RESUMO
BACKGROUND: The chicken has been a representative model organism to study embryonic development in birds, however important differences exist among this class of species. As a representative of one of oldest existing clades of birds, the African ostrich (Struthio camelus), has the largest body among birds, and has two toes. Our purpose is to establish the corresponding stages in ostrich embryo development that match the well-established HH system of the chicken to facilitate comparative studies between the ostrich and other birds to better understand differences in development. RESULTS: Here we describe in detail the middle period of embryonic development using microscopic images and skeletal staining. We found that clear morphological differentiation between the ostrich and the chicken begins at stage 26. Bird limb cartilage first form in stage 25, while the development of the limb skeletons differs after stage 31. Calcification of limb skeletons in the chicken was completed faster. The first and second toes of the ostrich disappear at stages 36 and 38, respectively. CONCLUSIONS: This study should greatly aid ostrich-related developmental and morphological research and provide a reference for studying the development and evolution of avian limb skeletons, including molecular research. Questions that can now be addressed include studies into the fusion of tarsometatarsal skeleton, ossification, and digit loss.
Assuntos
Struthioniformes , Animais , Struthioniformes/anatomia & histologia , Galinhas , Dedos do Pé , Desenvolvimento EmbrionárioRESUMO
The normal stages of embryonic development for wild-type Xenopus laevis were established by Nieuwkoop and Faber in 1956, a milestone in the history of understanding embryonic development. However, this work lacked photographic images and staining for skeleton structures from the corresponding stages. Here, we provide high-quality images of embryonic morphology and skeleton development to facilitate studies on amphibian development. On the basis of the classical work, we selected the albino mutant of X. laevis as the observation material to restudy embryonic development in this species. The lower level of pigmentation makes it easier to interpret histochemical experiments. At 23°C, albino embryos develop at the same rate as wild-type embryos, which can be divided into 66 stages as they develop into adults in about 58 days. We described the complete embryonic development system for X. laevis, supplemented with pictures of limb and skeleton development that are missing from previous studies, and summarized the characteristics and laws of limb and skeleton development. Our study should aid research into the development of X. laevis and the evolution of amphibians.
Assuntos
Desenvolvimento Embrionário , Organogênese , Animais , Xenopus laevisRESUMO
Atrial fibrillation (AF) is the most common type of arrhythmia in cardiovascular diseases. Atrial fibrosis is an important pathophysiological contributor to AF. This study aimed to investigate the role of the clustered miR-23b-3p and miR-27b-3p in atrial fibrosis. Human atrial fibroblasts (HAFs) were isolated from atrial appendage tissue of patients with sinus rhythm. A cell model of atrial fibrosis was achieved in Ang-II-induced HAFs. Cell proliferation and migration were detected. We found that miR-23b-3p and miR-27b-3p were markedly increased in atrial appendage tissues of AF patients and in Ang-II-treated HAFs. Overexpression of miR-23b-3p and miR-27b-3p enhanced the expression of collagen, type I, alpha 1 (COL1A1), COL3A1 and ACTA2 in HAFs without significant effects on their proliferation and migration. Luciferase assay showed that miR-23b-3p and miR-27b-3p targeted two different sites in 3'-UTR of transforming growth factor (TGF)-ß1 receptor 3 (TGFBR3) respectively. Consistently, TGFBR3 siRNA could increase fibrosis-related genes expression, along with the Smad1 inactivation and Smad3 activation in HAFs. Additionally, overexpression of TGFBR3 could alleviate the increase of COL1A1, COL3A1 and ACTA2 in HAFs after transfection with miR-23b-3p and miR-27b-3p respectively. Moreover, Smad3 was activated in HAFs in response to Ang-II treatment and inactivation of Smad3 attenuated up-regulation of miR-23b-3p and miR-27b-3p in Ang-II-treated HAFs. Taken together, these results suggest that the clustered miR-23b-3p and miR-27b-3p consistently promote atrial fibrosis by targeting TGFBR3 to activate Smad3 signalling in HAFs, suggesting that miR-23b-3p and miR-27b-3p are potential therapeutic targets for atrial fibrosis.
