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1.
Pharmacol Ther ; : 108006, 2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34582836

RESUMO

"Modern" vaping involving battery-operated electronic devices began approximately one dozen years and has quickly evolved into a multibillion dollar industry providing products to an estimated 50 million users worldwide. Originally developed as an alternative to traditional cigarette smoking, vaping now appeals to a diverse demographic including substantial involvement of young people who often have never used cigarettes. The rapid rise of vaping fueled by multiple factors has understandably outpaced understanding of biological effects, made even more challenging due to wide ranging individual user habits and preferences. Consequently while vaping-related research gathers momentum, vaping-associated pathological injury (VAPI) has been established by clinical case reports with severe cases manifesting as acute respiratory distress syndrome (ARDS) with examples of right ventricular cardiac failure. Therefore, basic scientific studies are desperately needed to understand the impact of vaping upon the lungs as well as cardiopulmonary structure and function. Experimental models that capture fundamental characteristics of vaping-induced ARDS are essential to study pathogenesis and formulate recommendations to mitigate harmful effects attributable to ingredients or equipment. So too, treatment strategies to promote recovery from vaping-associated damage require development and testing at the preclinical level. This review summarizes the back story of vaping leading to present day conundrums with particular emphasis upon VAPI-associated ARDS and prioritization of research goals.

2.
Nat Biomed Eng ; 5(8): 880-896, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34426676

RESUMO

Fibroblasts can be directly reprogrammed into cardiomyocytes, endothelial cells or smooth muscle cells. Here we report the reprogramming of mouse tail-tip fibroblasts simultaneously into cells resembling these three cell types using the microRNA mimic miR-208b-3p, ascorbic acid and bone morphogenetic protein 4, as well as the formation of tissue-like structures formed by the directly reprogrammed cells. Implantation of the formed cardiovascular tissue into the infarcted hearts of mice led to the migration of reprogrammed cells to the injured tissue, reducing regional cardiac strain and improving cardiac function. The migrated endothelial cells and smooth muscle cells contributed to vessel formation, and the migrated cardiomyocytes, which initially displayed immature characteristics, became mature over time and formed gap junctions with host cardiomyocytes. Direct reprogramming of somatic cells to make cardiac tissue may aid the development of applications in cell therapy, disease modelling and drug discovery for cardiovascular diseases.


Assuntos
Células Endoteliais/transplante , Coração/fisiologia , Infarto do Miocárdio/terapia , Miócitos de Músculo Liso/transplante , Regeneração , Animais , Ácido Ascórbico/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Reprogramação Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Junções Comunicantes/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Neovascularização Fisiológica , Transcriptoma
3.
Cardiovasc Res ; 117(1): 201-211, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32176281

RESUMO

AIMS: Telomere attrition in cardiomyocytes is associated with decreased contractility, cellular senescence, and up-regulation of proapoptotic transcription factors. Pim1 is a cardioprotective kinase that antagonizes the aging phenotype of cardiomyocytes and delays cellular senescence by maintaining telomere length, but the mechanism remains unknown. Another pathway responsible for regulating telomere length is the transforming growth factor beta (TGFß) signalling pathway where inhibiting TGFß signalling maintains telomere length. The relationship between Pim1 and TGFß has not been explored. This study delineates the mechanism of telomere length regulation by the interplay between Pim1 and components of TGFß signalling pathways in proliferating A549 cells and post-mitotic cardiomyocytes. METHODS AND RESULTS: Telomere length was maintained by lentiviral-mediated overexpression of PIM1 and inhibition of TGFß signalling in A549 cells. Telomere length maintenance was further demonstrated in isolated cardiomyocytes from mice with cardiac-specific overexpression of PIM1 and by pharmacological inhibition of TGFß signalling. Mechanistically, Pim1 inhibited phosphorylation of Smad2, preventing its translocation into the nucleus and repressing expression of TGFß pathway genes. CONCLUSION: Pim1 maintains telomere lengths in cardiomyocytes by inhibiting phosphorylation of the TGFß pathway downstream effectors Smad2 and Smad3, which prevents repression of telomerase reverse transcriptase. Findings from this study demonstrate a novel mechanism of telomere length maintenance and provide a potential target for preserving cardiac function.

