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1.
Cell Mol Life Sci ; 80(8): 241, 2023 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-37543540

RESUMO

Spinal muscular atrophy (SMA) is a neurodegenerative disorder caused by mutations in the SMN1 gene resulting in reduced levels of the SMN protein. Nusinersen, the first antisense oligonucleotide (ASO) approved for SMA treatment, binds to the SMN2 gene, paralogue to SMN1, and mediates the translation of a functional SMN protein. Here, we used longitudinal high-resolution mass spectrometry (MS) to assess both global proteome and metabolome in cerebrospinal fluid (CSF) from ten SMA type 3 patients, with the aim of identifying novel readouts of pharmacodynamic/response to treatment and predictive markers of treatment response. Patients had a median age of 33.5 [29.5; 38.25] years, and 80% of them were ambulant at time of the enrolment, with a median HFMSE score of 37.5 [25.75; 50.75]. Untargeted CSF proteome and metabolome were measured using high-resolution MS (nLC-HRMS) on CSF samples obtained before treatment (T0) and after 2 years of follow-up (T22). A total of 26 proteins were found to be differentially expressed between T0 and T22 upon VSN normalization and LIMMA differential analysis, accounting for paired replica. Notably, key markers of the insulin-growth factor signaling pathway were upregulated after treatment together with selective modulation of key transcription regulators. Using CombiROC multimarker signature analysis, we suggest that detecting a reduction of SEMA6A and an increase of COL1A2 and GRIA4 might reflect therapeutic efficacy of nusinersen. Longitudinal metabolome profiling, analyzed with paired t-Test, showed a significant shift for some aminoacid utilization induced by treatment, whereas other metabolites were largely unchanged. Together, these data suggest perturbation upon nusinersen treatment still sustained after 22 months of follow-up and confirm the utility of CSF multi-omic profiling as pharmacodynamic biomarker for SMA type 3. Nonetheless, validation studies are needed to confirm this evidence in a larger sample size and to further dissect combined markers of response to treatment.


Assuntos
Multiômica , Atrofia Muscular Espinal , Humanos , Estudos Retrospectivos , Seguimentos , Proteoma , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo
2.
Biomedicines ; 11(5)2023 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-37238925

RESUMO

Spinal muscular atrophy (SMA) is a neuromuscular disease resulting from mutations or deletions in SMN1 that lead to progressive death of alpha motor neurons, ultimately leading to severe muscle weakness and atrophy, as well as premature death in the absence of treatment. Recent approval of SMN-increasing medications as SMA therapy has altered the natural course of the disease. Thus, accurate biomarkers are needed to predict SMA severity, prognosis, drug response, and overall treatment efficacy. This article reviews novel non-targeted omics strategies that could become useful clinical tools for patients with SMA. Proteomics and metabolomics can provide insights into molecular events underlying disease progression and treatment response. High-throughput omics data have shown that untreated SMA patients have different profiles than controls. In addition, patients who clinically improved after treatment have a different profile than those who did not. These results provide a glimpse on potential markers that could assist in identifying therapy responders, in tracing the course of the disease, and in predicting its outcome. These studies have been restricted by the limited number of patients, but the approaches are feasible and can unravel severity-specific neuro-proteomic and metabolic SMA signatures.

