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Sodium glucose cotransporter 2 inhibitors (SGLT2i) constitute the only medication class that consistently prevents or attenuates human heart failure (HF) independent of ejection fraction. We have suggested earlier that the protective mechanisms of the SGLT2i Empagliflozin (EMPA) are mediated through reductions in the sodium hydrogen exchanger 1 (NHE1)-nitric oxide (NO) pathway, independent of SGLT2. Here, we examined the role of SGLT2, NHE1 and NO in a murine TAC/DOCA model of HF. SGLT2 knockout mice only showed attenuated systolic dysfunction without having an effect on other signs of HF. EMPA protected against systolic and diastolic dysfunction, hypertrophy, fibrosis, increased Nppa/Nppb mRNA expression and lung/liver edema. In addition, EMPA prevented increases in oxidative stress, sodium calcium exchanger expression and calcium/calmodulin-dependent protein kinase II activation to an equal degree in WT and SGLT2 KO animals. In particular, while NHE1 activity was increased in isolated cardiomyocytes from untreated HF, EMPA treatment prevented this. Since SGLT2 is not required for the protective effects of EMPA, the pathway between NHE1 and NO was further explored in SGLT2 KO animals. In vivo treatment with the specific NHE1-inhibitor Cariporide mimicked the protection by EMPA, without additional protection by EMPA. On the other hand, in vivo inhibition of NOS with L-NAME deteriorated HF and prevented protection by EMPA. In conclusion, the data support that the beneficial effects of EMPA are mediated through the NHE1-NO pathway in TAC/DOCA-induced heart failure and not through SGLT2 inhibition.
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The release of extracellular vesicles (EVs) is increased under cellular stress and cardiomyocyte damaging conditions. However, whether the cardiomyocyte-derived EVs eventually reach the systemic circulation and whether their number in the bloodstream reflects cardiac injury, remains unknown. Wild type C57B/6 and conditional transgenic mice expressing green fluorescent protein (GFP) by cardiomyocytes were studied in lipopolysaccharide (LPS)-induced systemic inflammatory response syndrome (SIRS). EVs were separated both from platelet-free plasma and from the conditioned medium of isolated cardiomyocytes of the left ventricular wall. Size distribution and concentration of the released particles were determined by Nanoparticle Tracking Analysis. The presence of GFP + cardiomyocyte-derived circulating EVs was monitored by flow cytometry and cardiac function was assessed by echocardiography. In LPS-treated mice, systemic inflammation and the consequent cardiomyopathy were verified by elevated plasma levels of TNFα, GDF-15, and cardiac troponin I, and by a decrease in the ejection fraction. Furthermore, we demonstrated elevated levels of circulating small- and medium-sized EVs in the LPS-injected mice. Importantly, we detected GFP+ cardiomyocyte-derived EVs in the circulation of control mice, and the number of these circulating GFP+ vesicles increased significantly upon intraperitoneal LPS administration (P = 0.029). The cardiomyocyte-derived GFP+ EVs were also positive for intravesicular troponin I (cTnI) and muscle-associated glycogen phosphorylase (PYGM). This is the first direct demonstration that cardiomyocyte-derived EVs are present in the circulation and that the increased number of cardiac-derived EVs in the blood reflects cardiac injury in LPS-induced systemic inflammation (SIRS).
Assuntos
Movimento Celular , Vesículas Extracelulares/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/patologia , Síndrome de Resposta Inflamatória Sistêmica/patologia , Animais , Movimento Celular/efeitos dos fármacos , Clusterina/metabolismo , Vesículas Extracelulares/efeitos dos fármacos , Glicogênio Fosforilase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Integrases/metabolismo , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Fenótipo , Síndrome de Resposta Inflamatória Sistêmica/sangue , Síndrome de Resposta Inflamatória Sistêmica/fisiopatologia , Tamoxifeno/farmacologia , Troponina I/metabolismoRESUMO
Lipid-lowering drugs have been shown to have cardioprotective effects but may have hidden cardiotoxic properties. Therefore, here we aimed to investigate if chronic treatment with the novel lipid-lowering drug bempedoic acid (BA) exerts hidden cardiotoxic and/or cardioprotective effects in a rat model of acute myocardial infarction (AMI). Wistar rats were orally treated with BA or its vehicle for 28 days, anesthetized and randomized to three different groups (vehicle + ischemia/reperfusion (I/R), BA + I/R, and positive control vehicle + ischemic preconditioning (IPC)) and subjected to cardiac 30 min ischemia and 120 min reperfusion. IPC was performed by 3 × 5 min I/R cycles before ischemia. Myocardial function, area at risk, infarct size and arrhythmias were analyzed. Chronic BA pretreatment did not influence cardiac function or infarct size as compared to the vehicle group, while the positive control IPC significantly reduced the infarct size. The incidence of reperfusion-induced arrhythmias was significantly reduced by BA and IPC. This is the first demonstration that BA treatment does not show cardioprotective effect although moderately reduces the incidence of reperfusion-induced arrhythmias. Furthermore, BA does not show hidden cardiotoxic effect in rats with AMI, showing its safety in the ischemic/reperfused heart.
