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1.
Circ Res ; 2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34623174

RESUMO

Rationale: After myocardial ischemic injury, improper phagocytic clearance of dying cardiac cells and the ensuing lack of inflammation resolution results in adverse cardiac remodeling and dysfunction that might lead to heart failure. Therefore, therapeutic strategies to ameliorate immune cell phagocytic function is critical for augmenting cardiac repair after injury. Objective: To determine if mesenchymal stem cell-derived exosomes (MSC-Exo) act as opsonin for apoptotic cells and/or trigger "eat me" phagocytic signaling in resident/recruited phagocytes after myocardial ischemic injury. Methods and Results: We evaluated MSC-Exo-mediated opsonization of apoptotic cardiomyocytes; and invitro and invivo effects of milk fat globule- epidermal growth factor-factor VIII (MFGE8)-deficient mouse MSC-Exo on macrophage engulfment of apoptotic cardiomyocytes and its implications on cardiac remodeling, repair and function. Microscopy and FACS analyses show that opsonization of apoptotic cardiomyocytes with MSC-Exo enhances their engulfment by macrophages. Furthermore, pre-incubation of macrophages with MSC-Exo reprogrammed the signaling pathways involved in phagocytosis and expression of pro-reparative cytokines. Protein analysis of MSC-Exo reveals expression of MFGE8, a glycoprotein which bridges externalized phosphatidylserine (PS) on the apoptotic cell surface to alphaVbeta3 or alphaVbeta5 integrins on the phagocyte. Most intriguingly, siRNA inhibition of MFGE8 significantly reduced the MSC-Exo-mediated augmentation of dead cell engulfment, associated signaling and pro-reparative phenotype. After myocardial ischemic injury, intramyocardial administration of MSC-Exo increases macrophage uptake of apoptotic bodies in the border zone of infarct and is associated with reduced proinflammatory response, increase in neovascularization, lower infarct size and an improvement in cardiac function and MFGE8-deficient MSC-Exo administration failed to protect mice against MI. Conclusions: Our data demonstrates that exosome-associated MFGE8 on one hand enhances opsonization of dead cells and on the other activates phagocytic signaling thus augmenting removal of apoptotic cells, resolution of inflammation and therefore efficient cardiac recovery after injury.

2.
Front Cardiovasc Med ; 8: 737826, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34485421

RESUMO

Doxorubicin (DOX, an anthracycline) is a widely used chemotherapy agent against various forms of cancer; however, it is also known to induce dose-dependent cardiotoxicity leading to adverse complications. Investigating the underlying molecular mechanisms and strategies to limit DOX-induced cardiotoxicity might have potential clinical implications. Our previous study has shown that expression of microRNA-377 (miR-377) increases in cardiomyocytes (CMs) after cardiac ischemia-reperfusion injury in mice, but its specific role in DOX-induced cardiotoxicity has not been elucidated. In the present study, we investigated the effect of anti-miR-377 on DOX-induced cardiac cell death, remodeling, and dysfunction. We evaluated the role of miR-377 in CM apoptosis, its target analysis by RNA sequencing, and we tested the effect of AAV9-anti-miR-377 on DOX-induced cardiotoxicity and mortality. DOX administration in mice increases miR-377 expression in the myocardium. miR-377 inhibition in cardiomyocyte cell line protects against DOX-induced cell death and oxidative stress. Furthermore, RNA sequencing and Gene Ontology (GO) analysis revealed alterations in a number of cell death/survival genes. Intriguingly, we observed accelerated mortality and enhanced myocardial remodeling in the mice pretreated with AAV9-anti-miR-377 followed by DOX administration as compared to the AAV9-scrambled-control-pretreated mice. Taken together, our data suggest that in vitro miR-377 inhibition protects against DOX-induced cardiomyocyte cell death. On the contrary, in vivo administration of AAV9-anti-miR-377 increases mortality in DOX-treated mice.