3.
Biomedicines ; 10(9)2022 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-36140183

RESUMO

Extracorporeal membrane oxygenation (ECMO) is a lifesaving support for respiratory and cardiovascular failure. However, ECMO induces a systemic inflammatory response syndrome that can lead to various complications, including endothelial dysfunction in the cerebral circulation. We aimed to investigate whether ECMO-associated endothelial dysfunction also affected coronary circulation. Ten-day-old piglets were randomized to undergo either 8 h of veno-arterial ECMO (n = 5) or no treatment (Control, n = 5). Hearts were harvested and coronary arteries were dissected and mounted as 3 mm rings in organ baths for isometric force measurement. Following precontraction with the thromboxane prostanoid (TP) receptor agonist U46619, concentration−response curves to the endothelium-dependent vasodilator bradykinin (BK) and the nitric oxide (NO) donor (endothelium-independent vasodilator) sodium nitroprusside (SNP) were performed. Relaxation to BK was studied in the absence or presence of the NO synthase inhibitor Nω-nitro-L-arginine methyl ester HCl (L-NAME). U46619-induced contraction and SNP-induced relaxation were similar in control and ECMO coronary arteries. However, BK-induced relaxation was significantly impaired in the ECMO group (30.4 ± 2.2% vs. 59.2 ± 2.1%; p < 0.0001). When L-NAME was present, no differences in BK-mediated relaxation were observed between the control and ECMO groups. Taken together, our data suggest that ECMO exposure impairs endothelium-derived NO-mediated coronary relaxation. However, there is a NO-independent component in BK-induced relaxation that remains unaffected by ECMO. In addition, the smooth muscle cell response to exogenous NO is not altered by ECMO exposure.

4.
J Cell Mol Med ; 26(17): 4678-4685, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35880500

RESUMO

Becker muscular dystrophy (BMD) is an X-linked neuromuscular disorder due to mutation in the DMD gene, encoding dystrophin. Despite a wide clinical variability, BMD is characterized by progressive muscle degeneration and proximal muscle weakness. Interestingly, a dysregulated expression of muscle-specific microRNAs (miRNAs), called myomirs, has been found in patients affected with muscular dystrophies, although few studies have been conducted in BMD. We analysed the serum expression levels of a subset of myomirs in a cohort of 29 ambulant individuals affected by BMD and further classified according to the degree of alterations at muscle biopsy and in 11 age-matched healthy controls. We found a significant upregulation of serum miR-1, miR-133a, miR-133b and miR-206 in our cohort of BMD patients, supporting the role of these miRNAs in the pathophysiology of the disease, and we identified serum cut-off levels discriminating patients from healthy controls, confiming the potential of circulating miRNAs as promising noninvasive biomarkers. Moreover, serum levels of miR-133b were found to be associated with fibrosis at muscle biopsy and with patients' motor performances, suggesting that miR-133b might be a useful prognostic marker for BMD patients. Taken together, our data showed that these serum myomirs may represent an effective tool that may support stratification of BMD patients, providing the opportunity of both monitoring disease progression and assessing the treatment efficacy in the context of clinical trials.


Assuntos
MicroRNA Circulante , MicroRNAs , Distrofia Muscular de Duchenne , Biomarcadores , Progressão da Doença , Humanos , MicroRNAs/genética , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo
5.
Int J Mol Sci ; 22(17)2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34502539

RESUMO

Muscular dystrophies are a heterogeneous group of inherited diseases characterized by the progressive degeneration and weakness of skeletal muscles, leading to disability and, often, premature death. To date, no effective therapies are available to halt or reverse the pathogenic process, and meaningful treatments are urgently needed. From this perspective, it is particularly important to establish reliable in vitro models of human muscle that allow the recapitulation of disease features as well as the screening of genetic and pharmacological therapies. We herein review and discuss advances in the development of in vitro muscle models obtained from human induced pluripotent stem cells, which appear to be capable of reproducing the lack of myofiber proteins as well as other specific pathological hallmarks, such as inflammation, fibrosis, and reduced muscle regenerative potential. In addition, these platforms have been used to assess genetic correction strategies such as gene silencing, gene transfer and genome editing with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), as well as to evaluate novel small molecules aimed at ameliorating muscle degeneration. Furthermore, we discuss the challenges related to in vitro drug testing and provide a critical view of potential therapeutic developments to foster the future clinical translation of preclinical muscular dystrophy studies.


