RESUMO
Mounting evidence shows that cell therapy provides therapeutic benefits in experimental and clinical settings of chronic heart failure. However, direct cardiac delivery of cells via transendocardial injection is logistically complex, expensive, entails risks, and is not amenable to multiple dosing. Intravenous administration would be a more convenient and clinically applicable route for cell therapy. Thus, we determined whether intravenous infusion of three widely used cell types improves left ventricular (LV) function and structure and compared their efficacy. Rats with a 30-day-old myocardial infarction (MI) received intravenous infusion of vehicle (PBS) or 1 of 3 types of cells: bone marrow mesenchymal stromal cells (MSCs), cardiac mesenchymal cells (CMCs), and c-kit-positive cardiac cells (CPCs), at a dose of 12 × 106 cells. Rats were followed for 35 days after treatment to determine LV functional status by serial echocardiography and hemodynamic studies. Blood samples were collected for Hemavet analysis to determine inflammatory cell profile. LV ejection fraction (EF) dropped ≥ 20 points in all hearts at 30 days after MI and deteriorated further at 35-day follow-up in the vehicle-treated group. In contrast, deterioration of EF was halted in rats that received MSCs and attenuated in those that received CMCs or CPCs. None of the 3 types of cells significantly altered scar size, myocardial content of collagen or CD45-positive cells, or Hemavet profile. This study demonstrates that a single intravenous administration of 3 types of cells in rats with chronic ischemic cardiomyopathy is effective in attenuating the progressive deterioration in LV function. The extent of LV functional improvement was greatest with CPCs, intermediate with CMCs, and least with MSCs.
Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Infarto do Miocárdio/terapia , Administração Intravenosa , Aloenxertos , Animais , Masculino , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Ratos , Ratos Endogâmicos F344RESUMO
RATIONALE: The beta-O-linkage of N-acetylglucosamine (i.e., O-GlcNAc) to proteins is a pro-adaptive response to cellular insults. To this end, increased protein O-GlcNAcylation improves short-term survival of cardiomyocytes subjected to acute injury. This observation has been repeated by multiple groups and in multiple models; however, whether increased protein O-GlcNAcylation plays a beneficial role in more chronic settings remains an open question. OBJECTIVE: Here, we queried whether increasing levels of cardiac protein O-GlcNAcylation would be beneficial during infarct-induced heart failure. METHODS AND RESULTS: To achieve increased protein O-GlcNAcylation, we targeted Oga, the gene responsible for removing O-GlcNAc from proteins. Here, we generated mice with cardiomyocyte-restricted, tamoxifen-inducible haploinsufficient Oga gene. In the absence of infarction, we observed a slight reduction in ejection fraction in Oga deficient mice. Overall, Oga reduction had no major impact on ventricular function. In additional cohorts, mice of both sexes and both genotypes were subjected to infarct-induced heart failure and followed for up to four weeks, during which time cardiac function was assessed via echocardiography. Contrary to our prediction, the Oga deficient mice exhibited exacerbated-not improved-cardiac function at one week following infarction. When the observation was extended to 4 wk post-MI, this acute exacerbation was lost. CONCLUSIONS: The present findings, coupled with our previous work, suggest that altering the ability of cardiomyocytes to either add or remove O-GlcNAc modifications to proteins exacerbates early infarct-induced heart failure. We speculate that more nuanced approaches to regulating O-GlcNAcylation are needed to understand its role-and, in particular, the possibility of cycling, in the pathophysiology of the failing heart.