4.
J Food Prot ; 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34525192

RESUMO

The presence of pesticide residues in fruit has been of extensive concern worldwide. In this paper, pesticide residues in litchi samples in China and their dietary exposure risks were evaluated. Fifty-seven pesticides in 150 litchi samples were measured by GC and UPLC/MS-MS. Seventeen different pesticides were detected and 70.7% of samples contained one or more pesticide residues. The most frequently detected pesticide was diflubenzuron with a detection rate of 36%. Only carbofuran  in one sample exceeded its MRL and the exceedance was 125%. In dietary exposure assessments, all the values below LOD were calculated as 0, 1/2 LOD and LOD separately and the results demonstrated that the chronic dietary risks were extremely low. Acute exposure to carbofurn was 11.08% of the acute reference dose and acute exposure to all other pesticides was below 2% of the acute reference dose. The findings indicated that the occurrence of pesticide residues in litchis should not be considered a public health problem.

5.
Adv Healthc Mater ; : e2100968, 2021 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-34369107

RESUMO

Vascular atresia are often treated via transcatheter recanalization or surgical vascular anastomosis due to congenital malformations or coronary occlusions. The cellular response to vascular anastomosis or recanalization is, however, largely unknown and current techniques rely on restoration rather than optimization of flow into the atretic arteries. An improved understanding of cellular response post anastomosis may result in reduced restenosis. Here, an in vitro platform is used to model anastomosis in pulmonary arteries (PAs) and for procedural planning to reduce vascular restenosis. Bifurcated PAs are bioprinted within 3D hydrogel constructs to simulate a reestablished intervascular connection. The PA models are seeded with human endothelial cells and perfused at physiological flow rate to form endothelium. Particle image velocimetry and computational fluid dynamics modeling show close agreement in quantifying flow velocity and wall shear stress within the bioprinted arteries. These data are used to identify regions with greatest levels of shear stress alterations, prone to stenosis. Vascular geometry and flow hemodynamics significantly affect endothelial cell viability, proliferation, alignment, microcapillary formation, and metabolic bioprofiles. These integrated in vitro-in silico methods establish a unique platform to study complex cardiovascular diseases and can lead to direct clinical improvements in surgical planning for diseases of disturbed flow.

6.
Theranostics ; 11(16): 7879-7895, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335970

RESUMO

Rationale: Previous studies have shown that human embryonic stem cell-derived cardiomyocytes improved myocardial recovery when administered to infarcted pig and non-human primate hearts. However, the engraftment of intramyocardially delivered cells is poor and the effectiveness of clinically relevant doses of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in large animal models of myocardial injury remains unknown. Here, we determined whether thymosin ß4 (Tb4) could improve the engraftment and reparative potency of transplanted hiPSC-CMs in a porcine model of myocardial infarction (MI). Methods: Tb4 was delivered from injected gelatin microspheres, which extended the duration of Tb4 administration for up to two weeks in vitro. After MI induction, pigs were randomly distributed into 4 treatment groups: the MI Group was injected with basal medium; the Tb4 Group received gelatin microspheres carrying Tb4; the CM Group was treated with 1.2 × 108 hiPSC-CMs; and the Tb4+CM Group received both the Tb4 microspheres and hiPSC-CMs. Myocardial recovery was assessed by cardiac magnetic resonance imaging (MRI), arrhythmogenesis was monitored with implanted loop recorders, and tumorigenesis was evaluated via whole-body MRI. Results: In vitro, 600 ng/mL of Tb4 protected cultured hiPSC-CMs from hypoxic damage by upregulating AKT activity and BcL-XL and promoted hiPSC-CM and hiPSC-EC proliferation. In infarcted pig hearts, hiPSC-CM transplantation alone had a minimal effect on myocardial recovery, but co-treatment with Tb4 significantly enhanced hiPSC-CM engraftment, induced vasculogenesis and the proliferation of cardiomyocytes and endothelial cells, improved left ventricular systolic function, and reduced infarct size. hiPSC-CM implantation did not increase incidence of ventricular arrhythmia and did not induce tumorigenesis in the immunosuppressed pigs. Conclusions: Co-treatment with Tb4-microspheres and hiPSC-CMs was safe and enhanced the reparative potency of hiPSC-CMs for myocardial repair in a large-animal model of MI.