Assuntos
Diferenciação Celular/fisiologia , Descoberta de Drogas/métodos , Terapia Genética/métodos , Células-Tronco Pluripotentes Induzidas/fisiologia , Células Musculares/fisiologia , Distrofias Musculares/terapia , Animais , Distrofina/genética , Distrofina/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células Musculares/citologia , Distrofias Musculares/genética , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/terapia
7.
J Cell Mol Med ; 24(5): 3034-3039, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32032473

RESUMO

The antisense oligonucleotide Nusinersen has been recently licensed to treat spinal muscular atrophy (SMA). Since SMA type 3 is characterized by variable phenotype and milder progression, biomarkers of early treatment response are urgently needed. We investigated the cerebrospinal fluid (CSF) concentration of neurofilaments in SMA type 3 patients treated with Nusinersen as a potential biomarker of treatment efficacy. The concentration of phosphorylated neurofilaments heavy chain (pNfH) and light chain (NfL) in the CSF of SMA type 3 patients was evaluated before and after six months since the first Nusinersen administration, performed with commercially available enzyme-linked immunosorbent assay (ELISA) kits. Clinical evaluation of SMA patients was performed with standardized motor function scales. Baseline neurofilament levels in patients were comparable to controls, but significantly decreased after six months of treatment, while motor functions were only marginally ameliorated. No significant correlation was observed between the change in motor functions and that of neurofilaments over time. The reduction of neurofilament levels suggests a possible early biochemical effect of treatment on axonal degeneration, which may precede changes in motor performance. Our study mandates further investigations to assess neurofilaments as a marker of treatment response.


Assuntos
Proteínas de Neurofilamentos/líquido cefalorraquidiano , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos/administração & dosagem , Atrofias Musculares Espinais da Infância/tratamento farmacológico , Adolescente , Adulto , Idade de Início , Idoso , Biomarcadores/líquido cefalorraquidiano , Pré-Escolar , Feminino , Humanos , Filamentos Intermediários/metabolismo , Masculino , Pessoa de Meia-Idade , Oligonucleotídeos/efeitos adversos , Oligonucleotídeos Antissenso/efeitos adversos , Atrofias Musculares Espinais da Infância/líquido cefalorraquidiano , Atrofias Musculares Espinais da Infância/patologia , Resultado do Tratamento
8.
Front Pediatr ; 7: 444, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31709212

RESUMO

Incidental azygos vein cannulation has been reported in a few cases of neonatal extracorporeal membrane oxygenation (ECMO). This complication is described in the literature mainly in pathological conditions wherein increased central venous pressure dilates the superior vena cava (SVC), i.e., right congenital diaphragmatic hernia (CDH) or pulmonary hypertension. Azygos vein cannulation should always be suspected in cases of impaired venous return and circuit failure. Although rare, it hinders proper venous aspiration of the ECMO circuit and generally requires repositioning or replacement of the venous cannula or conversion to central cannulation. In this report, we describe a newborn with severe right CDH who required ECMO assistance, wherein incidental cannulation of the azygos vein resulted in successful functioning of the circuit because of the concomitant presence of isolated interruption of the inferior vena cava and azygos continuation. To the best of our knowledge, this is the first report of successful neonatal ECMO despite azygos vein cannulation in a patient with such rare physiology.

9.
Ther Adv Neurol Disord ; 11: 1756285618754501, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29434670

RESUMO

Spinal muscular atrophy (SMA) is a progressive, recessively inherited neuromuscular disease, characterized by the degeneration of lower motor neurons in the spinal cord and brainstem, which leads to weakness and muscle atrophy. SMA currently represents the most common genetic cause of infant death. SMA is caused by the lack of survival motor neuron (SMN) protein due to mutations, which are often deletions, in the SMN1 gene. In the absence of treatments able to modify the disease course, a considerable burden falls on patients and their families. Greater knowledge of the molecular basis of SMA pathogenesis has fuelled the development of potential therapeutic approaches, which are illustrated here. Nusinersen, a modified antisense oligonucleotide that modulates the splicing of the SMN2 mRNA transcript, is the first approved drug for all types of SMA. Moreover, the first gene therapy clinical trial using adeno-associated virus (AAV) vectors encoding SMN reported positive results in survival and motor milestones achievement. In addition, other strategies are in the pipeline, including modulation of SMN2 transcripts, neuroprotection, and targeting an increasing number of other peripheral targets, including the skeletal muscle. Based on this premise, it is reasonable to expect that therapeutic approaches aimed at treating SMA will soon be changed, and improved, in a meaningful way. We discuss the challenges with regard to the development of novel treatments for patients with SMA, and depict the current and future scenarios as the field enters into a new era of promising effective treatments.

