RESUMO
Objective- Calcific aortic valve (AV) disease, characterized by AV sclerosis and calcification, is a major cause of death in the aging population; however, there are no effective medical therapies other than valve replacement. AV calcification preferentially occurs on the fibrosa side, exposed to disturbed flow (d-flow), whereas the ventricularis side exposed to predominantly stable flow remains protected by unclear mechanisms. Here, we tested the role of novel flow-sensitive UBE2C (ubiquitin E2 ligase C) and microRNA-483-3p (miR-483) in flow-dependent AV endothelial function and AV calcification. Approach and Results- Human AV endothelial cells and fresh porcine AV leaflets were exposed to stable flow or d-flow. We found that UBE2C was upregulated by d-flow in human AV endothelial cells in the miR-483-dependent manner. UBE2C mediated OS-induced endothelial inflammation and endothelial-mesenchymal transition by increasing the HIF-1α (hypoxia-inducible factor-1α) level. UBE2C increased HIF-1α by ubiquitinating and degrading its upstream regulator pVHL (von Hippel-Lindau protein). These in vitro findings were corroborated by immunostaining studies using diseased human AV leaflets. In addition, we found that reduction of miR-483 by d-flow led to increased UBE2C expression in human AV endothelial cells. The miR-483 mimic protected against endothelial inflammation and endothelial-mesenchymal transition in human AV endothelial cells and calcification of porcine AV leaflets by downregulating UBE2C. Moreover, treatment with the HIF-1α inhibitor (PX478) significantly reduced porcine AV calcification in static and d-flow conditions. Conclusions- These results suggest that miR-483 and UBE2C and pVHL are novel flow-sensitive anti- and pro-calcific AV disease molecules, respectively, that regulate the HIF-1α pathway in AV. The miR-483 mimic and HIF-1α pathway inhibitors may serve as potential therapeutics of calcific AV disease.
Assuntos
Estenose da Valva Aórtica/etiologia , Valva Aórtica/patologia , Calcinose/etiologia , Células Endoteliais/metabolismo , Hemorreologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , MicroRNAs/genética , Enzimas de Conjugação de Ubiquitina/biossíntese , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Animais , Valva Aórtica/metabolismo , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Calcinose/metabolismo , Calcinose/patologia , Adesão Celular , Transdiferenciação Celular , Células Cultivadas , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Inflamação , MicroRNAs/agonistas , Monócitos/fisiologia , Compostos de Mostarda/farmacologia , Oligonucleotídeos/farmacologia , Técnicas de Cultura de Órgãos , Fenilpropionatos/farmacologia , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Reologia , Estresse Mecânico , Suínos , Enzimas de Conjugação de Ubiquitina/fisiologia , UbiquitinaçãoRESUMO
BACKGROUND AND AIMS: Hypoxia inducible factor 1α (HIF1α) plays a critical role in atherosclerosis as demonstrated in endothelial-targeted HIF1α -deficient mice. However, it has not been shown if specific pharmacological inhibitors of HIF1α can be used as potential drugs for atherosclerosis. PX-478 is a selective inhibitor of HIF1α, which was used to reduce cancer and obesity in animal models. Here, we tested whether PX-478 can be used to inhibit atherosclerosis. METHODS: We first tested PX-478 in human aortic endothelial cells (HAEC) and found that it significantly inhibited expression of HIF1α and its targets, including Collagen I. Next, two independent atherosclerosis models, C57BL/6 mice treated with AAV-PCSK9 and ApoE-/- mice, were used to test the efficacy of PX-478. Both mouse models were fed a Western diet for 3 months with bi-weekly treatment with PX-478 (40 mg/kg) or saline. RESULTS: PX-478 treatment reduced atherosclerotic plaque burden in the aortic trees in both mouse models, while plaque burden in the aortic sinus was reduced in the AAV-PCSK9 mouse model, but not in the ApoE-/- mice. Russell-Movat's Pentachrome and Picrosirius Red staining showed a significant reduction in extracellular matrix remodeling and collagen maturation, respectively, in the PX-478-treated mice. As expected, PX-478 treatment reduced diet-induced weight-gain and abdominal adipocyte hypertrophy. Interestingly, PX-478 reduced plasma LDL cholesterol by 69% and 30% in AAV-PCSK9 and ApoE-/- mice, respectively. To explore the cholesterol-lowering mechanisms, we carried out an RNA sequencing study using the liver tissues from the ApoE-/- mouse study. We found 450 genes upregulated and 381 genes downregulated by PX-478 treatment in the liver. Further, gene ontology analysis showed that PX-478 treatment upregulated fatty acid and lipid catabolic pathways, while downregulating lipid biosynthesis and plasma lipoprotein particle remodeling processes. Of interest, Cfd, Elovl3, and Insig2 were some of the most downregulated genes by PX-478, and have been implicated in fat storage, fatty acid elongation, and cholesterol metabolism. The downregulation of Cfd, Elovl3, and Insig2 was further validated by qPCR in the liver tissues of ApoE-/- mice treated with PX-478. CONCLUSIONS: These results suggest that PX-478 is a potential anti-atherogenic drug, which targets vascular endothelium and hepatic cholesterol pathways.
