RESUMO
Cardiomyocytes (CMs) have little regenerative capacity. After myocardial infarction (MI), scar formation and myocardial remodeling proceed in the infarct and non-infarct areas, respectively, leading to heart failure (HF). Prolonged activation of cardiac fibroblasts (CFs) and inflammatory cells may contribute to this process; however, therapies targeting these cell types remain lacking. Cardiac reprogramming converts CFs into induced CMs, reduces fibrosis, and improves cardiac function in chronic MI through the overexpression of Mef2c/Gata4/Tbx5/Hand2 (MGTH). However, whether cardiac reprogramming reduces inflammation in infarcted hearts remains unclear. Moreover, the mechanism through which MGTH overexpression in CFs affects inflammatory cells remains unknown. Here, we showed that inflammation persists in the myocardium until three months after MI, which can be reversed with cardiac reprogramming. Single-cell RNA sequencing demonstrated that CFs expressed pro-inflammatory genes and exhibited strong intercellular communication with inflammatory cells, including macrophages, in chronic MI. Cardiac reprogramming suppressed the inflammatory profiles of CFs and reduced the relative ratios and pro-inflammatory signatures of cardiac macrophages. Moreover, fluorescence-activated cell sorting analysis (FACS) revealed that cardiac reprogramming reduced the number of chemokine receptor type 2 (CCR2)-positive inflammatory macrophages in the non-infarct areas in chronic MI, thereby restoring myocardial remodeling. Thus, cardiac reprogramming reduced the number of inflammatory macrophages to exacerbate cardiac function after MI.
Assuntos
Infarto do Miocárdio , Humanos , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Macrófagos/metabolismo , Inflamação/metabolismo , Fibroblastos/metabolismoRESUMO
BACKGROUND: The mechanisms underlying pulmonary hypertension (PH) remain largely unknown; further, why advanced vascular remodeling preferentially occurs in arterioles is yet to be answered. VEGF (vascular endothelial growth factor) regulates angiogenesis through Flk1 (fetal liver kinase 1) and Flt1 (fms-like tyrosine kinase 1) on endothelial cells (ECs), which may be related to PH pathogenesis. However, spatiotemporal expression patterns of Flk1 and Flt1 in the pulmonary vascular system and the role of endothelial Flk1 in PH development remain poorly understood. METHODS: We analyzed multiple reporter mice, including Flk1-GFP (green fluorescent protein) bacterial artificial chromosome transgenic (Tg), Flt1-DsRed bacterial artificial chromosome Tg, and Flk1-GFP/Flt1-DsRed double Tg mice, to determine the spatiotemporal expression of Flk1 and Flt1 in hypoxia-induced PH. We also used Cdh5CreERT2/Flk1f/f/Tomato (Flk1-KO [knockout]) mice to induce EC-specific Flk1 deletion and lineage tracing in chronic hypoxia. RESULTS: Flk1 was specifically expressed in the ECs of small pulmonary vessels, including arterioles. Conversely, Flt1 was more broadly expressed in the ECs of large- to small-sized vessels in adult mouse lungs. Intriguingly, Flk1+ ECs were transiently increased in hypoxia with proliferation, whereas Flt1 expression was unchanged. Flk1-KO mice did not exhibit pulmonary vascular remodeling nor PH in normoxia; however, the arteriolar ECs changed to a cuboidal shape with protrusion. In hypoxia, Flk1 deletion exacerbated EC dysfunction and reduced their number via apoptosis. Additionally, Flk1 deletion promoted medial thickening and neointimal formation in arterioles and worsened PH. Mechanistically, lineage tracing revealed that neointimal cells were derived from Flk1-KO ECs. Moreover, RNA sequencing in pulmonary ECs demonstrated that Flk1 deletion and hypoxia synergistically activated multiple pathways, including cell cycle, senescence/apoptosis, and cytokine/growth factor, concomitant with suppression of cell adhesion and angiogenesis, to promote vascular remodeling. CONCLUSIONS: Flk1 and Flt1 were differentially expressed in pulmonary ECs. Flk1 deficiency and hypoxia jointly dysregulated arteriolar ECs to promote vascular remodeling. Thus, dysfunction of Flk1+ ECs may contribute to the pathogenesis of advanced vascular remodeling in pulmonary arterioles.