Assuntos
Fibrilação Atrial/genética , MicroRNAs/genética , Proteoglicanas/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Angiotensina II/genética , Fibrilação Atrial/fisiopatologia , Proliferação de Células/genética , Colágeno Tipo III/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/genética , Fibrose/fisiopatologia , Regulação da Expressão Gênica/genética , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Humanos , Síndrome do Nó Sinusal/congênito , Transdução de Sinais/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta1/genéticaRESUMO
BACKGROUND: Ca2+ plays an important role in the regulation of vasoconstriction. Ca2+ signaling is regulated by a number of Ca2+-handling proteins. However, whether differences in Ca2+ handling affect the regulation of vasoconstriction in different arteries remains elusive. OBJECTIVE: To determine whether differences in Ca2+ handling affect the response to vasoconstrictors in different arteries. METHODS: Arterial ring contraction was measured using a Multi Myograph System. Vascular smooth muscle cells (VSMCs) were digested with type 2 collagenase in DMEM, then intracellular calcium concentration was measured with the Ca2+ probe fluo-4/AM in the isolated cells. Calcium-related proteins were assayed by Western blotting. RESULTS: Phenylephrine did not induce -coronary arterial contraction. There were differences in -5-hydroxytryptamine, 9,11-dideoxy-11a,9a-epoxymethano-prostaglandin F2a, and endothelin 1-induced vasoconstriction in different solutions between coronary and renal arteries. Vasoconstrictions in the presence of Bay K8644 were stronger in coronary than in renal arteries. Store-operated calcium (SOC) channels could mediate Ca2+ influx in VSMCs of both groups. SOC channels did not participate in the contraction of coronary arteries. In addition, there were significant differences in the expressions of receptors and ion channels between the two groups. CONCLUSIONS: Ca2+ handling contributed to the different responses to vasoconstrictors between coronary and renal arteries.
Assuntos
Sinalização do Cálcio , Cálcio , Vasos Coronários/metabolismo , Artéria Renal/metabolismo , Vasoconstrição , Animais , Sinalização do Cálcio/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Técnicas In Vitro , Masculino , Ratos Wistar , Artéria Renal/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
Endoplasmic reticulum (ER) stress is mediated by disturbance of Ca2+ homeostasis. The store-operated calcium (SOC) channel is the primary Ca2+ channel in non-excitable cells, but its participation in agent-induced ER stress is not clear. In this study, the effects of tunicamycin on Ca2+ influx in human umbilical vein endothelial cells (HUVECs) were observed with the fluorescent probe Fluo-4 AM. The effect of tunicamycin on the expression of the unfolded protein response (UPR)-related proteins BiP and CHOP was assayed by western blotting with or without inhibition of Orai1. Tunicamycin induced endothelial dysfunction by activating ER stress. Orai1 expression and the influx of extracellular Ca2+ in HUVECs were both upregulated during ER stress. The SOC channel inhibitor SKF96365 reversed tunicamycin-induced endothelial cell dysfunction by inhibiting ER stress. Regulation of tunicamycin-induced ER stress by Orai1 indicates that modification of Orai1 activity may have therapeutic value for conditions with ER stress-induced endothelial dysfunction.
RESUMO
Hypertension is a main risk factor for atrial fibrillation, but the direct effects of hydrostatic pressure on the atrial fibrosis are still unknown. The present study investigated whether hydrostatic pressure is responsible for atrial fibrosis, and addressed a potential role of the Smad pathway in this pathology. Biochemical assays were used to study regulation and expression of fibrotic factors in spontaneously hypertensive rats (SHRs) and Wistar rats, and in cardiac fibroblasts (CFs) cultured under standard (0 mmHg) and elevated (20, 40 mmHg) hydrostatic pressure. Levels of atrial fibrosis and protein expression of fibrotic factors Col-1A1/-3A1, TGF-ß1, and MMP-2 in SHRs' left atrial tissues were higher than those in Wistar rats. Exposure to elevated pressure was associated with the proliferation of CFs. The protein expression of Col-1A1/-3A1, TGF-ß1, and MMP-2 in CFs was also up-regulated in a pressure-dependent manner. The proliferation of CFs and increased expressions of fibrotic markers induced by elevated hydrostatic pressure could be reversed by the Smad3 inhibitor naringenin. The activation of Smad3 pathway was also stimulated by elevated hydrostatic pressure. These results demonstrate that CF secretory function and proliferation can be up-regulated by exposure to elevated pressure, and that Smad3 may modulate CF activation induced by high hydrostatic pressure.