5.
Dis Model Mech ; 13(11)2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33188007

RESUMO

Cardiomyopathy is a leading cause of early mortality in Duchenne muscular dystrophy (DMD). There is a need to gain a better understanding of the molecular pathogenesis for the development effective therapies. Exosomes (exo) are secreted vesicles and exert effects via their RNA, lipid and protein cargo. The role of exosomes in disease pathology is unknown. Exosomes derived from stem cells have demonstrated cardioprotection in the murine DMD heart. However, it is unknown how the disease status of the donor cell type influences exosome function. Here, we sought to determine the phenotypic responses of DMD cardiomyocytes (DMD-iCMs) after long-term exposure to DMD cardiac exosomes (DMD-exo). DMD-iCMs were vulnerable to stress, evidenced by production of reactive oxygen species, the mitochondrial membrane potential and cell death levels. Long-term exposure to non-affected exosomes (N-exo) was protective. By contrast, long-term exposure to DMD-exo was not protective, and the response to stress improved with inhibition of DMD-exo secretion in vitro and in vivo The microRNA (miR) cargo, but not exosome surface peptides, was implicated in the pathological effects of DMD-exo. Exosomal surface profiling revealed N-exo peptides associated with PI3K-Akt signaling. Transcriptomic profiling identified unique changes with exposure to either N- or DMD-exo. Furthermore, DMD-exo miR cargo regulated injurious pathways, including p53 and TGF-beta. The findings reveal changes in exosomal cargo between healthy and diseased states, resulting in adverse outcomes. Here, DMD-exo contained miR changes, which promoted the vulnerability of DMD-iCMs to stress. Identification of these molecular changes in exosome cargo and effectual phenotypes might shed new light on processes underlying DMD cardiomyopathy.This article has an associated First Person interview with the first author of the paper.

6.
Cells ; 9(9)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32878131

RESUMO

Enhancing cardiomyocyte survival is crucial to blunt deterioration of myocardial structure and function following pathological damage. PIM1 (Proviral Insertion site in Murine leukemia virus (PIM) kinase 1) is a cardioprotective serine threonine kinase that promotes cardiomyocyte survival and antagonizes senescence through multiple concurrent molecular signaling cascades. In hematopoietic stem cells, PIM1 interacts with the receptor tyrosine kinase c-Kit upstream of the ERK (Extracellular signal-Regulated Kinase) and Akt signaling pathways involved in cell proliferation and survival. The relationship between PIM1 and c-Kit activity has not been explored in the myocardial context. This study delineates the interaction between PIM1 and c-Kit leading to enhanced protection of cardiomyocytes from stress. Elevated c-Kit expression is induced in isolated cardiomyocytes from mice with cardiac-specific overexpression of PIM1. Co-immunoprecipitation and proximity ligation assay reveal protein-protein interaction between PIM1 and c-Kit. Following treatment with Stem Cell Factor, PIM1-overexpressing cardiomyocytes display elevated ERK activity consistent with c-Kit receptor activation. Functionally, elevated c-Kit expression confers enhanced protection against oxidative stress in vitro. This study identifies the mechanistic relationship between PIM1 and c-Kit in cardiomyocytes, demonstrating another facet of cardioprotection regulated by PIM1 kinase.


Assuntos
Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Animais , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-pim-1/biossíntese , Proteínas Proto-Oncogênicas c-pim-1/genética , Regulação para Cima
7.
Nat Commun ; 11(1): 3955, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769998