Assuntos
Hipertensão Pulmonar , Remodelação Vascular , Animais , Camundongos , Células Endoteliais/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Hipóxia/complicações , Hipóxia/genética , Hipóxia/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Cardiac transcription factors (TFs) directly reprogram fibroblasts into induced cardiomyocytes (iCMs), where MEF2C acts as a pioneer factor with GATA4 and TBX5 (GT). However, the generation of functional and mature iCMs is inefficient, and the molecular mechanisms underlying this process remain largely unknown. Here, we found that the overexpression of transcriptionally activated MEF2C via fusion of the powerful MYOD transactivation domain combined with GT increased the generation of beating iCMs by 30-fold. Activated MEF2C with GT generated iCMs that were transcriptionally, structurally, and functionally more mature than those generated by native MEF2C with GT. Mechanistically, activated MEF2C recruited p300 and multiple cardiogenic TFs to cardiac loci to induce chromatin remodeling. In contrast, p300 inhibition suppressed cardiac gene expression, inhibited iCM maturation, and decreased the beating iCM numbers. Splicing isoforms of MEF2C with similar transcriptional activities did not promote functional iCM generation. Thus, MEF2C/p300-mediated epigenetic remodeling promotes iCM maturation.
Assuntos
Montagem e Desmontagem da Cromatina , Fatores de Transcrição MEF2 , Miócitos Cardíacos , Fatores de Transcrição de p300-CBP , Epigênese Genética , Epigenômica , Fibroblastos , Fatores de Transcrição MEF2/genética , Fatores de Transcrição de p300-CBP/genéticaRESUMO
BACKGROUND: Because adult cardiomyocytes have little regenerative capacity, resident cardiac fibroblasts (CFs) synthesize extracellular matrix after myocardial infarction (MI) to form fibrosis, leading to cardiac dysfunction and heart failure. Therapies that can regenerate the myocardium and reverse fibrosis in chronic MI are lacking. The overexpression of cardiac transcription factors, including Mef2c/Gata4/Tbx5/Hand2 (MGTH), can directly reprogram CFs into induced cardiomyocytes (iCMs) and improve cardiac function under acute MI. However, the ability of in vivo cardiac reprogramming to repair chronic MI with established scars is undetermined. METHODS: We generated a novel Tcf21iCre/reporter/MGTH2A transgenic mouse system in which tamoxifen treatment could induce both MGTH and reporter expression in the resident CFs for cardiac reprogramming and fibroblast lineage tracing. We first tested the efficacy of this transgenic system in vitro and in vivo for acute MI. Next, we analyzed in vivo cardiac reprogramming and fusion events under chronic MI using Tcf21iCre/Tomato/MGTH2A and Tcf21iCre/mTmG/MGTH2A mice, respectively. Microarray and single-cell RNA sequencing were performed to determine the mechanism of cardiac repair by in vivo reprogramming. RESULTS: We confirmed the efficacy of transgenic in vitro and in vivo cardiac reprogramming for acute MI. In chronic MI, in vivo cardiac reprogramming converted ≈2% of resident CFs into iCMs, in which a majority of iCMs were generated by means of bona fide cardiac reprogramming rather than by fusion with cardiomyocytes. Cardiac reprogramming significantly improved myocardial contraction and reduced fibrosis in chronic MI. Microarray analyses revealed that the overexpression of MGTH activated cardiac program and concomitantly suppressed fibroblast and inflammatory signatures in chronic MI. Single-cell RNA sequencing demonstrated that resident CFs consisted of 7 subclusters, in which the profibrotic CF population increased under chronic MI. Cardiac reprogramming suppressed fibroblastic gene expression in chronic MI by means of conversion of profibrotic CFs to a quiescent antifibrotic state. MGTH overexpression induced antifibrotic effects partly by suppression of Meox1, a central regulator of fibroblast activation. CONCLUSIONS: These results demonstrate that cardiac reprogramming could repair chronic MI by means of myocardial regeneration and reduction of fibrosis. These findings present opportunities for the development of new therapies for chronic MI and heart failure.
