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1.
J Am Coll Cardiol ; 82(4): 317-332, 2023 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-37468187

RESUMO

BACKGROUND: Visceral obesity is directly linked to increased cardiovascular risk, including heart failure. OBJECTIVES: This study explored the ability of human epicardial adipose tissue (EAT)-derived microRNAs (miRNAs) to regulate the myocardial redox state and clinical outcomes. METHODS: This study screened for miRNAs expressed and released from human EAT and tested for correlations with the redox state in the adjacent myocardium in paired EAT/atrial biopsy specimens from patients undergoing cardiac surgery. Three miRNAs were then tested for causality in an in vitro model of cardiomyocytes. At a clinical level, causality/directionality were tested using genome-wide association screening, and the underlying mechanisms were explored using human biopsy specimens, as well as overexpression of the candidate miRNAs and their targets in vitro and in vivo using a transgenic mouse model. The final prognostic value of the discovered targets was tested in patients undergoing cardiac surgery, followed up for a median of 8 years. RESULTS: EAT miR-92a-3p was related to lower oxidative stress in human myocardium, a finding confirmed by using genetic regulators of miR-92a-3p in the human heart and EAT. miR-92a-3p reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase-derived superoxide (O2.-) by targeting myocardial expression of WNT5A, which regulated Rac1-dependent activation of NADPH oxidases. Finally, high miR-92a-3p levels in EAT were independently related with lower risk of adverse cardiovascular events. CONCLUSIONS: EAT-derived miRNAs exert paracrine effects on the human heart. Indeed miR-92a-3p suppresses the wingless-type MMTV integration site family, member 5a/Rac1/NADPH oxidase axis and improves the myocardial redox state. EAT-derived miR-92a-3p is related to improved clinical outcomes and is a rational therapeutic target for the prevention and treatment of obesity-related heart disease.


Assuntos
Estudo de Associação Genômica Ampla , MicroRNAs , Humanos , Camundongos , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Miocárdio/metabolismo , Oxirredução , Camundongos Transgênicos , Tecido Adiposo/metabolismo
3.
Lancet Digit Health ; 4(10): e705-e716, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36038496

RESUMO

BACKGROUND: Direct evaluation of vascular inflammation in patients with COVID-19 would facilitate more efficient trials of new treatments and identify patients at risk of long-term complications who might respond to treatment. We aimed to develop a novel artificial intelligence (AI)-assisted image analysis platform that quantifies cytokine-driven vascular inflammation from routine CT angiograms, and sought to validate its prognostic value in COVID-19. METHODS: For this prospective outcomes validation study, we developed a radiotranscriptomic platform that uses RNA sequencing data from human internal mammary artery biopsies to develop novel radiomic signatures of vascular inflammation from CT angiography images. We then used this platform to train a radiotranscriptomic signature (C19-RS), derived from the perivascular space around the aorta and the internal mammary artery, to best describe cytokine-driven vascular inflammation. The prognostic value of C19-RS was validated externally in 435 patients (331 from study arm 3 and 104 from study arm 4) admitted to hospital with or without COVID-19, undergoing clinically indicated pulmonary CT angiography, in three UK National Health Service (NHS) trusts (Oxford, Leicester, and Bath). We evaluated the diagnostic and prognostic value of C19-RS for death in hospital due to COVID-19, did sensitivity analyses based on dexamethasone treatment, and investigated the correlation of C19-RS with systemic transcriptomic changes. FINDINGS: Patients with COVID-19 had higher C19-RS than those without (adjusted odds ratio [OR] 2·97 [95% CI 1·43-6·27], p=0·0038), and those infected with the B.1.1.7 (alpha) SARS-CoV-2 variant had higher C19-RS values than those infected with the wild-type SARS-CoV-2 variant (adjusted OR 1·89 [95% CI 1·17-3·20] per SD, p=0·012). C19-RS had prognostic value for in-hospital mortality in COVID-19 in two testing cohorts (high [≥6·99] vs low [<6·99] C19-RS; hazard ratio [HR] 3·31 [95% CI 1·49-7·33], p=0·0033; and 2·58 [1·10-6·05], p=0·028), adjusted for clinical factors, biochemical biomarkers of inflammation and myocardial injury, and technical parameters. The adjusted HR for in-hospital mortality was 8·24 (95% CI 2·16-31·36, p=0·0019) in patients who received no dexamethasone treatment, but 2·27 (0·69-7·55, p=0·18) in those who received dexamethasone after the scan, suggesting that vascular inflammation might have been a therapeutic target of dexamethasone in COVID-19. Finally, C19-RS was strongly associated (r=0·61, p=0·00031) with a whole blood transcriptional module representing dysregulation of coagulation and platelet aggregation pathways. INTERPRETATION: Radiotranscriptomic analysis of CT angiography scans introduces a potentially powerful new platform for the development of non-invasive imaging biomarkers. Application of this platform in routine CT pulmonary angiography scans done in patients with COVID-19 produced the radiotranscriptomic signature C19-RS, a marker of cytokine-driven inflammation driving systemic activation of coagulation and responsible for adverse clinical outcomes, which predicts in-hospital mortality and might allow targeted therapy. FUNDING: Engineering and Physical Sciences Research Council, British Heart Foundation, Oxford BHF Centre of Research Excellence, Innovate UK, NIHR Oxford Biomedical Research Centre, Wellcome Trust, Onassis Foundation.