Assuntos
Infarto do Miocárdio/terapia , Miócitos Cardíacos/metabolismo , Timosina/farmacologia , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , China , Modelos Animais de Doenças , Células Endoteliais/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/patologia , Regeneração , Transplante de Células-Tronco/métodos , Suínos , Timosina/metabolismo , Timosina/fisiologia
7.
Theranostics ; 11(16): 7995-8007, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335976

RESUMO

Rationale: The conserved long non-coding RNA (lncRNA) myocardial infarction associate transcript (Miat) was identified for its multiple single-nucleotide polymorphisms that are strongly associated with susceptibility to MI, but its role in cardiovascular biology remains elusive. Here we investigated whether Miat regulates cardiac response to pathological hypertrophic stimuli. Methods: Both an angiotensin II (Ang II) infusion model and a transverse aortic constriction (TAC) model were used in adult WT and Miat-null knockout (Miat-KO) mice to induce pathological cardiac hypertrophy. Heart structure and function were evaluated by echocardiography and histological assessments. Gene expression in the heart was evaluated by RNA sequencing (RNA-seq), quantitative real-time RT-PCR (qRT-PCR), and Western blotting. Primary WT and Miat-KO mouse cardiomyocytes were isolated and used in Ca2+ transient and contractility measurements. Results: Continuous Ang II infusion for 4 weeks induced concentric hypertrophy in WT mice, but to a lesser extent in Miat-KO mice. Surgical TAC for 6 weeks resulted in decreased systolic function and heart failure in WT mice but not in Miat-KO mice. In both models, Miat-KO mice displayed reduced heart-weight to tibia-length ratio, cardiomyocyte cross-sectional area, cardiomyocyte apoptosis, and cardiac interstitial fibrosis and a better-preserved capillary density, as compared to WT mice. In addition, Ang II treatment led to significantly reduced mRNA and protein expression of the Ca2+ cycling genes Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and ryanodine receptor 2 (RyR2) and a dramatic increase in global RNA splicing events in the left ventricle (LV) of WT mice, and these changes were largely blunted in Miat-KO mice. Consistently, cardiomyocytes isolated from Miat-KO mice demonstrated more efficient Ca2+ cycling and greater contractility. Conclusions: Ablation of Miat attenuates pathological hypertrophy and heart failure, in part, by enhancing cardiomyocyte contractility.


Assuntos
Insuficiência Cardíaca/genética , Miócitos Cardíacos/metabolismo , RNA Longo não Codificante/genética , Angiotensina II/farmacologia , Animais , Apoptose , Cardiomegalia/genética , Modelos Animais de Doenças , Ecocardiografia , Fibrose , Masculino , Camundongos , Camundongos Knockout , Infarto do Miocárdio/patologia , RNA Longo não Codificante/metabolismo
8.
Adv Sci (Weinh) ; 8(18): e2004629, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34319658

RESUMO

Angiogenesis is essential for vascular development. The roles of regulatory long noncoding RNAs (lncRNAs) in mediating angiogenesis remain under-explored. Human embryonic stem cell-derived mesenchymal stem cells (hES-MSCs) are shown to exert more potent cardioprotective effects against cardiac ischemia than human bone marrow-derived MSCs (hBM-MSCs), associated with enhanced neovascularization. The purpose of this study is to search for angiogenic lncRNAs enriched in hES-MSCs, and investigate their roles and mechanisms. AC103746.1 is one of the most highly expressed intergenic lncRNAs detected in hES-MSCs versus hBM-MSCs, and named as SCDAL (stem cell-derived angiogenic lncRNA). SCDAL knockdown significantly reduce the angiogenic potential and reparative effects of hES-MSCs in the infarcted hearts, while overexpression of SCDAL in either hES-MSCs or hBM-MSCs exhibits augmented angiogenesis and cardiac function recovery. Mechanistically, SCDAL induces growth differentiation factor 6 (GDF6) expression via direct interaction with SNF5 at GDF6 promoter. Secreted GDF6 promotes endothelial angiogenesis via non-canonical vascular endothelial growth factor receptor 2 activation. Furthermore, SCDAL-GDF6 is expressed in human endothelial cells, and directly enhances endothelial angiogenesis in vitro and in vivo. Thus, these findings uncover a previously unknown lncRNA-dependent regulatory circuit for angiogenesis. Targeted intervention of the SCDAL-GDF6 pathway has potential as a therapy for ischemic heart diseases.