Assuntos
Fibrilação Atrial/etiologia , Remodelamento Atrial , Pressão Sanguínea , Fibroblastos/metabolismo , Átrios do Coração/metabolismo , Hipertensão/complicações , Proteína Smad3/metabolismo , Animais , Fibrilação Atrial/metabolismo , Fibrilação Atrial/patologia , Fibrilação Atrial/fisiopatologia , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Colágeno Tipo III/metabolismo , Modelos Animais de Doenças , Fibroblastos/patologia , Fibrose , Átrios do Coração/patologia , Átrios do Coração/fisiopatologia , Pressão Hidrostática , Hipertensão/metabolismo , Hipertensão/patologia , Hipertensão/fisiopatologia , Metaloproteinase 2 da Matriz/metabolismo , Ratos Endogâmicos SHR , Ratos Wistar , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismoRESUMO
BACKGROUND: Sudden cardiac death (SCD) induced by malignant ventricular tachycardia (MVT) among young adults with right ventricular cardiomyopathy/dysplasia (ARVC/D) is a devastating event. Parts of ARVC/D patients have a mutation in genes encoding components of cardiac desmosomes, such as desmoglein-2 (DSG2), plakophilin-2 and desmoplakin. CASE PRESENTATION: Here we report a potentially pathogenic mutation in the DSG2 gene, which was identified in a family with ARVC/D using Whole Exome Sequencing (WES) and Sanger Sequencing. In all, Patient III:1 with ARVC/D carried the compound heterozygous mutations of DSG2 p.F531C and KCNE5 p.D92E/E93X, which were both inherited from her mother (II:2), who died of SCD. Carriers of DSG2p.F531C showed various phenotypes, such as ARVC/D, SCD, MVT and dilated cardiomyopathy. For III:1, there were significant low-voltage regions in the inferior-apical, inferior-lateral wall of the right ventricular epicardium and outflow tracts of the right ventricle. Under the guidance of a three-dimensional mapping system, MVT was successfully ablated with an epicardial-endocardial approach targeting for late, double or fragmental potentials after implantable cardioverter-defibrillator (ICD) electrical storms. No VT recurrence was observed during the one year of follow-up. CONCLUSIONS: When coexisting with heterozygous KCNE5 p.D92E/E93X, heterozygous DSG2 p.F531C as a genetic background was found to predispose to ARVC/D, SCD and MVT, which were successfully ablated using an epicardial-endocardial approach.
Assuntos
Displasia Arritmogênica Ventricular Direita/complicações , Morte Súbita Cardíaca/etiologia , Morte Súbita/etiologia , Desmogleína 2/genética , Mutação/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Adulto , Displasia Arritmogênica Ventricular Direita/genética , Feminino , Heterozigoto , Humanos , MasculinoRESUMO
BACKGROUND: This study was designed to identify the pathogenic mutation in a Chinese family with arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) using whole genome sequencing (WGS). METHODS AND RESULTS: Probands II:1 and II:2 underwent routine examinations for diagnosis. Genomic DNA was extracted from the peripheral blood of family members and analyzed using WGS. A total of 60,285 single-nucleotide polymorphisms (SNP) and 13,918 insertions/deletions (InDel) occurring in the exonic regions of genes and predisposing to cardiomyopathies and arrhythmias were identified. When filtered using the 1000 Genomes Project (2014 version), NHLBI ESP6500, and ExAC databases, 12 missense SNP and 2 InDel in exonic regions remained, the allele frequencies of which were <0.01 or unknown. The potentially pathogenic mutations that occurred in the genes DSG2, PKP4, PRKAG2, FOXD4, CTTN, and DMD, which were identified by SIFT or PolyPhen-2 software as "damaging," were validated using Sanger sequencing. Probands II:1 and II:2 shared an extremely rare homozygous mutation in the DSG2 (p.F531C) gene, which was also demonstrated using intersection analysis of WGS data from probands II:1 and II:2. Electron microscopy and histological staining of myocardial biopsies showed widened and destroyed intercalated discs, and interrupted, atrophic, and disarranged myocardial fibers, and hyperplastic interstitial fibers, collagen fibers, and adipocytes were infiltrated and invaded. CONCLUSIONS: A homozygous mutation of DSG2 p.F531C was identified as the pathogenic mutation in patients with ARVC/D involving both ventricles, as a result of widened and impaired intercalated discs, interrupted myocardial fibers, and abnormally hyperplastic interstitial fibers, collagen fibers, and adipocytes.
Assuntos
Displasia Arritmogênica Ventricular Direita/diagnóstico por imagem , Displasia Arritmogênica Ventricular Direita/genética , Desmogleína 2/genética , Miocárdio/patologia , Adolescente , Adulto , Povo Asiático/genética , Ecocardiografia , Eletrocardiografia , Frequência do Gene , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Mutação , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Genoma , Adulto JovemRESUMO
MicroRNA-1 (miR-1) is approved involved in cardiac hypertrophy, but the underlying molecular mechanisms of miR-1 in cardiac hypertrophy are not well elucidated. The present study aimed to investigate the potential role of miR-1 in modulating CDKs-Rb pathway during cardiomyocyte hypertrophy. A rat model of hypertrophy was established with abdominal aortic constriction, and a cell model of hypertrophy was also achieved based on PE-promoted neonatal rat ventricular cardiomyocytes (NRVCs). We demonstrated that miR-1 expression was markedly decreased in hypertrophic myocardium and hypertrophic cardiomyocytes. Dual luciferase reporter assays revealed that miR-1 interacted with the 3'UTR of CDK6, and miR-1 was verified to inhibit CDK6 expression at the posttranscriptional level. CDK6 protein expression was observed increased in hypertrophic myocardium and hypertrophic cardiomyocytes. Morover, miR-1 mimic, in parallel to CDK6 siRNA, could inhibit PE-induced hypertrophy of NRVCs, with decreases in cell size, newly transcribed RNA, expressions of ANF and ß-MHC, and the phosphorylated pRb. Taken together, our results reveal that derepression of CDK6 and activation of Rb pathway contributes to the effect of attenuation of miR-1 on provoking cardiomyocyte hypertrophy.
