) in Stx-mediated pathophysiology. The changes in gene expression required a catalytically active Stx A subunit and were mediated by enhanced transcription and mRNA stability. Stx also enhanced the association of CXCR4, CXCR7, and SDF1 mRNAs with ribosomes. In a mouse model of Stx-mediated pathology, we noted changes in plasma and tissue content of CXCR4, CXCR7, and SDF-1 after Stx exposure. Furthermore, inhibition of the CXCR4/SDF-1 interaction decreased endothelial activation and organ injury and improved animal survival. Finally, in children infected with E. coli O157:H7, plasma SDF-1 levels were elevated in individuals who progressed to HUS. Collectively, these data implicate the CXCR4/CXCR7/SDF-1 pathway in Stx-mediated pathogenesis and suggest novel therapeutic strategies for prevention and/or treatment of complications associated with E. coli O157:H7 infection.
Asunto(s)
Quimiocina CXCL12/metabolismo , Síndrome Hemolítico-Urémico/etiología , Síndrome Hemolítico-Urémico/fisiopatología , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Toxinas Shiga/toxicidad , Animales , Línea Celular , Quimiocina CXCL12/genética , Niño , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Infecciones por Escherichia coli/complicaciones , Escherichia coli O157/metabolismo , Escherichia coli O157/patogenicidad , Expresión Génica/efectos de los fármacos , Síndrome Hemolítico-Urémico/patología , Humanos , Riñón/patología , Riñón/fisiopatología , Ratones , Análisis por Micromatrices , Análisis de Secuencia por Matrices de Oligonucleótidos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Receptores CXCR/genética , Receptores CXCR4/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
To elucidate the role of endothelial NO synthase (eNOS)-derived NO during mammalian embryogenesis, we assessed the expression of the eNOS gene during development. Using transgenic eNOS promoter/reporter mice (with beta-galactosidase and green fluorescent protein reporters), in situ cRNA hybridization, and immunohistochemistry to assess transcription, steady-state mRNA levels, and protein expression, respectively, we noted that eNOS expression in the developing cardiovascular system was highly restricted to endothelial cells of medium- and large-sized arteries and the endocardium. The onset of transcription of the native eNOS gene and reporters coincided with the establishment of robust, unidirectional blood flow at embryonic day 9.5, as assessed by Doppler ultrasound biomicroscopy. Interestingly, reporter transgene expression and native eNOS mRNA were also observed in discrete regions of the developing skeletal musculature and the apical ectodermal ridge of developing limbs, suggesting a role for eNOS-derived NO in limb development. In vitro studies of promoter/reporter constructs indicated that similar eNOS promoter regions operate in both embryonic skeletal muscle and vascular endothelial cells. In summary, transcriptional activity of the eNOS gene in the murine circulatory system occurred following the establishment of embryonic blood flow. Thus, the eNOS gene is a late-onset gene in endothelial ontogeny.
Asunto(s)
Sistema Cardiovascular/enzimología , Embrión de Mamíferos/enzimología , Desarrollo Embrionario/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Animales , Velocidad del Flujo Sanguíneo/fisiología , Sistema Cardiovascular/embriología , Células Endoteliales/enzimología , Ratones , Ratones Transgénicos , Músculo Esquelético/embriología , Músculo Esquelético/enzimología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo III , Especificidad de Órganos/fisiología , Regiones Promotoras Genéticas/fisiología , Transcripción Genética/fisiologíaRESUMEN
Inhibiting the actions of VEGF is a new therapeutic paradigm in cancer management with antiangiogenic therapy also under intensive investigation in a range of nonmalignant diseases characterized by pathological angiogenesis. However, the effects of VEGF inhibition on organs that constitutively express it in adulthood, such as the kidney, are mostly unknown. Accordingly, we examined the effect of VEGF inhibition on renal structure and function under physiological conditions and in the setting of the common renal stressors: hypertension and activation of the renin-angiotensin system. When compared with normotensive Sprague-Dawley (SD) rats, glomerular VEGF mRNA was increased 2-fold in transgenic (mRen-2)27 rats that overexpress renin with spontaneously hypertensive rat (SHR) kidneys showing VEGF expression levels that were intermediate between them. Administration of either an orally active inhibitor of the type 2 VEGF receptor (VEGFR-2) tyrosine kinase or a VEGF neutralizing antibody to TGR(mRen-2)27 rats resulted in loss of glomerular endothelial cells and transformation to a malignant hypertensive phenotype with severe glomerulosclerosis. VEGFR-2 kinase inhibition treatment was well tolerated in SDs and SHRs; although even in these animals there was detectable endothelial cell loss and rise in albuminuria. Mild mesangial expansion was also noted in hypertensive SHR, but not in SD rats. These studies illustrate: (i) VEGF has a role in the maintenance of glomerular endothelial integrity under physiological circumstances, (ii) glomerular VEGF is increased in response to hypertension and activation of the renin-angiotensin system, and (iii) VEGF signaling plays a protective role in the setting of these renal stressors.
