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1.
Physiol Genomics ; 54(8): 296-304, 2022 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-35759450

RESUMEN

Fibro-adipogenic progenitor cells (FAPs) are a population of stem cells in skeletal muscle that play multiple roles in muscle repair and regeneration through their complex secretome; however, it is not well understood how the FAP secretome is altered with muscle disuse atrophy. Previous work suggests that the inflammatory cytokine IL-1ß is increased in FAPs with disuse and denervation. Inflammasome activation and IL-1ß secretion are also known to stimulate the release of extracellular vesicles (EVs). Here, we examined the microRNA (miRNA) cargo of FAP-derived, platelet-derived growth factor receptor A (PDGFRα+) EVs from hindlimb muscles of wild-type and IL-1ß KO mice after 14 days of single-hindlimb immobilization. Hindlimb muscles were isolated from mice following the immobilization period, and PDGFRα+ extracellular vesicles were isolated using size-exclusion chromatography and immunoprecipitation. Microarrays were performed to detect changes in miRNAs with unloading and IL-1ß deficiency. Results indicate that the PDGFRα+, FAP-derived EVs show a significant increase in miRNAs, such as miR-let-7c, miR-let-7b, miR-181a, and miR-124. These miRNAs have previously been demonstrated to play important roles in cellular senescence and muscle atrophy. Furthermore, the expression of these same miRNAs was not significantly altered in FAP-derived EVs isolated from the immobilized IL-1ß KO. These data suggest that disuse-related activation of IL-1ß can mediate the miRNA cargo of FAP-derived EVs, contributing directly to the release of senescence- and atrophy-related miRNAs. Therapies targeting FAPs in settings associated with muscle disuse atrophy may therefore have the potential to preserve muscle function and enhance muscle recovery.


Asunto(s)
Vesículas Extracelulares , Interleucina-1beta/metabolismo , MicroARNs , Trastornos Musculares Atróficos , Animales , Vesículas Extracelulares/metabolismo , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Músculo Esquelético/metabolismo , Trastornos Musculares Atróficos/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Células Madre/metabolismo
2.
Circ Res ; 124(1): 161-169, 2019 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-30605412

RESUMEN

On March 1 and 2, 2018, the National Institutes of Health 2018 Progenitor Cell Translational Consortium, Cardiovascular Bioengineering Symposium, was held at the University of Alabama at Birmingham. Convergence of life sciences and engineering to advance the understanding and treatment of heart failure was the theme of the meeting. Over 150 attendees were present, and >40 scientists presented their latest work on engineering human functional myocardium for disease modeling, drug development, and heart failure research. The scientists, engineers, and physicians in the field of cardiovascular sciences met and discussed the most recent advances in their work and proposed future strategies for overcoming the major roadblocks of cardiovascular bioengineering and therapy. Particular emphasis was given for manipulation and using of stem/progenitor cells, biomaterials, and methods to provide molecular, chemical, and mechanical cues to cells to influence their identity and fate in vitro and in vivo. Collectively, these works are profoundly impacting and progressing toward deciphering the mechanisms and developing novel treatments for left ventricular dysfunction of failing hearts. Here, we present some important perspectives that emerged from this meeting.


Asunto(s)
Disciplinas de las Ciencias Biológicas , Ingeniería Biomédica , Investigación Biomédica , Insuficiencia Cardíaca , Comunicación Interdisciplinaria , Animales , Conducta Cooperativa , Difusión de Innovaciones , Corazón/fisiopatología , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Cardíaca/terapia , Humanos , Miocardio/metabolismo , Miocardio/patología , Recuperación de la Función , Regeneración
3.
Acta Pharmacol Sin ; 42(4): 529-535, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32601364

RESUMEN

Duchenne muscular dystrophy (DMD) is a progressive disease characterized by skeletal muscle atrophy, respiratory failure, and cardiomyopathy. Our previous studies have shown that transplantation with allogeneic myogenic progenitor-derived exosomes (MPC-Exo) can improve cardiac function in X-linked muscular dystrophy (Mdx) mice. In the present study we explored the molecular mechanisms underlying this beneficial effect. We quantified gene expression in the hearts of two strains of Mdx mice (D2.B10-DmdMdx/J and Utrntm1Ked-DmdMdx/J). Two days after MPC-Exo or control treatment, we performed unbiased next-generation RNA-sequencing to identify differentially expressed genes (DEGs) in treated Mdx hearts. Venn diagrams show a set of 780 genes that were ≥2-fold upregulated, and a set of 878 genes that were ≥2-fold downregulated, in both Mdx strains following MPC-Exo treatment as compared with control. Gene ontology (GO) and protein-protein interaction (PPI) network analysis showed that these DEGs were involved in a variety of physiological processes and pathways with a complex connection. qRT-PCR was performed to verify the upregulated ATP2B4 and Bcl-2 expression, and downregulated IL-6, MAPK8 and Wnt5a expression in MPC-Exo-treated Mdx hearts. Western blot analysis verified the increased level of Bcl-2 and decreased level of IL-6 protein in MPC-Exo-treated Mdx hearts compared with control treatment, suggesting that anti-apoptotic and anti-inflammatory effects might be responsible for heart function improvement by MPC-Exo. Based on these findings, we believed that these DEGs might be therapeutic targets that can be explored to develop new strategies for treating DMD.