9.
Essays Biochem ; 65(3): 429-439, 2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34223619

RESUMO

Three-dimensional (3D) bioprinting is rapidly evolving, offering great potential for manufacturing functional tissue analogs for use in diverse biomedical applications, including regenerative medicine, drug delivery, and disease modeling. Biomaterials used as bioinks in printing processes must meet strict physiochemical and biomechanical requirements to ensure adequate printing fidelity, while closely mimicking the characteristics of the native tissue. To achieve this goal, nanomaterials are increasingly being investigated as a robust tool to functionalize bioink materials. In this review, we discuss the growing role of different nano-biomaterials in engineering functional bioinks for a variety of tissue engineering applications. The development and commercialization of these nanomaterial solutions for 3D bioprinting would be a significant step towards clinical translation of biofabrication.

10.
FASEB J ; 35(8): e21772, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34252225

RESUMO

Genetic deletion of Src associated in mitosis of 68kDa (Sam68), a pleiotropic adaptor protein prevents high-fat diet-induced weight gain and insulin resistance. To clarify the role of Sam68 in energy metabolism in the adult stage, we generated an inducible Sam68 knockout mice. Knockout of Sam68 was induced at the age of 7-10 weeks, and then we examined the metabolic profiles of the mice. Sam68 knockout mice gained less body weight over time and at 34 or 36 weeks old, had smaller fat mass without changes in food intake and absorption efficiency. Deletion of Sam68 in mice elevated thermogenesis, increased energy expenditure, and attenuated core-temperature drop during acute cold exposure. Furthermore, we examined younger Sam68 knockout mice at 11 weeks old before their body weights deviate, and confirmed increased energy expenditure and thermogenic gene program. Thus, Sam68 is essential for the control of adipose thermogenesis and energy homeostasis in the adult.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Metabolismo Energético , Termogênese , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Masculino , Camundongos , Camundongos Knockout , Proteínas de Ligação a RNA/metabolismo
11.
Nat Commun ; 12(1): 3340, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099657

RESUMO

Hepatic gluconeogenesis is essential for glucose homeostasis and also a therapeutic target for type 2 diabetes, but its mechanism is incompletely understood. Here, we report that Sam68, an RNA-binding adaptor protein and Src kinase substrate, is a novel regulator of hepatic gluconeogenesis. Both global and hepatic deletions of Sam68 significantly reduce blood glucose levels and the glucagon-induced expression of gluconeogenic genes. Protein, but not mRNA, levels of CRTC2, a crucial transcriptional regulator of gluconeogenesis, are >50% lower in Sam68-deficient hepatocytes than in wild-type hepatocytes. Sam68 interacts with CRTC2 and reduces CRTC2 ubiquitination. However, truncated mutants of Sam68 that lack the C- (Sam68ΔC) or N-terminal (Sam68ΔN) domains fails to bind CRTC2 or to stabilize CRTC2 protein, respectively, and transgenic Sam68ΔN mice recapitulate the blood-glucose and gluconeogenesis profile of Sam68-deficient mice. Hepatic Sam68 expression is also upregulated in patients with diabetes and in two diabetic mouse models, while hepatocyte-specific Sam68 deficiencies alleviate diabetic hyperglycemia and improves insulin sensitivity in mice. Thus, our results identify a role for Sam68 in hepatic gluconeogenesis, and Sam68 may represent a therapeutic target for diabetes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Gluconeogênese/fisiologia , Fígado/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Glicemia/metabolismo , Proteínas de Ligação a DNA , Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica , Glucagon/metabolismo , Gluconeogênese/genética , Glucose/metabolismo , Hepatócitos/metabolismo , Homeostase , Humanos , Hiperglicemia , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Regulação para Cima
12.
Stem Cell Res ; 53: 102365, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34087994