Assuntos
Infarto do Miocárdio/patologia , Miocárdio/enzimologia , N-Acetilglucosaminiltransferases/genética , Disfunção Ventricular/etiologia , Animais , Ecocardiografia , Feminino , Glicosilação , Haploinsuficiência , Coração/fisiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Infarto do Miocárdio/complicações , Miocárdio/metabolismo , Miocárdio/patologia , N-Acetilglucosaminiltransferases/deficiência , N-Acetilglucosaminiltransferases/metabolismo , Tamoxifeno/farmacologia , Regulação para Cima , Função Ventricular/efeitos dos fármacosAssuntos
Adenosina Desaminase/fisiologia , Adenosina/metabolismo , Coração/embriologia , Inosina/metabolismo , Edição de RNA/fisiologia , Adenosina Desaminase/genética , Animais , Apoptose , Proliferação de Células , Sobrevivência Celular , Cruzamentos Genéticos , Desaminação , Deleção de Genes , Coração/crescimento & desenvolvimento , Camundongos , Miócitos Cardíacos/fisiologiaRESUMO
Several post-translational modifications figure prominently in ventricular remodeling. The beta-O-linkage of N-acetylglucosamine (O-GlcNAc) to proteins has emerged as an important signal in the cardiovascular system. Although there are limited insights about the regulation of the biosynthetic pathway that gives rise to the O-GlcNAc post-translational modification, much remains to be elucidated regarding the enzymes, such as O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which regulate the presence/absence of O-GlcNAcylation. Recently, we showed that the transcription factor, E2F1, could negatively regulate OGT and OGA expression in vitro. The present study sought to determine whether E2f1 deletion would improve post-infarct ventricular function by de-repressing expression of OGT and OGA. Male and female mice were subjected to non-reperfused myocardial infarction (MI) and followed for 1 or 4 week. MI significantly increased E2F1 expression. Deletion of E2f1 alone was not sufficient to alter OGT or OGA expression in a naïve setting. Cardiac dysfunction was significantly attenuated at 1-week post-MI in E2f1-ablated mice. During chronic heart failure, E2f1 deletion also attenuated cardiac dysfunction. Despite the improvement in function, OGT and OGA expression was not normalized and protein O-GlcNAcyltion was not changed at 1-week post-MI. OGA expression was significantly upregulated at 4-week post-MI but overall protein O-GlcNAcylation was not changed. As an alternative explanation, we also performed guided transcriptional profiling of predicted targets of E2F1, which indicated potential differences in cardiac metabolism, angiogenesis, and apoptosis. E2f1 ablation increased heart size and preserved remote zone capillary density at 1-week post-MI. During chronic heart failure, cardiomyocytes in the remote zone of E2f1-deleted hearts were larger than wildtype. These data indicate that, overall, E2f1 exerts a deleterious effect on ventricular remodeling. Thus, E2f1 deletion improves ventricular remodeling with limited impact on enzymes regulating O-GlcNAcylation.
Assuntos
Fator de Transcrição E2F1/deficiência , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Função Ventricular Esquerda , Remodelação Ventricular , Animais , Capilares/metabolismo , Capilares/patologia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Modelos Animais de Doenças , Fator de Transcrição E2F1/genética , Feminino , Deleção de Genes , Glicosilação , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , N-Acetilglucosaminiltransferases/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Preclinical investigations support the concept that donor cells more oriented towards a cardiovascular phenotype favor repair. In light of this philosophy, we previously identified HDAC1 as a mediator of cardiac mesenchymal cell (CMC) cardiomyogenic lineage commitment and paracrine signaling potency in vitro-suggesting HDAC1 as a potential therapeutically exploitable target to enhance CMC cardiac reparative capacity. In the current study, we examined the effects of pharmacologic HDAC1 inhibition, using the benzamide class 1 isoform-selective HDAC inhibitor entinostat (MS-275), on CMC cardiomyogenic lineage commitment and CMC-mediated myocardial repair in vivo. Human CMCs pre-treated with entinostat or DMSO diluent control were delivered intramyocardially in an athymic nude rat model of chronic ischemic cardiomyopathy 30 days after a reperfused myocardial infarction. Indices of cardiac function were assessed by echocardiography and left ventricular (LV) Millar conductance catheterization 35 days after treatment. Compared with naïve CMCs, entinostat-treated CMCs exhibited heightened capacity for myocyte-like differentiation in vitro and superior ability to attenuate LV remodeling and systolic dysfunction in vivo. The improvement in CMC therapeutic efficacy observed with entinostat pre-treatment was not associated with enhanced donor cell engraftment, cardiomyogenesis, or vasculogenesis, but instead with more efficient inhibition of myocardial fibrosis and greater increase in myocyte size. These results suggest that HDAC inhibition enhances the reparative capacity of CMCs, likely via a paracrine mechanism that improves ventricular compliance and contraction and augments myocyte growth and function.
