RESUMEN
BACKGROUND: There is increased evidence that the effects of stem cells can mostly be duplicated by administration of their secretome which might streamline the translation towards the clinics. METHODS: The 12-patient SECRET-HF phase 1 trial has thus been designed to determine the feasibility and safety of repeated intravenous injections of the extracellular vesicle (EV)-enriched secretome of cardiovascular progenitor cells differentiated from pluripotent stem cells in severely symptomatic patients with drug-refractory left ventricular (LV) dysfunction secondary to non-ischemic dilated cardiomyopathy. Here we report the case of the first treated patient (baseline NYHA class III; LV Ejection Fraction:25%) in whom a dose of 20 × 109 particles/kg was intravenously infused three times three weeks apart. FINDINGS: In addition to demonstrating the feasibility of producing a cardiac cell secretome compliant with Good Manufacturing Practice standards, this case documents the excellent tolerance of its repeated delivery, without any adverse events during or after infusions. Six months after the procedure, the patient is in NYHA Class II with improved echo parameters, a reduced daily need for diuretics (from 240 mg to 160 mg), no firing from the previously implanted automatic internal defibrillator and no alloimmunization against the drug product, thereby supporting its lack of immunogenicity. INTERPRETATION: The rationale underlying the intravenous route is that the infused EV-enriched secretome may act by rewiring endogenous immune cells, both circulating and in peripheral organs, to take on a reparative phenotype. These EV-modified immune cells could then traffic to the heart to effect tissue repair, including mitigation of inflammation which is a hallmark of cardiac failure. FUNDING: This trial is funded by the French Ministry of Health (Programme Hospitalier de Recherche CliniqueAOM19330) and the "France 2030" National Strategy Program (ANR-20-F2II-0003). It is sponsored by Assistance Publique-Hôpitaux de Paris.
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Insuficiencia Cardíaca , Secretoma , Humanos , Insuficiencia Cardíaca/terapia , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/etiología , Secretoma/metabolismo , Masculino , Vesículas Extracelulares/metabolismo , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
AIMS: The cardioprotective effects of human induced pluripotent stem cell-derived cardiovascular progenitor cells (CPC) are largely mediated by the paracrine release of extracellular vesicles (EV). We aimed to assess the immunological behaviour of EV-CPC, which is a prerequisite for their clinical translation. METHODS AND RESULTS: Flow cytometry demonstrated that EV-CPC expressed very low levels of immune relevant molecules including HLA Class I, CD80, CD274 (PD-L1), and CD275 (ICOS-L); and moderate levels of ligands of the natural killer (NK) cell activating receptor, NKG2D. In mixed lymphocyte reactions, EV-CPC neither induced nor modulated adaptive allogeneic T cell immune responses. They also failed to induce NK cell degranulation, even at high concentrations. These in vitro effects were confirmed in vivo as repeated injections of EV-CPC did not stimulate production of immunoglobulins or affect the interferon (IFN)-γ responses from primed splenocytes. In a mouse model of chronic heart failure, intra-myocardial injections of EV-CPC, 3 weeks after myocardial infarction, decreased both the number of cardiac pro-inflammatory Ly6Chigh monocytes and circulating levels of pro-inflammatory cytokines (IL-1α, TNF-α, and IFN-γ). In a model of acute infarction, direct cardiac injection of EV-CPC 2 days after infarction reduced pro-inflammatory macrophages, Ly6Chigh monocytes, and neutrophils in heart tissue as compared to controls. EV-CPC also reduced levels of pro-inflammatory cytokines IL-1α, IL-2, and IL-6, and increased levels of the anti-inflammatory cytokine IL-10. These effects on human macrophages and monocytes were reproduced in vitro; EV-CPC reduced the number of pro-inflammatory monocytes and M1 macrophages, while increasing the number of anti-inflammatory M2 macrophages. CONCLUSIONS: EV-CPC do not trigger an immune response either in in vitro human allogeneic models or in immunocompetent animal models. The capacity for orienting the response of monocyte/macrophages towards resolution of inflammation strengthens the clinical attractiveness of EV-CPC as an acellular therapy for cardiac repair.