Asunto(s)
Cardiomiopatías/terapia , Exosomas/trasplante , Distrofia Muscular de Duchenne/terapia , Miocardio/metabolismo , Animales , Cardiomiopatías/etiología , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Regulación hacia Abajo/fisiología , Perfilación de la Expresión Génica , Ontología de Genes , Masculino , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/complicaciones , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Mapas de Interacción de Proteínas , RNA-Seq , Regulación hacia Arriba/fisiología
4.
J Biol Chem ; 294(21): 8577-8591, 2019 05 24.
Artículo en Inglés | MEDLINE | ID: mdl-30971429

RESUMEN

Enhancer of zeste homolog 2 (EZH2), an epigenetic regulator that plays a key role in cell differentiation and oncogenesis, was reported to promote adipogenic differentiation in vitro by catalyzing trimethylation of histone 3 lysine 27. However, inhibition of EZH2 induced lipid accumulation in certain cancer and hepatocyte cell lines. To address this discrepancy, we investigated the role of EZH2 in adipogenic differentiation and lipid metabolism using primary human and mouse preadipocytes and adipose-specific EZH2 knockout (KO) mice. We found that the EZH2-selective inhibitor GSK126 induced lipid accumulation in human adipocytes, without altering adipocyte differentiation marker gene expression. Moreover, adipocyte-specific EZH2 KO mice, generated by crossing EZH2 floxed mice with adiponectin-Cre mice, displayed significantly increased body weight, adipose tissue mass, and adipocyte cell size and reduced very low-density lipoprotein (VLDL) levels, as compared with littermate controls. These phenotypic alterations could not be explained by differences in feeding behavior, locomotor activity, metabolic energy expenditure, or adipose lipolysis. In addition, human adipocytes treated with either GSK126 or vehicle exhibited comparable rates of glucose-stimulated triglyceride accumulation and fatty acid uptake. Mechanistically, lipid accumulation induced by GSK126 in adipocytes was lipoprotein-dependent, and EZH2 inhibition or gene deletion promoted lipoprotein-dependent lipid uptake in vitro concomitant with up-regulated apolipoprotein E (ApoE) gene expression. Deletion of ApoE blocked the effects of GSK126 to promote lipoprotein-dependent lipid uptake in murine adipocytes. Collectively, these results indicate that EZH2 inhibition promotes lipoprotein-dependent lipid accumulation via inducing ApoE expression in adipocytes, suggesting a novel mechanism of lipid regulation by EZH2.


Asunto(s)
Adipocitos/metabolismo , Apolipoproteínas E/metabolismo , Diferenciación Celular , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Lipogénesis , Lipólisis , Adipocitos/citología , Animales , Apolipoproteínas E/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Humanos , Lipoproteínas VLDL/genética , Lipoproteínas VLDL/metabolismo , Ratones , Regulación hacia Arriba
5.
Circ Res ; 122(10): 1395-1408, 2018 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-29618597

RESUMEN

RATIONALE: Vascular calcification (VC) is a marker of the severity of atherosclerotic disease. Hormones play important roles in regulating calcification; estrogen and parathyroid hormones exert opposing effects, the former alleviating VC and the latter exacerbating it. To date no treatment strategies have been developed to regulate clinical VC. OBJECTIVE: The objective of this study was to investigate the effect of growth hormone-releasing hormone (GHRH) and its agonist (GHRH-A) on the blocking of VC in a mouse model. METHODS AND RESULTS: Young adult osteoprotegerin-deficient mice were given daily subcutaneous injections of GHRH-A (MR409) for 4 weeks. Significant reductions in calcification of the aortas of MR409-treated mice were paralleled by markedly lower alkaline phosphatase activity and a dramatic reduction in the expression of transcription factors, including the osteogenic marker gene Runx2 and its downstream factors, osteonectin and osteocalcin. The mechanism of action of GHRH-A was dissected in smooth muscle cells isolated from human and mouse aortas. Calcification of smooth muscle cells induced by osteogenic medium was inhibited in the presence of GHRH or MR409, as evidenced by reduced alkaline phosphatase activity and Runx2 expression. Inhibition of calcification by MR409 was partially reversed by MIA602, a GHRH antagonist, or a GHRH receptor-selective small interfering RNA. Treatment with MR409 induced elevated cytosolic cAMP and its target, protein kinase A which in turn blocked nicotinamide adenine dinucleotide phosphate oxidase activity and reduced production of reactive oxygen species, thus blocking the phosphorylation of nuclear factor κB (p65), a key intermediate in the ligand of receptor activator for nuclear factor-κ B-Runx2/alkaline phosphatase osteogenesis program. A protein kinase A-selective small interfering RNA or the chemical inhibitor H89 abolished these beneficial effects of MR409. CONCLUSIONS: GHRH-A controls osteogenesis in smooth muscle cells by targeting cross talk between protein kinase A and nuclear factor κB (p65) and through the suppression of reactive oxygen species production that induces the Runx2 gene and alkaline phosphatase. Inflammation-mediated osteogenesis is thereby blocked. GHRH-A may represent a new pharmacological strategy to regulate VC.