Assuntos
Histona Desacetilase 1/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/patologia , Animais , Benzamidas/farmacologia , Fibrose , Xenoenxertos , Humanos , Células-Tronco Mesenquimais/metabolismo , Piridinas/farmacologia , Ratos , Ratos Nus , Recuperação de Função FisiológicaRESUMO
BACKGROUND: We have recently found that 3 repeated doses (12×106 each) of c-kitPOS cardiac progenitor cells (CPCs) were markedly more effective than a single dose of 12×106 cells in alleviating postinfarction left ventricular dysfunction and remodeling. However, since the single-dose group received only one third of the total number of CPCs given to the multiple-dose group, it is unknown whether the superior therapeutic efficacy was caused by repeated treatments per se or by administration of a higher total number of CPCs. This issue has major clinical implications because multiple cell injections in patients pose significant challenges, which would be obviated by using 1 large injection. Accordingly, we determined whether the beneficial effects of 3 repeated CPC doses can be recapitulated by 1 large dose containing the same total number of cells. METHODS AND RESULTS: Rats with a 30-day-old myocardial infarction received 3 echo-guided intraventricular infusions, 35 days apart, of vehicle-vehicle-vehicle, 36×106 CPCs-vehicle-vehicle, or 3 equal doses of 12×106 CPCs. Infusion of a single, large dose of CPCs (36×106 cells) produced an initial improvement in left ventricular function, but no further improvement was observed after the second and third infusions (both vehicle). In contrast, each of the 3 doses of CPCs (12×106) caused a progressive improvement in left ventricular function, the cumulative magnitude of which was greater than with a single dose. Unlike the single dose, repeated doses reduced collagen content and immune cell infiltration. CONCLUSIONS: Three repeated doses of CPCs are superior to 1 dose even though the total number of cells infused is the same, possibly because of greater antifibrotic and anti-inflammatory actions.
Assuntos
Infarto do Miocárdio/cirurgia , Miocárdio/patologia , Miócitos Cardíacos/transplante , Transplante de Células-Tronco/métodos , Função Ventricular Esquerda , Remodelação Ventricular , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Colágeno/metabolismo , Modelos Animais de Doenças , Feminino , Fibrose , Hemodinâmica , Masculino , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Fenótipo , Ratos Endogâmicos F344 , Recuperação de Função Fisiológica , Fatores de TempoRESUMO
BACKGROUND: The authors previously reported that the c-kit-positive (c-kitPOS) cells isolated from slowly adhering (SA) but not from rapidly adhering (RA) fractions of cardiac mesenchymal cells (CMCs) are effective in preserving left ventricular (LV) function after myocardial infarction (MI). OBJECTIVES: This study evaluated whether adherence to plastic alone, without c-kit sorting, was sufficient to isolate reparative CMCs. METHODS: RA and SA CMCs were isolated from mouse hearts, expanded in vitro, characterized, and evaluated for therapeutic efficacy in mice subjected to MI. RESULTS: Morphological and phenotypic analysis revealed that murine RA and SA CMCs are indistinguishable; nevertheless, transcriptome analysis showed that they possess fundamentally different gene expression profiles related to factors that regulate post-MI LV remodeling and repair. A similar population of SA CMCs was isolated from porcine endomyocardial biopsy samples. In mice given CMCs 2 days after MI, LV ejection fraction 28 days later was significantly increased in the SA CMC group (31.2 ± 1.0% vs. 24.7 ± 2.2% in vehicle-treated mice; p < 0.05) but not in the RA CMC group (24.1 ± 1.2%). Histological analysis showed reduced collagen deposition in the noninfarcted region in mice given SA CMCs (7.6 ± 1.5% vs. 14.5 ± 2.8% in vehicle-treated mice; p < 0.05) but not RA CMCs (11.7 ± 1.7%), which was associated with reduced infiltration of inflammatory cells (14.1 ± 1.6% vs. 21.3 ± 1.5% of total cells in vehicle and 19.3 ± 1.8% in RA CMCs; p < 0.05). Engraftment of SA CMCs was negligible, which implies a paracrine mechanism of action. CONCLUSIONS: We identified a novel population of c-kit-negative reparative cardiac cells (SA CMCs) that can be isolated with a simple method based on adherence to plastic. SA CMCs exhibited robust reparative properties and offered numerous advantages, appearing to be more suitable than c-kitPOS cardiac progenitor cells for widespread clinical therapeutic application.