RESUMO

Cellular therapy to treat heart failure is an ongoing focus of intense research, but progress toward structural and functional recovery remains modest. Engineered augmentation of established cellular effectors overcomes impediments to enhance reparative activity. Such 'next generation' implementation includes delivery of combinatorial cell populations exerting synergistic effects. Concurrent isolation and expansion of three distinct cardiac-derived interstitial cell types from human heart tissue, previously reported by our group, prompted design of a 3D structure that maximizes cellular interaction, allows for defined cell ratios, controls size, enables injectability, and minimizes cell loss. Herein, mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs) and c-Kit+ cardiac interstitial cells (cCICs) when cultured together spontaneously form scaffold-free 3D microenvironments termed CardioClusters. scRNA-Seq profiling reveals CardioCluster expression of stem cell-relevant factors, adhesion/extracellular-matrix molecules, and cytokines, while maintaining a more native transcriptome similar to endogenous cardiac cells. CardioCluster intramyocardial delivery improves cell retention and capillary density with preservation of cardiomyocyte size and long-term cardiac function in a murine infarction model followed 20 weeks. CardioCluster utilization in this preclinical setting establish fundamental insights, laying the framework for optimization in cell-based therapeutics intended to mitigate cardiomyopathic damage.


Assuntos
Microambiente Celular , Miocárdio/patologia , Cicatrização , Animais , Animais Recém-Nascidos , Capilares/patologia , Agregação Celular , Morte Celular , Linhagem da Célula , Tamanho Celular , Citoproteção , Células Progenitoras Endoteliais/citologia , Feminino , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Humanos , Processamento de Imagem Assistida por Computador , Recém-Nascido , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos NOD , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/citologia , Estresse Oxidativo , Comunicação Parácrina , Ratos Sprague-Dawley , Transcrição Genética
9.
J Am Heart Assoc ; 9(1): e013452, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31902324

RESUMO

Background CardioChimeras produced by fusion of murine c-kit+ cardiac interstitial cells with mesenchymal stem cells promote superior structural and functional recovery in a mouse model of myocardial infarction compared with either precursor cell alone or in combination. Creation of human CardioChimeras (hCCs) represents the next step in translational development of this novel cell type, but new challenges arise when working with c-kit+ cardiac interstitial cells isolated and expanded from human heart tissue samples. The objective of the study was to establish a reliable cell fusion protocol for consistent optimized creation of hCCs and characterize fundamental hCC properties. Methods and Results Cell fusion was induced by incubating human c-kit+ cardiac interstitial cells and mesenchymal stem cells at a 2:1 ratio with inactivated Sendai virus. Hybrid cells were sorted into 96-well microplates for clonal expansion to derive unique cloned hCCs, which were then characterized for various cellular and molecular properties. hCCs exhibited enhanced survival relative to the parent cells and promoted cardiomyocyte survival in response to serum deprivation in vitro. Conclusions The generation of hCC is demonstrated and validated in this study, representing the next step toward implementation of a novel cell product for therapeutic development. Feasibility of creating human hybrid cells prompts consideration of multiple possibilities to create novel chimeric cells derived from cells with desirable traits to promote healing in pathologically damaged myocardium.


Assuntos
Fusão Celular , Células Híbridas/fisiologia , Células-Tronco Mesenquimais/fisiologia , Miocárdio/citologia , Animais , Biomarcadores/metabolismo , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Diploide , Humanos , Miócitos Cardíacos/fisiologia , Fenótipo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Ratos , Fatores de Tempo
10.
Stem Cells Transl Med ; 9(5): 620-635, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31891237

RESUMO

Cardiac interstitial cells (CICs) perform essential roles in myocardial biology through preservation of homeostasis as well as response to injury or stress. Studies of murine CIC biology reveal remarkable plasticity in terms of transcriptional reprogramming and ploidy state with important implications for function. Despite over a decade of characterization and in vivo utilization of adult c-Kit+ CIC (cCIC), adaptability and functional responses upon delivery to adult mammalian hearts remain poorly understood. Limitations of characterizing cCIC biology following in vitro expansion and adoptive transfer into the adult heart were circumvented by delivery of the donated cells into early cardiogenic environments of embryonic, fetal, and early postnatal developing hearts. These three developmental stages were permissive for retention and persistence, enabling phenotypic evaluation of in vitro expanded cCICs after delivery as well as tissue response following introduction to the host environment. Embryonic blastocyst environment prompted cCIC integration into trophectoderm as well as persistence in amniochorionic membrane. Delivery to fetal myocardium yielded cCIC perivascular localization with fibroblast-like phenotype, similar to cCICs introduced to postnatal P3 heart with persistent cell cycle activity for up to 4 weeks. Fibroblast-like phenotype of exogenously transferred cCICs in fetal and postnatal cardiogenic environments is consistent with inability to contribute directly toward cardiogenesis and lack of functional integration with host myocardium. In contrast, cCICs incorporation into extra-embryonic membranes is consistent with fate of polyploid cells in blastocysts. These findings provide insight into cCIC biology, their inherent predisposition toward fibroblast fates in cardiogenic environments, and remarkable participation in extra-embryonic tissue formation.