Assuntos
COVID-19 , SARS-CoV-2 , Angiografia , Inteligência Artificial , COVID-19/diagnóstico por imagem , Citocinas , Humanos , Inflamação/diagnóstico por imagem , Estudos Prospectivos , Medicina Estatal , Tomografia Computadorizada por Raios X
4.
Cell Mol Life Sci ; 78(23): 7355-7378, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34698884

RESUMO

The age-related vasculature alteration is the prominent risk factor for vascular diseases (VD), namely, atherosclerosis, abdominal aortic aneurysm, vascular calcification (VC) and pulmonary arterial hypertension (PAH). The chronic sterile low-grade inflammation state, alias inflammaging, characterizes elderly people and participates in VD development. MicroRNA34-a (miR-34a) is emerging as an important mediator of inflammaging and VD. miR-34a increases with aging in vessels and induces senescence and the acquisition of the senescence-associated secretory phenotype (SASP) in vascular smooth muscle (VSMCs) and endothelial (ECs) cells. Similarly, other VD risk factors, including dyslipidemia, hyperglycemia and hypertension, modify miR-34a expression to promote vascular senescence and inflammation. miR-34a upregulation causes endothelial dysfunction by affecting ECs nitric oxide bioavailability, adhesion molecules expression and inflammatory cells recruitment. miR-34a-induced senescence facilitates VSMCs osteoblastic switch and VC development in hyperphosphatemia conditions. Conversely, atherogenic and hypoxic stimuli downregulate miR-34a levels and promote VSMCs proliferation and migration during atherosclerosis and PAH. MiR34a genetic ablation or miR-34a inhibition by anti-miR-34a molecules in different experimental models of VD reduce vascular inflammation, senescence and apoptosis through sirtuin 1 Notch1, and B-cell lymphoma 2 modulation. Notably, pleiotropic drugs, like statins, liraglutide and metformin, affect miR-34a expression. Finally, human studies report that miR-34a levels associate to atherosclerosis and diabetes and correlate with inflammatory factors during aging. Herein, we comprehensively review the current knowledge about miR-34a-dependent molecular and cellular mechanisms activated by VD risk factors and highlight the diagnostic and therapeutic potential of modulating its expression in order to reduce inflammaging and VD burn and extend healthy lifespan.


Assuntos
Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Sistema Cardiovascular/patologia , Senescência Celular/fisiologia , MicroRNAs/genética , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/patologia , Aterosclerose/genética , Aterosclerose/patologia , Células Endoteliais/metabolismo , Humanos , Inflamação/genética , Inflamação/patologia , Músculo Liso Vascular/patologia , Hipertensão Arterial Pulmonar/genética , Hipertensão Arterial Pulmonar/patologia , Calcificação Vascular/genética , Calcificação Vascular/patologia
5.
Eur Heart J ; 42(48): 4947-4960, 2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34293101