Assuntos
Adesão Celular , Separação Celular/métodos , Células-Tronco Mesenquimais/metabolismo , Miocárdio/citologia , Animais , Células Cultivadas , Perfilação da Expressão Gênica , Camundongos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Regeneração , SuínosRESUMO
The myocardial response to pressure overload involves coordination of multiple transcriptional, posttranscriptional, and metabolic cues. The previous studies show that one such metabolic cue, O-GlcNAc, is elevated in the pressure-overloaded heart, and the increase in O-GlcNAcylation is required for cardiomyocyte hypertrophy in vitro. Yet, it is not clear whether and how O-GlcNAcylation participates in the hypertrophic response in vivo. Here, we addressed this question using patient samples and a preclinical model of heart failure. Protein O-GlcNAcylation levels were increased in myocardial tissue from heart failure patients compared with normal patients. To test the role of OGT in the heart, we subjected cardiomyocyte-specific, inducibly deficient Ogt (i-cmOgt -/-) mice and Ogt competent littermate wild-type (WT) mice to transverse aortic constriction. Deletion of cardiomyocyte Ogt significantly decreased O-GlcNAcylation and exacerbated ventricular dysfunction, without producing widespread changes in metabolic transcripts. Although some changes in hypertrophic and fibrotic signaling were noted, there were no histological differences in hypertrophy or fibrosis. We next determined whether significant differences were present in i-cmOgt -/- cardiomyocytes from surgically naïve mice. Interestingly, markers of cardiomyocyte dedifferentiation were elevated in Ogt-deficient cardiomyocytes. Although no significant differences in cardiac dysfunction were apparent after recombination, it is possible that such changes in dedifferentiation markers could reflect a larger phenotypic shift within the Ogt-deficient cardiomyocytes. We conclude that cardiomyocyte Ogt is not required for cardiomyocyte hypertrophy in vivo; however, loss of Ogt may exert subtle phenotypic differences in cardiomyocytes that sensitize the heart to pressure overload-induced ventricular dysfunction.
Assuntos
Cardiomegalia/metabolismo , Insuficiência Cardíaca/metabolismo , Miócitos Cardíacos/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Animais , Apoptose , Modelos Animais de Doenças , Humanos , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
We have recently demonstrated that repeated administrations of c-kitPOS cardiac progenitor cells (CPCs) have cumulative beneficial effects in rats with old myocardial infarction (MI), resulting in markedly greater improvement in left ventricular (LV) function compared with a single administration. To determine whether this paradigm applies to other species and cell types, mice with a 3-week-old MI received one or three doses of cardiac mesenchymal cells (CMCs), a novel cell type that we have recently described. CMCs or vehicle were infused percutaneously into the LV cavity, 14 days apart. Compared with vehicle-treated mice, the single-dose group exhibited improved LV ejection fraction (EF) after the 1st infusion (consisting of CMCs) but not after the 2nd and 3rd (vehicle). In contrast, in the multiple-dose group, LV EF improved after each CMC infusion, so that at the end of the study, LV EF averaged 35.5 ± 0.7% vs. 32.7 ± 0.6% in the single-dose group (P < 0.05). The multiple-dose group also exhibited less collagen in the non-infarcted region vs. the single-dose group. Engraftment and differentiation of CMCs were negligible in both groups, indicating paracrine effects. These results demonstrate that, in mice with ischemic cardiomyopathy, the beneficial effects of three doses of CMCs are significantly greater than those of one dose, supporting the concept that multiple treatments are necessary to properly evaluate the full therapeutic potential of cell therapy. Thus, the repeated-treatment paradigm is not limited to c-kit POS CPCs or to rats, but applies to other cell types and species. The generalizability of this concept dramatically augments its significance.