11.
J Muscle Res Cell Motil ; 41(4): 363-373, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-31863324

RESUMO

Researchers continue to develop therapeutic products for the repair and replacement of myocardial tissue that demonstrates contractility equivalent to normal physiologic states. As clinical trials focused on pure adult stem cell populations undergo meta-analysis for preclinical through clinical design, the field of tissue engineering is emerging as a new clinical frontier to repair the myocardium and improve cardiac output. This review will first discuss the three primary tissue engineering product themes that are advancing in preclinical to clinical models: (1) cell-free scaffolds, (2) scaffold-free cellular, and (3) hybrid cell and scaffold products. The review will then focus on the products that have advanced from preclinical models to clinical trials. In advancing the cardiac regenerative medicine field, long-term gains towards discovering an optimal product to generate functional myocardial tissue and eliminate heart failure may be achieved.

12.
Trends Cardiovasc Med ; 30(6): 338-343, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-31515053

RESUMO

Cardiovascular disease remains the primary cause of death in the United States and in most nations worldwide, despite ongoing intensive efforts to promote cardiac health and treat heart failure. Replacing damaged myocardium represents perhaps the most promising treatment strategy, but also the most challenging given that the adult mammalian heart is notoriously resistant to endogenous repair. Cardiac regeneration following pathologic challenge would require proliferation of surviving tissue, expansion and differentiation of resident progenitors, or transdifferentiation of exogenously applied progenitor cells into functioning myocardium. Adult cardiomyocyte proliferation has been the focus of investigation for decades, recently enjoying a renaissance of interest as a therapeutic strategy for reversing cardiomyocyte loss due in large part to ongoing controversies and frustrations with myocardial cell therapy outcomes. The promise of cardiac cell therapy originated with reports of resident adult cardiac stem cells that could be isolated, expanded and reintroduced into damaged myocardium, producing beneficial effects in preclinical animal models. Despite modest functional improvements, Phase I clinical trials using autologous cardiac derived cells have proven safe and effective, setting the stage for an ongoing multi-center Phase II trial combining autologous cardiac stem cell types to enhance beneficial effects. This overview will examine the history of these two approaches for promoting cardiac repair and attempt to provide context for current and future directions in cardiac regenerative research.


Assuntos
Cardiopatias/cirurgia , Miocárdio/patologia , Miócitos Cardíacos/transplante , Regeneração , Transplante de Células-Tronco , Animais , Diferenciação Celular , Proliferação de Células , Senescência Celular , Cardiopatias/metabolismo , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Humanos , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fenótipo , Recuperação de Função Fisiológica , Resultado do Tratamento
13.
Stem Cell Res Ther ; 10(1): 373, 2019 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-31801634