10.
Mol Neurobiol ; 55(4): 2789-2813, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28455693

RESUMO

Amyotrophic lateral sclerosis (ALS) is a neurological disease characterized by the progressive loss of cortical, bulbar, and spinal motor neurons (MNs). The cardinal manifestation of ALS is a progressive paralysis which leads to death within a time span of 3 to 5 years after disease onset. Despite similar final output of neuronal death, the underlying pathogenic causes are various and no common cause of neuronal damage has been identified to date. Inflammation-mediated neuronal injury is increasingly recognized as a major factor that promotes disease progression and amplifies the MN death-inducing processes. The neuroimmune activation is not only a physiological reaction to cell-autonomous death but is an active component of nonautonomous cell death. Such injury-perpetuating phenomenon is now proved to be a common mechanism in many human disorders characterized by progressive neurodegeneration. Therefore, it represents an interesting therapeutic target. To date, no single cell population has been proved to play a major role. The existing evidence points to a complex cross talk between resident immune cells and nonresident cells, like monocytes and T lymphocytes, and to a dysregulation in cytokine profile and in phenotype commitment. After a summary of the most important mechanisms involved in the inflammatory reaction in ALS, this review will focus on novel therapeutic tools that rely on tackling inflammation to improve motor function and survival. Herein, completed, ongoing, or planned clinical trials, which aim to modify the rapidly fatal course of this disease, are discussed. Anti-inflammatory compounds that are currently undergoing preclinical study and novel suitable molecular targets are also mentioned.


Assuntos
Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/terapia , Inflamação/patologia , Terapia de Alvo Molecular , Animais , Citocinas/metabolismo , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Humanos
11.
Nucleic Acids Res ; 45(1): 461-469, 2017 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-27899589

RESUMO

Aptamers directed against human thrombin can selectively bind to two different exosites on the protein surface. The simultaneous use of two DNA aptamers, HD1 and HD22, directed to exosite I and exosite II respectively, is a very powerful approach to exploit their combined affinity. Indeed, strategies to link HD1 and HD22 together have been proposed in order to create a single bivalent molecule with an enhanced ability to control thrombin activity. In this work, the crystal structures of two ternary complexes, in which thrombin is sandwiched between two DNA aptamers, are presented and discussed. The structures shed light on the cross talk between the two exosites. The through-bond effects are particularly evident at exosite II, with net consequences on the HD22 structure. Moreover, thermodynamic data on the binding of the two aptamers are also reported and analyzed.


Assuntos
Aptâmeros de Nucleotídeos/química , Trombina/química , Aptâmeros de Nucleotídeos/síntese química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Termodinâmica , Trombina/antagonistas & inibidores
12.
J Cell Mol Med ; 19(9): 2058-66, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26095024

RESUMO

Spinal muscular atrophy with respiratory distress (SMARD1) is an autosomal recessive neuromuscular disease caused by mutations in the IGHMBP2 gene, encoding the immunoglobulin µ-binding protein 2, leading to motor neuron degeneration. It is a rare and fatal disease with an early onset in infancy in the majority of the cases. The main clinical features are muscular atrophy and diaphragmatic palsy, which requires prompt and permanent supportive ventilation. The human disease is recapitulated in the neuromuscular degeneration (nmd) mouse. No effective treatment is available yet, but novel therapeutical approaches tested on the nmd mouse, such as the use of neurotrophic factors and stem cell therapy, have shown positive effects. Gene therapy demonstrated effectiveness in SMA, being now at the stage of clinical trial in patients and therefore representing a possible treatment for SMARD1 as well. The significant advancement in understanding of both SMARD1 clinical spectrum and molecular mechanisms makes ground for a rapid translation of pre-clinical therapeutic strategies in humans.