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Proliferación Celular , Vesículas Extracelulares/trasplante , Insuficiencia Cardíaca/cirugía , Células Madre Pluripotentes Inducidas/trasplante , Infarto del Miocardio/cirugía , Miocardio/inmunología , Miocitos Cardíacos/trasplante , Regeneración , Animales , Línea Celular , Técnicas de Cocultivo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Vesículas Extracelulares/inmunología , Vesículas Extracelulares/metabolismo , Insuficiencia Cardíaca/inmunología , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/metabolismo , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Monocitos/inmunología , Monocitos/metabolismo , Infarto del Miocardio/inmunología , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/inmunología , Miocitos Cardíacos/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Fenotipo , RatasRESUMEN
INTRODUCTION: In left atrial (LA) strain-derived two-dimensional speckle tracking echocardiography, the reference intervals in healthy dogs can provide useful information to evaluate the LA function in dogs with heart disease. ANIMALS: Six laboratory beagles and 120 privately owned dogs without cardiac diseases were recruited. MATERIALS AND METHODS: The LA strain and strain rate (SR) and echocardiographic indices were obtained in dogs who underwent standard echocardiography and offline analysis for LA strain and SR measurement by speckle tracking echocardiography. RESULTS: The intra-observer within-day variations of strain variables showed adequate repeatability (coefficient of variation <20%). The mean values of strain were 25.37 for the LA reservoir function, 11.06 for the LA conduit function, and 14.17 for the LA booster-pump function; the strain was significantly correlated with the LA fractional volume change at each phasic function. The left atrial longitudinal strain during early ventricular diastole showed moderate correlation with the peak velocity of early diastolic transmitral flow (r = 0.5560) and ratio of peak velocity of early diastolic transmitral flow to peak velocity of late transmitral flow (r = 0.5515). In multiple regression analysis, only age was significantly related to the strain/SR and volumetric change indices, indicating conduit function. CONCLUSIONS: Left atrial speckle tracking echocardiographic analysis provided useful information to assess the LA function in healthy dogs. The influencing factors on strain and SR variables including the age, body weight, and heart rate should be considered in interpretation of these parameters in a clinical setting.
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Función del Atrio Izquierdo/fisiología , Perros/fisiología , Ecocardiografía/veterinaria , Atrios Cardíacos/diagnóstico por imagen , Animales , Femenino , Masculino , Variaciones Dependientes del ObservadorRESUMEN
Hepatocellular carcinoma (HCC) is the most common primary liver tumour in dogs. However, the clinical features and risk factors of HCC have not been confirmed. The objective of this study was to investigate the clinical features and risk factors for canine HCC. Medical records of 44 dogs diagnosed with HCC at Hokkaido University Veterinary Teaching Hospital between 2013 and 2017 were retrospectively reviewed. All dogs evaluated at the teaching hospital during the study period were used as the reference population for breed, age, sex predispositions or possible related factors for HCC, including concurrent disorders. Clinical characteristics of HCC were determined using propensity score matching analysis. The prevalence of HCC diagnosis was 0.96%. Multivariate analysis revealed that dogs diagnosed with HCC were significantly older (odds ratio [OR], 1.20; 95% confidence intervals [CI], 1.07-1.33) than the reference population. Welsh Corgis (OR, 3.68; 95% CI, 1.56-8.67) and Beagles (OR, 4.33; 95% CI, 1.58-11.90) were significantly predisposed to HCC. Twenty-seven of 44 dogs with HCC had at least one concurrent disorder. The most common concurrent disorder was hyperadrenocorticism (n = 10), and the adjusted odds of hyperadrenocorticism in dogs with HCC were 4.13 higher than those of the reference population (95% CI, 1.95-8.76). Propensity score matching analysis revealed that thrombocytosis (n = 30/43), increased alanine aminotransferase (n = 41/44), increased alkaline phosphatase (n = 42/44), and hypercalcemia (n = 13/32) were significantly associated with HCC diagnosis. The results of this study suggest that Welsh Corgis and Beagles are breeds with a predisposition for HCC and that hyperadrenocorticism might be a potential risk factor.