RESUMO

AIMS: Recent clinical trials indicate that sodium-glucose cotransporter 2 (SGLT2) inhibitors improve cardiovascular outcomes in heart failure patients, but the underlying mechanisms remain unknown. We explored the direct effects of canagliflozin, an SGLT2 inhibitor with mild SGLT1 inhibitory effects, on myocardial redox signalling in humans. METHODS AND RESULTS: Study 1 included 364 patients undergoing cardiac surgery. Right atrial appendage biopsies were harvested to quantify superoxide (O2.-) sources and the expression of inflammation, fibrosis, and myocardial stretch genes. In Study 2, atrial tissue from 51 patients was used ex vivo to study the direct effects of canagliflozin on NADPH oxidase activity and nitric oxide synthase (NOS) uncoupling. Differentiated H9C2 and primary human cardiomyocytes (hCM) were used to further characterize the underlying mechanisms (Study 3). SGLT1 was abundantly expressed in human atrial tissue and hCM, contrary to SGLT2. Myocardial SGLT1 expression was positively associated with O2.- production and pro-fibrotic, pro-inflammatory, and wall stretch gene expression. Canagliflozin reduced NADPH oxidase activity via AMP kinase (AMPK)/Rac1signalling and improved NOS coupling via increased tetrahydrobiopterin bioavailability ex vivo and in vitro. These were attenuated by knocking down SGLT1 in hCM. Canagliflozin had striking ex vivo transcriptomic effects on myocardial redox signalling, suppressing apoptotic and inflammatory pathways in hCM. CONCLUSIONS: We demonstrate for the first time that canagliflozin suppresses myocardial NADPH oxidase activity and improves NOS coupling via SGLT1/AMPK/Rac1 signalling, leading to global anti-inflammatory and anti-apoptotic effects in the human myocardium. These findings reveal a novel mechanism contributing to the beneficial cardiac effects of canagliflozin.


Assuntos
Canagliflozina , Inibidores do Transportador 2 de Sódio-Glicose , Canagliflozina/metabolismo , Canagliflozina/farmacologia , Humanos , Miocárdio , Miócitos Cardíacos/metabolismo , Oxirredução , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia
6.
J Am Coll Cardiol ; 77(20): 2494-2513, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34016263

RESUMO

BACKGROUND: Obesity is associated with increased cardiovascular risk; however, the potential role of dysregulations in the adipose tissue (AT) metabolome is unknown. OBJECTIVES: The aim of this study was to explore the role of dysregulation in the AT metabolome on vascular redox signaling and cardiovascular outcomes. METHODS: A screen was conducted for metabolites differentially secreted by thoracic AT (ThAT) and subcutaneous AT in obese patients with atherosclerosis (n = 48), and these metabolites were then linked with dysregulated vascular redox signaling in 633 patients undergoing coronary bypass surgery. The underlying mechanisms were explored in human aortic endothelial cells, and their clinical value was tested against hard clinical endpoints. RESULTS: Because ThAT volume was associated significantly with arterial oxidative stress, there were significant differences in sphingolipid secretion between ThAT and subcutaneous AT, with C16:0-ceramide and derivatives being the most abundant species released within adipocyte-derived extracellular vesicles. High ThAT sphingolipid secretion was significantly associated with reduced endothelial nitric oxide bioavailability and increased superoxide generated in human vessels. Circulating C16:0-ceramide correlated positively with ThAT ceramides, dysregulated vascular redox signaling, and increased systemic inflammation in 633 patients with atherosclerosis. Exogenous C16:0-ceramide directly increased superoxide via tetrahydrobiopterin-mediated endothelial nitric oxide synthase uncoupling and dysregulated protein phosphatase 2 in human aortic endothelial cells. High plasma C16:0-ceramide and its glycosylated derivative were independently related with increased risk for cardiac mortality (adjusted hazard ratios: 1.394; 95% confidence interval: 1.030 to 1.886; p = 0.031 for C16:0-ceramide and 1.595; 95% confidence interval: 1.042 to 2.442; p = 0.032 for C16:0-glycosylceramide per 1 SD). In a randomized controlled clinical trial, 1-year treatment of obese patients with the glucagon-like peptide-1 analog liraglutide suppressed plasma C16:0-ceramide and C16:0-glycosylceramide changes compared with control subjects. CONCLUSIONS: These results demonstrate for the first time in humans that AT-derived ceramides are modifiable regulators of vascular redox state in obesity, with a direct impact on cardiac mortality in advanced atherosclerosis. (The Interaction Between Appetite Hormones; NCT02094183).


Assuntos
Tecido Adiposo/metabolismo , Artérias/metabolismo , Aterosclerose/metabolismo , Ceramidas/metabolismo , Obesidade/metabolismo , Aterosclerose/complicações , Aterosclerose/mortalidade , Estudos de Casos e Controles , Endotélio Vascular/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Técnicas In Vitro , Liraglutida , Metabolômica , Obesidade/complicações , Estresse Oxidativo , Ensaios Clínicos Controlados Aleatórios como Assunto , Esfingolipídeos/metabolismo , Superóxidos/metabolismo
8.
Int J Mol Sci ; 21(12)2020 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-32585876