Assuntos
Atrofia Muscular Espinal/patologia , Atrofia Muscular Espinal/terapia , Síndrome do Desconforto Respiratório do Recém-Nascido/patologia , Síndrome do Desconforto Respiratório do Recém-Nascido/terapia , Animais , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Terapia Genética , Humanos , Atrofia Muscular Espinal/etiologia , Síndrome do Desconforto Respiratório do Recém-Nascido/etiologia , Transplante de Células-Tronco
13.
Clin Ther ; 37(3): 668-80, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-25666449

RESUMO

PURPOSE: Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease in adults. It is almost invariably lethal within a few years after the onset of symptoms. No effective treatment is currently available beyond supportive care and riluzole, a putative glutamate release blocker linked to modestly prolonged survival. This review provides a general overview of preclinical and clinical advances during recent years and summarizes the literature regarding emerging therapeutic approaches, focusing on their molecular targets. METHODS: A systematic literature review of PubMed was performed, identifying key clinical trials involving molecular therapies for ALS. In addition, the ALS Therapy Development Institute website was carefully analyzed, and a selection of ALS clinical trials registered at ClinicalTrials.gov has been included. FINDINGS: In the last several years, strategies have been developed to understand both the genetic and molecular mechanisms of ALS. Several therapeutic targets have been actively pursued, including kinases, inflammation inhibitors, silencing of key genes, and modulation or replacement of specific cell populations. The majority of ongoing clinical trials are investigating the safety profiles and tolerability of pharmacologic, gene, and cellular therapies, and have begun to assess their effects on ALS progression. IMPLICATIONS: Currently, no therapeutic effort seems to be efficient, but recent findings in ALS could help accelerate the discovery of an effective treatment for this disease.


Assuntos
Esclerose Lateral Amiotrófica/terapia , Riluzol/uso terapêutico , Adulto , Esclerose Lateral Amiotrófica/fisiopatologia , Progressão da Doença , Humanos , Resultado do Tratamento
14.
PLoS One ; 8(1): e53748, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23341992

RESUMO

AIMS: the adult zebrafish heart regenerates spontaneously after injury and has been used to study the mechanisms of cardiac repair. However, no zebrafish model is available that mimics ischemic injury in mammalian heart. We developed and characterized zebrafish cardiac injury induced by hypoxia/reoxygenation (H/R) and the regeneration that followed it. METHODS AND RESULTS: adult zebrafish were kept either in hypoxic (H) or normoxic control (C) water for 15 min; thereafter fishes were returned to C water. Within 2-6 hours (h) after reoxygenation there was evidence of cardiac oxidative stress by dihydroethidium fluorescence and protein nitrosylation, as well as of inflammation. We used Tg(cmlc2:nucDsRed) transgenic zebrafish to identify myocardial cell nuclei. Cardiomyocyte apoptosis and necrosis were evidenced by TUNEL and Acridine Orange (AO) staining, respectively; 18 h after H/R, 9.9±2.6% of myocardial cell nuclei were TUNEL(+) and 15.0±2.5% were AO(+). At the 30-day (d) time point myocardial cell death was back to baseline (n = 3 at each time point). We evaluated cardiomyocyte proliferation by Phospho Histone H3 (pHH3) or Proliferating Cell Nuclear Antigen (PCNA) expression. Cardiomyocyte proliferation was apparent 18-24 h after H/R, it achieved its peak 3-7d later, and was back to baseline at 30d. 7d after H/R 17.4±2.3% of all cardiomyocytes were pHH3(+) and 7.4±0.6% were PCNA(+) (n = 3 at each time point). Cardiac function was assessed by 2D-echocardiography and Ventricular Diastolic and Systolic Areas were used to compute Fractional Area Change (FAC). FAC decreased from 29.3±2.0% in normoxia to 16.4±1.8% at 18 h after H/R; one month later ventricular function was back to baseline (n = 12 at each time point). CONCLUSIONS: zebrafish exposed to H/R exhibit evidence of cardiac oxidative stress and inflammation, myocardial cell death and proliferation. The initial decrease in ventricular function is followed by full recovery. This model more closely mimics reperfusion injury in mammals than other cardiac injury models.