Assuntos
Transplante de Células-Tronco Mesenquimais/métodos , Infarto do Miocárdio , Animais , Modelos Animais de Doenças , Ecocardiografia , Feminino , Imuno-Histoquímica , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/citologia , Distribuição AleatóriaRESUMO
BACKGROUND: Immune cell-mediated inflammation is an essential process for mounting a repair response after myocardial infarction (MI). The sympathetic nervous system is known to regulate immune system function through ß-adrenergic receptors (ßARs); however, their role in regulating immune cell responses to acute cardiac injury is unknown. METHODS: Wild-type (WT) mice were irradiated followed by isoform-specific ßAR knockout (ßARKO) or WT bone-marrow transplantation (BMT) and after full reconstitution underwent MI surgery. Survival was monitored over time, and alterations in immune cell infiltration after MI were examined through immunohistochemistry. Alterations in splenic function were identified through the investigation of altered adhesion receptor expression. RESULTS: ß2ARKO BMT mice displayed 100% mortality resulting from cardiac rupture within 12 days after MI compared with ≈20% mortality in WT BMT mice. ß2ARKO BMT mice displayed severely reduced post-MI cardiac infiltration of leukocytes with reciprocally enhanced splenic retention of the same immune cell populations. Splenic retention of the leukocytes was associated with an increase in vascular cell adhesion molecule-1 expression, which itself was regulated via ß-arrestin-dependent ß2AR signaling. Furthermore, vascular cell adhesion molecule-1 expression in both mouse and human macrophages was sensitive to ß2AR activity, and spleens from human tissue donors treated with ß-blocker showed enhanced vascular cell adhesion molecule-1 expression. The impairments in splenic retention and cardiac infiltration of leukocytes after MI were restored to WT levels via lentiviral-mediated re-expression of ß2AR in ß2ARKO bone marrow before transplantation, which also resulted in post-MI survival rates comparable to those in WT BMT mice. CONCLUSIONS: Immune cell-expressed ß2AR plays an essential role in regulating the early inflammatory repair response to acute myocardial injury by facilitating cardiac leukocyte infiltration.