RESUMO

The senescence of vascular smooth muscle cells (VSMCs), characterized by the acquisition of senescence-associated secretory phenotype (SASP), is relevant for VSMCs osteoblastic differentiation and vascular calcification (VC). MicroRNA-34a (miR-34a) is a driver of such phenomena and could play a role in vascular inflammaging. Herein, we analyzed the relationship between miR-34a and the prototypical SASP component IL6 in in vitro and in vivo models. miR-34a and IL6 levels increased and positively correlated in aortas of 21 months-old male C57BL/6J mice and in human aortic smooth muscle cells (HASMCs) isolated from donors of different age and undergone senescence. Lentiviral overexpression of miR-34a in HASMCs enhanced IL6 secretion. HASMCs senescence and calcification accelerated after exposure to conditioned medium of miR-34a-overexpressing cells. Analysis of miR-34a-induced secretome revealed enhancement of several pro-inflammatory cytokines and chemokines, including IL6, pro-senescent growth factors and matrix-degrading molecules. Moreover, induction of aortas medial calcification and concomitant IL6 expression, with an overdose of vitamin D, was reduced in male C57BL/6J Mir34a-/- mice. Finally, a positive correlation was observed between circulating miR-34a and IL6 in healthy subjects of 20-90 years. Hence, the vascular age-associated miR-34a promotes VSMCs SASP activation and contributes to arterial inflammation and dysfunctions such as VC.


Assuntos
Senescência Celular , Interleucina-6/metabolismo , MicroRNAs/genética , Músculo Liso Vascular/patologia , Calcificação Vascular/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Feminino , Voluntários Saudáveis , Humanos , Interleucina-6/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Músculo Liso Vascular/metabolismo , Calcificação Vascular/genética , Calcificação Vascular/metabolismo , Adulto Jovem
9.
Sci Transl Med ; 12(541)2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350133

RESUMO

Recent clinical trials have revealed that aggressive insulin treatment has a neutral effect on cardiovascular risk in patients with diabetes despite improved glycemic control, which may suggest confounding direct effects of insulin on the human vasculature. We studied 580 patients with coronary atherosclerosis undergoing coronary artery bypass surgery (CABG), finding that high endogenous insulin was associated with reduced nitric oxide (NO) bioavailability ex vivo in vessels obtained during surgery. Ex vivo experiments with human internal mammary arteries and saphenous veins obtained from 94 patients undergoing CABG revealed that both long-acting insulin analogs and human insulin triggered abnormal responses of post-insulin receptor substrate 1 downstream signaling ex vivo, independently of systemic insulin resistance status. These abnormal responses led to reduced NO bioavailability, activation of NADPH oxidases, and uncoupling of endothelial NO synthase. Treatment with an oral dipeptidyl peptidase 4 inhibitor (DPP4i) in vivo or DPP4i administered to vessels ex vivo restored physiological insulin signaling, reversed vascular insulin responses, reduced vascular oxidative stress, and improved endothelial function in humans. The detrimental effects of insulin on vascular redox state and endothelial function as well as the insulin-sensitizing effect of DPP4i were also validated in high-fat diet-fed ApoE-/- mice treated with DPP4i. High plasma DPP4 activity and high insulin were additively related with higher cardiac mortality in patients with coronary atherosclerosis undergoing CABG. These findings may explain the inability of aggressive insulin treatment to improve cardiovascular outcomes, raising the question whether vascular insulin sensitization with DPP4i should precede initiation of insulin treatment and continue as part of a long-term combination therapy.


Assuntos
Aterosclerose , Dipeptidil Peptidase 4 , Animais , Ponte de Artéria Coronária , Humanos , Insulina/uso terapêutico , Camundongos , Oxirredução
10.
Cardiovasc Res ; 116(8): 1458-1472, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31688894