Assuntos
Traumatismos Cardíacos/fisiopatologia , Coração/fisiopatologia , Hipóxia/fisiopatologia , Miocárdio/metabolismo , Oxigênio/metabolismo , Regeneração , Animais , Apoptose , Proliferação de Células , Traumatismos Cardíacos/metabolismo , Traumatismos Cardíacos/patologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Hipóxia/metabolismo , Hipóxia/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/patologia , Estresse Oxidativo , Recuperação de Função Fisiológica , Peixe-Zebra
15.
Nat Immunol ; 12(8): 796-803, 2011 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-21706005

RESUMO

MicroRNAs are small noncoding RNAs that regulate gene expression post-transcriptionally. Here we applied microRNA profiling to 17 human lymphocyte subsets to identify microRNA signatures that were distinct among various subsets and different from those of mouse lymphocytes. One of the signature microRNAs of naive CD4+ T cells, miR-125b, regulated the expression of genes encoding molecules involved in T cell differentiation, including IFNG, IL2RB, IL10RA and PRDM1. The expression of synthetic miR-125b and lentiviral vectors encoding the precursor to miR-125b in naive lymphocytes inhibited differentiation to effector cells. Our data provide an 'atlas' of microRNA expression in human lymphocytes, define subset-specific signatures and their target genes and indicate that the naive state of T cells is enforced by microRNA.


Assuntos
Linfócitos T CD4-Positivos/imunologia , MicroRNAs/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Biologia Computacional/métodos , Citometria de Fluxo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Humanos , Camundongos , MicroRNAs/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Arterioscler Thromb Vasc Biol ; 31(7): 1589-97, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21527751

RESUMO

OBJECTIVE: The vascular competence of human-derived hematopoietic progenitors for postnatal vascularization is still poorly characterized. It is unclear whether, in the absence of ischemia, hematopoietic progenitors participate in neovascularization and whether they play a role in new blood vessel formation by incorporating into developing vessels or by a paracrine action. METHODS AND RESULTS: In the present study, human cord blood-derived CD34(+) (hCD34(+)) cells were transplanted into pre- and postgastrulation zebrafish embryos and in an adult vascular regeneration model induced by caudal fin amputation. When injected before gastrulation, hCD34(+) cells cosegregated with the presumptive zebrafish hemangioblasts, characterized by Scl and Gata2 expression, in the anterior and posterior lateral mesoderm and were involved in early development of the embryonic vasculature. These morphogenetic events occurred without apparent lineage reprogramming, as shown by CD45 expression. When transplanted postgastrulation, hCD34(+) cells were recruited into developing vessels, where they exhibited a potent paracrine proangiogenic action. Finally, hCD34(+) cells rescued vascular defects induced by Vegf-c in vivo targeting and enhanced vascular repair in the zebrafish fin amputation model. CONCLUSIONS: These results indicate an unexpected developmental ability of human-derived hematopoietic progenitors and support the hypothesis of an evolutionary conservation of molecular pathways involved in endothelial progenitor differentiation in vivo.