Assuntos
Insuficiência Cardíaca , Infarto do Miocárdio , Camundongos , Animais , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fibrose , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Fibroblastos/metabolismo , Reprogramação CelularRESUMO
While the vascular healing process after drug-eluting stent implantation is not fully elucidated, it is generally accepted that macrophages play an important role in inflammation. It is also known that macrophages involved in the pathogenesis of atherosclerosis may stem from several origins, that is, monocyte-derived macrophages versus resident macrophages. However, little is known about the role of human macrophages on reperfusion of culprit coronary arteries in patients with atherosclerotic disease who have sustained acute coronary syndrome. In this present case report, we describe the presence of cluster of differentiation (CD) 163-positive macrophages in close proximity to the stent struts at the luminal site 72 hours after drug-eluting stent implantation in the culprit coronary lesion for ST elevation myocardial infarction by postmortem examination.
Assuntos
Stents Farmacológicos , Infarto do Miocárdio com Supradesnível do Segmento ST , Humanos , Macrófagos , Polímeros , Infarto do Miocárdio com Supradesnível do Segmento ST/cirurgia , Sirolimo/uso terapêutico , Stents , Resultado do TratamentoRESUMO
In this study, deep learning (DL)-based estimation of the Flory-Huggins χ parameter of A-B diblock copolymers from two-dimensional cross-sectional images of three-dimensional (3D) phase-separated structures were investigated. 3D structures with random networks of phase-separated domains were generated from real-space self-consistent field simulations in the 25-40 χN range for chain lengths (N) of 20 and 40. To confirm that the prepared data can be discriminated using DL, image classification was performed using the VGG-16 network. We comprehensively investigated the performances of the learned networks in the regression problem. The generalization ability was evaluated from independent images with the unlearned χN. We found that, except for large χN values, the standard deviation values were approximately 0.1 and 0.5 for A-component fractions of 0.2 and 0.35, respectively. The images for larger χN values were more difficult to distinguish. In addition, the learning performances for the 4-class problem were comparable to those for the 8-class problem, except when the χN values were large. This information is useful for the analysis of real experimental image data, where the variation of samples is limited.
Assuntos
Fator de Transcrição GATA4/biossíntese , Infarto do Miocárdio/genética , Miócitos Cardíacos/metabolismo , Proteínas com Domínio T/biossíntese , Animais , Fusão Celular , Reprogramação Celular/fisiologia , Feminino , Fatores de Transcrição MEF2/biossíntese , Masculino , Camundongos , Miócitos Cardíacos/citologiaRESUMO
Adult hearts have limited regenerative capacity. Hence, after acute myocardial infarction (MI), dead myocardial tissues are digested by immune cells and replaced by fibrosis, leading to ventricular remodeling and heart failure at the chronic stage. Direct reprogramming of the cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) with cardiac transcription factors, including Gata4, Mef2c, and Tbx5 (GMT), may have significant potential for cardiac repair. Sendai virus (SeV) vectors expressing GMT have been reported to reprogram the mouse cardiac fibroblasts into iCMs without any risk of insertional mutagenesis. In vivo reprogramming improved the cardiac function after acute MI in immunodeficient mice. However, it is unknown whether the newly generated iCMs could exist in infarct hearts for a prolonged period and SeV-GMT can improve cardiac function after MI at the chronic stage in immunocompetent mice. Here, we show that SeV vectors efficiently infect CFs in vivo and reprogram them into iCMs, which existed for at least four weeks after MI, in fibroblast-linage tracing mice. Moreover, SeV-GMT improved cardiac function and reduced fibrosis and collagen I expression at 12 weeks after MI in immunocompetent mice. Thus, direct cardiac reprogramming with SeV vectors could be a promising therapy for MI.