RESUMO

AIMS: Increased Ankyrin Repeat Domain 1 (ANKRD1) levels linked to gain of function mutations have been associated to total anomalous pulmonary venous return and adult cardiomyopathy occurrence in humans. The link between increased ANKRD1 level and cardiac structural and functional disease is not understood. To get insight into this problem, we have generated a gain of function ANKRD1 mouse model by overexpressing ANKRD1 in the myocardium. METHODS AND RESULTS: Ankrd1 is expressed non-homogeneously in the embryonic myocardium, with a dynamic nucleo-sarcomeric localization in developing cardiomyocytes. ANKRD1 transgenic mice present sinus venosus defect, which originates during development by impaired remodelling of early embryonic heart. Adult transgenic hearts develop diastolic dysfunction with preserved ejection fraction, which progressively evolves into heart failure, as shown histologically and haemodynamically. Transgenic cardiomyocyte structure, sarcomeric assembly, and stability are progressively impaired from embryonic to adult life. Postnatal transgenic myofibrils also present characteristic functional alterations: impaired compliance at neonatal stage and impaired lusitropism in adult hearts. Altogether, our combined analyses suggest that impaired embryonic remodelling and adult heart dysfunction in ANKRD1 transgenic mice present a common ground of initial cardiomyocyte defects, which are exacerbated postnatally. Molecular analysis showed transient activation of GATA4-Nkx2.5 transcription in early transgenic embryos and subsequent dynamic transcriptional modulation within titin gene. CONCLUSIONS: ANKRD1 is a fine mediator of cardiomyocyte response to haemodynamic load in the developing and adult heart. Increased ANKRD1 levels are sufficient to initiate an altered cellular phenotype, which is progressively exacerbated into a pathological organ response by the high ventricular workload during postnatal life. Our study defines for the first time a unifying picture for ANKRD1 role in heart development and disease and provides the first mechanistic link between ANKRD1 overexpression and cardiac disease onset.


Assuntos
Comunicação Interatrial/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Disfunção Ventricular Esquerda/metabolismo , Função Ventricular Esquerda , Animais , Diástole , Feminino , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Comunicação Interatrial/genética , Comunicação Interatrial/patologia , Comunicação Interatrial/fisiopatologia , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Masculino , Camundongos Transgênicos , Proteínas Musculares/genética , Miocárdio/patologia , Proteínas Nucleares/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Repressoras/genética , Regulação para Cima , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologia
11.
Sci Transl Med ; 11(510)2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31534019

RESUMO

Obesity is associated with changes in the secretome of adipose tissue (AT), which affects the vasculature through endocrine and paracrine mechanisms. Wingless-related integration site 5A (WNT5A) and secreted frizzled-related protein 5 (SFRP5), adipokines that regulate noncanonical Wnt signaling, are dysregulated in obesity. We hypothesized that WNT5A released from AT exerts endocrine and paracrine effects on the arterial wall through noncanonical RAC1-mediated Wnt signaling. In a cohort of 1004 humans with atherosclerosis, obesity was associated with increased WNT5A bioavailability in the circulation and the AT, higher expression of WNT5A receptors Frizzled 2 and Frizzled 5 in the human arterial wall, and increased vascular oxidative stress due to activation of NADPH oxidases. Plasma concentration of WNT5A was elevated in patients with coronary artery disease compared to matched controls and was independently associated with calcified coronary plaque progression. We further demonstrated that WNT5A induces arterial oxidative stress and redox-sensitive migration of vascular smooth muscle cells via Frizzled 2-mediated activation of a previously uncharacterized pathway involving the deubiquitinating enzyme ubiquitin-specific protease 17 (USP17) and the GTPase RAC1. Our study identifies WNT5A and its downstream vascular signaling as a link between obesity and vascular disease pathogenesis, with translational implications in humans.


Assuntos
Tecido Adiposo/metabolismo , Vasos Sanguíneos/metabolismo , Endopeptidases/metabolismo , NADPH Oxidases/metabolismo , Obesidade/metabolismo , Transdução de Sinais , Proteína Wnt-5a/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Tecido Adiposo/efeitos dos fármacos , Animais , Artérias/metabolismo , Artérias/patologia , Aterosclerose/sangue , Aterosclerose/complicações , Aterosclerose/patologia , Vasos Sanguíneos/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ligantes , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Obesidade/complicações , Oxidantes/toxicidade , Oxirredução , Transdução de Sinais/efeitos dos fármacos , Doenças Vasculares/complicações , Doenças Vasculares/metabolismo , Proteína Wnt-5a/sangue
13.
Arterioscler Thromb Vasc Biol ; 38(9): 2079-2090, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30026277