Assuntos
Nadadeiras de Animais/irrigação sanguínea , Antígenos CD34/análise , Diferenciação Celular , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Células Endoteliais/transplante , Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Neovascularização Fisiológica , Peixe-Zebra , Amputação Cirúrgica , Nadadeiras de Animais/cirurgia , Animais , Animais Geneticamente Modificados , Células CACO-2 , Diferenciação Celular/efeitos dos fármacos , Movimento Celular , Células Endoteliais/imunologia , Sangue Fetal/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/imunologia , Humanos , Comunicação Parácrina , Fenótipo , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Regeneração , Transdução de Sinais , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
17.
Am J Physiol Heart Circ Physiol ; 300(5): H1875-84, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21357510

RESUMO

The efficacy of cardiac repair by stem cell administration relies on a successful functional integration of injected cells into the host myocardium. Safety concerns have been raised about the possibility that stem cells may induce foci of arrhythmia in the ischemic myocardium. In a previous work (36), we showed that human cord blood CD34(+) cells, when cocultured on neonatal mouse cardiomyocytes, exhibit excitation-contraction coupling features similar to those of cardiomyocytes, even though no human genes were upregulated. The aims of the present work are to investigate whether human CD34(+) cells, isolated after 1 wk of coculture with neonatal ventricular myocytes, possess molecular and functional properties of cardiomyocytes and to discriminate, using a reporter gene system, whether cardiac differentiation derives from a (trans)differentiation or a cell fusion process. Umbilical cord blood CD34(+) cells were isolated by a magnetic cell sorting method, transduced with a lentiviral vector carrying the enhanced green fluorescent protein (EGFP) gene, and seeded onto primary cultures of spontaneously beating rat neonatal cardiomyocytes. Cocultured EGFP(+)/CD34(+)-derived cells were analyzed for their electrophysiological features at different time points. After 1 wk in coculture, EGFP(+) cells, in contact with cardiomyocytes, were spontaneously contracting and had a maximum diastolic potential (MDP) of -53.1 mV, while those that remained isolated from the surrounding myocytes did not contract and had a depolarized resting potential of -11.4 mV. Cells were then resuspended and cultured at low density to identify EGFP(+) progenitor cell derivatives. Under these conditions, we observed single EGFP(+) beating cells that had acquired an hyperpolarization-activated current typical of neonatal cardiomyocytes (EGFP(+) cells, -2.24 ± 0.89 pA/pF; myocytes, -1.99 ± 0.63 pA/pF, at -125 mV). To discriminate between cell autonomous differentiation and fusion, EGFP(+)/CD34(+) cells were cocultured with cardiac myocytes infected with a red fluorescence protein-lentiviral vector; under these conditions we found that 100% of EGFP(+) cells were also red fluorescent protein positive, suggesting cell fusion as the mechanism by which cardiac functional features are acquired.


Assuntos
Antígenos CD34/metabolismo , Comunicação Celular/fisiologia , Fusão Celular/métodos , Sangue Fetal/citologia , Miócitos Cardíacos/citologia , Células-Tronco/citologia , Células-Tronco/imunologia , Animais , Antígenos CD34/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Modelos Animais , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Ratos , Células-Tronco/fisiologia
18.
J Electromyogr Kinesiol ; 19(5): e290-300, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18801662

RESUMO

The aim of the present study was to clarify the impact of long term (1 year) resistance training (RT) on structure and function of single muscle fibres of vastus lateralis in young female subjects. Five young women (age: 25.4+/-6.2 year) performed exercise sessions at 60% of single subject own repetition maximum (1 RM) 1h twice a week. Maximum voluntary force was determined pre- and post-RT and was found to significantly increase post-RT ensuring a successful impact of RT on muscle performance in vivo. Needle muscle biopsy samples were obtained both pre- and post-RT and the following determinations were performed: myosin heavy chain isoform (MHC) distribution of the whole muscle samples by SDS-PAGE; cross sectional area (CSA), specific force (Po/CSA) and maximum shortening velocity (Vo) of a large population (n=358) of single skinned muscle fibres classified on the basis of MHC isoform composition by SDS-PAGE. The results suggest that the long duration of RT can determine a significant increase in specific force (Po/CSA) and unloaded shortening velocity (Vo) of single muscle fibres in female subjects, whereas no muscle fibre hypertrophy and no shift in MHC isoform content was observed.