Assuntos
Cardiomegalia/metabolismo , Quinase 6 Dependente de Ciclina/fisiologia , MicroRNAs/metabolismo , Miócitos Cardíacos/patologia , Animais , Quinase 6 Dependente de Ciclina/genética , Regulação para Baixo , Masculino , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-DawleyRESUMO
Cyclins/retinoblastoma protein (pRb) pathway participates in cardiomyocyte hypertrophy. MicroRNAs (miRNAs), the endogenous small non-coding RNAs, were recognized to play significant roles in cardiac hypertrophy. But, it remains unknown whether cyclin/Rb pathway is modulated by miRNAs during cardiac hypertrophy. This study investigates the potential role of microRNA-16 (miR-16) in modulating cyclin/Rb pathway during cardiomyocyte hypertrophy. An animal model of hypertrophy was established in a rat with abdominal aortic constriction (AAC), and in a mouse with transverse aortic constriction (TAC) and in a mouse with subcutaneous injection of phenylephrine (PE) respectively. In addition, a cell model of hypertrophy was also achieved based on PE-promoted neonatal rat ventricular cardiomyocyte and based on Ang-II-induced neonatal mouse ventricular cardiomyocyte respectively. We demonstrated that miR-16 expression was markedly decreased in hypertrophic myocardium and hypertrophic cardiomyocytes in rats and mice. Overexpression of miR-16 suppressed rat cardiac hypertrophy and hypertrophic phenotype of cultured cardiomyocytes, and inhibition of miR-16 induced a hypertrophic phenotype in cardiomyocytes. Expressions of cyclins D1, D2 and E1, and the phosphorylated pRb were increased in hypertrophic myocardium and hypertrophic cardiomyocytes, but could be reversed by enforced expression of miR-16. Cyclins D1, D2 and E1, not pRb, were further validated to be modulated post-transcriptionally by miR-16. In addition, the signal transducer and activator of transcription-3 and c-Myc were activated during myocardial hypertrophy, and inhibitions of them prevented miR-16 attenuation. Therefore, attenuation of miR-16 provoke cardiomyocyte hypertrophy via derepressing the cyclins D1, D2 and E1, and activating cyclin/Rb pathway, revealing that miR-16 might be a target to manage cardiac hypertrophy.
Assuntos
Cardiomegalia/genética , Ciclina D1/metabolismo , Ciclina D2/metabolismo , Ciclinas/metabolismo , MicroRNAs/genética , Animais , Aorta Abdominal/cirurgia , Linhagem Celular , Ciclina D1/biossíntese , Ciclina D2/biossíntese , Ciclinas/biossíntese , Modelos Animais de Doenças , Ativação Enzimática , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/biossíntese , Miócitos Cardíacos/patologia , Fenilefrina/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas c-myc , Ratos , Ratos Sprague-Dawley , Proteína do Retinoblastoma/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
OBJECTIVE: This study was designed to evaluate the pharmacokinetics (PK) and safety of eptifibatide in healthy Chinese volunteers and provide information for the further study in the Chinese population. METHODS: 30 healthy volunteers (15 male) were enrolled in the study and divided into three dose groups (45 µg x kg⻹, 90 µg x kg⻹, and 180 µg x kg⻹). Plasma and urine samples were drawn after one single-bolus administration and measured by LC-MS/MS. The plasma and urine data were analyzed simultaneously by the population approach using the NONMEM software and evaluated by the visual predicted check (VPC) and bootstraping. The PK profiles of dose regimens approved for a Western population in the Chinese population were simulated. RESULTS: A two-compartment model adequately described the PK profiles of eptifibatide. The clearance (CL) and the distribution volume (V1) of the central compartment were 0.128 L x h⻹ x kg⻹ and 0.175 L x kg⻹, respectively. The clearance (Q) and V2of the peripheral compartment were 0.0988 L x h⻹ x kg⻹ and 0.147 L x kg⻹, respectively. The elimination fraction from plasma to urine (F0) was 17.2%. No covariates were found to have a significant effect. Inter-individual variabilites were all within 33.9%. The VPC plots and bootstrap results indicated good precision and prediction of the model. The simulations of the approved regimens in the Chinese population showed much lower steady-state concentrations than the target concentration obtained from the Western clinical trials. No severe safety events were found in this study. CONCLUSIONS: The PK model of eptifibatide was established and could provide PK information for further studies in the Chinese population.
