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AIMS: Proprotein convertase subtilisin/kexin type-9 (PCSK9), a molecular determinant of low-density lipoprotein (LDL) receptor (LDLR) fate, has emerged as a promising therapeutic target for atherosclerotic cardiovascular diseases. However, the precise mechanism by which PCSK9 regulates the internalization and lysosomal degradation of LDLR is unknown. Recently, we identified adenylyl cyclase-associated protein 1 (CAP1) as a receptor for human resistin whose globular C-terminus is structurally similar to the C-terminal cysteine-rich domain (CRD) of PCSK9. Herein, we investigated the role of CAP1 in PCSK9-mediated lysosomal degradation of LDLR and plasma LDL cholesterol (LDL-C) levels. METHODS AND RESULTS: The direct binding between PCSK9 and CAP1 was confirmed by immunoprecipitation assay, far-western blot, biomolecular fluorescence complementation, and surface plasmon resonance assay. Fine mapping revealed that the CRD of PCSK9 binds with the Src homology 3 binding domain (SH3BD) of CAP1. Two loss-of-function polymorphisms found in human PCSK9 (S668R and G670E in CRD) were attributed to a defective interaction with CAP1. siRNA against CAP1 reduced the PCSK9-mediated degradation of LDLR in vitro. We generated CAP1 knock-out mice and found that the viable heterozygous CAP1 knock-out mice had higher protein levels of LDLR and lower LDL-C levels in the liver and plasma, respectively, than the control mice. Mechanistic analysis revealed that PCSK9-induced endocytosis and lysosomal degradation of LDLR were mediated by caveolin but not by clathrin, and they were dependent on binding between CAP1 and caveolin-1. CONCLUSION: We identified CAP1 as a new binding partner of PCSK9 and a key mediator of caveolae-dependent endocytosis and lysosomal degradation of LDLR.
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Aterosclerose/genética , Proteínas de Transporte/genética , LDL-Colesterol/sangue , Mutação , Pró-Proteína Convertase 9/genética , Receptores de LDL/sangue , Animais , Aterosclerose/metabolismo , Proteínas de Transporte/metabolismo , DNA/genética , Análise Mutacional de DNA , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Pró-Proteína Convertase 9/metabolismoRESUMO
Induced pluripotent stem cells (iPSCs) have opened up unprecedented opportunities for novel therapeutic options for precision medicine. Hematopoietic stem cell (HSC) donor pools with previously determined HLA types may be ideal sources for iPSC production. Based on the HLA distribution of cryopreserved cord blood units (CBUs) and registered bone marrow (BM) donors, we estimated how much of the Korean population could be covered by HLA-homozygous iPSCs. We analyzed a total of 143,866 Korean HSC donors (27,904 CBUs and 115,962 BM donors). Each donor sample was typed for the HLA-A, -B, and -DRB1 alleles at low to intermediate resolution by DNA-based molecular techniques: PCR sequence-specific oligonucleotide (PCR-SSOP), PCR with sequence-specific primers (PCR-SSP) and PCR with sequence-based typing (PCR-SBT). We also identified individuals possessing homozygous HLA haplotypes by direct counting. The matching probabilities for zero-mismatch transplantation were calculated for 143,866 Koreans and 50 million potential Korean patients. Among the HSC donor pool, 17 HLA-A alleles, 41 HLA-B alleles, and 13 HLA-DRB1 alleles, as well as 128 homozygous HLA-A-B-DRB1 haplotypes, were identified at serologic equivalents, and those haplotypes cumulatively matched 93.20% of the 143,866 Korean donors as zero HLA-mismatch iPSC sources. Among the combinations of 2,056 haplotypes with frequencies ≥ 0.001% in a population of 50 million, those 128 homozygous haplotypes can provide 93.65% coverage for potential Korean recipients. Haplobanking of a reasonable number of HLA-A, -B, and -DRB1 homozygous iPSC lines derived from CBUs and cells of registered BM donors may be an efficient option for allogenic iPSC therapy.