Assuntos
Precondicionamento Isquêmico Miocárdico , Infarto do Miocárdio , Isquemia Miocárdica , Traumatismo por Reperfusão Miocárdica , Animais , Ratos , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/prevenção & controle , Cardiotoxicidade , Lipídeos/uso terapêutico , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Ratos WistarRESUMO
BACKGROUND: Cardiac cell lines and primary cells are widely used in cardiovascular research. Despite increasing number of publications using these models, comparative characterization of these cell lines has not been performed, therefore, their limitations are undetermined. We aimed to compare cardiac cell lines to primary cardiomyocytes and to mature cardiac tissues in a systematic manner. METHODS AND RESULTS: Cardiac cell lines (H9C2, AC16, HL-1) were differentiated with widely used protocols. Left ventricular tissue, neonatal primary cardiomyocytes, and human induced pluripotent stem cell-derived cardiomyocytes served as reference tissue or cells. RNA expression of cardiac markers (e.g. Tnnt2, Ryr2) was markedly lower in cell lines compared to references. Differentiation induced increase in cardiac- and decrease in embryonic markers however, the overall transcriptomic profile and annotation to relevant biological processes showed consistently less pronounced cardiac phenotype in all cell lines in comparison to the corresponding references. Immunocytochemistry confirmed low expressions of structural protein sarcomeric alpha-actinin, troponin I and caveolin-3 in cell lines. Susceptibility of cell lines to sI/R injury in terms of viability as well as mitochondrial polarization differed from the primary cells irrespective of their degree of differentiation. CONCLUSION: Expression patterns of cardiomyocyte markers and whole transcriptomic profile, as well as response to sI/R, and to hypertrophic stimuli indicate low-to-moderate similarity of cell lines to primary cells/cardiac tissues regardless their differentiation. Low resemblance of cell lines to mature adult cardiac tissue limits their potential use. Low translational value should be taken into account while choosing a particular cell line to model cardiomyocytes.
Assuntos
Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Animais , Biomarcadores/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Fenótipo , TranscriptomaRESUMO
Patients diagnosed with metastatic uveal melanoma (MUM) have a poor survival prognosis. Unfortunately for this rare disease, there is no known cure and suitable therapeutic options are limited. HDAC6 inhibitors (HDAC6i) are currently in clinical trials for other cancers and show potential beneficial effects against tumor cell survival in vitro and in vivo. In MUM cells, HDAC6i show an anti-proliferative effect in vitro and in preclinical xenograft models. The use of HDAC6 inhibitors as a treatment option for MUM should be explored further. Therefore, this review discusses (1) what is known about HDAC6i in MUM and (2) whether HDAC6 inhibitors offer a potential therapeutic option for MUM.
Assuntos
Melanoma , Segunda Neoplasia Primária , Neoplasias Uveais , Desacetilase 6 de Histona , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Neoplasias Uveais/tratamento farmacológico , Neoplasias Uveais/patologiaRESUMO
Cardiac stromal cells (CSCs) contain a pool of cells with supportive and paracrine functions. Various types of mesenchymal stromal cells (MSCs) can influence CSCs in the cardiac niche through their paracrine activity. Ischaemia/reperfusion (I/R) leads to cell death and reduction of the paracrine activity of CSCs. The forced co-expression of telomerase reverse transcriptase (TERT) and myocardin (MYOCD), known to potentiate anti-apoptotic, pro-survival and pro-angiogenic activities of MSCs isolated from the adipose tissue (AT-MSCs), may increase CSC survival, favouring their paracrine activities. We aimed at investigating the hypothesis that CSCs feature improved resistance to simulated I/R (SI/R) and increased commitment towards the cardiovascular lineage when preconditioned with conditioned media (CM) or extracellular vesicles (EV) released from AT-MSCs overexpressing TERT and MYOCD (T/M AT-MSCs). Murine CSCs were isolated with the cardiosphere (CSps) isolation technique. T/M AT-MSCs and their secretome improved spontaneous intracellular calcium changes and ryanodine receptor expression in aged CSps. The cytoprotective effect of AT-MSCs was tested in CSCs subjected to SI/R. SI/R induced cell death as compared to normoxia (28 ± 4 vs 10 ± 3%, P = .02). Pre-treatment with CM (15 ± 2, P = .02) or with the EV-enriched fraction (10 ± 1%, P = .02) obtained from mock-transduced AT-MSCs in normoxia reduced cell death after SI/R. The effect was more pronounced with CM (7 ± 1%, P = .01) or the EV-enriched fraction (2 ± 1%, P = .01) obtained from T/M AT-MSCs subjected to SI/R. In parallel, we observed lower expression of the apoptosis marker cleaved caspase-3 and higher expression of cardiac and vascular markers eNOS, sarcomeric α-actinin and cardiac actin. The T/M AT-MSCs secretome exerts a cytoprotective effect and promotes development of CSCs undergoing SI/R towards a cardiovascular phenotype.