Assuntos
Povo Asiático , Simulação por Computador , Modelos Biológicos , Peptídeos/administração & dosagem , Peptídeos/farmacocinética , Inibidores da Agregação Plaquetária/administração & dosagem , Inibidores da Agregação Plaquetária/farmacocinética , Ocidente , População Branca , Adolescente , Adulto , Área Sob a Curva , China , Cromatografia Líquida , Cálculos da Dosagem de Medicamento , Eptifibatida , Feminino , Meia-Vida , Voluntários Saudáveis , Humanos , Masculino , Taxa de Depuração Metabólica , Segurança do Paciente , Peptídeos/efeitos adversos , Peptídeos/sangue , Peptídeos/urina , Inibidores da Agregação Plaquetária/efeitos adversos , Inibidores da Agregação Plaquetária/sangue , Inibidores da Agregação Plaquetária/urina , Medição de Risco , Software , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
Despite the fact that tissue engineered heart valves (TEHV) hold great promise for heart valve disease treatment, one of the challenges is to find suitable seeding cells. Bone marrow derived mesenchymal stem cells (MSCs) were considered to be one of the best seed cell sources. In this study we propose a novel approach to promote stem cell differentiation into the seed cells of TEHV, valvular interstitial cells (VICs). Newly induced MSCs (iMSCs) were created from a co-culture niche in which healthy human donor derived MSCs were co-cultured with cardiac fibroblasts (H9C2 cell line). Then iMSCs were transfected with either a mock vector (iMSCs(mock) ) as controls or with a vector that overexpresses thefibroblast inducible factor 14 (Fn14) gene (iMSCs(Fn14) ). Immunofluorescence staining was performed to assay VIC differentiation. Western blot analysis was performed to analyze the involved signaling pathway. The results demonstrate that the expression of α-smooth muscle actin (SMA) was significantly higher in iMSCs(Fn14) as compared with iMSC(mock) , and MSC, and also had higher co-alignment of α-actinin and stress fiber (F-actin) in bundles. Additionally, increased biosynthesis of extracellular matrix (ECM) proteins including collagen I, collagen III, and fibronection were observed in iMSCs(Fn14) in comparison with iMSCs(mock) . These data observed in iMSCs(Fn14) were in accordance with VIC phenotype from normal heart valves. In addition, the PI3K/Akt signaling pathway was activated in iMSCs(Fn14) which allowed higher Akt phosphorylation (p-Akt) levels and SMA levels, whereas, it was attenuated by LY294002 (PI3K/Akt inhibitor). These new findings of the effect of Fn14 on VIC-like cell differentiation may provide a novel therapeutic strategy for heart valve disease treatment.
Assuntos
Diferenciação Celular/fisiologia , Valvas Cardíacas/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Receptores do Fator de Necrose Tumoral/metabolismo , Linhagem Celular , Técnicas de Cocultura , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Fibroblastos/citologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Células-Tronco Mesenquimais/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Receptores do Fator de Necrose Tumoral/genética , Transdução de Sinais/fisiologia , Receptor de TWEAK , Engenharia Tecidual , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
The beneficial effects of mesenchymal stem cells (MSCs) in cardiac cell therapy are greatly limited due to poor survival after transplantation into ischemic hearts. Here, we investigated whether caspase 8 small hairpin RNA (shRNA) modification enhance human MSCs (hMSCs) survival and improve infarcted heart function. Recombinant adenovirus encoding pre-miRNA-155-designed caspase 8 shRNA was prepared to inhibit caspase 8 expression in hMSCs. The effect of caspase 8 shRNA modification on protecting hMSCs from apoptosis under the conditions of serum deprivation and hypoxia was tested by Annexin V/PI staining and caspase 8 activity assay. The caspase 8 shRNA-modified and superparamagnetic iron oxide (SPIO)-labeled hMSCs were injected into the border zone of the infarcted region of rat heart. Echocardiography and Masson trichrome staining were performed to assess heart function and cardiac fibrosis. Our results showed that adenovirus-mediated caspase 8 shRNA could efficiently inhibit caspase 8 expression in hMSCs. Knock-down of caspase 8 expression lead to inhibition of hMSCs apoptosis, reduction of caspase 8 activity and up-regulations of HGF, IGF-1 and Bcl-2. Transplantation of caspase 8 shRNA-modified hMSCs could significantly improve infracted heart function, attenuate cardiac fibrosis. Consistently, the rate of cardiomyocyte apoptosis and caspase 8 activity were significantly decreased, and the survival rate of transplanted hMSCs was markedly elevated in the myocardium receiving caspase 8 shRNA-modified hMSCs transplantation. Together, our findings implicated the therapeutic potential of caspase 8 shRNA-modified hMSCs in improving the infarcted heart function.