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Células-Tronco Pluripotentes Induzidas , Alelos , Medula Óssea , Sangue Fetal , Cadeias HLA-DRB1/genética , Haplótipos , Células-Tronco Hematopoéticas , Teste de Histocompatibilidade , Humanos , Sistema de Registros , Doadores de TecidosAssuntos
Diferenciação Celular , Linhagem da Célula , Coração/embriologia , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Receptores de Peptídeos/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Organogênese , Células-Tronco Pluripotentes/efeitos dos fármacos , Receptores de Peptídeos/genética , Transdução de Sinais , Fatores de TempoRESUMO
BACKGROUND: Cell-based therapies to augment endothelial cells (ECs) hold great therapeutic promise. Here, we report a novel approach to generate functional ECs directly from adult fibroblasts. METHODS AND RESULTS: Eleven candidate genes that are key regulators of endothelial development were selected. Green fluorescent protein (GFP)-negative skin fibroblasts were prepared from Tie2-GFP mice and infected with lentiviruses allowing simultaneous overexpression of all 11 factors. Tie2-GFP(+) cells (0.9%), representing Tie2 gene activation, were detected by flow cytometry. Serial stepwise screening revealed 5 key factors (Foxo1, Er71, Klf2, Tal1, and Lmo2) that were required for efficient reprogramming of skin fibroblasts into Tie2-GFP(+) cells (4%). This reprogramming strategy did not involve pluripotency induction because neither Oct4 nor Nanog was expressed after 5 key factor transduction. Tie2-GFP(+) cells were isolated using fluorescence-activated cell sorting and designated as induced ECs (iECs). iECs exhibited endothelium-like cobblestone morphology and expressed EC molecular markers. iECs possessed endothelial functions such as Bandeiraea simplicifolia-1 lectin binding, acetylated low-density lipoprotein uptake, capillary formation on Matrigel, and nitric oxide production. The epigenetic profile of iECs was similar to that of authentic ECs because the promoters of VE-cadherin and Tie2 genes were demethylated. mRNA profiling showed clustering of iECs with authentic ECs and highly enriched endothelial genes in iECs. In a murine model of hind-limb ischemia, iEC implantation increased capillary density and enhanced limb perfusion, demonstrating the in vivo viability and functionality of iECs. CONCLUSIONS: We demonstrated the first direct conversion of adult fibroblasts to functional ECs. These results suggest a novel therapeutic modality for cell therapy in ischemic vascular disease.
Assuntos
Células Endoteliais/citologia , Fibroblastos/citologia , Terapia Genética/métodos , Isquemia/terapia , Doenças Vasculares/terapia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores Etários , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Fibroblastos/fisiologia , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Proteínas de Fluorescência Verde/genética , Membro Posterior/irrigação sanguínea , Isquemia/patologia , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Nus , Camundongos Transgênicos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Pele/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Doenças Vasculares/patologiaRESUMO
AIMS: The roles of peroxisome proliferator-activated receptor (PPAR)-δ in vascular biology are mainly unknown. We investigated the effects of PPAR-δ activation on the paracrine networks between endothelial progenitor cells (EPCs) and endothelial cells (ECs)/skeletal muscle. METHODS AND RESULTS: Treatment of EPCs with GW501516, a PPAR-δ agonist, induced specifically matrix metallo-proteinase (MMP)-9 by direct transcriptional activation. Subsequently, this increased-MMP-9 broke down insulin-like growth factor-binding protein (IGFBP)-3, resulting in IGF-1 receptor (IGF-1R) activation in surrounding target cells. Treatment of conditioned medium from GW501516-stimulated EPCs enhanced the number and functions of human umbilical vein ECs and C2C12 myoblasts via MMP-9-mediated IGF-1R activation. Systemic administration of GW501516 in mice increased MMP-9 expression in EPCs, and augmented IGFBP-3 degradation in serum. In a mouse hindlimb ischaemia model, systemic treatment of GW501516 or local transplantation of GW501516-treated EPCs induced IGF-1R phosphorylation in ECs and skeletal muscle in the ischaemic limbs, leading to augmented angiogenesis and skeletal muscle regeneration. It also enhanced wound healing with increased angiogenesis in a mouse skin punch wound model. These pro-angiogenic and muscle-regenerating effects were abolished by MMP-9 knock-out. CONCLUSION: Our results suggest that PPAR-δ is a crucial modulator of angio-myogenesis via the paracrine effects of EPCs, and its agonist is a good candidate as a therapeutic drug for patients with peripheral vascular diseases.