RESUMO

Objective- Vascular calcification (VC) is age dependent and a risk factor for cardiovascular and all-cause mortality. VC involves the senescence-induced transdifferentiation of vascular smooth muscle cells (SMCs) toward an osteochondrogenic lineage resulting in arterial wall mineralization. miR-34a increases with age in aortas and induces vascular SMC senescence through the modulation of its target SIRT1 (sirtuin 1). In this study, we aimed to investigate whether miR-34a regulates VC. Approach and Results- We found that miR-34a and Runx2 (Runt-related transcription factor 2) expression correlates in young and old mice. Mir34a+/+ and Mir34a-/- mice were treated with vitamin D, and calcium quantification revealed that Mir34a deficiency reduces soft tissue and aorta medial calcification and the upregulation of the VC Sox9 (SRY [sex-determining region Y]-box 9) and Runx2 and the senescence p16 and p21 markers. In this model, miR-34a upregulation was transient and preceded aorta mineralization. Mir34a-/- SMCs were less prone to undergo senescence and under osteogenic conditions deposited less calcium compared with Mir34a+/+ cells. Furthermore, unlike in Mir34a+/+ SMC, the known VC inhibitors SIRT1 and Axl (AXL receptor tyrosine kinase) were only partially downregulated in calcifying Mir34a-/- SMC. Strikingly, constitutive miR-34a overexpression to senescence-like levels in human aortic SMCs increased calcium deposition and enhanced Axl and SIRT1 decrease during calcification. Notably, we also showed that miR-34a directly decreased Axl expression in human aortic SMC, and restoration of its levels partially rescued miR-34a-dependent growth arrest. Conclusions- miR-34a promotes VC via vascular SMC mineralization by inhibiting cell proliferation and inducing senescence through direct Axl and SIRT1 downregulation, respectively. This miRNA could be a good therapeutic target for the treatment of VC.


Assuntos
Senescência Celular/fisiologia , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Sirtuína 1/metabolismo , Calcificação Vascular , Adulto , Envelhecimento/patologia , Animais , Aorta/metabolismo , Proliferação de Células , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação para Baixo , Humanos , Masculino , Camundongos , Camundongos Knockout , Músculo Liso Vascular/citologia , Fatores de Transcrição SOX9/metabolismo , Regulação para Cima , Adulto Jovem , Receptor Tirosina Quinase Axl
14.
Biochim Biophys Acta Mol Basis Dis ; 1863(11): 2693-2704, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28716707

RESUMO

Myocardial infarction (MI) is a major health burden worldwide. Extracellular High mobility group box 1 (HMGB1) regulates tissue healing after injuries. The reduced form of HMGB1 (fr-HMGB1) exerts chemotactic activity by binding CXCL12 through CXCR4, while the disulfide form, (ds-HMGB1), induces cytokines expression by TLR4. Here, we assessed the role of HMGB1 redox forms and the non-oxidizable mutant (3S) on human cardiac fibroblast (hcFbs) functions and cardiac remodeling after infarction. Among HMGB1 receptors, hcFbs express CXCR4. Fr-HMGB1 and 3S, but not ds-HMGB1, promote hcFbs migration through Src activation, while none of HMGB1 redox forms induces proliferation or inflammatory mediators. 3S is more effective than fr-HMGB1 in stimulating hcFbs migration and Src phosphorylation being active at lower concentrations and in oxidizing conditions. Notably, chemotaxis toward both proteins is CXCR4-dependent but, in contrast to fr-HMGB1, 3S does not require CXCL12 since hcFbs migration persists in the presence of the CXCL12/CXCR4 inhibitor AMD3100 or an anti-CXCL12 antibody. Interestingly, 3S interacts with CXCR4 and induces a different receptor conformation than CXCL12. Mice undergoing MI and receiving 3S exhibit adverse LV remodeling owing to an excessive collagen deposition promoted by a higher number of myofibroblasts. On the contrary, fr-HMGB1 ameliorates cardiac performance enhancing neoangiogenesis and reducing the infarcted area and fibrosis. Altogether, our results demonstrate that non-oxidizable HMGB1 induce a sustained cardiac fibroblasts migration despite the redox state of the environment and by altering CXCL12/CXCR4 axis. This affects proper cardiac remodeling after an infarction.


Assuntos
Movimento Celular , Quimiocina CXCL12/metabolismo , Fibroblastos/metabolismo , Proteína HMGB1/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Receptores CXCR4/metabolismo , Feminino , Fibroblastos/patologia , Humanos , Masculino , Infarto do Miocárdio/patologia , Miocárdio/patologia , Oxirredução
15.
J Mol Cell Cardiol ; 81: 62-70, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25640160