Assuntos
Adenoviridae , Caspase 8 , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/enzimologia , MicroRNAs , Infarto do Miocárdio/terapia , Adulto , Animais , Apoptose/genética , Caspase 8/biossíntese , Caspase 8/genética , Feminino , Técnicas de Silenciamento de Genes/métodos , Humanos , Masculino , MicroRNAs/biossíntese , MicroRNAs/genética , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Ratos , Ratos Sprague-Dawley , Transdução GenéticaRESUMO
BACKGROUND: Among those with heart failure (HF), women are more likely to develop depression than men. Few studies have focused on the outcomes of female patients with HF with depressive symptoms. METHODS AND RESULTS: A total of 506 female patients with HF with preserved ejection fraction were included in this secondary analysis from the TOPCAT (Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist) cohort, and 439 female patients with HF with reduced ejection fraction were included from the HF-ACTION (Heart Failure: A Controlled Trial Investigating Outcomes of Exercise Training) cohort. Depressive symptoms were measured using the Patient Health Questionnaire-9 and Beck Depression Inventory-II. The depression class was categorized by severity, and the change in clinical depression class was defined as aggravated (1-grade increase) or improved (1-grade decrease). The prognostic value of depressive symptoms was determined by using multivariable Cox proportional hazards models. Female patients with improved depressive symptoms had worse depressive status at baseline and lower baseline Kansas City Cardiomyopathy Questionnaire scores. Depression class at the 12-month visit and depression class change were the dominant prognostic factors for cardiovascular death in female patients with HF with preserved ejection fraction (hazard ratio [HR], 1.43 [95% CI, 1.02-2.01], P=0.036; HR, 1.71 [95% CI, 1.14-2.55], P=0.009). Among the patients with HF with reduced ejection fraction, both the depression class at baseline and depression class change had significant prognostic effects on cardiovascular death (HR, 3.30 [95% CI, 1.70-6.39], P<0.001; HR, 2.21 [95% CI, 1.28-3.80], P=0.004). However, the prognostic value of depressive assessments for hospitalization in patients with HF is unclear. CONCLUSIONS: In female patients with HF with reduced ejection fraction, the depression class at baseline was most strongly associated with cardiovascular death, whereas in female patients with HF with preserved ejection fraction, the change in depression class exhibited a more significant prognostic trend.
Assuntos
Depressão , Insuficiência Cardíaca , Volume Sistólico , Humanos , Feminino , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/psicologia , Insuficiência Cardíaca/mortalidade , Insuficiência Cardíaca/complicações , Volume Sistólico/fisiologia , Depressão/diagnóstico , Depressão/psicologia , Depressão/epidemiologia , Idoso , Pessoa de Meia-Idade , Prognóstico , Função Ventricular Esquerda , Fatores de Risco , Fatores Sexuais , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Modelos de Riscos Proporcionais , Fatores de TempoRESUMO
The T-type Ca(2+) current (I(Ca,T)) plays an important role in the pathogenesis of atrial fibrillation (AF). The present study sought to investigate the role of macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, in the regulation of T-type Ca(2+) channels (TCCs) in atrial myocytes. We used the whole-cell voltage-clamp technique and biochemical assays to study the regulation and expression of I(Ca,T) in atrial myocytes. Gene levels of the α1G and α1H subunit of TCCs were decreased in human atrial tissue of patients with AF. In cultured atrium-derived myocytes (HL-1 cells), mouse recombinant MIF (20 or 40 nm, 24 h) suppressed peak I(Ca,T) in a concentration-dependent manner, impaired the voltage-dependent activation of I(Ca,T) and downregulated TCC α1G and α1H mRNA. The Src inhibitors genistein and PP1 significantly enhanced I(Ca,T). The reduction of I(Ca,T) and TCC subunit mRNA induced by recombinant MIF could be reversed by genistein and PP1. The TCC α1G associated with Src in HL-1 cells and mouse cardiomycytes. Macrophage migration inhibitory factor is involved in the pathogenesis of AF, probably by decreasing the T-type calcium current in atrium-derived myocytes through impairment of channel function and activation of c-Src kinases, representing a potential pathogenic mechanism in atrial fibrillation.