Assuntos
Biomarcadores/metabolismo , Doenças Cardiovasculares/terapia , Coração/crescimento & desenvolvimento , Células-Tronco Mesenquimais/citologia , Proteínas Nucleares/metabolismo , Traumatismo por Reperfusão/complicações , Telomerase/metabolismo , Transativadores/metabolismo , Animais , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Telomerase/genética , Transativadores/genéticaRESUMO
Cardiomyopathy resistant to treatment is the most serious adverse effect of doxorubicin (dox). The mechanisms of dox-induced cardiomyopathy (DCM) have been extensively studied in dilated forms of DCM. However, efficient treatment did not emerge. The aim of the present work was to revisit the experimental model of DCM in rats, to define phenotype/s and associate them to the changes in cardiac transcriptome. Male Wistar rats equipped with radiotelemetry device, were randomized in DOX group (5 mg/0,5 mL/kg, IV dox; n = 18) and CONT group (0,5 mL/kg IV saline; n = 6). Echocardiography, autonomic spectral markers and baroreceptor reflex evaluation was performed prior to, and after treatment. Blood samples were collected at the end of experimentation. Cardiac, renal and hepatic tissues were analysed post-mortem by histology. Changes in expression of key cardiac genes affected by dox were assessed by RT-qPCR. Phenotypes were identified by clustering non-redundant features using four different algorithms averaged by evidence accumulation cluster technique. The results emphasize the existence of two major phenotypes of DCM with comparably high mortality rates: phenotype 1 characterized by, left ventricular (LV) dilatation, thinning of LV posterior wall, reduced LV ejection fraction (LVEF) and fractional shortening (LVFS), decreased HR variability (HRV), decreased baroreceptor effectiveness index (BEI) and increased NT-proBNP; and phenotype 2 with LV hypertrophy - increased LV mass, preserved LVEF, LVFS, no changes in HRV and BEI and moderate NT-proBNP increase. Both phenotypes exhibited a genetic shift to a new-born program.
Assuntos
Antibióticos Antineoplásicos/toxicidade , Cardiomiopatias/classificação , Cardiomiopatias/genética , Mapeamento Cromossômico/métodos , Doxorrubicina/toxicidade , Animais , Cardiomiopatias/induzido quimicamente , Masculino , Distribuição Aleatória , Ratos , Ratos WistarRESUMO
Cardioprotective medications are still unmet clinical needs. We have previously identified several cardioprotective microRNAs (termed ProtectomiRs), the mRNA targets of which may reveal new drug targets for cardioprotection. Here we aimed to identify key molecular targets of ProtectomiRs and confirm their association with cardioprotection in a translational pig model of acute myocardial infarction (AMI). By using a network theoretical approach, we identified 882 potential target genes of 18 previously identified protectomiRs. The Rictor gene was the most central and it was ranked first in the protectomiR-target mRNA molecular network with the highest node degree of 5. Therefore, Rictor and its targeting microRNAs were further validated in heart samples obtained from a translational pig model of AMI and cardioprotection induced by pre- or postconditioning. Three out of five Rictor-targeting pig homologue of rat ProtectomiRs showed significant upregulation in postconditioned but not in preconditioned pig hearts. Rictor was downregulated at the mRNA and protein level in ischemic postconditioning but not in ischemic preconditioning. This is the first demonstration that Rictor is the central molecular target of ProtectomiRs and that decreased Rictor expression may regulate ischemic postconditioning-, but not preconditioning-induced acute cardioprotection. We conclude that Rictor is a potential novel drug target for acute cardioprotection.