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Carcinoma Hepatocelular/veterinaria , Enfermedades de los Perros/epidemiología , Neoplasias Hepáticas/veterinaria , Hiperfunción de las Glándulas Suprarrenales/veterinaria , Animales , Carcinoma Hepatocelular/epidemiología , Estudios de Casos y Controles , Enfermedades de los Perros/sangre , Enfermedades de los Perros/etiología , Perros , Femenino , Japón/epidemiología , Neoplasias Hepáticas/epidemiología , Masculino , Linaje , Prevalencia , Registros/veterinaria , Estudios RetrospectivosRESUMEN
Aims: We have shown that extracellular vesicles (EVs) secreted by embryonic stem cell-derived cardiovascular progenitor cells (Pg) recapitulate the therapeutic effects of their parent cells in a mouse model of chronic heart failure (CHF). Our objectives are to investigate whether EV released by more readily available cell sources are therapeutic, whether their effectiveness is influenced by the differentiation state of the secreting cell, and through which mechanisms they act. Methods and results: The total EV secreted by human induced pluripotent stem cell-derived cardiovascular progenitors (iPSC-Pg) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were isolated by ultracentrifugation and characterized by Nanoparticle Tracking Analysis, western blot, and cryo-electron microscopy. In vitro bioactivity assays were used to evaluate their cellular effects. Cell and EV microRNA (miRNA) content were assessed by miRNA array. Myocardial infarction was induced in 199 nude mice. Three weeks later, mice with left ventricular ejection fraction (LVEF) ≤ 45% received transcutaneous echo-guided injections of iPSC-CM (1.4 × 106, n = 19), iPSC-Pg (1.4 × 106, n = 17), total EV secreted by 1.4 × 106 iPSC-Pg (n = 19), or phosphate-buffered saline (control, n = 17) into the peri-infarct myocardium. Seven weeks later, hearts were evaluated by echocardiography, histology, and gene expression profiling, blinded to treatment group. In vitro, EV were internalized by target cells, increased cell survival, cell proliferation, and endothelial cell migration in a dose-dependent manner and stimulated tube formation. Extracellular vesicles were rich in miRNAs and most of the 16 highly abundant, evolutionarily conserved miRNAs are associated with tissue-repair pathways. In vivo, EV outperformed cell injections, significantly improving cardiac function through decreased left ventricular volumes (left ventricular end systolic volume: -11%, P < 0.001; left ventricular end diastolic volume: -4%, P = 0.002), and increased LVEF (+14%, P < 0.0001) relative to baseline values. Gene profiling revealed that EV-treated hearts were enriched for tissue reparative pathways. Conclusion: Extracellular vesicles secreted by iPSC-Pg are effective in the treatment of CHF, possibly, in part, through their specific miRNA signature and the associated stimulation of distinct cardioprotective pathways. The processing and regulatory advantages of EV could make them effective substitutes for cell transplantation.
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Vesículas Extracelulares/trasplante , Insuficiencia Cardíaca/terapia , Animales , Proliferación Celular , Supervivencia Celular , Células Madre Embrionarias/ultraestructura , Vesículas Extracelulares/genética , Insuficiencia Cardíaca/patología , Humanos , Ratones Desnudos , MicroARNs/análisis , Infarto del Miocardio/patología , Infarto del Miocardio/terapia , Miocitos Cardíacos/ultraestructura , Células Madre Pluripotentes/ultraestructura , Resultado del TratamientoRESUMEN