Asunto(s)
Fragmentos de Péptidos/uso terapéutico , Calcificación Vascular/prevención & control , Fosfatasa Alcalina/biosíntesis , Fosfatasa Alcalina/genética , Animales , Aorta/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Medios de Cultivo/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Hormona Liberadora de Hormona del Crecimiento , Trasplante de Corazón , Humanos , Isoquinolinas/farmacología , Ratones , Ratones Endogámicos C57BL , Osteogénesis , Osteoprotegerina/deficiencia , Fragmentos de Péptidos/farmacología , ARN Interferente Pequeño/genética , Receptores de Neuropéptido/antagonistas & inhibidores , Receptores de Neuropéptido/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/antagonistas & inhibidores , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Sulfonamidas/farmacología , Factor de Transcripción ReIA/metabolismo , Calcificación Vascular/fisiopatología
6.
J Biol Chem ; 292(15): 6312-6324, 2017 04 14.
Artículo en Inglés | MEDLINE | ID: mdl-28242765

RESUMEN

Impaired adipogenic differentiation during diet-induced obesity (DIO) promotes adipocyte hypertrophy and inflammation, thereby contributing to metabolic disease. Adenomatosis polyposis coli down-regulated 1 (APCDD1) has recently been identified as an inhibitor of Wnt signaling, a key regulator of adipogenic differentiation. Here we report a novel role for APCDD1 in adipogenic differentiation via repression of Wnt signaling and an epigenetic linkage between miR-130 and APCDD1 in DIO. APCDD1 expression was significantly up-regulated in mature adipocytes compared with undifferentiated preadipocytes in both human and mouse subcutaneous adipose tissues. siRNA-based silencing of APCDD1 in 3T3-L1 preadipocytes markedly increased the expression of Wnt signaling proteins (Wnt3a, Wnt5a, Wnt10b, LRP5, and ß-catenin) and inhibited the expression of adipocyte differentiation markers (CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ)) and lipid droplet accumulation, whereas adenovirus-mediated overexpression of APCDD1 enhanced adipogenic differentiation. Notably, DIO mice exhibited reduced APCDD1 expression and increased Wnt expression in both subcutaneous and visceral adipose tissues and impaired adipogenic differentiation in vitro Mechanistically, we found that miR-130, whose expression is up-regulated in adipose tissues of DIO mice, could directly target the 3'-untranslated region of the APCDD1 gene. Furthermore, transfection of an miR-130 inhibitor in preadipocytes enhanced, whereas an miR-130 mimic blunted, adipogenic differentiation, suggesting that miR-130 contributes to impaired adipogenic differentiation during DIO by repressing APCDD1 expression. Finally, human subcutaneous adipose tissues isolated from obese individuals exhibited reduced expression of APCDD1, C/EBPα, and PPARγ compared with those from non-obese subjects. Taken together, these novel findings suggest that APCDD1 positively regulates adipogenic differentiation and that its down-regulation by miR-130 during DIO may contribute to impaired adipogenic differentiation and obesity-related metabolic disease.


Asunto(s)
Adipocitos/metabolismo , Diferenciación Celular , Silenciador del Gen , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Proteínas de la Membrana/biosíntesis , Obesidad/metabolismo , Vía de Señalización Wnt , Células 3T3-L1 , Adipocitos/patología , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Dieta/efectos adversos , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Proteínas de la Membrana/genética , Ratones , Obesidad/inducido químicamente , Obesidad/genética , Obesidad/patología , Proteínas Wnt/genética , Proteínas Wnt/metabolismo
7.
Acta Pharmacol Sin ; 39(7): 1100-1109, 2018 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-29565037

RESUMEN

Recent advancements in genome-wide analyses and RNA-sequencing technologies led to the discovery of small noncoding RNAs, such as microRNAs (miRs), as well as both linear long noncoding RNAs (lncRNAs) and circular long noncoding RNAs (circRNAs). The importance of miRs and lncRNAs in the treatment, prognosis and diagnosis of cardiovascular diseases (CVDs) has been extensively reported. We also previously reviewed their implications in therapies and as biomarkers for CVDs. More recently, circRNAs have also emerged as important regulators in CVDs. CircRNAs are circular genome products that are generated by back splicing of specific regions of pre-messenger RNAs (pre-mRNAs). Growing interest in circRNAs led to the discovery of a wide array of their pathophysiological functions. CircRNAs have been shown to be key regulators of CVDs such as myocardial infarction, atherosclerosis, cardiomyopathy and cardiac fibrosis. Accordingly, circRNAs have been recently proposed as potential therapeutic targets and biomarkers for CVDs. In this review, we summarize the current state of the literature on circRNAs, starting with their biogenesis and global mechanisms of actions. We then provide a synopsis of their involvement in various CVDs. Lastly, we emphasize the great potential of circRNAs as biomarkers for the early detection of CVDs, and discuss several patents and recent papers that highlight the utilization of circRNAs as promising biomarkers.


Asunto(s)
Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/tratamiento farmacológico , ARN Largo no Codificante/sangre , Animales , Biomarcadores/sangre , Humanos
8.
Int J Mol Sci ; 17(3): 356, 2016 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-26978351

RESUMEN

Protein-coding genes account for only a small part of the human genome; in fact, the vast majority of transcripts are comprised of non-coding RNAs (ncRNAs) including long ncRNAs (lncRNAs) and small ncRNAs, microRNAs (miRs). Accumulating evidence indicates that ncRNAs could play critical roles in regulating many cellular processes which are often implicated in health and disease. For example, ncRNAs are aberrantly expressed in cancers, heart diseases, and many other diseases. LncRNAs and miRs are therefore novel and promising targets to be developed into biomarkers for diagnosis and prognosis as well as treatment options. The interaction between lncRNAs and miRs as well as its pathophysiological significance have recently been reported. Mechanistically, it is believed that lncRNAs exert "sponge-like" effects on various miRs, which subsequently inhibits miR-mediated functions. This crosstalk between two types of ncRNAs frequently contributes to the pathogenesis of the disease. In this review, we provide a summary of the recent studies highlighting the interaction between these ncRNAs and the effects of this interaction on disease pathogenesis and regulation.