RESUMO

Cardiovascular disease, until now, is still the leading cause of death in the United States. Due to the limited regenerative capacity of adult hearts, the damage caused by heart injury cannot be reversed and eventually progress into heart failure. In need of cardiovascular disease treatment, many therapies aimed at either cell transplantation or cell regeneration have been proposed. Direct reprogramming of somatic cells into induced cardiomyocytes (iCMs) is considered to be a promising strategy for regenerative medicine. The induction of cardiomyocytes from non-myocytes can be achieved efficiently via ectopic expression of reprogramming factors both in vitro and in vivo in the mouse model, however, the generation of human induced cardiomyocyte-like cells (hiCMs) remains challenging. The inefficiency of hiCMs production called for the identification of the additional epigenetic memories in non-myocytes which might be damping the hiCM reprogramming. Here, we conducted an unbiased loss-of-function screening focusing on epigenetic regulators and identified enhancer of zeste homolog 2 (EZH2) as an important epigenetic barrier during hiCM reprogramming. We found that the removal of EZH2 via genetic knockdown or treatment of EZH2 selective degrader significantly increased the hiCM reprogramming efficiency and led to profound activation of cardiac genes and repression of collagen and extracellular matrix genes. Furthermore, EZH2 inhibitors targeting its catalytic activity also promotes hiCM reprogramming, suggesting that EZH2 may restrain cardiac conversion through H3K27me3-mediated gene repression. Indeed, genomic profiling of H3K27me3 revealed a subset of cardiac genes that remain repressed with high levels of H3K27me3 despite of the delivery of the reprogramming factors. Inhibition of EZH2, however, leads to reduced H3K27me3 occupancy and robust activation of these cardiac genes. Taken together, our data suggested that EZH2 inhibition facilitates the activation of cardiac genes in fibroblasts and eases the production of hiCMs.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste , Repressão Epigenética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Fibroblastos/metabolismo , Histonas/metabolismo , Humanos , Miócitos Cardíacos/metabolismo , Ativação Transcricional
13.
Front Bioeng Biotechnol ; 9: 674260, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34178964

RESUMO

Human induced-pluripotent stem cells (hiPSCs) can be efficiently differentiated into cardiomyocytes (hiPSC-CMs) via the GiWi method, which uses small-molecule inhibitors of glycogen synthase kinase (GSK) and tankyrase to first activate and then suppress Wnt signaling. However, this method is typically conducted in 6-well culture plates with two-dimensional (2D) cell sheets, and consequently, cannot be easily scaled to produce the large numbers of hiPSC-CMs needed for clinical applications. Cell suspensions are more suitable than 2D systems for commercial biomanufacturing, and suspended hiPSCs form free-floating aggregates (i.e., spheroids) that can also be differentiated into hiPSC-CMs. Here, we introduce a protocol for differentiating suspensions of hiPSC spheroids into cardiomyocytes that is based on the GiWi method. After optimization based on cardiac troponin T staining, the purity of hiPSC-CMs differentiated via our novel protocol exceeded 98% with yields of about 1.5 million hiPSC-CMs/mL and less between-batch purity variability than hiPSC-CMs produced in 2D cultures; furthermore, the culture volume could be increased ∼10-fold to 30 mL with no need for re-optimization, which suggests that this method can serve as a framework for large-scale hiPSC-CM production.