RESUMO

BACKGROUND: Delivery of stem cells to the failing heart is a promising therapeutic strategy. However, the improvement in cardiac function in animal studies has not fully translated to humans. To help bridge the gap between species, we investigated the effects of adult human cardiac stem cells (hCSCs) on contractile function of human engineered cardiac tissues (hECTs) as a species-specific model of the human myocardium. METHODS: Human induced pluripotent stem cell-derived cardiomyoctes (hCMs) were mixed with Collagen/Matrigel to fabricate control hECTs, with an experimental group of hCSC-supplemented hECT fabricated using a 9:1 ratio of hCM to hCSC. Functional testing was performed starting on culture day 6, under spontaneous conditions and also during electrical pacing from 0.25 to 1.0 Hz, measurements repeated at days 8 and 10. hECTs were then frozen and processed for gene analysis using a Nanostring assay with a cardiac targeted custom panel. RESULTS: The hCSC-supplemented hECTs displayed a twofold higher developed force vs. hCM-only controls by day 6, with approximately threefold higher developed stress and maximum rates of contraction and relaxation during pacing at 0.75 Hz. The spontaneous beat rate characteristics were similar between groups, and hCSC supplementation did not adversely impact beat rate variability. The increased contractility persisted through days 8 and 10, albeit with some decrease in the magnitude of the difference of the force by day 10, but with developed stress still significantly higher in hCSC-supplemented hECT; these findings were confirmed with multiple hCSC and hCM cell lines. The force-frequency relationship, while negative for both, control (- 0.687 Hz- 1; p = 0.013 vs. zero) and hCSC-supplemented (- 0.233 Hz- 1;p = 0.067 vs. zero) hECTs, showed a significant rectification in the regression slope in hCSC-supplemented hECT (p = 0.011 vs. control). Targeted gene exploration (59 genes) identified a total of 14 differentially expressed genes, with increases in the ratios of MYH7/MHY6, MYL2/MYL7, and TNNI3/TNNI1 in hCSC-supplemented hECT versus controls. CONCLUSIONS: For the first time, hCSC supplementation was shown to significantly improve human cardiac tissue contractility in vitro, without evidence of proarrhythmic effects, and was associated with increased expression of markers of cardiac maturation. These findings provide new insights about adult cardiac stem cells as contributors to functional improvement of human myocardium.


Assuntos
Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Miócitos Cardíacos/fisiologia , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Diferenciação Celular , Colágeno/química , Combinação de Medicamentos , Estimulação Elétrica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Laminina/química , Miocárdio/citologia , Miócitos Cardíacos/citologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Proteoglicanas/química , Transcriptoma , Troponina I/genética , Troponina I/metabolismo
14.
Regen Med ; 14(9): 805-810, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31464566

RESUMO

Transcriptional profiling continues to produce phenotypical data essential for understanding of basic cardiac biology and required to improve efficiency of cardiac regenerative and therapeutic approaches after injury. Accurate interpretation of cardiac transcriptional data comes with the unique challenges of heart biology including cardiomyocyte morphology, cryopreservation of limited samples and adequate selection of transcriptional platform at a single-cell resolution. Consequently, development and implementation of novel transcriptional platforms and creative bioinformatic analysis are essential to resolve standing questions in the field of cardiac regenerative medicine. Targeted bioinformatic approaches, advancing technological access, increase technical availability and fostering communication between interdisciplinary groups is critical to improve therapeutic approaches and to overcome challenges inherent to transcriptomic cardiac research.


Assuntos
Biologia Computacional , Perfilação da Expressão Gênica , Miócitos Cardíacos , Medicina Regenerativa , Transcriptoma , Humanos
15.
Trends Endocrinol Metab ; 30(8): 557-567, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31262545

RESUMO

Ability to promote completion of mitotic cycling of adult mammalian cardiomyocytes remains an intractable and vexing challenge, despite being one of the most sought after 'holy grails' of cardiovascular research. While some of the struggle is attributable to adult cardiomyocytes themselves that are notoriously post-mitotic, another contributory factor rests with difficulty in definitive tracking of adult cardiomyocyte cell cycle and lack of rigorous measures to track proliferation in situ. This review summarizes past, present, and future directions to promote adult mammalian cardiomyocyte cell cycle progression, proliferation, and renewal. Establishing relationship(s) between cardiomyocyte cell cycle progression and cellular biological properties is sorely needed to understand the mechanistic basis for cardiomyocyte cell cycle withdrawal to enhance cardiomyocyte cell cycle progression and mitosis.