Assuntos
MicroRNAs/metabolismo , Terapia de Alvo Molecular , Infarto do Miocárdio/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Animais , Cardiotônicos , Pós-Condicionamento Isquêmico , Precondicionamento Isquêmico Miocárdico , Ratos , SuínosRESUMO
Helium inhalation induces cardioprotection against ischemia/reperfusion injury, the cellular mechanism of which remains not fully elucidated. Extracellular vesicles (EVs) are cell-derived, nano-sized membrane vesicles which play a role in cardioprotective mechanisms, but their function in helium conditioning (HeC) has not been studied so far. We hypothesized that HeC induces fibroblast-mediated cardioprotection via EVs. We isolated neonatal rat cardiac fibroblasts (NRCFs) and exposed them to glucose deprivation and HeC rendered by four cycles of 95% helium + 5% CO2 for 1 h, followed by 1 h under normoxic condition. After 40 h of HeC, NRCF activation was analyzed with a Western blot (WB) and migration assay. From the cell supernatant, medium extracellular vesicles (mEVs) were isolated with differential centrifugation and analyzed with WB and nanoparticle tracking analysis. The supernatant from HeC-treated NRCFs was transferred to naïve NRCFs or immortalized human umbilical vein endothelial cells (HUVEC-TERT2), and a migration and angiogenesis assay was performed. We found that HeC accelerated the migration of NRCFs and did not increase the expression of fibroblast activation markers. HeC tended to decrease mEV secretion of NRCFs, but the supernatant of HeC or the control NRCFs did not accelerate the migration of naïve NRCFs or affect the angiogenic potential of HUVEC-TERT2. In conclusion, HeC may contribute to cardioprotection by increasing fibroblast migration but not by releasing protective mEVs or soluble factors from cardiac fibroblasts.
Assuntos
Movimento Celular/efeitos dos fármacos , Micropartículas Derivadas de Células/fisiologia , Fibroblastos/efeitos dos fármacos , Hélio/farmacologia , Miocárdio/citologia , Animais , Animais Recém-Nascidos , Linhagem Celular , Movimento Celular/fisiologia , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/ultraestrutura , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Neovascularização Fisiológica/efeitos dos fármacos , Ratos WistarRESUMO
Unexpected cardiac adverse effects are the leading causes of discontinuation of clinical trials and withdrawal of drugs from the market. Since the original observations in the mid-90s, it has been well established that cardiovascular risk factors and comorbidities (such as ageing, hyperlipidaemia, and diabetes) and their medications (e.g. nitrate tolerance, adenosine triphosphate-dependent potassium inhibitor antidiabetic drugs, statins, etc.) may interfere with cardiac ischaemic tolerance and endogenous cardioprotective signalling pathways. Indeed drugs may exert unwanted effects on the diseased and treated heart that is hidden in the healthy myocardium. Hidden cardiotoxic effects may be due to (i) drug-induced enhancement of deleterious signalling due to ischaemia/reperfusion injury and/or the presence of risk factors and/or (ii) inhibition of cardioprotective survival signalling pathways, both of which may lead to ischaemia-related cell death and/or pro-arrhythmic effects. This led to a novel concept of 'hidden cardiotoxicity', defined as cardiotoxity of a drug that manifests only in the diseased heart with e.g. ischaemia/reperfusion injury and/or in the presence of its major comorbidities. Little is known on the mechanism of hidden cardiotoxocity, moreover, hidden cardiotoxicity cannot be revealed by the routinely used non-clinical cardiac safety testing methods on healthy animals or tissues. Therefore, here, we emphasize the need for development of novel cardiac safety testing platform involving combined experimental models of cardiac diseases (especially myocardial ischaemia/reperfusion and ischaemic conditioning) in the presence and absence of major cardiovascular comorbidities and/or cotreatments.
Assuntos
Cardiotoxicidade/prevenção & controle , Cardiotoxinas , Desenvolvimento de Medicamentos/normas , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Segurança do Paciente , Animais , Comorbidade , Cardiopatias/induzido quimicamente , Cardiopatias/prevenção & controle , Humanos , CamundongosRESUMO
Little is known about the mechanism of prediabetes-induced cardiac dysfunction. Therefore, we aimed to explore key molecular changes with transcriptomic and bioinformatics approaches in a prediabetes model showing heart failure with preserved ejection fraction phenotype. To induce prediabetes, Long-Evans rats were fed a high-fat diet for 21 weeks and treated with a single low-dose streptozotocin at week 4. Small RNA-sequencing, in silico microRNA (miRNA)-mRNA target prediction, Gene Ontology analysis, and target validation with qRT-PCR were performed in left ventricle samples. From the miRBase-annotated 752 mature miRNA sequences expression of 356 miRNAs was detectable. We identified two upregulated and three downregulated miRNAs in the prediabetic group. We predicted 445 mRNA targets of the five differentially expressed miRNAs and selected 11 mRNAs targeted by three differentially expressed miRNAs, out of which five mRNAs were selected for validation. Out of these five targets, downregulation of three mRNAs i.e., Juxtaposed with another zinc finger protein 1 (Jazf1); RAP2C, member of RAS oncogene family (Rap2c); and Zinc finger with KRAB and SCAN domains 1 (Zkscan1) were validated. This is the first demonstration that prediabetes alters cardiac miRNA expression profile. Predicted targets of differentially expressed miRNAs include Jazf1, Zkscan1, and Rap2c mRNAs. These transcriptomic changes may contribute to the diastolic dysfunction and may serve as drug targets.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/genética , Miocárdio/metabolismo , Estado Pré-Diabético/genética , Animais , Biologia Computacional , Modelos Animais de Doenças , Regulação para Baixo/genética , Perfilação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , MicroRNAs/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Long-Evans , Reprodutibilidade dos Testes , Regulação para Cima/genéticaRESUMO