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Células Endoteliais/citologia , Fator de Crescimento Insulin-Like I/metabolismo , Metaloproteinase 9 da Matriz/biossíntese , PPAR delta/fisiologia , Células-Tronco/citologia , Análise de Variância , Animais , Proliferação de Células , Células Endoteliais/metabolismo , Xenoenxertos , Membro Posterior/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Isquemia/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Camundongos Nus , Monócitos/citologia , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citologia , Neovascularização Fisiológica/fisiologia , PPAR delta/agonistas , Fosforilação , Receptor IGF Tipo 1/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Transplante de Células-Tronco , Células-Tronco/metabolismo , Tiazóis/farmacologiaRESUMO
Resistin plays an important role in the pathophysiology of obesity-mediated insulin resistance in mice. However, the biology of resistin in humans is quite different from that in rodents. Therefore, the association between resistin and insulin resistance remains unclear in humans. Here, we tested whether and how the endocannabinoid system (ECS) control circulating peripheral blood mononuclear cells (PBMCs) that produce resistin and infiltrate into the adipose tissue, heart, skeletal muscle, and liver, resulting in inflammation and insulin resistance. Using human PBMCs, we investigate whether the ECS is connected to human resistin. To test whether the ECS regulates inflammation and insulin resistance in vivo, we used 2 animal models such as "humanized" nonobese diabetic/Shi-severe combined immunodeficient interleukin-2Rγ (null) (NOG) mice and "humanized" resistin mouse models, which mimic human body. In human atheromatous plaques, cannabinoid 1 receptor (CB1R)-positive macrophage was colocalized with the resistin expression. In addition, resistin was exclusively expressed in the sorted CB1R-positive cells from human PBMCs. In CB1R-positive cells, endocannabinoid ligands induced resistin expression via the p38-Sp1 pathway. In both mouse models, a high-fat diet increased the accumulation of endocannabinoid ligands in adipose tissue, which recruited the CB1R-positive cells that secrete resistin, leading to adipose tissue inflammation and insulin resistance. This phenomenon was suppressed by CB1R blockade or in resistin knockout mice. Interestingly, this process was accompanied by mitochondrial change that was induced by resistin treatment. These results provide important insights into the ECS-resistin axis, leading to the development of metabolic diseases. Therefore, the regulation of resistin via the CB1R could be a potential therapeutic strategy for cardiometabolic diseases.
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BACKGROUND: Edge restenosis is not an unusual finding after implantation of drug-eluting stents (DES). We hypothesized that mechanical stress imposed on the stent edge would cause vessel wall injury and inflammation, which may consequently lead to edge restenosis. METHODS AND RESULTS: In total, 1,496 patients were implanted with a sirolimus-eluting stent (SES), paclitaxel-eluting stent (PES) or zotarolimus-eluting stent (ZES) in Seoul National University Hospital between 2007 and 2009. Binary restenosis occurred in 161 lesions in 119 patients. We retrospectively compared the 3 DES with regard to the percentage of edge stent restenosis among all cases of restenosis. We also evaluated the maximal, minimal, and Δ (maximal angle-minimal angle) angles. The percentage of edge restenosis was higher for SES than for ZES (37.5% vs. 16.7%, P=0.017). Maximal angle at the proximal edge was 64.82°±33.46° for 26 stents with proximal edge restenosis compared with 31.84°±31.51° for 89 stents without proximal edge restenosis (P=0.001). The Δ angle was also significantly different between the 2 groups (14.81°±15.98° vs. 7.60°±8.86°, P=0.035). Similar findings were observed for distal edge restenosis. Both the maximal angle (39.09°±21.04° vs. 22.71°±22.83°, P=0.010) and Δ angle (20.23°±15.39° vs. 9.18°±9.66°, P=0.016) at the distal edge were significantly different between the 2 groups. CONCLUSIONS: Physical stress determined by angulation at the stent edge segment and biomechanical properties of the DES can be considered as one of the plausible mechanisms for edge stent restenosis.
Assuntos
Stents Farmacológicos/efeitos adversos , Oclusão de Enxerto Vascular/patologia , Oclusão de Enxerto Vascular/fisiopatologia , Idoso , Feminino , Seguimentos , Oclusão de Enxerto Vascular/metabolismo , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Loss of cardiomyocytes impairs cardiac function after myocardial infarction (MI). Recent studies suggest that cardiac stem/progenitor cells could repair the damaged heart. However, cardiac progenitor cells are difficult to maintain in terms of purity and multipotency when propagated in two-dimensional culture systems. Here, we investigated a new strategy that enhances potency and enriches progenitor cells. We applied the repeated sphere formation strategy (cardiac explant â primary cardiosphere (CS) formation â sphere-derived cells (SDCs) in adherent culture condition â secondary CS formation by three-dimensional culture). Cells in secondary CS showed higher differentiation potentials than SDCs. When transplanted into the infarcted myocardium, secondary CSs engrafted robustly, improved left ventricular (LV) dysfunction, and reduced infarct sizes more than SDCs did. In addition to the cardiovascular differentiation of transplanted secondary CSs, robust vascular endothelial growth factor (VEGF) synthesis and secretion enhanced neovascularization in the infarcted myocardium. Microarray pathway analysis and blocking experiments using E-selectin knock-out hearts, specific chemicals, and small interfering RNAs (siRNAs) for each pathway revealed that E-selectin was indispensable to sphere initiation and ERK/Sp1/VEGF autoparacrine loop was responsible for sphere maturation. These results provide a simple strategy for enhancing cellular potency for cardiac repair. Furthermore, this strategy may be implemented to other types of stem/progenitor cell-based therapy.