Asunto(s)
Enfermedad/genética , MicroARNs/genética , ARN Largo no Codificante/genética , Progresión de la Enfermedad , Regulación de la Expresión Génica , Humanos
9.
J Mol Cell Cardiol ; 81: 49-53, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25655934

RESUMEN

The G protein-coupled receptor CXCR4 and its ligand stromal-cell derived factor 1 (SDF-1) play a crucial role in directing progenitor cell (PC) homing to ischemic tissue. The Src family protein kinases (SFK) can be activated by, and serve as effectors of, G proteins. In this study we sought to determine whether SFK play a role in SDF-1/CXCR4-mediated PC homing. First, we investigated whether SDF-1/CXCR4 signaling activates SFK. Bone-marrow mononuclear cells (BM MNCs) were isolated from WT and BM-specific CXCR4-KO mice and treated with SDF-1 and/or CXCR4 antagonist AMD3100. SDF-1 treatment rapidly induced phosphorylation (activation) of hematopoietic Src (i.e., Lyn, Fgr, and Hck) in WT cells but not in AMD3100-treated cells or CXCR4-KO cells. Then, we investigated whether SFK are involved in SDF-1/CXCR4-mediated PC chemotaxis. In a combined chemotaxis and endothelial-progenitor-cell (EPC) colony assay, Src inhibitor SU6656 dose-dependently inhibited the SDF-1-induced migration of colony-forming EPCs. Next, we investigated whether SFK play a role in SDF-1/CXCR4-mediated BM PC homing to the ischemic heart. BM MNCs from CXCR4BAC:eGFP reporter mice were i.v. injected into WT and SDF-1BAC:SDF1-RFP transgenic mice following surgically-induced myocardial infarction (MI). eGFP(+) MNCs and eGFP(+)c-kit(+) PCs that were recruited in the infarct border zone in SDF-1BAC:SDF1-RFP recipients were significantly more than that in WT recipients. Treatments of mice with SU6656 significantly reduced eGFP(+) and eGFP(+)c-kit(+) cell recruitment in both WT and SDF-1BAC:RFP recipients and abrogated the difference between the two groups. Remarkably, PCs isolated from BM-specific C-terminal Src kinase (CSK)-KO (Src activated) mice were recruited more efficiently than PCs from WT PCs in the WT recipients. In conclusion, SFK are activated by SDF-1/CXCR4 signaling and play an essential role in SDF-1/CXCR4-mediated BM PC chemotactic response and ischemic cardiac recruitment.


Asunto(s)
Células de la Médula Ósea/metabolismo , Quimiocina CXCL12/genética , Células Madre Mesenquimatosas/metabolismo , Isquemia Miocárdica/genética , Receptores CXCR4/genética , Familia-src Quinasas/genética , Animales , Bencilaminas , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/patología , Quimiocina CXCL12/antagonistas & inhibidores , Quimiocina CXCL12/metabolismo , Quimiotaxis/genética , Ciclamas , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Compuestos Heterocíclicos/farmacología , Indoles/farmacología , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/patología , Ratones , Ratones Noqueados , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patología , Miocardio/metabolismo , Miocardio/patología , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptores CXCR4/deficiencia , Transducción de Señal , Sulfonamidas/farmacología , Familia-src Quinasas/metabolismo
10.
J Mol Cell Cardiol ; 60: 68-71, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23603666

RESUMEN

The growth of new blood vessels after ischemic injury requires endothelial cells (ECs) to divide and proliferate, and the E2F transcription factors are key regulators of the genes responsible for cell-cycle progression; however, the specific roles of individual E2Fs in ECs are largely unknown. To determine the roles of E2F2 and E2F3 in EC proliferation and the angiogenic response to ischemic injury, hind-limb ischemia was surgically induced in E2F2(-/-) mice, endothelial-specific E2F3-knockout (EndoE2F3(∆/∆)) mice, and their littermates with wild-type E2F2 and E2F3 expression. Two weeks later, Laser-Doppler perfusion measurements, capillary density, and endothelial proliferation were significantly greater in E2F2(-/-) mice and significantly lower in EndoE2F3(∆/∆) mice than in their littermates, and EndoE2F3(∆/∆) mice also developed toe and limb necrosis. The loss of E2F2 expression was associated with increases in the proliferation and G1/S-phase gene expression of isolated ECs, while the loss of E2F3 expression led to declines in these parameters. Thus E2F2 impairs, and endothelial E2F3 promotes, the angiogenic response to peripheral ischemic injury through corresponding changes in EC cell-cycle progression.


Asunto(s)
Factor de Transcripción E2F2/metabolismo , Factor de Transcripción E2F3/metabolismo , Células Endoteliales/metabolismo , Fase G1 , Miembro Posterior/irrigación sanguínea , Isquemia/metabolismo , Neovascularización Patológica/metabolismo , Fase S , Animales , Factor de Transcripción E2F2/genética , Factor de Transcripción E2F3/genética , Células Endoteliales/patología , Isquemia/patología , Ratones , Ratones Mutantes , Neovascularización Patológica/genética , Neovascularización Patológica/patología
11.
Circ Res ; 107(9): 1083-93, 2010 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-20847314