Assuntos
Doenças da Aorta , Aterosclerose , Placa Aterosclerótica , Animais , Doenças da Aorta/tratamento farmacológico , Doenças da Aorta/genética , Doenças da Aorta/prevenção & controle , Apolipoproteínas E/genética , Aterosclerose/tratamento farmacológico , Aterosclerose/genética , Aterosclerose/prevenção & controle , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Hipóxia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Compostos de Mostarda , Fenilpropionatos , Pró-Proteína Convertase 9/metabolismoRESUMO
Calcific aortic valve disease (CAVD) is a major cause of morbidity in the aging population, but the underlying mechanisms of its progression remain poorly understood. Aortic valve calcification preferentially occurs on the fibrosa, which is subjected to disturbed flow. The side-specific progression of the disease is characterized by inflammation, calcific lesions, and extracellular matrix (ECM) degradation. Here, we explored the role of mechanosensitive microRNA-181b and its downstream targets in human aortic valve endothelial cells (HAVECs). Mechanistically, miR-181b is upregulated in OS and fibrosa, and it targets TIMP3, SIRT1, and GATA6, correlated with increased gelatinase/MMP activity. Overexpression of miR-181b led to decreased TIMP3 and exacerbated MMP activity as shown by gelatinase assay, and miR-181b inhibition decreased gelatinase activity through the repression of TIMP3 levels. Luciferase assay showed specific binding of miR-181b to the TIMP3 gene. Overexpression of miR-181b in HAVECs subjected to either LS or OS increased MMP activity, and miR-181b inhibition abrogated shear-sensitive MMP activity. These studies suggest that targeting this shear-dependent miRNA may provide a novel noninvasive treatment for CAVD.
Assuntos
Valva Aórtica/metabolismo , Calcinose/metabolismo , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Doenças das Valvas Cardíacas/metabolismo , Mecanotransdução Celular , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Regiões 3' não Traduzidas , Valva Aórtica/patologia , Sítios de Ligação , Calcinose/genética , Calcinose/patologia , Células Cultivadas , Células Endoteliais/patologia , Gelatinases/metabolismo , Regulação da Expressão Gênica , Doenças das Valvas Cardíacas/genética , Doenças das Valvas Cardíacas/patologia , Humanos , MicroRNAs/genética , Estresse Mecânico , Inibidor Tecidual de Metaloproteinase-3/genéticaRESUMO
SIGNIFICANCE: Currently, calcific aortic valve disease (CAVD) is only treatable through surgical intervention because the specific mechanisms leading to the disease remain unclear. In this review, we explore the forces and structure of the valve, as well as the mechanosensors and downstream signaling in the valve endothelium known to contribute to inflammation and valve dysfunction. RECENT ADVANCES: While the valvular structure enables adaptation to dynamic hemodynamic forces, these are impaired during CAVD, resulting in pathological systemic changes. Mechanosensing mechanisms-proteins, sugars, and membrane structures-at the surface of the valve endothelial cell relay mechanical signals to the nucleus. As a result, a large number of mechanosensitive genes are transcribed to alter cellular phenotype and, ultimately, induce inflammation and CAVD. Transforming growth factor-ß signaling and Wnt/ß-catenin have been widely studied in this context. Importantly, NADPH oxidase and reactive oxygen species/reactive nitrogen species signaling has increasingly been recognized to play a key role in the cellular response to mechanical stimuli. In addition, a number of valvular microRNAs are mechanosensitive and may regulate the progression of CAVD. CRITICAL ISSUES: While numerous pathways have been described in the pathology of CAVD, no treatment options are available to avoid surgery for advanced stenosis and calcification of the aortic valve. More work must be focused on this issue to lead to successful therapies for the disease. FUTURE DIRECTIONS: Ultimately, a more complete understanding of the mechanisms within the aortic valve endothelium will lead us to future therapies important for treatment of CAVD without the risks involved with valve replacement or repair. Antioxid. Redox Signal. 25, 401-414.