14.
J Sci Food Agric ; 2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34081336

RESUMO

BACKGROUND: Half of all apple production worldwide comes from China. However, the geographic authentication of Chinese apples has not been well studied. We highlight the multi-element-based geographical discrimination of apples from the southwest cold highlands (SCH) of China. 565 samples from the SCH (138) and others (427) were obtained, and the content of fifteen elements were applied to construct models for discrimination. RESULTS: The SCH apples from 2017 to 2019 had higher concentrations of Mn, Zn, Cr, Cd, Se, Pb, and Fe, but lower concentrations of Na, B, Ni, and P. With sufficient training, linear discriminant analysis (LDA) discriminated the SCH, and the testing accuracy averaged 92.5% and 92.2%. Nonlinear discrimination models were more suitable than the linear models. Optimized random forest analysis was the model with the best fit, and with averaged training and testing it obtained a level of accuracy of 98.2% and 98.5%. CONCLUSION: The multielement-based discrimination of SCH apples could aid further studies of geographical origins. © 2021 Society of Chemical Industry.

15.
Front Pharmacol ; 12: 673621, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34149424

RESUMO

Mammalian cardiomyocytes exit the cell cycle during the perinatal period, and although cardiomyocytes differentiated from human induced-pluripotent stem cells (hiPSC-CMs) are phenotypically immature, their intrinsic cell-cycle activity remains limited. Thus, neither endogenous cardiomyocytes nor the small number of transplanted hiPSC-CMs that are engrafted by infarcted hearts can remuscularize the myocardial scar. microRNAs are key regulators of cardiomyocyte proliferation, and when adeno-associated viruses coding for microRNA-199a (miR-199a) expression were injected directly into infarcted pig hearts, measures of cardiac function and fibrosis significantly improved, but the treatment was also associated with lethal arrhythmia. For the studies reported here, the same vector (AAV6-miR-199a) was transduced into hiPSC-CMs, and the cells were subsequently evaluated in a mouse model of myocardial infarction. AAV6-mediated miR-199a overexpression increased proliferation in both cultured and transplanted hiPSC-CMs, and measures of left ventricular ejection fraction, fractional shortening, and scar size were significantly better in mice treated with miR-199a-overexpressing hiPSC-CMs than with hiPSC-CMs that had been transduced with a control vector. Furthermore, although this investigation was not designed to characterize the safety of transplanted AAV6-miR-199a-transduced hiPSC-CMs, there was no evidence of sudden death. Collectively, these results support future investigations of miR-199a-overexpressing hiPSC-CMs in large animals.

16.
Circulation ; 144(3): 210-228, 2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-33951921

RESUMO

BACKGROUND: Human induced pluripotent stem cells with normal (wild-type) or upregulated (overexpressed) levels of CCND2 (cyclin D2) expression were differentiated into cardiomyocytes (CCND2WTCMs or CCND2OECMs, respectively) and injected into infarcted pig hearts. METHODS: Acute myocardial infarction was induced by a 60-minute occlusion of the left anterior descending coronary artery. Immediately after reperfusion, CCND2WTCMs or CCND2OECMs (3×107 cells each) or an equivalent volume of the delivery vehicle was injected around the infarct border zone area. RESULTS: The number of the engrafted CCND2OECMs exceeded that of the engrafted CCND2WTCMs from 6- to 8-fold, rising from 1 week to 4 weeks after implantation. In contrast to the treatment with the CCND2WTCMs or the delivery vehicle, the administration of CCND2OECM was associated with significantly improved left ventricular function, as revealed by magnetic resonance imaging. This correlated with reduction of infarct size, fibrosis, ventricular hypertrophy, and cardiomyocyte apoptosis, and increase of vascular density and arterial density, as per histologic analysis of the treated hearts. Expression of cell proliferation markers (eg, Ki67, phosphorylated histone 3, and Aurora B kinase) was also significantly upregulated in the recipient cardiomyocytes from the CCND2OECM-treated than from the CCND2WTCM-treated pigs. The cell proliferation rate and the hypoxia tolerance measured in cultured human induced pluripotent stem cell cardiomyocytes were significantly greater after treatment with exosomes isolated from the CCND2OECMs (CCND2OEExos) than from the CCND2WTCMs (CCND2WTExos). As demonstrated by our study, CCND2OEExos can also promote the proliferation activity of postnatal rat and adult mouse cardiomyocytes. A bulk miRNA sequencing analysis of CCND2OEExos versus CCND2WTExos identified 206 and 91 miRNAs that were significantly upregulated and downregulated, respectively. Gene ontology enrichment analysis identified significant differences in the expression profiles of miRNAs from various functional categories and pathways, including miRNAs implicated in cell-cycle checkpoints (G2/M and G1/S transitions), or the mechanism of cytokinesis. CONCLUSIONS: We demonstrated that enhanced potency of CCND2OECMs promoted myocyte proliferation in both grafts and recipient tissue in a large mammal acute myocardial infarction model. These results suggest that CCND2OECMs transplantation may be a potential therapeutic strategy for the repair of infarcted hearts.