RESUMEN

RATIONALE: The mobilization of bone marrow (BM) progenitor cells (PCs) is largely governed by interactions between stromal cell-derived factor (SDF)-1 and CXC chemokine receptor (CXCR)4. Ischemic injury disrupts the SDF-1-CXCR4 interaction and releases BM PCs into the peripheral circulation, where the mobilized cells are recruited to the injured tissue and contribute to vessel growth. BM PCs can also be mobilized by the pharmacological CXCR4 antagonist AMD3100, but the other components of the SDF-1-CXCR4 signaling pathway are largely unknown. c-kit, a membrane-bound tyrosine kinase and the receptor for stem cell factor, has also been shown to play a critical role in BM PC mobilization and ischemic tissue repair. OBJECTIVE: To investigate the functional interaction between SDF-1-CXCR4 signaling and c-kit activity in BM PC mobilization. METHODS AND RESULTS: AMD3100 administration failed to mobilize BM PCs in mice defective in c-kit kinase activity or in mice transplanted with BM cells that expressed a constitutively active c-kit mutant. Furthermore, BM levels of phosphorylated (phospho)-c-kit declined after AMD3100 administration and after CXCR4 deletion. In cells adhering to culture plates coated with vascular cell adhesion molecule 1, SDF-1 and stem cell factor increased phospho-c-kit levels, and AMD3100 treatment suppressed SDF-1-induced, but not SCF-induced, c-kit phosphorylation. SDF-1-induced c-kit phosphorylation also required the activation of Src nonreceptor tyrosine kinase: pretreatment of cells with a selective Src inhibitor blocked both c-kit phosphorylation and the interaction between c-kit and phospho-Src. CONCLUSIONS: These findings indicate that the regulation of BM PC trafficking by SDF-1 and CXCR4 is dependent on Src-mediated c-kit phosphorylation.


Asunto(s)
Células de la Médula Ósea/fisiología , Movimiento Celular/fisiología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptores CXCR4/fisiología , Células Madre/fisiología , Animales , Bencilaminas , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/enzimología , Línea Celular , Movimiento Celular/genética , Quimiocina CXCL12/fisiología , Ciclamas , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Compuestos Heterocíclicos/farmacología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-kit/deficiencia , Proteínas Proto-Oncogénicas c-kit/genética , Receptores CXCR4/deficiencia , Receptores CXCR4/genética , Células Madre/efectos de los fármacos , Células Madre/enzimología , Familia-src Quinasas/fisiología
12.
Circ Res ; 104(10): 1209-16, 2009 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-19407239

RESUMEN

Myocardial infarction rapidly depletes the endogenous cardiac progenitor cell pool, and the inefficient recruitment of exogenously administered progenitor cells limits the effectiveness of cardiac cell therapy. Recent reports indicate that interactions between the CXC chemokine stromal cell-derived factor 1 and its receptor CXC chemokine receptor 4 (CXCR4) critically mediate the ischemia-induced recruitment of bone marrow-derived circulating stem/progenitor cells, but the expression of CXCR4 in cardiac progenitor cells is very low. Here, we studied the influence of hypoxia on CXCR4 expression in cardiac progenitor cells, on the recruitment of intravenously administered cells to ischemic heart tissue, and on the preservation of heart function in a murine myocardial infarction model. We found that hypoxic preconditioning increased CXCR4 expression in CLK (cardiosphere-derived, Lin(-)c-kit(+) progenitor) cells and markedly augmented CLK cell migration (in vitro) and recruitment (in vivo) to the ischemic myocardium. Four weeks after surgically induced myocardial infarction, infarct size and heart function were significantly better in mice administered hypoxia-preconditioned CLK cells than in mice treated with cells cultured under normoxic conditions. Furthermore, these effects were largely abolished by the addition of a CXCR4 inhibitor, indicating that the benefits of hypoxic preconditioning are mediated by the stromal cell-derived factor 1/CXCR4 axis, and that therapies targeting this axis may enhance cardiac-progenitor cell-based regenerative therapy.


Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Hipoxia/metabolismo , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Infarto del Miocardio/metabolismo , Infarto del Miocardio/terapia , Receptores CXCR4/metabolismo , Animales , Movimiento Celular/fisiología , Células Cultivadas , Quimiocina CXCL12/metabolismo , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/patología , Miocardio/metabolismo , Miocardio/patología , Fenotipo , Transducción de Señal/fisiología
13.
Circulation ; 120(13): 1213-21, 2009 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-19752322

RESUMEN

BACKGROUND: Recent studies have identified a polymorphism in the endothelin-converting enzyme (ECE)-1b promoter (-338C/A) that is strongly associated with hypertension in women. The polymorphism is located in a consensus binding sequence for the E2F family of transcription factors. E2F proteins are crucially involved in cell-cycle regulation, but their roles in cardiovascular function are poorly understood. Here, we investigated the potential role of E2F2 in blood pressure regulation. METHODS AND RESULTS: Tail-cuff measurements of systolic and diastolic blood pressures were significantly higher in E2F2-null (E2F2(-/-)) mice than in their wild-type littermates, and in ex vivo ring assays, aortas from the E2F2(-/-) mice exhibited significantly greater contractility in response to big endothelin-1. Big endothelin-1 is activated by ECE-1, and mRNA levels of ECE-1b, the repressive ECE-1 isoform, were significantly lower in E2F2(-/-) mice than in wild-type mice. In endothelial cells, chromatin immunoprecipitation assays confirmed that E2F2 binds the ECE-1b promoter, and promoter-reporter assays indicated that E2F2 activates ECE-1b transcription. Furthermore, loss or downregulation of E2F2 led to a decline in ECE-1b levels, to higher levels of the membranous ECE-1 isoforms (ie, ECE-1a, -1c, and -1d), and to deregulated ECE-1 activity. Finally, Sam68 coimmunoprecipitated with E2F2, occupied the ECE-1b promoter (chromatin immunoprecipitation), and repressed E2F2-mediated ECE-1b promoter activity (promoter-reporter assays). CONCLUSIONS: Our results identify a cell-cycle-independent mechanism by which E2F2 regulates endothelial function, arterial contractility, and blood pressure.