BACKGROUND: Cell-based therapies are being explored as a therapeutic option for patients with chronic heart failure following myocardial infarction. Extracellular vesicles (EV), including exosomes and microparticles, secreted by transplanted cells may orchestrate their paracrine therapeutic effects. We assessed whether post-infarction administration of EV released by human embryonic stem cell-derived cardiovascular progenitors (hESC-Pg) can provide equivalent benefits to administered hESC-Pg and whether hESC-Pg and EV treatments activate similar endogenous pathways. METHODS: Mice underwent surgical occlusion of their left coronary arteries. After 2-3 weeks, 95 mice included in the study were treated with hESC-Pg, EV, or Minimal Essential Medium Alpha Medium (alpha-MEM; vehicle control) delivered by percutaneous injections under echocardiographic guidance into the peri-infarct myocardium. functional and histologic end-points were blindly assessed 6 weeks later, and hearts were processed for gene profiling. Genes differentially expressed between control hearts and hESC-Pg-treated and EV-treated hearts were clustered into functionally relevant pathways. RESULTS: At 6 weeks after hESC-Pg administration, treated mice had significantly reduced left ventricular end-systolic (-4.20 ± 0.96 µl or -7.5%, p = 0.0007) and end-diastolic (-4.48 ± 1.47 µl or -4.4%, p = 0.009) volumes compared with baseline values despite the absence of any transplanted hESC-Pg or human embryonic stem cell-derived cardiomyocytes in the treated mouse hearts. Equal benefits were seen with the injection of hESC-Pg-derived EV, whereas animals injected with alpha-MEM (vehicle control) did not improve significantly. Histologic examination suggested a slight reduction in infarct size in hESC-Pg-treated animals and EV-treated animals compared with alpha-MEM-treated control animals. In the hESC-Pg-treated and EV-treated groups, heart gene profiling identified 927 genes that were similarly upregulated compared with the control group. Among the 49 enriched pathways associated with these up-regulated genes that could be related to cardiac function or regeneration, 78% were predicted to improve cardiac function through increased cell survival and/or proliferation or DNA repair as well as pathways related to decreased fibrosis and heart failure. CONCLUSIONS: In this post-infarct heart failure model, either hESC-Pg or their secreted EV enhance recovery of cardiac function and similarly affect cardiac gene expression patterns that could be related to this recovery. Although the mechanisms by which EV improve cardiac function remain to be determined, these results support the idea that a paracrine mechanism is sufficient to effect functional recovery in cell-based therapies for post-infarction-related chronic heart failure.
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Insuficiencia Cardíaca , Animales , Enfermedad Crónica , Células Madre Embrionarias , Vesículas Extracelulares , Humanos , Ratones , Infarto del Miocardio , Miocardio , Miocitos CardíacosRESUMEN
BACKGROUND: In humans with heart disease, vitamin D deficiency is associated with disease progression and a poor prognosis. A recent study showed that serum 25-hydroxyvitamin D [25(OH)D] concentration, the hallmark of vitamin D status, was lower in dogs with heart failure than in normal dogs, and a low concentration was associated with poor outcome in dogs with heart failure. OBJECTIVES: To elucidate the vitamin D status of dogs with chronic valvular heart disease (CVHD) at different stages of disease severity. ANIMALS: Forty-three client-owned dogs with CVHD. METHODS: In this cross-sectional study, dogs were divided into 3 groups (14 dogs in Stage B1, 17 dogs in Stage B2, and 12 dogs in Stage C/D) according to ACVIM guidelines. Dogs underwent clinical examination including echocardiography. Serum 25(OH)D concentrations were measured in each dog. RESULTS: Serum 25(OH)D concentration was significantly lower in Stage B2 (median, 33.2 nmol/L; range, 4.9-171.7 nmol/L) and C/D (13.1 nmol/L; 4.9-58.1 nmol/L) than in Stage B1 (52.5 nmol/L; 33.5-178.0 nmol/L) and was not significantly different between Stage B2 and Stage C/D. Among clinical variables, there were significant negative correlations between 25(OH)D concentration and both left atrial-to-aortic root ratio and left ventricular end-diastolic diameter normalized for body weight. CONCLUSIONS AND CLINICAL IMPORTANCE: These results indicate that vitamin D status is associated with the degree of cardiac remodeling, and the serum 25(OH)D concentration begins to decrease before the onset of heart failure in dogs with CVHD.