Swiprosin-1 (EFhD2) is a molecule that triggers structural adaptation of isolated adult rat cardiomyocytes to cell culture conditions by initiating a process known as cell spreading. This process mimics central aspects of cardiac remodeling, as it occurs subsequent to myocardial infarction. However, expression of swiprosin-1 in cardiac tissue and its regulation in vivo has not yet been addressed. The expression of swiprosin-1 was analyzed in mice, rat, and pig hearts undergoing myocardial infarction or ischemia/reperfusion with or without cardiac protection by ischemic pre- and postconditioning. In mouse hearts, swiprosin-1 protein expression was increased after 4 and 7 days in myocardial infarct areas specifically in cardiomyocytes as verified by immunoblotting and histology. In rat hearts, swiprosin-1 mRNA expression was induced within 7 days after ischemia/reperfusion but this induction was abrogated by conditioning. As in cultured cardiomyocytes, the expression of swiprosin-1 was associated with a coinduction of arrestin-2, suggesting a common mechanism of regulation. Rno-miR-32-3p and rno-miR-34c-3p were associated with the regulation pattern of both molecules. Moreover, induction of swiprosin-1 and ssc-miR-34c was also confirmed in the infarct zone of pigs. In summary, our data show that up-regulation of swiprosin-1 appears in the postischemic heart during cardiac remodeling and repair in different species.
Assuntos
Remodelamento Atrial/genética , Proteínas de Ligação ao Cálcio/biossíntese , Regulação da Expressão Gênica , Precondicionamento Isquêmico Miocárdico , Proteínas dos Microfilamentos/biossíntese , Infarto do Miocárdio/genética , Traumatismo por Reperfusão/genética , Remodelação Ventricular/genética , Animais , Remodelamento Atrial/fisiologia , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Camundongos , MicroRNAs/biossíntese , MicroRNAs/genética , Proteínas dos Microfilamentos/genética , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Traumatismo por Reperfusão/metabolismo , Suínos , Remodelação Ventricular/fisiologia , beta-Arrestina 1/biossíntese , beta-Arrestina 1/genéticaRESUMO
The administration of immune checkpoint inhibitors (ICIs) often leads to immune-related adverse events. However, their effect on auditory function is largely unexplored. Thorough preclinical studies have not been published yet, only sporadic cases and pharmacovigilance reports suggest their significance. Here we investigated the effect of anti-PD-1 antibody treatment (4 weeks, intraperitoneally, 200 µg/mouse, 3 times/week) on hearing function and cochlear morphology in C57BL/6J mice. ICI treatment did not influence the hearing thresholds in click or tone burst stimuli at 4-32 kHz frequencies measured by auditory brainstem response. The number and morphology of spiral ganglion neurons were unaltered in all cochlear turns. The apical-middle turns (<32 kHz) showed preservation of the inner and outer hair cells (OHCs), whilst ICI treatment mitigated the age-related loss of OHCs in the basal turn (>32 kHz). The number of Iba1-positive macrophages has also increased moderately in this high frequency region. We conclude that a 4-week long ICI treatment does not affect functional and morphological integrity of the inner ear in the most relevant hearing range (4-32 kHz; apical-middle turns), but a noticeable preservation of OHCs and an increase in macrophage activity appeared in the >32 kHz basal part of the cochlea.
Assuntos
Anticorpos Monoclonais/farmacologia , Limiar Auditivo/efeitos dos fármacos , Cóclea/efeitos dos fármacos , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Audição , Inibidores de Checkpoint Imunológico/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gânglio Espiral da Cóclea/efeitos dos fármacosRESUMO
Cardioprotection against ischemia/reperfusion injury is still an unmet clinical need. The transient activation of Toll-like receptors (TLRs) has been implicated in cardioprotection, which may be achieved by treatment with blood-derived extracellular vesicles (EVs). However, since the isolation of EVs from blood takes considerable effort, the aim of our study was to establish a cellular model from which cardioprotective EVs can be isolated in a well-reproducible manner. EV release was induced in HEK293 cells with calcium ionophore A23187. EVs were characterized and cytoprotection was assessed in H9c2 and AC16 cell lines. Cardioprotection afforded by EVs and its mechanism were investigated after 16 h simulated ischemia and 2 h reperfusion. The induction of HEK293 cells by calcium ionophore resulted in the release of heterogenous populations of EVs. In H9c2 and AC16 cells, stressEVs induced the downstream signaling of TLR4 and heme oxygenase 1 (HO-1) expression in H9c2 cells. StressEVs decreased necrosis due to simulated ischemia/reperfusion injury in H9c2 and AC16 cells, which was independent of TLR4 induction, but not that of HO-1. Calcium ionophore-induced EVs exert cytoprotection by inducing HO-1 in a TLR4-independent manner.