Assuntos
Canais de Cálcio Tipo T/fisiologia , Oxirredutases Intramoleculares/fisiologia , Fatores Inibidores da Migração de Macrófagos/fisiologia , Adulto , Idoso , Animais , Fibrilação Atrial , Proteína Tirosina Quinase CSK , Linhagem Celular , Feminino , Genisteína/farmacologia , Átrios do Coração/citologia , Humanos , Oxirredutases Intramoleculares/farmacologia , Fatores Inibidores da Migração de Macrófagos/farmacologia , Masculino , Camundongos , Pessoa de Meia-Idade , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Proteínas Recombinantes/farmacologia , Quinases da Família src/biossínteseRESUMO
Scavenger receptor A (SR-A) is a key transmembrane receptor in the endocytosis of lipids and contributes to the pathogenesis of atherosclerosis. To assess its role in hyperlipidemic chronic kidney disease, wild-type and SR-A-deficient (knockout) mice underwent uninephrectomy followed by either normal or high-fat diet. After 16 weeks of diet intervention, hyperlipidemic wild-type mice presented characteristic features of progressive nephropathy: albuminuria, renal fibrosis, and overexpression of transforming growth factor (TGF)-ß1/Smad. These changes were markedly diminished in hyperlipidemic knockout mice and attributed to reduced renal lipid retention, oxidative stress, and CD11c(+) cell infiltration. In vitro, overexpression of SR-A augmented monocyte chemoattractant protein-1 release and TGF-ß1/Smad activation in HK-2 cells exposed to oxidized low-density lipoprotein. SR-A knockdown prevented lipid-induced cell injury. Moreover, wild-type to knockout bone marrow transplantation resulted in renal fibrosis in uninephrectomized mice following 16 weeks of the high-fat diet. In contrast, knockout to wild-type bone marrow transplantation led to markedly reduced albuminuria, CD11c(+) cell infiltration, and renal fibrosis compared to wild-type to SR-A knockout or wild-type to wild-type bone marrow transplanted mice, without difference in plasma lipid levels. Thus, SR-A on circulating leukocytes rather than resident renal cells predominantly mediates lipid-induced kidney injury.
Assuntos
Gorduras na Dieta/metabolismo , Hiperlipidemias/complicações , Nefropatias/prevenção & controle , Rim/metabolismo , Leucócitos/metabolismo , Receptores Depuradores Classe A/deficiência , Albuminúria/etiologia , Albuminúria/metabolismo , Albuminúria/prevenção & controle , Animais , Transplante de Medula Óssea , Antígeno CD11c/metabolismo , Linhagem Celular , Quimiocina CCL2/metabolismo , Doença Crônica , Gorduras na Dieta/sangue , Modelos Animais de Doenças , Progressão da Doença , Fibrose , Hiperlipidemias/etiologia , Rim/patologia , Nefropatias/etiologia , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Camundongos , Camundongos Knockout , Nefrectomia , Estresse Oxidativo , Interferência de RNA , Receptores Depuradores Classe A/genética , Receptores Depuradores Classe A/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Fatores de Tempo , Transfecção , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Diabetic patients continue to develop inflammation and cardiovascular complication even after achieving glycemic control, suggesting a "metabolic memory". Metabolic memory is a major challenge in the treatment of diabetic complication, and the mechanisms underlying metabolic memory are not clear. Recent studies suggest a link between chromatin histone methylation and metabolic memory. In this study, we tested whether histone 3 lysine-9 tri-methylation (H3K9me3), a key epigenetic chromatin marker, was involved in high glucose (HG)-induced inflammation and metabolic memory. Incubating cardiomyocyte cells in HG resulted in increased levels of inflammatory cytokine IL-6 mRNA when compared with myocytes incubated in normal culture media, whereas mannitol (osmotic control) has no effect. Chromatin immunoprecipitation (ChIP) assays showed that H3K9me3 levels were significantly decreased at the promoters of IL-6. Immunoblotting demonstrated that protein levels of the H3K9me3 methyltransferase, Suv39h1, were also reduced after HG treatment. HG-induced apoptosis, mitochondrial dysfunction and cytochrome-c release were reversible. However, the effects of HG on the expression of IL-6 and the levels of H3K9me3 were irreversible after the removal of HG from the culture. These results suggest that HG-induced sustained inflammatory phenotype and epigenetic histone modification, rather than HG-induced mitochondrial dysfunction and apoptosis, are main mechanisms responsible for metabolic memory. In conclusion, our data demonstrate that HG increases expression of inflammatory cytokine and decreases the levels of histone-3 methylation at the cytokine promoter, and suggest that modulating histone 3 methylation and inflammatory cytokine expression may be a useful strategy to prevent metabolic memory and cardiomyopathy in diabetic patients.