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Enfermedades de los Perros/sangre , Enfermedades de las Válvulas Cardíacas/veterinaria , Vitamina D/análogos & derivados , Animales , Enfermedad Crónica , Perros , Femenino , Enfermedades de las Válvulas Cardíacas/sangre , Masculino , Vitamina D/sangreRESUMEN
In eutherian mammals, one X-chromosome in every XX somatic cell is transcriptionally silenced through the process of X-chromosome inactivation (XCI). Females are thus functional mosaics, where some cells express genes from the paternal X, and the others from the maternal X. The relative abundance of the two cell populations (X-inactivation pattern, XIP) can have significant medical implications for some females. In mice, the 'choice' of which X to inactivate, maternal or paternal, in each cell of the early embryo is genetically influenced. In humans, the timing of XCI choice and whether choice occurs completely randomly or under a genetic influence is debated. Here, we explore these questions by analysing the distribution of XIPs in large populations of normal females. Models were generated to predict XIP distributions resulting from completely random or genetically influenced choice. Each model describes the discrete primary distribution at the onset of XCI, and the continuous secondary distribution accounting for changes to the XIP as a result of development and ageing. Statistical methods are used to compare models with empirical data from Danish and Utah populations. A rigorous data treatment strategy maximises information content and allows for unbiased use of unphased XIP data. The Anderson-Darling goodness-of-fit statistics and likelihood ratio tests indicate that a model of genetically influenced XCI choice better fits the empirical data than models of completely random choice.
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Cromosomas Humanos X/metabolismo , Inactivación del Cromosoma X/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Femenino , Humanos , Recién Nacido , Persona de Mediana Edad , Adulto JovenRESUMEN
The gene dosage inequality between females with two X-chromosomes and males with one is compensated for by X-chromosome inactivation (XCI), which ensures the silencing of one X in every somatic cell of female mammals. XCI in humans results in a mosaic of two cell populations: those expressing the maternal X-chromosome and those expressing the paternal X-chromosome. We have previously shown that the degree of mosaicism (the X-inactivation pattern) in a Canadian family is directly related to disease severity in female carriers of the X-linked recessive bleeding disorder, haemophilia A. The distribution of X-inactivation patterns in this family was consistent with a genetic trait having a co-dominant mode of inheritance, suggesting that XCI choice may not be completely random. To identify genetic elements that could be responsible for biased XCI choice, a linkage analysis was undertaken using an approach tailored to accommodate the continuous nature of the X-inactivation pattern phenotype in the Canadian family. Several X-linked regions were identified, one of which overlaps with a region previously found to be linked to familial skewed XCI. SA2, a component of the cohesin complex is identified as a candidate gene that could participate in XCI through its association with CTCF.
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Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Cromosomas Humanos X/genética , Inactivación del Cromosoma X/genética , Canadá , Factor VIII/genética , Femenino , Ligamiento Genético , Genotipo , Hemofilia A/genética , Humanos , Masculino , Mutación/genética , Linaje , CohesinasRESUMEN
Factor VIII gene, F8, mutations cause haemophilia A (HA), an X-linked recessive disorder. Expression in heterozygous females has been ascribed to skewed X-chromosome inactivation (XCI). To investigate the cause of HA in three heterozygous females within an Atlantic Canadian kindred, the proband (severely affected girl, FVIII activity: 2%) and 17 relatives across three generations were studied. F8 genotype, FVIII activity, XCI ratio (XCIR) (paternal active X: maternal active X), karyotype, submegabase resolution tiling set array competitive genome hybridization (competitive genomic hybridization (SMRT)), and microsatellite analyses were utilized. A positive linear relationship between FVIII activity and percentage-activated normal X-chromosome was found in HA heterozygous females (R(2)=0.87). All affected, but no unaffected females, had an XCIR skewed toward activation of the mutant X-chromosome (proband 92:8, SD 2). Unexpectedly, high numbers of females have dramatically skewed XCIRs (>80:20 or <20:80) (P<0.05). The distribution of XCIR frequencies within this family was significantly different than predicted by normal population data or models of random XCI (P<0.025), with more females having higher degrees of skewing. Known causes of skewing, such as chromosomal abnormalities, selection against deleterious alleles, and X-inactive-specific transcript mutations, are not consistent with our results. This study shows that FVIII activity in HA heterozygous females can be directly related to XCI skewing, and that low FVIII activity in females in this family is due to unfavourable XCI skewing. Further, the findings suggest that these XCI ratios are genetically influenced, consistent with a novel heritable human X controlling element (XCE) functioning similarly to the mouse Xce.