Assuntos
Exossomos/metabolismo , Heme Oxigenase-1/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Calcimicina/farmacologia , Ionóforos de Cálcio/farmacologia , Morte Celular , Exossomos/efeitos dos fármacos , Células HEK293 , Heme Oxigenase-1/genética , Humanos , Camundongos , Ratos , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
RATIONALE: Understanding mechanisms of the therapeutic effects of stem/progenitor cells, among which adipose tissue-derived mesenchymal stromal cells (AT-MSCs), has important implications for clinical use. Since the majority of such cells die within days or weeks after transplantation and do not persist in the transplanted organ or tissue, their effects appear to be largely mediated by paracrine signaling pathways, and are enhanced by overexpression of the antisenescent protein telomerase reverse transcriptase (TERT), and the anti-apoptotic transcription factor myocardin (MYOCD). AIM: By a proteomic approach combining two-dimensional gel electrophoresis (2DE) with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/TOF) mass spectrometry, we aimed at analyzing how soluble and vesicular secretomes of aged murine AT-MSCs and their angiogenic function are modulated by the overexpression of TERT and MYOCD. METHODS: We cultured murine mock-transduced AT-MSCs and "rejuvenated" AT-MSCs overexpressing TERT and MYOCD (rTMAT-MSCs) harvested from 1-year-old male C57BL/6 mice. We established proteomes from 3 mock-transduced AT-MSCs and rTMAT-MSCs cultures in serum-free conditions, as well as their corresponding conditioned medium (CM) and exosome-enriched fractions (Exo+). RESULTS AND CONCLUSIONS: Proteomic analysis revealed a 2-fold increase of matrix metalloproteinase-2 (MMP-2) and its inhibitor metalloproteinase inhibitor 2 (TIMP2) in the CM - but not in the Exoâ¯+â¯- of rTMAT-MSCs as compared to mock-transduced AT-MSCs. At the functional level, rTMAT-MSCs-CM, and - to a lesser extent - its Exoâ¯+â¯fraction, increased tube formation of human vein endothelial cells (HUVECs), which could be blocked by anti-MMP2 and enhanced by anti-TIMP2 antibodies, respectively. Altogether, our results identify MMP2 and its inhibitor TIMP2 as novel candidates by which rTMAT-MSCs can support angiogenesis. Our strategy also illustrates the usefulness of comparative targeted proteomic approach to decipher molecular pathways underlying rTMAT-MSCs properties.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Proteínas Nucleares/metabolismo , Proteômica/métodos , Telomerase/metabolismo , Transativadores/metabolismo , Tecido Adiposo/citologia , Animais , Exossomos/genética , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Inibidor Tecidual de Metaloproteinase-2/metabolismoRESUMO
Although the application of cardioprotective ischaemia/reperfusion (I/R) stimuli after myocardial infarction (MI) is a promising concept for salvaging the myocardium, translation to a clinical scenario has not fulfilled expectations. We have previously shown that in pigs, ischaemic postconditioning (IPostC) reduces myocardial oedema and microvascular obstruction (MVO), without influencing myocardial infarct size. In the present study, we analyzed the mechanisms underlying the IPostC-induced microvascular protection by transcriptomic analysis, followed by pathway analysis. Closed-chest reperfused MI was induced by 90 min percutaneous balloon occlusion of the left anterior descending coronary artery, followed by balloon deflation in anaesthetised pigs. Animals were randomised to IPostC (n = 8), MI (non-conditioned, n = 8), or Control (sham-operated, n = 4) groups. After three hours or three days follow-up, myocardial tissue samples were harvested and subjected to RNA-seq analysis. Although the transcriptome analysis revealed similar expression between IPostC and MI in transcripts involved in cardioprotective pathways, we identified gene expression changes responding to IPostC at the three days follow-up. Focal adhesion signaling, downregulated genes participating in cardiomyopathy and activation of blood cells may have critical consequences for microvascular protection. Specific analyses of the gene subsets enriched in the endothelium of the infarcted area, revealed strong deregulation of transcriptional functional clusters, DNA processing, replication and repair, cell proliferation, and focal adhesion, suggesting sustentative function in the endothelial cell layer protection and integrity. The spatial and time-dependent transcriptome analysis of porcine myocardium supports a protective effect of IPostC on coronary microvasculature post-MI.