Assuntos
Epigênese Genética , Glucose/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Miócitos Cardíacos/metabolismo , Linhagem Celular , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Humanos , Inflamação/genética , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Metilação , Miócitos Cardíacos/efeitos dos fármacos , Regiões Promotoras GenéticasRESUMO
We investigated whether transplantation of bone marrow mesenchymal stem cells (BMSC) with induced BMSC (iBMSC) or uninduced BMSC (uBMSC) into the myocardium could improve the performance of post-infarcted rat hearts. BMSCs were specified by flowcytometry. IBMSCs were cocultured with rat cardiomyocyte before transplantation. Cells were injected into borders of cardiac scar tissue 1 week after experimental infarction. Cardiac performance was evaluated by echocardiography at 1, 2, and 4 weeks after cellular or PBS injection. Langendorff working-heart and histological studies were performed 4 weeks after treatment. Myogenesis was detected by quantitative PCR and immunofluorescence. Echocardiography showed a nearly normal ejection fraction (EF) in iBMSC-treated rats and all sham control rats but a lower EF in all PBS-treated animals. The iBMSC-treated heart, assessed by echocardiography, improved fractional shortening compared with PBS-treated hearts. The coronary flow (CF) was decreased obviously in PBS and uBMSC-treated groups, but recovered in iBMSC-treated heart at 4 weeks (P < 0.01). Immunofluorescent microscopy revealed co-localization of Superparamagnetic iron oxide (SPIO)-labeled transplanted cells with cardiac markers for cardiomyocytes, indicating regeneration of damaged myocardium. These data provide strong evidence that iBMSC implantation is of more potential to improve infarcted cardiac performance than uBMSC treatment. It will open new promising therapeutic opportunities for patients with post-infarction heart failure.
Assuntos
Transplante de Medula Óssea , Coração/fisiologia , Transplante de Células-Tronco Mesenquimais , Infarto do Miocárdio/terapia , Animais , Diferenciação Celular/fisiologia , Primers do DNA/genética , Ecocardiografia , Citometria de Fluxo , Masculino , Microscopia de Fluorescência , Desenvolvimento Muscular/fisiologia , Miócitos Cardíacos/transplante , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-DawleyRESUMO
Increasing evidence has shown that circular RNAs (circRNAs) participate in the process of cardiac remodeling. CircRNA circ_0036176 originating from the back-splicing of exon 2 to exon4 of myosin IXA (Myo9a) gene was shown to be increased in the myocardium of patients with heart failure (HF) and riched in exosomes from human AC16 cardiomyocytes with overexpression of circ_0036176. Proliferation activity was inhibited in mCFs subjected to exosomal circ_0036176 treatment and in mCFs with overexpression of circ_0036176. Interestingly, circ_0036176 contains an IRES element and an ORF of 627 nt encoding a 208-amino acid protein (termed as Myo9a-208). Myo9a-208 was shown to mediate the inhibitory effect of circ_0036176 on CFs proliferation, and miR-218-5p could inhibit Myo9a-208 expression by binding to circ_0036176, resulting in abolishing the effect of circ_0036176 on inactivating cyclin/Rb signal and suppressing CFs proliferation. Our findings suggest that circ_0036176 inhibits mCFs proliferation by translating Myo9a-208 protein to suppress cyclin/Rb pathway.
Assuntos
Fibroblastos , MicroRNAs , Miocárdio , RNA Circular , Proliferação de Células , Ciclinas , Fibroblastos/metabolismo , Humanos , MicroRNAs/genética , Miocárdio/citologia , RNA Circular/genéticaRESUMO
Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine associated with the atherosclerotic process and atherosclerotic plaque stability. MIF was shown to be highly expressed in advanced atherosclerotic lesions. Neutralizing MIF with a blocking antibody induced a regression of established atherosclerotic lesions. In this study, we investigated the mechanism underlying the proangiogenic effect of MIF in human umbilical vein endothelial cells (HUVECs). We showed that MIF induced the expression of angiogenesis-related genes in HUVECs. We also showed that MIF induced tube formation of HUVECs in vitro and in vivo. Angiotensin II (Ang II) could specifically up-regulate MIF expression in HUVECs. Using a luciferase reporter assay, we demonstrated that the AP-1 response element in the 5'-UTR of the MIF gene played a role in Ang II-induced MIF expression. Small hairpin RNA (shRNA) targeting c-Jun, a component of AP-1, and the AP-1 inhibitor CHX both efficiently inhibited MIF expression. The consistent result of electrophoretic mobility shift assay (EMSA) showed that Ang II specifically increased AP-1 activation in HUVECs. Our results suggest that AP-1 mediates Ang II-induced MIF expression which contributes to atherosclerotic plaque destabilization in human endothelial cells.