Asunto(s)
Presión Sanguínea/fisiología , Factor de Transcripción E2F2/genética , Factor de Transcripción E2F2/metabolismo , Activación Transcripcional/fisiología , Vasoconstricción/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Aorta/fisiología , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Ciclo Celular/fisiología , Membrana Celular/metabolismo , Regulación hacia Abajo/fisiología , Endotelina-1/farmacología , Enzimas Convertidoras de Endotelina , Femenino , Masculino , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Ratones , Ratones Mutantes , Regiones Promotoras Genéticas/fisiología , Proteínas de Unión al ARN/metabolismo , Vasoconstricción/efectos de los fármacos
14.
Zhonghua Xin Xue Guan Bing Za Zhi ; 37(8): 692-5, 2009 Aug.
Artículo en Zh | MEDLINE | ID: mdl-20021919

RESUMEN

OBJECTIVE: To observe the effect of intracoronary transfer of autologous HO-1 overexpressed MSCs in porcine model of myocardial ischemia (1 h)/reperfusion. METHODS: Apoptosis was assayed and cytokine concentrations in supernatant were measured in cells exposed to hypoxia-reoxygen in vitro. In vivo, Chinese male mini-pigs were allocated to the following treatment groups: control group (saline), MSCs group (MSCs), MSCs transfected with pcDNA3.1-nHO-1 (HO-1-MSCs). 1 x 10(7) of autologous stem cells or identical volume of saline was injected intracoronary into porcine hearts 1 h after ischemia. MRI assay and postmortem analysis were assessed 3 months after stem cell transplantation. RESULTS: In vitro, cell apoptosis rate post hypoxia-reoxygen was significantly reduced in HO-1-MSCs group (30.30% +/- 7.64%) compared with that in MSCs group (56.93% +/- 4.68%, P < 0.001) and LacZ-MSCs group (55.88% +/- 4.38%, P < 0.001), VEGF was also significantly upregulated in HO-1-MSCs group [(768.44 +/- 78.38) pg/ml] compared with that in MSCs group [(555.27 +/- 67.67) pg/ml, P < 0.001] and LacZ-MSCs group [(522.97 +/- 71.45) pg/ml, P < 0.001]. In vivo, cardiac function was significantly improved in both MSCs transplantation groups compared to saline group (all P < 0.05 vs.saline) and the left ventricular ejection fraction was significantly higher in HO-1-MSCs group compared with that in MSCs group at 3 months after transplantation (53.50% +/- 2.09% vs. 49.54% +/- 2.74%, P = 0.017), capillary density in the peri-infarct area was also significantly higher in HO-1-MSC group than that in MSCs group [(14.59 +/- 2.39)/HPF vs. (11.78 +/- 2.48)/HPF, P = 0.033]. CONCLUSIONS: Efficacy of HO-1 overexpressed MSCs on improving cardiac function and promoting angiogenesis was greater than those by MSCs in this porcine ischemia/reperfusion model.


Asunto(s)
Hemo-Oxigenasa 1/genética , Trasplante de Células Madre Mesenquimatosas , Infarto del Miocardio/terapia , Transfección , Animales , Apoptosis , Células Cultivadas , Vectores Genéticos , Masculino , Isquemia Miocárdica/terapia , Porcinos , Porcinos Enanos
15.
Adv Drug Deliv Rev ; 60(2): 160-72, 2008 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-18031863

RESUMEN

Gene modification of cells prior to their transplantation, especially stem cells, enhances their survival and increases their function in cell therapy. Like the Trojan horse, the gene-modified cell has to gain entrance inside the host's walls and survive and deliver its transgene products. Using cellular, molecular and gene manipulation techniques the transplanted cell can be protected in a hostile environment from immune rejection, inflammation, hypoxia and apoptosis. Genetic engineering to modify cells involves constructing modules of functional gene sequences. They can be simple reporter genes or complex cassettes with gene switches, cell specific promoters and multiple transgenes. We discuss methods to deliver and construct gene cassettes with viral and non-viral delivery, siRNA, and conditional Cre/Lox P. We review the current uses of gene-modified stem cells in cardiovascular disease, diabetes, neurological diseases, (including Parkinson's, Alzheimer's and spinal cord injury repair), bone defects, hemophilia, and cancer.


Asunto(s)
Técnicas de Transferencia de Gen , Trasplante de Células Madre/métodos , Células Madre/metabolismo , Animales , Diabetes Mellitus Tipo 1/terapia , Fracturas Óseas/terapia , Terapia Genética/métodos , Cardiopatías/terapia , Humanos , Neoplasias/terapia , Células Madre/citología
16.
Chin Med J (Engl) ; 121(6): 544-50, 2008 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-18364144