Assuntos
Pós-Condicionamento Isquêmico , Microvasos/patologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Transcrição Gênica , Animais , Tamanho Celular , Análise por Conglomerados , Modelos Animais de Doenças , Adesões Focais/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Análise de Sobrevida , SuínosRESUMO
Feeding rats with high-fat diet (HFD) with a single streptozotocin (STZ) injection induced obesity, slightly elevated fasting blood glucose and impaired glucose and insulin tolerance, and caused cardiac hypertrophy and mild diastolic dysfunction as published before by Koncsos et al. in 2016. Here we aimed to explore the renal consequences in the same groups of rats. Male Long-Evans rats were fed normal chow (CON; n = 9) or HFD containing 40% lard and were administered STZ at 20 mg/kg (i.p.) at week four (prediabetic rats, PRED, n = 9). At week 21 blood and urine samples were taken and kidney and liver samples were collected for histology, immunohistochemistry and for analysis of gene expression. HFD and STZ increased body weight and visceral adiposity and plasma leptin concentration. Despite hyperleptinemia, plasma C-reactive protein concentration decreased in PRED rats. Immunohistochemistry revealed elevated collagen IV protein expression in the glomeruli, and Lcn2 mRNA expression increased, while Il-1ß mRNA expression decreased in both the renal cortex and medulla in PRED vs. CON rats. Kidney histology, urinary protein excretion, plasma creatinine, glomerular Feret diameter, desmin protein expression, and cortical and medullary mRNA expression of TGF-ß1, Nrf2, and PPARγ were similar in CON and PRED rats. Reduced AMPKα phosphorylation of the autophagy regulator Akt was the first sign of liver damage, while plasma lipid and liver enzyme concentrations were similar. In conclusion, glomerular collagen deposition and increased lipocalin-2 expression were the early signs of kidney injury, while most biomarkers of inflammation, oxidative stress and fibrosis were negative in the kidneys of obese, prediabetic rats with mild heart and liver injury.
Assuntos
Colágeno/metabolismo , Glomérulos Renais/lesões , Glomérulos Renais/metabolismo , Lipocalina-2/metabolismo , Obesidade/metabolismo , Estado Pré-Diabético/metabolismo , Tecido Adiposo/metabolismo , Animais , Biomarcadores/metabolismo , Peso Corporal , Dieta Hiperlipídica , Fibrose , Regulação da Expressão Gênica , Inflamação/genética , Inflamação/patologia , Glomérulos Renais/patologia , Lipídeos/sangue , Fígado/enzimologia , Fígado/patologia , Fígado/fisiopatologia , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Obesidade/sangue , Estresse Oxidativo/genética , Fosforilação , Fosfosserina/metabolismo , Estado Pré-Diabético/sangue , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Long-Evans , EstreptozocinaRESUMO
Microglia are highly dynamic cells in the brain. Their functional diversity and phenotypic versatility brought microglial energy metabolism into the focus of research. Although it is known that microenvironmental cues shape microglial phenotype, their bioenergetic response to local nutrient availability remains unclear. In the present study effects of energy substrates on the oxidative and glycolytic metabolism of primary - and BV-2 microglial cells were investigated. Cellular oxygen consumption, glycolytic activity, the levels of intracellular ATP/ADP, autophagy, mTOR phosphorylation, apoptosis and cell viability were measured in the absence of nutrients or in the presence of physiological energy substrates: glutamine, glucose, lactate, pyruvate or ketone bodies. All of the oxidative energy metabolites increased the rate of basal and maximal respiration. However, the addition of glucose decreased microglial oxidative metabolism and glycolytic activity was enhanced. Increased ATP/ADP ratio and cell viability, activation of the mTOR and reduction of autophagic activity were observed in glutamine-supplemented media. Moreover, moderate and transient oxidation of ketone bodies was highly enhanced by glutamine, suggesting that anaplerosis of the TCA-cycle could stimulate ketone body oxidation. It is concluded that microglia show high metabolic plasticity and utilize a wide range of substrates. Among them glutamine is the most efficient metabolite. To our knowledge these data provide the first account of microglial direct metabolic response to nutrients under short-term starvation and demonstrate that microglia exhibit versatile metabolic machinery. Our finding that microglia have a distinct bioenergetic profile provides a critical foundation for specifying microglial contributions to brain energy metabolism.