17.
Front Cell Dev Biol ; 9: 670504, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33937272

RESUMO

Engineered cardiac tissues fabricated from human induced pluripotent stem cells (hiPSCs) show promise for ameliorating damage from myocardial infarction, while also restoring function to the damaged left ventricular (LV) myocardium. For these constructs to reach their clinical potential, they need to be of a clinically relevant volume and thickness, and capable of generating synchronous and forceful contraction to assist the pumping action of the recipient heart. Design prerequisites include a structure thickness sufficient to produce a beneficial contractile force, prevascularization to overcome diffusion limitations and sufficient structural development to allow for maximal cell communication. Previous attempts to meet these prerequisites have been hindered by lack of oxygen and nutrient transport due to diffusion limits (100-200 µm) resulting in necrosis. This study employs a layer-by-layer (LbL) fabrication method to produce cardiac tissue constructs that meet these design prerequisites and mimic normal myocardium in form and function. Thick (>2 mm) cardiac tissues created from hiPSC-derived cardiomyocytes, -endothelial cells (ECs) and -fibroblasts (FBs) were assessed, in vitro, over a 4-week period for viability (<6% necrotic cells), cell morphology and functionality. Functional performance assessment showed enhanced t-tubule network development, gap junction communication as well as previously unseen, physiologically relevant conduction velocities (CVs) (>30 cm/s). These results demonstrate that LbL fabrication can be utilized successfully to create prevascularized, functional cardiac tissue constructs from hiPSCs for potential therapeutic applications.

18.
Front Bioeng Biotechnol ; 9: 599928, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33842440

RESUMO

The billions of cardiomyocytes lost to acute myocardial infarction (MI) cannot be replaced by the limited regenerative capacity of adult mammalian hearts, and despite decades of research, there are still no clinically effective therapies for remuscularizing and restoring damaged myocardial tissue. Although the majority of the cardiac mass is composed of cardiomyocytes, cardiac fibroblasts (CFs) are one type of most numerous cells in the heart and the primary drivers of fibrosis, which prevents ventricular rupture immediately after MI but the fibrotic scar expansion and LV dilatation can eventually lead to heart failure. However, embryonic CFs produce cytokines that can activate proliferation in cultured cardiomyocytes, and the structural proteins produced by CFs may regulate cardiomyocyte cell-cycle activity by modulating the stiffness of the extracellular matrix (ECM). CFs can also be used to generate induced-pluripotent stem cells and induced cardiac progenitor cells, both of which can differentiate into cardiomyocytes and vascular cells, but cardiomyocytes appear to be more readily differentiated from iPSCs that have been reprogrammed from CFs than from other cell types. Furthermore, the results from recent studies suggest that cultured CFs, as well as the CFs present in infarcted hearts, can be reprogrammed directly into cardiomyocytes. This finding is very exciting as should we be able to successfully increase the efficiency of this reprogramming, we could remuscularize the injured ventricle and restore the LV function without need the transplantation of cells or cell products. This review summarizes the role of CFs in the innate response to MI and how their phenotypic plasticity and involvement in ECM production might be manipulated to improve cardiac performance in injured hearts.