Assuntos
Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/fisiologia , Resistência Física/fisiologia , Treinamento Resistido/métodos , Adaptação Fisiológica/fisiologia , Adulto , Feminino , Humanos , Articulação do Joelho/fisiologia , Estudos Longitudinais , Estresse Mecânico
19.
Eur J Appl Physiol ; 104(5): 885-93, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18677504

RESUMO

Four elderly women (78 +/- 4.3 years) were resistance trained (RT) for one year and needle biopsies of the vastus lateralis muscle were taken pre- and post-RT. A training intensity of 60% one-repetition maximum (1RM) was adopted for the entire duration of the study. Following RT, significant gain in isometric force of the quadriceps muscles was observed and MHC isoform distribution of vastus lateralis muscle showed a very significant shift from MHC-1 and MHC-2X towards MHC-2A, i.e. a bi-directional shift. A large population (n = 310) of individual skinned muscle fibres were dissected from biopsy samples and used for determination of cross-sectional area (CSA), specific force (Po/CSA) and unloaded shortening velocity (Vo) at 12 degrees C and maximum activation. Fibres were classified on the basis of their MHC isoform content determined by SDS-PAGE in three pure fibre types (1, 2A and 2X) and two hybrid fibre types (1-2A and 2AX). Whereas the CSA of individual muscle fibres did not change, Po/CSA was significantly higher in both type 1 and type 2A fibres post training. Vo of type 1 fibres was significantly higher post-training, whereas no change in Vo was observed in type 2A fibres. The number of pure type 2X fibres was very low especially post-training and did not enable a statistically significant comparison. The data suggest that in elderly women moderate and prolonged RT: (1) can determine similar adaptations in MHC distribution of skeletal muscle to those expected in young subjects; (2) can improve function of muscle fibres.


Assuntos
Contração Muscular , Fibras Musculares Esqueléticas/fisiologia , Força Muscular , Músculo Quadríceps/fisiologia , Treinamento Resistido , Adaptação Fisiológica , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Fibras Musculares Esqueléticas/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Isoformas de Proteínas , Músculo Quadríceps/citologia , Músculo Quadríceps/metabolismo
20.
J Clin Invest ; 116(11): 2945-54, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17080195

RESUMO

NF-kappaB is a major pleiotropic transcription factor modulating immune, inflammatory, cell survival, and proliferative responses, yet the relevance of NF-kappaB signaling in muscle physiology and disease is less well documented. Here we show that muscle-restricted NF-kappaB inhibition in mice, through targeted deletion of the activating kinase inhibitor of NF-kappaB kinase 2 (IKK2), shifted muscle fiber distribution and improved muscle force. In response to denervation, IKK2 depletion protected against atrophy, maintaining fiber type, size, and strength, increasing protein synthesis, and decreasing protein degradation. IKK2-depleted mice with a muscle-specific transgene expressing a local Igf-1 isoform (mIgf-1) showed enhanced protection against muscle atrophy. In response to muscle damage, IKK2 depletion facilitated skeletal muscle regeneration through enhanced satellite cell activation and reduced fibrosis. Our results establish IKK2/NF-kappaB signaling as an important modulator of muscle homeostasis and suggest a combined role for IKK inhibitors and growth factors in the therapy of muscle diseases.


Assuntos
Quinase I-kappa B/metabolismo , Força Muscular , Músculo Esquelético/citologia , Músculo Esquelético/enzimologia , Regeneração , Animais , Fibrose , Deleção de Genes , Quinase I-kappa B/deficiência , Quinase I-kappa B/genética , Camundongos , Camundongos Transgênicos , Músculo Esquelético/inervação , Atrofia Muscular/enzimologia , Atrofia Muscular/genética , Atrofia Muscular/patologia , NF-kappa B/metabolismo , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
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