Assuntos
Técnicas de Cultura de Células , Infarto do Miocárdio/terapia , Transplante de Células-Tronco , Células-Tronco/citologia , Disfunção Ventricular Esquerda/terapia , Animais , Diferenciação Celular , Selectina E/genética , Selectina E/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Expressão Gênica , Humanos , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Masculino , Camundongos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Neovascularização Fisiológica , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/genética , Transdução de Sinais , Células-Tronco/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/patologiaRESUMO
Telomerase activity, not detectable in somatic cells but frequently activated during carcinogenesis, confers immortality to tumors. Mechanisms governing expression of the catalytic subunit hTERT, the limiting factor for telomerase activity, still remain unclear. We previously proposed a model in which the binding of the transcription factor CTCF to the two first exons of hTERT results in transcriptional inhibition in normal cells. This inhibition is abrogated, however, by methylation of CTCF binding sites in 85% of tumors. Here, we showed that hTERT was unmethylated in testicular and ovarian tumors and in derivative cell lines. We demonstrated that CTCF and its paralogue, BORIS/CTCFL, were both present in the nucleus of the same cancer cells and bound to the first exon of hTERT in vivo. Moreover, exogenous BORIS expression in normal BORIS-negative cells was sufficient to activate hTERT transcription with an increasing number of cell passages. Thus, expression of BORIS was sufficient to allow hTERT transcription in normal cells and to counteract the inhibitory effect of CTCF in testicular and ovarian tumor cells. These results define an important contribution of BORIS to immortalization during tumorigenesis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/genética , Telomerase/genética , Neoplasias Testiculares/genética , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular , Metilação de DNA , Éxons , Feminino , Regulação Enzimológica da Expressão Gênica , Células HeLa , Humanos , Masculino , Neoplasias Ovarianas/metabolismo , Neoplasias Testiculares/metabolismoRESUMO
Recently, single cell RNA sequencing (scRNA-seq) technology has enabled the discovery of novel or rare subtypes of cells and their characteristics. This technique has advanced unprecedented biomedical research by enabling the profiling and analysis of the transcriptomes of single cells at high resolution and throughput. Thus, scRNA-seq has contributed to recent advances in cardiovascular research by the generation of cell atlases of heart and blood vessels and the elucidation of mechanisms involved in cardiovascular development and diseases. This review summarizes the overall workflow of the scRNA-seq technique itself and key findings in the cardiovascular development and diseases based on the previous studies. In particular, we focused on how the single-cell sequencing technology can be utilized in clinical field and precision medicine to treat specific diseases.
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Hematopoiesis is regulated by crosstalk between long-term repopulating hematopoietic stem cells (LT-HSCs) and supporting niche cells in the bone marrow (BM). Here, we describe the role of KAI1, which is mainly expressed on LT-HSCs and rarely on other hematopoietic stem-progenitor cells (HSPCs), in nichemediated LT-HSC maintenance. KAI1 activates TGF-ß1/Smad3 signal in LT-HSCs, leading to the induction of CDK inhibitors and inhibition of the cell cycle. The KAI1-binding partner DARC is expressed on macrophages and stabilizes KAI1 on LT-HSCs, promoting their quiescence. Conversely, when DARC+ BM macrophages were absent, the level of surface KAI1 on LT-HSCs decreases, leading to cell-cycle entry, proliferation, and differentiation. Thus, KAI1 acts as a functional surface marker of LTHSCs that regulates dormancy through interaction with DARCexpressing macrophages in the BM stem cell niche. Recently, we showed very special and rare macrophages expressing α-SMA+ COX2+ & DARC+ induce not only dormancy of LTHSC through interaction of KAI1-DARC but also protect HSCs by down-regulating ROS through COX2 signaling. In the near future, the strategy to combine KAI1-positive LT-HSCs and α-SMA/Cox2/DARC triple-positive macrophages will improve the efficacy of stem cell transplantation after the ablative chemo-therapy for hematological disorders including leukemia. [BMB Reports 2023; 56(9): 482-487].