RESUMO

Age-associated cardiovascular diseases are at least partially ascribable to vascular cell senescence. Replicative senescence (RS) and stress-induced premature senescence (SIPS) are provoked respectively by endogenous (telomere erosion) and exogenous (H2O2, UV) stimuli resulting in cell cycle arrest in G1 and G2 phases. In both scenarios, mitochondria-derived ROS are important players in senescence initiation. We aimed to define whether a mtDNA-transcribed long-non-coding-RNA (lncRNA), ASncmtRNA-2, has a role in vascular aging and senescence. Aortas of old mice, characterized by increased senescence, showed an increment in ASncmtRNA-2 expression. In vitro analysis of Endothelial Cells (EC) and Vascular Smooth Muscle Cells (VSMC) established that ASncmtRNA-2 is induced in EC, but not in VSMC, during RS. Surprisingly, ASncmtRNA-2 is not upregulated in two different EC SIPS scenarios, treated with H2O2 and UV. The p16 gene displayed similar ASncmtRNA-2 expression patterns, suggesting a possible co-regulation of the two genes. Interestingly, the expression of two miRNAs, hsa-miR-4485 and hsa-miR-1973, with perfect homology to the double strand region of ASncmtRNA-2 and originating at least in part from a mitochondrial transcript, was induced in RS, opening to the possibility that this lncRNA functions as a non-canonical precursor of these miRNAs. Cell cycle analysis of EC transiently over-expressing ASncmtRNA-2 revealed an accumulation of EC in the G2/M phase, but not in the G1 phase. We propose that ASncmtRNA-2 in EC might be involved in the RS establishment by participating in the cell cycle arrest in G2/M phase, possibly through the production of hsa-miR-4485 and hsa-miR-1973. This article is part of a Special Issue entitled: Mitochondria.


Assuntos
Envelhecimento/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Mitocôndrias/metabolismo , Miócitos de Músculo Liso/metabolismo , RNA Longo não Codificante/genética , RNA/genética , Envelhecimento/genética , Animais , Aorta/citologia , Aorta/metabolismo , Sequência de Bases , Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Humanos , Peróxido de Hidrogênio/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Mitocôndrias/genética , Dados de Sequência Molecular , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos da radiação , RNA/metabolismo , RNA Longo não Codificante/metabolismo , RNA Mitocondrial , Transdução de Sinais , Raios Ultravioleta
16.
J Gerontol A Biol Sci Med Sci ; 70(11): 1304-11, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25352462

RESUMO

Arterial aging is a major risk factor for the occurrence of cardiovascular diseases. The aged artery is characterized by endothelial dysfunction and vascular smooth muscle cells altered physiology together with low-grade chronic inflammation. MicroRNA-34a (miR-34a) has been recently implicated in cardiac, endothelial, and endothelial progenitor cell senescence; however, its contribution to aging-associated vascular smooth muscle cells phenotype has not been explored so far. We found that miR-34a was highly expressed in aortas isolated from old mice. Moreover, its well-known target, the longevity-associated protein SIRT1, was significantly downregulated during aging in both endothelial cells and vascular smooth muscle cells. Increased miR-34a as well as decreased SIRT1 expression was also observed in replicative-senescent human aortic smooth muscle cells. miR-34a overexpression in proliferative human aortic smooth muscle cells caused cell cycle arrest along with enhanced p21 protein levels and evidence of cell senescence. Furthermore, miR-34a ectopic expression induced pro-inflammatory senescence-associated secretory phenotype molecules. Finally, SIRT1 protein significantly decreased upon miR-34a overexpression and restoration of its levels rescued miR-34a-dependent human aortic smooth muscle cells senescence, but not senescence-associated secretory phenotype factors upregulation. Taken together, our findings suggest that aging-associated increase of miR-34a expression levels, by promoting vascular smooth muscle cells senescence and inflammation through SIRT1 downregulation and senescence-associated secretory phenotype factors induction, respectively, may lead to arterial dysfunctions.


Assuntos
Aorta/metabolismo , Senescência Celular/fisiologia , MicroRNAs/metabolismo , Miócitos de Músculo Liso/fisiologia , Sirtuína 1/metabolismo , Animais , Aorta/patologia , Técnicas de Cultura de Células , Células Endoteliais/fisiologia , Humanos , Camundongos , MicroRNAs/genética , Sirtuína 1/genética
17.
Circ Res ; 115(4): 432-41, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24963028