Assuntos
Ruptura Cardíaca/etiologia , Leucócitos/metabolismo , Infarto do Miocárdio/complicações , Receptores Adrenérgicos beta 2/fisiologia , Idoso , Idoso de 80 Anos ou mais , Animais , Modelos Animais de Doenças , Feminino , Vetores Genéticos/uso terapêutico , Humanos , Macrófagos/metabolismo , Masculino , Metoprolol/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Quimera por Radiação , Receptores Adrenérgicos beta 2/deficiência , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes de Fusão/metabolismo , Baço/metabolismo , Baço/patologia , Esplenectomia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
BACKGROUND: MicroRNA (miR) dysregulation in the myocardium has been implicated in cardiac remodeling after injury or stress. OBJECTIVES: The aim of this study was to explore the role of miR in human CD34(+) cell (hCD34(+)) dysfunction in vivo after transplantation into the myocardium under ischemia-reperfusion (I-R) conditions. METHODS: In response to inflammatory stimuli, the miR array profile of endothelial progenitor cells was analyzed using a polymerase chain reaction-based miR microarray. miR-377 expression was assessed in myocardial tissue from human patients with heart failure (HF). We investigated the effect of miR-377 inhibition on an hCD34(+) cell angiogenic proteome profile in vitro and on cardiac repair and function after I-R injury in immunodeficient mice. RESULTS: The miR array data from endothelial progenitor cells in response to inflammatory stimuli indicated changes in numerous miR, with a robust decrease in the levels of miR-377. Human cardiac biopsies from patients with HF showed significant increases in miR-377 expression compared with nonfailing control hearts. The proteome profile of hCD34(+) cells transfected with miR-377 mimics showed significant decrease in the levels of proangiogenic proteins versus nonspecific control-transfected cells. We also validated that serine/threonine kinase 35 is a target of miR-377 using a dual luciferase reporter assay. In a mouse model of myocardial I-R, intramyocardial transplantation of miR-377 silenced hCD34(+) cells in immunodeficient mice, promoting neovascularization (at 28 days, post-I-R) and lower interstitial fibrosis, leading to improved left ventricular function. CONCLUSIONS: These findings indicate that HF increased miR-377 expression in the myocardium, which is detrimental to stem cell function, and transplantation of miR-377 knockdown hCD34(+) cells into ischemic myocardium promoted their angiogenic ability, attenuating left ventricular remodeling and cardiac fibrosis.
Assuntos
Células Progenitoras Endoteliais/metabolismo , Insuficiência Cardíaca/metabolismo , MicroRNAs/metabolismo , Miocárdio/metabolismo , Traumatismo por Reperfusão/metabolismo , Adulto , Animais , Antígenos CD34 , Feminino , Coração , Humanos , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/citologia , Miocárdio/patologia , Neovascularização Fisiológica/fisiologia , Traumatismo por Reperfusão/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Although autophagy is an essential cellular salvage process to maintain cellular homeostasis, pathological autophagy can lead to cardiac abnormalities and ultimately heart failure. Therefore, a tight regulation of autophagic process would be important to treat chronic heart failure. Previously, we have shown that IL-10 strongly inhibited pressure overload-induced hypertrophy and heart failure, but role of IL-10 in regulation of pathological autophagy is unknown. Here we tested the hypothesis that IL-10 inhibits angiotensin II-induced pathological autophagy and this process, in part, leads to improve cardiac function. METHODS AND RESULTS: Chronic Ang II strongly induced mortality, cardiac dysfunction in IL-10 Knockout mice. IL-10 deletion exaggerated pathological autophagy in response to Ang II treatment. In isolated cardiac myocytes, IL-10 attenuated Ang II-induced pathological autophagy and activated Akt/mTORC1 signaling. Pharmacological or molecular inhibition of Akt and mTORC1 signaling attenuated IL-10 effects on Ang II-induced pathological autophagy. Furthermore, lysosomal inhibition in autophagic flux experiments further confirmed that IL-10 inhibits pathological autophagy via mTORC1 signaling. CONCLUSION: Our data demonstrate a novel role of IL-10 in regulation of pathological autophagy; thus can act as a potential therapeutic molecule for treatment of chronic heart disease.
Assuntos
Autofagia , Cardiomegalia/patologia , Interleucina-10/metabolismo , Angiotensina II/administração & dosagem , Animais , Animais Recém-Nascidos , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/efeitos dos fármacos , Proteína Beclina-1 , Cardiomegalia/complicações , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Deleção de Genes , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/patologia , Ventrículos do Coração/patologia , Ventrículos do Coração/ultraestrutura , Interleucina-10/deficiência , Interleucina-10/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
INTRODUCTION: Cardiovascular gene therapy is the third most popular application for gene therapy, representing 8.4% of all gene therapy trials as reported in 2012 estimates. Gene therapy in cardiovascular disease is aiming to treat heart failure from ischemic and non-ischemic causes, peripheral artery disease, venous ulcer, pulmonary hypertension, atherosclerosis and monogenic diseases, such as Fabry disease. AREAS COVERED: In this review, we will focus on elucidating current molecular targets for the treatment of ventricular dysfunction following myocardial infarction (MI). In particular, we will focus on the treatment of i) the clinical consequences of it, such as heart failure and residual myocardial ischemia and ii) etiological causes of MI (coronary vessels atherosclerosis, bypass venous graft disease, in-stent restenosis). EXPERT OPINION: We summarise the scheme of the review and the molecular targets either already at the gene therapy clinical trial phase or in the pipeline. These targets will be discussed below. Following this, we will focus on what we believe are the 4 prerequisites of success of any gene target therapy: safety, expression, specificity and efficacy (SESE).