Asunto(s)
Salud , Hipertensión/fisiopatología , Riñón/metabolismo , Riñón/fisiopatología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Línea Celular , Células Endoteliales/efectos de los fármacos , Células Endoteliales/ultraestructura , Regulación de la Expresión Génica , Humanos , Hipertensión/patología , Riñón/citología , Riñón/efectos de los fármacos , Pruebas de Función Renal , Masculino , Microscopía Electrónica de Transmisión , Fosforilación/efectos de los fármacos , Piperidinas/farmacología , Quinazolinas/farmacología , Ratas , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/inmunología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
This study was carried out to determine the role of increased vascular matrix metalloproteinase-2 (MMP-2) expression in the changes in systemic arterial contraction after prolonged hypoxia. Rats and mice were exposed to hypoxia (10% and 8% O(2), respectively) or normoxia (21% O(2)) for 16 h, 48 h, or 7 days. Aortae and mesenteric arteries were either mounted in organ bath myographs or frozen in liquid nitrogen. MMP-2 inhibition with cyclic CTTHWGFTLC (CTT) reduced contraction to phenylephrine (PE) in aortae and mesenteric arteries from rats exposed to hypoxia for 7 days but not in vessels from normoxic rats. Similarly, CTT reduced contraction to Big endothelin-1 (Big ET-1) in aortae from rats exposed to hypoxia for 7 days. Responses to PE were reduced in hypoxic MMP-2(-/-) mice compared with MMP-2(+/+) mice. Increased contraction to Big ET-1 after hypoxia was observed in MMP-2(+/+) mice but not in MMP-2(-/-) mice. Rat aortic MMP-2 and membrane type 1 (MT1)-MMP protein levels and MMP activity were increased after 7 days of hypoxia. Rat aortic MMP-2 and MT1-MMP mRNA levels were increased in the deep medial vascular smooth muscle. We conclude that hypoxic induction of MMP-2 expression potentiates contraction in systemic conduit and resistance arteries. This may preserve the capacity to regulate the systemic circulation in the transition between the alterations in vascular tone and structural remodeling that occurs during prolonged hypoxic epochs.
Asunto(s)
Arterias/fisiopatología , Endotelio Vascular/fisiopatología , Hipoxia/fisiopatología , Metaloproteinasa 2 de la Matriz/metabolismo , Músculo Liso Vascular/fisiopatología , Péptidos Cíclicos/farmacología , Vasoconstricción/efectos de los fármacos , Animales , Endotelio Vascular/efectos de los fármacos , Activación Enzimática , Masculino , Contracción Muscular/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Rapidly progressive glomerulonephritis (RPGN) is a clinical syndrome characterized by loss of renal function within days to weeks and by glomerular crescents on biopsy. The pathogenesis of this disease is unclear, but circulating factors are believed to have a major role. Here, we show that deletion of the Von Hippel-Lindau gene (Vhlh) from intrinsic glomerular cells of mice is sufficient to initiate a necrotizing crescentic glomerulonephritis and the clinical features that accompany RPGN. Loss of Vhlh leads to stabilization of hypoxia-inducible factor alpha subunits (HIFs). Using gene expression profiling, we identified de novo expression of the HIF target gene Cxcr4 (ref. 3) in glomeruli from both mice and humans with RPGN. The course of RPGN is markedly improved in mice treated with a blocking antibody to Cxcr4, whereas overexpression of Cxcr4 alone in podocytes of transgenic mice is sufficient to cause glomerular disease. Collectively, these results indicate an alternative mechanism for the pathogenesis of RPGN and glomerular disease in an animal model and suggest novel molecular pathways for intervention in this disease.
Asunto(s)
Glomerulonefritis/fisiopatología , Glomérulos Renales/metabolismo , Receptores CXCR4/biosíntesis , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proliferación Celular , Perfilación de la Expresión Génica , Glomerulonefritis/etiología , Glomerulonefritis/patología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Podocitos/citología , Podocitos/metabolismo , Regulación hacia ArribaRESUMEN
The basis for the endothelial cell-restricted expression of endothelial nitric-oxide synthase (eNOS) is not known. While transgenic promoter/reporter mice demonstrated endothelium cell-specific eNOS expression, we found robust expression of episomal eNOS promoter/reporter constructs in cell types that do not express the native eNOS transcript. To explore the mechanism underlying this differential activity pattern of chromatin-versus episome-based eNOS promoters, we examined the methylation status of 5'-regulatory sequences of the human eNOS gene. DNA methylation differed dramatically between endothelial and nonendothelial cell types, including vascular smooth muscle cells. This same cell type-specific methylation pattern was observed in vivo in endothelial and vascular smooth muscle cells of the mouse aorta at the native murine eNOS promoter. We addressed the functional consequences of methylation on eNOS transcription using transient transfection of in vitro methylated promoter/reporter constructs and found that methylated constructs exhibited a marked decrease in the synergistic action of Sp1, Sp3, and Ets1 on eNOS promoter activity. The addition of methyl-CpG-binding protein 2 further reduced the transcriptional activity of methylated eNOS constructs. Importantly, chromatin immunoprecipitation demonstrated the presence of Sp1, Sp3, and Ets1 at the native eNOS promoter in endothelial cells but not in vascular smooth muscle cells. Finally, robust expression of eNOS mRNA was induced in nonendothelial cell types following inhibition of DNA methyltransferase activity with 5-azacytidine, demonstrating the importance of DNA methylation-mediated repression. This report is the first to show that promoter DNA methylation plays an important role in the cell-specific expression of a constitutively expressed gene in the vascular endothelium.