19.
Sci Transl Med ; 13(584)2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33692129

RESUMO

Stem cell-derived small extracellular vesicles (sEVs) promote angiogenesis after myocardial infarction (MI). However, the components of sEVs that contribute to these effects and the safety and efficiency of engineered sEV treatment for MI remain unresolved. Here, we observed improved cardiac function, enhanced vascular density, and smaller infarct size in mice treated with the sEVs from hypoxia-preconditioned (HP) mesenchymal stem cells (MSCs) (HP-sEVs) than in mice treated with normoxia-preconditioned (N) MSCs (N-sEVs). MicroRNA profiling revealed a higher abundance of miR-486-5p in HP-sEVs than in N-sEVs, and miR-486-5p inactivation abolished the benefit of HP-sEV treatment, whereas miR-486-5p up-regulation enhanced the benefit of N-sEV treatment. Matrix metalloproteinase 19 (MMP19) abundance was lower in HP-sEV-treated than N-sEV-treated mouse hearts but was enriched in cardiac fibroblasts (CFs), and Mmp19 was identified as one of the target genes of miR-486-5p. Conditioned medium from CFs that overexpressed miR-486-5p or silenced MMP19 increased the angiogenic activity of endothelial cells; however, medium from CFs that simultaneously overexpressed Mmp19 and miR-486-5p abolished this effect. Mmp19 silencing in CFs reduced the cleavage of extracellular vascular endothelial growth factor (VEGF). Furthermore, miR-486-5p-overexpressing N-sEV treatment promoted angiogenesis and cardiac recovery without increasing arrhythmia complications in a nonhuman primate (NHP) MI model. Collectively, this study highlights the key role of sEV miR-486-5p in promoting cardiac angiogenesis via fibroblastic MMP19-VEGFA cleavage signaling. Delivery of miR-486-5p-engineered sEVs safely enhanced angiogenesis and cardiac function in an NHP MI model and may promote cardiac repair.


Assuntos
Vesículas Extracelulares , MicroRNAs , Infarto do Miocárdio , Animais , Células Endoteliais , Camundongos , MicroRNAs/genética , Primatas , Fator A de Crescimento do Endotélio Vascular
20.
Front Cardiovasc Med ; 8: 621781, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33718449

RESUMO

Tissue engineering combines principles of engineering and biology to generate living tissue equivalents for drug testing, disease modeling, and regenerative medicine. As techniques for reprogramming human somatic cells into induced pluripotent stem cells (iPSCs) and subsequently differentiating them into cardiomyocytes and other cardiac cells have become increasingly efficient, progress toward the development of engineered human cardiac muscle patch (hCMP) and heart tissue analogs has accelerated. A few pilot clinical studies in patients with post-infarction LV remodeling have been already approved. Conventional methods for hCMP fabrication include suspending cells within scaffolds, consisting of biocompatible materials, or growing two-dimensional sheets that can be stacked to form multilayered constructs. More recently, advanced technologies, such as micropatterning and three-dimensional bioprinting, have enabled fabrication of hCMP architectures at unprecedented spatiotemporal resolution. However, the studies working on various hCMP-based strategies for in vivo tissue repair face several major obstacles, including the inadequate scalability for clinical applications, poor integration and engraftment rate, and the lack of functional vasculature. Here, we review many of the recent advancements and key concerns in cardiac tissue engineering, focusing primarily on the production of hCMPs at clinical/industrial scales that are suitable for administration to patients with myocardial disease. The wide variety of cardiac cell types and sources that are applicable to hCMP biomanufacturing are elaborated. Finally, some of the key challenges remaining in the field and potential future directions to address these obstacles are discussed.

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