Assuntos
Sistema Cardiovascular , Terapia Genética/métodos , Infarto do Miocárdio/terapia , Animais , Arritmias Cardíacas/terapia , Cálcio/metabolismo , Perfilação da Expressão Gênica , Humanos , Isquemia Miocárdica/patologia , Miocárdio/patologia , Neovascularização Fisiológica , Receptores Adrenérgicos/metabolismo , Regeneração , Disfunção Ventricular/fisiopatologia , Disfunção Ventricular/terapiaRESUMO
Direct intercellular communication mediated by gap junctions (GJs) is a hallmark of normal cell and tissue physiology. In addition, GJs significantly contribute to physical cell-cell adhesion. Clearly, these cellular functions require precise modulation. Typically, GJs represent arrays of hundreds to thousands of densely packed channels, each one assembled from two half-channels (connexons), that dock head-on in the extracellular space to form the channel arrays that link neighboring cells together. Interestingly, docked GJ channels cannot be separated into connexons under physiological conditions, posing potential challenges to GJ channel renewal and physical cell-cell separation. We described previously that cells continuously-and effectively after treatment with natural inflammatory mediators-internalize their GJs in an endo-/exocytosis process that utilizes clathrin-mediated endocytosis components, thus enabling these critical cellular functions. GJ internalization generates characteristic cytoplasmic double-membrane vesicles, described and termed earlier annular GJs (AGJs) or connexosomes. Here, using expression of the major fluorescent-tagged GJ protein, connexin 43 (Cx43-GFP/YFP/mApple) in HeLa cells, analysis of endogenously expressed Cx43, ultrastructural analyses, confocal colocalization microscopy, pharmacological and molecular biological RNAi approaches depleting cells of key-autophagic proteins, we provide compelling evidence that GJs, following internalization, are degraded by autophagy. The ubiquitin-binding protein p62/sequestosome 1 was identified in targeting internalized GJs to autophagic degradation. While previous studies identified proteasomal and endo-/lysosomal pathways in Cx43 and GJ degradation, our study provides novel molecular and mechanistic insights into an alternative GJ degradation pathway. Its recent link to health and disease lends additional importance to this GJ degradation mechanism and to autophagy in general.
Assuntos
Autofagia , Endocitose , Junções Comunicantes/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores/metabolismo , Western Blotting , Membrana Celular/metabolismo , Conexina 43/metabolismo , Fluorescência , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Transporte Proteico , Interferência de RNA , Proteína Sequestossoma-1RESUMO
Double-membrane-spanning gap junction (GJ) channels cluster into two-dimensional arrays, termed plaques, to provide direct cell-to-cell communication. GJ plaques often contain circular, channel-free domains ( approximately 0.05-0.5 mum in diameter) identified >30 y ago and termed nonjunctional membrane (NM) domains. We show, by expressing the GJ protein connexin43 (Cx43) tagged with green fluorescent protein, or the novel photoconvertible fluorescent protein Dendra2, that NM domains appear to be remnants generated by the internalization of small GJ channel clusters that bud over time from central plaque areas. Channel clusters internalized within seconds forming endocytic double-membrane GJ vesicles ( approximately 0.18-0.27 mum in diameter) that were degraded by lysosomal pathways. Surprisingly, NM domains were not repopulated by surrounding channels and instead remained mobile, fused with each other, and were expelled at plaque edges. Quantification of internalized, photoconverted Cx43-Dendra2 vesicles indicated a GJ half-life of 2.6 h that falls within the estimated half-life of 1-5 h reported for GJs. Together with previous publications that revealed continuous accrual of newly synthesized channels along plaque edges and simultaneous removal of channels from plaque centers, our data suggest how the known dynamic channel replenishment of functional GJ plaques can be achieved. Our observations may have implications for the process of endocytic vesicle budding in general.