Assuntos
Ciclo Celular/fisiologia , Proliferação de Células/fisiologia , Miócitos Cardíacos/citologia , Animais , Ciclo Celular/genética , Proliferação de Células/genética , Senescência Celular/genética , Senescência Celular/fisiologia , Humanos , Miócitos Cardíacos/metabolismo
16.
Gene Ther ; 26(7-8): 324-337, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31239537

RESUMO

Advancement of stem cell-based treatment will involve next-generation approaches to enhance therapeutic efficacy which is often modest, particularly in the context of myocardial regenerative therapy. Our group has previously demonstrated the beneficial effect of genetic modification of cardiac stem cells with Pim-1 kinase overexpression to rejuvenate aged cells as well as potentiate myocardial repair. Despite these encouraging findings, concerns were raised regarding potential for oncogenic risk associated with Pim-1 kinase overexpression. Testing of Pim-1 engineered c-kit+ cardiac interstitial cells (cCIC) derived from heart failure patient samples for indices of oncogenic risk was undertaken using multiple assessments including soft agar colony formation, micronucleation, gamma-Histone 2AX foci, and transcriptome profiling. Collectively, findings demonstrate comparable phenotypic and biological properties of cCIC following Pim-1 overexpression compared with using baseline control cells with no evidence for oncogenic phenotype. Using a highly selective and continuous sensor for quantitative assessment of PIM1 kinase activity revealed a sevenfold increase in Pim-1 engineered vs. control cells. Kinase activity profiling using a panel of sensors for other kinases demonstrates elevation of IKKs), AKT/SGK, CDK1-3, p38, and ERK1/2 in addition to Pim-1 consistent with heightened kinase activity correlating with Pim-1 overexpression that may contribute to Pim-1-mediated effects. Enhancement of cellular survival, proliferation, and other beneficial properties to augment stem cell-mediated repair without oncogenic risk is a feasible, logical, and safe approach to improve efficacy and overcome current limitations inherent to cellular adoptive transfer therapeutic interventions.


Assuntos
Carcinogênese/genética , Terapia Genética/efeitos adversos , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/genética , Células-Tronco/metabolismo , Transcriptoma , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Testes para Micronúcleos , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Células-Tronco/patologia
17.
Commun Biol ; 2: 205, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31231694

RESUMO

Cardiomyocyte ploidy has been described but remains obscure in cardiac interstitial cells. Ploidy of c-kit+ cardiac interstitial cells was assessed using confocal, karyotypic, and flow cytometric technique. Notable differences were found between rodent (rat, mouse) c-kit+ cardiac interstitial cells possessing mononuclear tetraploid (4n) content, compared to large mammals (human, swine) with mononuclear diploid (2n) content. In-situ analysis, confirmed with fresh isolates, revealed diploid content in human c-kit+ cardiac interstitial cells and a mixture of diploid and tetraploid content in mouse. Downregulation of the p53 signaling pathway provides evidence why rodent, but not human, c-kit+ cardiac interstitial cells escape replicative senescence. Single cell transcriptional profiling reveals distinctions between diploid versus tetraploid populations in mouse c-kit+ cardiac interstitial cells, alluding to functional divergences. Collectively, these data reveal notable species-specific biological differences in c-kit+ cardiac interstitial cells, which could account for challenges in extrapolation of myocardial from preclinical studies to clinical trials.


Assuntos
Senescência Celular , Miócitos Cardíacos/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Tetraploidia , Animais , Proliferação de Células , Regulação para Baixo , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Cariotipagem , Leucócitos Mononucleares/citologia , Masculino , Camundongos , Microscopia Confocal , Ploidias , Ratos , Suínos
18.
Regen Med ; 14(6): 489-494, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31115258
19.
Autophagy ; 15(7): 1182-1198, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30741592