Assuntos
Metabolismo Energético/fisiologia , Glucose/metabolismo , Glutamina/metabolismo , Lactatos/metabolismo , Microglia/metabolismo , Piruvatos/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Metabolismo Energético/efeitos dos fármacos , Feminino , Glucose/farmacologia , Glutamina/farmacologia , Glicólise/efeitos dos fármacos , Lactatos/farmacologia , Masculino , Camundongos , Microglia/citologia , Microglia/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Piruvatos/farmacologiaRESUMO
BACKGROUND: Cardioprotective value of ischemic post- (IPostC), remote (RIC) conditioning in acute myocardial infarction (AMI) is unclear in clinical trials. To evaluate cardioprotection, most translational animal studies and clinical trials utilize necrotic tissue referred to the area at risk (AAR) by magnetic resonance imaging (MRI). However, determination of AAR by MRI' may not be accurate, since MRI-indices of microvascular damage, i.e., myocardial edema and microvascular obstruction (MVO), may be affected by cardioprotection independently from myocardial necrosis. Therefore, we assessed the effect of IPostC, RIC conditioning and ischemic preconditioning (IPreC; positive control) on myocardial necrosis, edema and MVO in a clinically relevant, closed-chest pig model of AMI. METHODS AND RESULTS: Acute myocardial infarction was induced by a 90-min balloon occlusion of the left anterior descending coronary artery (LAD) in domestic juvenile female pigs. IPostC (6 × 30 s ischemia/reperfusion after 90-min occlusion) and RIC (4 × 5 min hind limb ischemia/reperfusion during 90-min LAD occlusion) did not reduce myocardial necrosis as assessed by late gadolinium enhancement 3 days after reperfusion and by ex vivo triphenyltetrazolium chloride staining 3 h after reperfusion, however, the positive control, IPreC (3 × 5 min ischemia/reperfusion before 90-min LAD occlusion) did. IPostC and RIC attenuated myocardial edema as measured by cardiac T2-weighted MRI 3 days after reperfusion, however, AAR measured by Evans blue staining was not different among groups, which confirms that myocardial edema is not a measure of AAR, IPostC and IPreC but not RIC decreased MVO. CONCLUSION: We conclude that IPostC and RIC interventions may protect the coronary microvasculature even without reducing myocardial necrosis.
Assuntos
Cardiotônicos/metabolismo , Pós-Condicionamento Isquêmico , Precondicionamento Isquêmico Miocárdico , Imageamento por Ressonância Magnética/métodos , Microvasos/patologia , Traumatismo por Reperfusão Miocárdica/diagnóstico , Animais , Edema/patologia , Eletrocardiografia , Feminino , Testes de Função Cardíaca , Hemodinâmica , Traumatismo por Reperfusão Miocárdica/diagnóstico por imagem , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Necrose , Coloração e Rotulagem , Sus scrofaRESUMO
BACKGROUND: We have previously shown that efficiency of ischemic conditioning is diminished in hypercholesterolemia and that autophagy is necessary for cardioprotection. However, it is unknown whether isolated hypercholesterolemia disturbs autophagy or the mammalian target of rapamycin (mTOR) pathways. Therefore, we investigated whether isolated hypercholesterolemia modulates cardiac autophagy-related pathways or programmed cell death mechanisms such as apoptosis and necroptosis in rat heart. METHODS: Male Wistar rats were fed either normal chow (NORM; n = 9) or with 2% cholesterol and 0.25% cholic acid-enriched diet (CHOL; n = 9) for 12 weeks. CHOL rats exhibited a 41% increase in plasma total cholesterol level over that of NORM rats (4.09 mmol/L vs. 2.89 mmol/L) at the end of diet period. Animals were sacrificed, hearts were excised and briefly washed out. Left ventricles were snap-frozen for determination of markers of autophagy, mTOR pathway, apoptosis, and necroptosis by Western blot. RESULTS: Isolated hypercholesterolemia was associated with a significant reduction in expression of cardiac autophagy markers such as LC3-II, Beclin-1, Rubicon and RAB7 as compared to controls. Phosphorylation of ribosomal S6, a surrogate marker for mTOR activity, was increased in CHOL samples. Cleaved caspase-3, a marker of apoptosis, increased in CHOL hearts, while no difference in the expression of necroptotic marker RIP1, RIP3 and MLKL was detected between treatments. CONCLUSIONS: This is the first comprehensive analysis of autophagy and programmed cell death pathways of apoptosis and necroptosis in hearts of hypercholesterolemic rats. Our data show that isolated hypercholesterolemia suppresses basal cardiac autophagy and that the decrease in autophagy may be a result of an activated mTOR pathway. Reduced autophagy was accompanied by increased apoptosis, while cardiac necroptosis was not modulated by isolated hypercholesterolemia. Decreased basal autophagy and elevated apoptosis may be responsible for the loss of cardioprotection reported in hypercholesterolemic animals.