RESUMO

RATIONALE: The evolutionary conserved Tbx3/Tbx5 gene cluster encodes T-box transcription factors that play crucial roles in the development and homeostasis of the cardiac conduction system in human and mouse. Both genes are expressed in overlapping patterns and function in strictly tissue-specific and dose-dependent manners, yet, their regulation is poorly understood. OBJECTIVE: To analyze the mechanism underlying the complex regulation of the Tbx3/Tbx5 cluster. METHODS AND RESULTS: By probing the 3-dimensional architecture of the Tbx3/Tbx5 cluster using high-resolution circular chromosome conformation capture sequencing in vivo, we found that its regulatory landscape is in a preformed conformation similar in embryonic heart, limbs, and brain. Tbx3 and its flanking gene desert form a 1 Mbp loop between CCCTC-binding factor (CTCF)-binding sites that is separated from the neighboring Tbx5 loop. However, Ctcf inactivation did not result in transcriptional regulatory interaction between Tbx3 and Tbx5. Multiple sites within the Tbx3 locus contact the promoter, including sites corresponding to regions known to contain variations in the human genome influencing conduction. We identified an atrioventricular-specific enhancer and a pan-cardiac enhancer that contact the promoter and each other and synergize to activate transcription in the atrioventricular conduction system. CONCLUSIONS: We provide a high-resolution model of the 3-dimensional structure and function of the Tbx3/Tbx5 locus and show that the locus is organized in a preformed, permissive structure. The Tbx3 locus forms a CTCF-independent autonomous regulatory domain with multiple combinatorial regulatory elements that control the precise pattern of Tbx3 in the cardiac conduction system.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Sistema de Condução Cardíaco/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Região 3'-Flanqueadora , Animais , Sítios de Ligação , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Fator de Ligação a CCCTC , Cromossomos Artificiais Bacterianos , DNA Circular/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Sistema de Condução Cardíaco/embriologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Morfogênese , Família Multigênica , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Transdução de Sinais , Transcrição Gênica , Ativação Transcricional
18.
FEBS Lett ; 583(15): 2486-92, 2009 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-19589340

RESUMO

The ANKRD1/CARP gene encodes a muscle-specific protein which has been implicated in transcriptional regulation and myofibrillar assembly. Several features at both the mRNA and protein levels define ANKRD1 as a gene whose expression is tightly regulated, and deregulated expression of this protein has been recently associated to human congenital heart disease. It is therefore crucial to define the intracellular pathways that regulate the ANKRD1 protein's steady-state levels. Here, we show that ANKRD1 is a short-lived protein whose levels are tightly regulated by the 26S proteasome. In addition, a critical role for a putative PEST motif was established, although other degrons within the ANKRD1 protein are likely implicated in the control of its intracellular levels.


Assuntos
Proteínas Musculares/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Humanos , Proteínas Musculares/genética , Proteínas Nucleares/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética
19.
Hum Mutat ; 29(4): 468-74, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18273862

RESUMO

Total anomalous pulmonary venous return (TAPVR) is a congenital heart defect in which the pulmonary veins fail to enter the left atrium and drain instead into the right atrium or one of its venous tributaries. Although a genetic basis for TAPVR has long been recognized, no single gene involved in the pathogenesis of this disease has been identified to date. We previously reported a TAPVR patient bearing a de novo 10;21 balanced translocation. In this work, we cloned both translocation breakpoints from this patient and mapped the ANKRD1 gene, encoding a cardiac transcriptional regulator, 130 kb proximally to the breakpoint on chromosome 10. In situ hybridization analysis performed on murine embryos showed ANKRD1 expression in the developing pulmonary veins, suggesting a possible role for this gene in TAPVR pathogenesis. Moreover, ANKRD1 expression levels were found to be highly increased in lymphoblastoid cell lines derived from both the translocation-bearing proband and a second independent sporadic TAPVR patient, suggesting that disruption of the normal ANKRD1 expression pattern is associated with TAPVR. Finally, a nonconservative missense mutation in the ANKRD1 gene was found in a third sporadic TAPVR patient. In vitro calpain-mediated degradation assays, coupled to reporter gene analysis in transfected HeLa cells, strongly suggested that this mutation enhances both the stability of the ANKRD1/CARP protein and its transcriptional repression activity upon the cardiac-specific atrial natriuretic factor (ANF) promoter. Taken together, these results define ANKRD1 as a possible candidate gene for TAPVR pathogenesis.


Assuntos
Cardiopatias Congênitas/genética , Proteínas Musculares/genética , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Veias Pulmonares/anormalidades , Proteínas Repressoras/genética , Animais , Sequência de Bases , Linhagem Celular , Cromossomos Humanos Par 10/genética , Cromossomos Humanos Par 21/genética , DNA/genética , Feminino , Expressão Gênica , Células HeLa , Cardiopatias Congênitas/metabolismo , Humanos , Masculino , Camundongos , Proteínas Musculares/metabolismo , Proteínas Nucleares/metabolismo , Linhagem , Gravidez , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Transfecção , Translocação Genética
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