Assuntos
Reprogramação Celular , Vetores Genéticos , Infarto do Miocárdio/terapia , Vírus Sendai/genética , Animais , Doença Crônica , Colágeno Tipo I/metabolismo , Fibroblastos , Fibrose , Masculino , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/citologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/genéticaRESUMO
Direct cardiac reprogramming holds great potential for regenerative medicine. However, it remains inefficient, and induced cardiomyocytes (iCMs) generated in vitro are less mature than those in vivo, suggesting that undefined extrinsic factors may regulate cardiac reprogramming. Previous in vitro studies mainly used hard polystyrene dishes, yet the effect of substrate rigidity on cardiac reprogramming remains unclear. Thus, we developed a Matrigel-based hydrogel culture system to determine the roles of matrix stiffness and mechanotransduction in cardiac reprogramming. We found that soft matrix comparable with native myocardium promoted the efficiency and quality of cardiac reprogramming. Mechanistically, soft matrix enhanced cardiac reprogramming via inhibition of integrin, Rho/ROCK, actomyosin, and YAP/TAZ signaling and suppression of fibroblast programs, which were activated on rigid substrates. Soft substrate further enhanced cardiac reprogramming with Sendai virus vectors via YAP/TAZ suppression, increasing the reprogramming efficiency up to â¼15%. Thus, mechanotransduction could provide new targets for improving cardiac reprogramming.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Reprogramação Celular , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Actomiosina/metabolismo , Animais , Vetores Genéticos/metabolismo , Integrinas/metabolismo , Camundongos Transgênicos , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miosina Tipo II/metabolismo , Vírus Sendai/genética , Transdução de Sinais , Proteínas de Sinalização YAP , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Apparent permeability coefficients (P app) across a human intestinal epithelial Caco-2 cell monolayer were measured for a range of industrial/drug chemicals. A predictive equation for determining in vitro P app values of fifty-six substances was set up using multivariate regression analysis based on in silico-estimated physicochemical properties (molecular weights and water distribution coefficients for apical and basal pH environments) (râ¯=â¯0.77, pâ¯<⯠0.01). Predicted logP app values of a secondary set of 34 compounds were correlated with the measured values. Under the medicinal logP app values associated with their reported fraction absorbed, a significant inverse non-linear correlation was found between the logarithmic transformed values of observed P app values and reported hepatic no-observed-effect levels of industrial chemicals (r = -0.55, pâ¯<⯠0.01, n = 29). In vitro determination and/or in silico prediction of permeability across intestinal cells could be effective for estimating oral absorption as a putative indicator for hepatotoxicity.
RESUMO
The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium Thermochromatium tepidum requires Ca2+ for its thermal stability and characteristic absorption maximum at 915 nm. To explore the role of specific amino acid residues of the LH1 polypeptides in Ca-binding behavior, we constructed a genetic system for heterologously expressing the Tch. tepidum LH1 complex in an engineered Rhodobacter sphaeroides mutant strain. This system contained a chimeric pufBALM gene cluster (pufBA from Tch. tepidum and pufLM from Rba. sphaeroides) and was subsequently deployed for introducing site-directed mutations on the LH1 polypeptides. All mutant strains were capable of phototrophic (anoxic/light) growth. The heterologously expressed Tch. tepidum wild-type LH1 complex was isolated in a reaction center (RC)-associated form and displayed the characteristic absorption properties of this thermophilic phototroph. Spheroidene (the major carotenoid in Rba. sphaeroides) was incorporated into the Tch. tepidum LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per αß-subunit. The hybrid LH1-RC complexes expressed in Rba. sphaeroides were characterized using absorption, fluorescence excitation, and resonance Raman spectroscopy. Site-specific mutagenesis combined with spectroscopic measurements revealed that α-D49, ß-L46, and a deletion at position 43 of the α-polypeptide play critical roles in Ca binding in the Tch. tepidum LH1 complex; in contrast, α-N50 does not participate in Ca2+ coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated Tch. tepidum LH1-RC complex and have unambiguously identified the location of Ca2+ within this key antenna complex.