RESUMO

Cell-based therapies represent a very promising strategy to repair and regenerate the injured heart to prevent progression to heart failure. To date, these therapies have had limited success due to a lack of survival and retention of the infused cells. Therefore, it is important to increase our understanding of the biology of these cells and utilize this information to enhance their survival and function in the injured heart. Mitochondria are critical for progenitor cell function and survival. Here, we demonstrate the importance of mitochondrial autophagy, or mitophagy, in the differentiation process in adult cardiac progenitor cells (CPCs). We found that mitophagy was rapidly induced upon initiation of differentiation in CPCs. We also found that mitophagy was mediated by mitophagy receptors, rather than the PINK1-PRKN/PARKIN pathway. Mitophagy mediated by BNIP3L/NIX and FUNDC1 was not involved in regulating progenitor cell fate determination, mitochondrial biogenesis, or reprogramming. Instead, mitophagy facilitated the CPCs to undergo proper mitochondrial network reorganization during differentiation. Abrogating BNIP3L- and FUNDC1-mediated mitophagy during differentiation led to sustained mitochondrial fission and formation of donut-shaped impaired mitochondria. It also resulted in increased susceptibility to cell death and failure to survive the infarcted heart. Finally, aging is associated with accumulation of mitochondrial DNA (mtDNA) damage in cells and we found that acquiring mtDNA mutations selectively disrupted the differentiation-activated mitophagy program in CPCs. These findings demonstrate the importance of BNIP3L- and FUNDC1-mediated mitophagy as a critical regulator of mitochondrial network formation during differentiation, as well as the consequences of accumulating mtDNA mutations. Abbreviations: Baf: bafilomycin A1; BCL2L13: BCL2 like 13; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CPCs: cardiac progenitor cells; DM: differentiation media; DNM1L: dynamin 1 like; EPCs: endothelial progenitor cells; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; FUNDC1: FUN14 domain containing 1; HSCs: hematopoietic stem cells; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MFN1/2: mitofusin 1/2; MSCs: mesenchymal stem cells; mtDNA: mitochondrial DNA; OXPHOS: oxidative phosphorylation; PPARGC1A: PPARG coactivator 1 alpha; PHB2: prohibitin 2; POLG: DNA polymerase gamma, catalytic subunit; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TMRM: tetramethylrhodamine methyl ester.


Assuntos
Autofagossomos/metabolismo , Diferenciação Celular , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mitofagia , Mioblastos Cardíacos/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , DNA Polimerase gama/genética , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial/efeitos dos fármacos , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/genética , Mitofagia/efeitos dos fármacos , Mitofagia/genética , Mioblastos Cardíacos/efeitos dos fármacos , Infarto do Miocárdio , Biogênese de Organelas
20.
Stem Cells ; 37(4): 555-567, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30629785

RESUMO

Senescence-associated dysfunction deleteriously affects biological activities of human c-Kit+ cardiac progenitor cells (hCPCs), particularly under conditions of in vitro culture. In comparison, preservation of self-renewal and decreases in mitochondrial reactive oxygen species (ROS) are characteristics of murine CPCs in vivo that reside within hypoxic niches. Recapitulating hypoxic niche oxygen tension conditions of ∼1% O2 in vitro for expansion of hCPCs rather than typical normoxic cell culture conditions (21% O2 ) could provide significant improvement of functional and biological activities of hCPCs. hCPCs were isolated and expanded under permanent hypoxic (hCPC-1%) or normoxic (hCPC-21%) conditions from left ventricular tissue explants collected during left ventricular assist device implantation. hCPC-1% exhibit increased self-renewal and suppression of senescence characteristics relative to hCPC-21%. Oxidative stress contributed to higher susceptibility to apoptosis, as well as decreased mitochondrial function in hCPC-21%. Hypoxia prevented accumulation of dysfunctional mitochondria, supporting higher oxygen consumption rates and mitochondrial membrane potential. Mitochondrial ROS was an upstream mediator of senescence since treatment of hCPC-1% with mitochondrial inhibitor antimycin A recapitulated mitochondrial dysfunction and senescence observed in hCPC-21%. NAD+ /NADH ratio and autophagic flux, which are key factors for mitochondrial function, were higher in hCPC-1%, but hCPC-21% were highly dependent on BNIP3/NIX-mediated mitophagy to maintain mitochondrial function. Overall, results demonstrate that supraphysiological oxygen tension during in vitro expansion initiates a downward spiral of oxidative stress, mitochondrial dysfunction, and cellular energy imbalance culminating in early proliferation arrest of hCPCs. Senescence is inhibited by preventing ROS through hypoxic culture of hCPCs. Stem Cells 2019;37:555-567.


Assuntos
Senescência Celular/fisiologia , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Células-Tronco/metabolismo , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Humanos , Mitocôndrias
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