RESUMEN

BACKGROUND: Mesenchymal stem cells (MSCs) transplantation provides a new approach for myocardial repair. However, many important fundamental questions about MSCs transplantation remain unanswered. There is an urgent need to identify MSCs from the beating heart and analyze the efficacy of this new approach. This study aimed to localize the magnetically labeled MSCs (MR-MSCs) and monitor the restorative effects of MR-MSCs with magnetic resonance (MR) imaging. METHODS: Acute myocardial infarction (AMI) was created in swine by a balloon occlusion of the left anterior descending coronary artery. Cells were delivered via intracoronary infusion after myocardial infarction. Infarct size change and cardiac function were assessed with 3.0T MR scanner. The results were then confirmed by histological and western blot analysis. All statistical procedures were performed with Systat (SPSS version 12.01). RESULTS: A total of 26 swine were divided into four groups (sham-operated group, n=6; AMI group with PBS transplantation, n=6; labeled MSCs group, n=7; unlabeled MSCs group, n=7). MSCs, MR-MSCs (10(7) cells) or PBS were delivered by intracoronary injection after MI and serial cardiac MR imaging studies were performed at 0, 4 and 8 weeks after transplantation. MR imaging demonstrated MI size decreased after MSCs transplantation in labeled and unlabeled groups, however, increases were seen in the AMI group at 8 weeks after MI. The left ventricular ejection fraction (LVEF) was slightly increased in the AMI group ((41.87+/-2.45)% vs (39.04+/-2.80)%, P>0.05), but significantly improved in the MR-MSCs group ((56.85+/-1.29)% vs (40.67+/-2.00)%, P<0.05) and unlabeled group ((55.38+/-1.07)% vs (41.78+/-2.08)%, P<0.05) at 8 weeks after treatment. MR-MSCs were further confirmed by Prussian blue and immunofluorescent staining. Western blot analysis demonstrated that there was an increased expression of cardiomyocyte markers such as myosin heavy chain and troponin T in the MSCs treatment groups and the ratio of matrix metalloproteinase 2 to tissue inhibitor of metalloproteinase 1 decreased in the labeled group and unlabeled group compared with the AMI group and sham-operated group. CONCLUSION: Transplanted MR-MSCs can regenerate new myocardium and prevent remolding in an MI model at 2-month follow-up and represent a preferred method to better understand the mechanisms of stem cell therapy in future clinical studies.


Asunto(s)
Magnetismo , Trasplante de Células Madre Mesenquimatosas , Infarto del Miocardio/terapia , Animales , Western Blotting , Supervivencia Celular , Modelos Animales de Enfermedad , Imagen por Resonancia Magnética , Infarto del Miocardio/fisiopatología , Porcinos , Función Ventricular Izquierda
17.
Mol Aspects Med ; 60: 123-128, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-28965750

RESUMEN

Research into the biology of extracellular vesicles (EVs), including exosomes and microvesicles, has expanded significantly with advances in EV isolation techniques, a better understanding of the surface markers that characterize exosomes and microvesicles, and greater information derived from -omics approaches on the proteins, lipids, mRNAs, and microRNAs (miRNAs) transported by EVs. We have recently discovered a role for exosome-derived miRNAs in age-related bone loss and osteoarthritis, two conditions that impose a significant public health burden on the aging global population. Previous work has also revealed multiple roles for EVs and their miRNAs in muscle regeneration and congenital myopathies. Thus, EVs appear to be involved in a number of degenerative conditions that impact the musculoskeletal system, indicating that the musculoskeletal system is an excellent model for investigating the role of EVs in tissue maintenance and repair. This review highlights the role of EVs in bone, skeletal muscle, and joint health, including both normal tissue metabolism as well as tissue injury repair and regeneration. A consistent theme that emerges from study of musculoskeletal EVs is that various miRNAs appear to mediate a number of key pathological processes. These findings point to a potential therapeutic opportunity to target EV-derived miRNAs as a strategy for improving musculoskeletal function.


Asunto(s)
Vesículas Extracelulares/metabolismo , Enfermedades Musculoesqueléticas/metabolismo , Animales , Biomarcadores , Huesos/metabolismo , Diferenciación Celular/genética , Susceptibilidad a Enfermedades , Humanos , Artropatías/etiología , Artropatías/metabolismo , Artropatías/patología , Células Musculares/citología , Células Musculares/metabolismo , Osteoporosis/metabolismo , Regeneración/genética
18.
J Hypertens ; 25(1): 197-205, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17143192

RESUMEN

OBJECTIVES: Beta-blockers are widely used and effective for treating hypertension, acute myocardial infarction (MI) and heart failure, but they present side-effects mainly due to antagonism of beta2-adrenergic receptor (AR). Currently available beta-blockers are at best selective but not specific for beta1 or beta2-AR. METHODS: To specifically inhibit the expression of the beta1-AR, we developed a small interfering RNA (siRNA) targeted to beta1-AR. Three different sequences of beta1 siRNA were delivered into C6-2B cells with 90% efficiency. RESULTS: One of the three sequences reduced the level of beta1-AR mRNA by 70%. The siRNA was highly specific for beta1-AR inhibition with no overlap with beta2-AR. To test this in vivo, systemic injection of beta1 siRNA complexed with liposomes resulted in efficient delivery into the heart, lung, kidney and liver, and effectively reduced beta1-AR expression in the heart without altering beta2-AR. beta1 siRNA significantly lowered blood pressure of spontaneously hypertensive rats (SHR) for at least 12 days and reduced cardiac hypertrophy following a single injection. Pretreatment with beta1 siRNA 3 days before induction of MI in Wistar rats significantly improved cardiac function, as demonstrated by dP/dt and electrocardiogram following the MI. The protective mechanism involved reduction of cardiomyocyte apoptosis in the beta1 siRNA-treated hearts. CONCLUSIONS: The present study demonstrates the possibility of using siRNA for treating cardiovascular diseases and may represent a novel beta-blocker specific for beta1-AR.