Assuntos
Valva Aórtica/metabolismo , Endotélio/metabolismo , Regulação da Expressão Gênica , Mecanotransdução Celular , Resistência ao Cisalhamento , Animais , Valva Aórtica/anatomia & histologia , Valva Aórtica/citologia , Valva Aórtica/patologia , Estenose da Valva Aórtica/etiologia , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Estenose da Valva Aórtica/terapia , Biomarcadores , Calcinose/etiologia , Calcinose/metabolismo , Calcinose/patologia , Calcinose/terapia , Hemodinâmica , Humanos , Mecanorreceptores/metabolismo , Estresse MecânicoRESUMO
Aortic valve (AV) calcification is an inflammation driven process that occurs preferentially in the fibrosa. To explore the underlying mechanisms, we investigated if key microRNAs (miRNA) in the AV are differentially expressed due to disturbed blood flow (oscillatory shear (OS)) experienced by the fibrosa compared to the ventricularis. To identify the miRNAs involved, endothelial-enriched RNA was isolated from either side of healthy porcine AVs for microarray analysis. Validation using qPCR confirmed significantly higher expression of 7 miRNAs (miR-100, -130a, -181a/b, -199a-3p, -199a-5p, and -214) in the fibrosa versus the ventricularis. Upon bioinformatics analysis, miR-214 was selected for further investigation using porcine AV leaflets in an ex vivo shear system. Fibrosa and ventricularis sides were exposed to either oscillatory or unidirectional pulsatile shear for 2 days and 3 &7 days in regular and osteogenic media, respectively. Higher expression of miR-214, increased thickness of the fibrosa, and calcification was observed when the fibrosa was exposed to OS compared to the ventricularis. Silencing of miR-214 by anti-miR-214 in whole AV leaflets with the fibrosa exposed to OS significantly increased the protein expression of TGFß1 and moderately increased collagen content but did not affect AV calcification. Thus, miR-214 is identified as a side- and shear-dependent miRNA that regulates key mechanosensitive gene in AV such as TGFß1.
Assuntos
Valva Aórtica/patologia , Aterosclerose/patologia , MicroRNAs/análise , Animais , Modelos Animais de Doenças , Análise em Microsséries , Reação em Cadeia da Polimerase em Tempo Real , Estresse Mecânico , SuínosRESUMO
PURPOSE: To demonstrate the first use of a novel technology for quantifying suture forces on annuloplasty rings to better understand the mechanisms of ring dehiscence. DESCRIPTION: Force transducers were developed, attached to a size 24 Physio ring, and implanted in the mitral annulus of an ovine animal. Ring suture forces were measured after implantation and for cardiac cycles reaching peak left ventricular pressures (LVP) of 100, 125, and 150 mm Hg. EVALUATION: After implantation of the undersized ring to the flaccid annulus, the mean suture force was 2.0±0.6 N. During cyclic contraction, the anterior ring suture forces were greater than the posterior ring suture forces at peak LVPs of 100 mm Hg (4.9±2.0 N vs 2.1±1.1 N), 125 mm Hg (5.4±2.3 N vs 2.3±1.2 N), and 150 mm Hg (5.7±2.4 N vs 2.4±1.1 N). The largest force was 7.4 N at 150 mm Hg. CONCLUSIONS: The preliminary results demonstrate trends in annuloplasty suture forces and their variation with location and LVP. Future studies will significantly contribute to clinical knowledge by elucidating the mechanisms of ring dehiscence while improving annuloplasty ring design and surgical repair techniques.