Assuntos
Endocitose , Junções Comunicantes/metabolismo , Membranas Intracelulares/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Fluorescência , Junções Comunicantes/ultraestrutura , Células HeLa , Humanos , Membranas Intracelulares/ultraestrutura , Fusão de Membrana , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/ultraestrutura , Modelos Biológicos , Ratos , Vesículas Transportadoras/ultraestruturaRESUMO
During the inflammatory response, activation of G-protein coupled receptors (GPCRs) by inflammatory mediators rapidly leads to inhibition of gap junction intercellular communication (GJIC); however, the steps that lead to this inhibition are not known. Combining high-resolution fluorescence microscopy and functional assays, we found that activation of the GPCRs PAR-1 and ET(A/B) by their natural inflammatory mediator agonists, thrombin and endothelin-1, resulted in rapid and acute internalization of gap junctions (GJs) that coincided with the inhibition of GJIC followed by increased vascular permeability. The endocytosis protein clathrin and the scaffold protein ZO-1 appeared to be involved in GJ internalization, and ZO-1 was partially displaced from GJs during the internalization process. These findings demonstrate that GJ internalization is an efficient mechanism for modulating GJIC in inflammatory response.
Assuntos
Endotélio Vascular/metabolismo , Junções Comunicantes/metabolismo , Inflamação/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Clatrina/metabolismo , Endocitose , Endotelina-1/metabolismo , Endotelina-1/farmacologia , Endotélio Vascular/ultraestrutura , Junções Comunicantes/efeitos dos fármacos , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Permeabilidade , Fosfoproteínas/metabolismo , Receptor de Endotelina A/agonistas , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/agonistas , Receptor de Endotelina B/metabolismo , Receptor PAR-1/agonistas , Receptor PAR-1/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Trombina/metabolismo , Trombina/farmacologia , Proteína da Zônula de Oclusão-1RESUMO
Direct cell-cell communication mediated by plasma membrane-spanning gap junction (GJ) channels is vital to all aspects of cellular life. Obviously, GJ intercellular communication (GJIC) requires precise regulation, and it is known that controlled biosynthesis and degradation, and channel opening and closing (gating) are exploited. We discovered that cells internalize GJs in response to various stimuli. Here, we report that GJ internalization is a clathrin-mediated endocytic process that utilizes the vesicle-coat protein clathrin, the adaptor proteins adaptor protein complex 2 and disabled 2, and the GTPase dynamin. To our knowledge, we are first to report that the endocytic clathrin machinery can internalize double-membrane vesicles into cells.
Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Endocitose , Junções Comunicantes/metabolismo , Complexo 2 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose , Comunicação Celular , Clatrina/genética , Conexina 43/genética , Dinaminas/genética , Dinaminas/metabolismo , Células HeLa , Humanos , Interferência de RNA , Proteínas Supressoras de TumorRESUMO
Gap junction channels may be comprised of either connexin or pannexin proteins (innexins and pannexins). Membrane topologies of both families are similar, but sequence similarity is lacking. Recently, connexin-like sequences have been identified in mammalian and zebrafish genomes that have only four conserved cysteines in the extracellular domains (Cx23), a feature of the pannexins. Phylogenetic analyses of the non-canonical "C4" connexins reveal that these sequences are indeed connexins. Functional assays reveal that the Cx23 gap junctions are capable of sharing neurobiotin, and further, that Cx23 connexins form hemichannels in vitro.