Asunto(s)
Antihipertensivos/metabolismo , Presión Sanguínea , Hipertensión/metabolismo , Isquemia Miocárdica/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Animales , Antihipertensivos/uso terapéutico , Apoptosis , Línea Celular Tumoral , Modelos Animales de Enfermedad , Ventrículos Cardíacos/patología , Hipertensión/genética , Hipertensión/patología , Hipertensión/fisiopatología , Masculino , Ratones , Isquemia Miocárdica/genética , Isquemia Miocárdica/patología , Isquemia Miocárdica/fisiopatología , Miocardio/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéutico , Ratas , Ratas Endogámicas SHR , Ratas Wistar , Receptores Adrenérgicos beta 1/genética , Factores de Tiempo , Transfección , Función Ventricular Izquierda
19.
Regul Pept ; 125(1-3): 1-8, 2005 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-15582707

RESUMEN

A concentration gradient of stromal-cell-derived factor-1alpha (SDF-1alpha) is the major mechanism for homing of haematopoietic stem cells (HSCs) in bone marrow. We tested the hypothesis that a gene therapy using SDF-1alpha can enhance HSCs recruiting to the heart upon myocardial infarction (MI). Adult mice with surgically induced myocardial ischemia were injected intramyocardially with either saline (n=12) or SDF-1alpha plasmid (n=12) in 50 microl volume in the ischemic border zone of the infarcted heart 2 weeks after myocardial infarction. Donor Lin-c-kit+ HSCs from isogenic BalB/c mice were harvested, sorted through magnetic cell sorting (MACS) and labeled with PKH26 Red. Three days after plasmid or saline injection, 1x10(5) labeled cells were injected intravenously (i.v.) into saline mice (n=4) and SDF-1alpha plasmid mice (n=4). The hearts and other tissue were removed for Western blot assay 2 weeks after plasmid or saline treatment. The labeled Lin-c-kit+ cells were identified with immunofluoresent staining and endogenous c-kit+ cells were identified by immunohistochemical staining. In mice killed at 1 month postinfarct, Western blot showed higher levels of SDF-1alpha expression in SDF-1alpha-treated mouse ischemic hearts compared to saline-treated hearts and other tissues. In the SDF-1alpha plasmid-treated hearts, SDF-1alpha is overexpressed in the periinfarct zone. The labeled stem cells engrafted to the SDF-1alpha positive site in the myocardium. There was also evidence for endogenous stem cell recruiting. The density of c-kit+ cells in border zone, an index of endogenous stem cell mobilization, was significantly higher in the SDF-1alpha-treated group than in the saline group (14.63+/-1.068 cells/hpf vs. 11.31+/-0.65 cells/hpf, P=0.013) at 2 weeks after SDF-1alpha or saline treatment. Following myocardial infarction, treatment with SDF-1alpha recruits stem cells to damaged heart where they may have a role in repairing and regeneration. The gene therapy with an SDF-1alpha vector offers a promising therapeutic strategy for mobilizing stem cells to the ischemic myocardium.


Asunto(s)
Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Terapia Genética/métodos , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/citología , Isquemia , Miocardio/patología , Animales , Western Blotting , Células de la Médula Ósea/citología , Separación Celular , Células Cultivadas , Quimiocina CXCL12 , Humanos , Inmunohistoquímica , Isquemia/patología , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Plásmidos/metabolismo , Proteínas Proto-Oncogénicas c-kit/biosíntesis , Células Madre/citología , Factores de Tiempo
20.
J Cardiovasc Pharmacol Ther ; 10(4): 251-63, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16382261

RESUMEN

OBJECTIVES: The effect of a cardiac specific, hypoxia-regulated, human heme oxygenase-1 (hHO-1) vector to provide cardioprotection from ischemia-reperfusion injury was assessed. BACKGROUND: When myocardial ischemia and reperfusion is asymptomatic, the damaging effects are cumulative and patients miss timely treatment. A gene therapy approach that expresses therapeutic genes only when ischemia is experienced is a desirable strategy. We have developed a cardiac-specific, hypoxia-regulated gene therapy "vigilant vector'' system that amplifies cardioprotective gene expression. METHODS: Vigilant hHO-1 plasmids, LacZ plasmids, or saline (n = 40 per group) were injected into mouse heart 2 days in advance of ischemia-reperfusion injury. Animals were exposed to 60 minutes of ischemia followed by 24 hours of reperfusion. For that term (24 hours) effects, the protein levels of HO-1, inflammatory responses, apoptosis, and infarct size were determined. For long-term (3 week) effects, the left ventricular remodeling and recovery of cardiac function were assessed. RESULTS: Ischemia-reperfusion resulted in a timely overexpression of HO-1 protein. Infarct size at 24 hours after ischemia-reperfusion was significantly reduced in the HO-1-treated animals compared with the LacZ-treated group or saline-treated group (P < .001). The reduction of infarct size was accompanied by a decrease in lipid peroxidant activity, inflammatory cell infiltration, and proapoptotic protein level in ischemia-reperfusion-injured myocardium. The long-term study demonstrated that timely, hypoxia-induced HO-1 overexpression is beneficial in conserving cardiac function and attenuating left ventricle remodelling. CONCLUSIONS: The vigilant HO-1 vector provides a protective therapy in the heart for reducing cellular damage during ischemia-reperfusion injury and preserving heart function.


Asunto(s)
Terapia Genética , Hemo-Oxigenasa 1/biosíntesis , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/metabolismo , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Vectores Genéticos , Hemo-Oxigenasa 1/genética , Humanos , Hipoxia , Peroxidación de Lípido , Masculino , Ratones , Ratones Endogámicos BALB C , Daño por Reperfusión Miocárdica/terapia , Miocardio/enzimología , Plásmidos , Remodelación Ventricular
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