Assuntos
Hematopoese , Células-Tronco Hematopoéticas , Ciclo-Oxigenase 2/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Diferenciação Celular/fisiologia , MacrófagosRESUMO
The concept of reprogramming of somatic cells has opened a new era in regenerative medicine. Transduction of defined factors has successfully achieved pluripotency. However, during the generation process of induced pluripotent stem (iPS) cells, genetic manipulation of certain factors may cause tumorigenicity, which limits further application. We report that that a single transfer of embryonic stem (ES) cell-derived proteins into primarily cultured adult mouse fibroblasts, rather than repeated transfer or prolonged exposure to materials, can achieve full reprogramming up to the pluripotent state without the forced expression of ectopic transgenes. During the process, gene expression and epigenetic status were converted from somatic to ES-equivalent status. We verified that protein-based reprogramming was neither by the contamination of protein donor ES cell nor by DNA/RNA from donor ES cell. Protein-iPS cells were biologically and functionally very similar to ES cells and differentiated into 3 germ layers in vitro. Furthermore, protein-iPS cells possessed in vivo differentiation (well-differentiated teratoma formation) and development (chimeric mice generation and a tetraploid blastocyst complementation) potentials. Our results provide an alternative and safe strategy for the reprogramming of somatic cells that can be used to facilitate pluripotent stem cell-based cell therapy.
Assuntos
Desdiferenciação Celular , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteínas/administração & dosagem , Fosfatase Alcalina/metabolismo , Animais , Desdiferenciação Celular/efeitos dos fármacos , Desdiferenciação Celular/fisiologia , Diferenciação Celular , Células Cultivadas , Metilação de DNA , Transferência Embrionária , Células-Tronco Embrionárias/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Células-Tronco Pluripotentes/efeitos dos fármacos , Gravidez , Proteínas/isolamento & purificação , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: The benefit of complete (CR) vs. incomplete revascularization (IR) with drug-eluting stent (DES), unlike with bypass grafting, is not well established in patients with multivessel coronary artery disease (MVD). METHODS AND RESULTS: Consecutive patients from a single center DES registry who were newly diagnosed as having MVD and who underwent successful percutaneous coronary intervention between March 2003 and December 2009 were traced for the occurrence of death, myocardial infarction (MI), and repeat revascularization. Among 845 patients (337 with CR and 508 with IR), propensity score-matched 275 pairs were followed for a median of 3.9 years. The adjusted hazard ratio (HR) of CR [95% confidence interval] was 0.66 [0.34-1.28] for death; 0.51 [0.28-0.95] for death and MI; 0.84 [0.60-1.19] for death, MI, and repeat revascularization. The observed benefit of CR was also cardiac-specific. The adjusted HR of CR for cardiac death and MI was 0.39 [0.16-0.96]. In 3 subgroups of patients with diabetes (n=191), ejection fraction <55% (n=153) and estimated glomerular filtration rate (eGFR) <60 ml/min (n=170), the benefit of CR was pronounced with the adjusted HR for cardiac death and MI of 0.27 [0.08-0.93], 0.18 [0.05-0.68] and 0.27 [0.07-0.99], respectively. CONCLUSIONS: In MVD patients treated with DES, CR was associated with the long-term benefit in reducing any or cardiac death and MI. The main beneficiaries of CR were those with diabetes, low ejection fraction and low eGFR.
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Angioplastia Coronária com Balão/instrumentação , Doença da Artéria Coronariana/terapia , Stents Farmacológicos , Idoso , Angioplastia Coronária com Balão/efeitos adversos , Angioplastia Coronária com Balão/mortalidade , Doença da Artéria Coronariana/mortalidade , Doença da Artéria Coronariana/fisiopatologia , Diabetes Mellitus/epidemiologia , Feminino , Taxa de Filtração Glomerular , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/mortalidade , Razão de Chances , Pontuação de Propensão , Modelos de Riscos Proporcionais , Desenho de Prótese , Sistema de Registros , República da Coreia , Medição de Risco , Fatores de Risco , Volume Sistólico , Fatores de Tempo , Resultado do Tratamento , Função Ventricular EsquerdaRESUMO
Duffy antigen receptor for chemokines (DARC)/CD234, also known as atypical chemokine receptor 1 (ACKR1), is a seven-transmembrane domain protein expressed on erythrocytes, vascular endothelium, and a subset of epithelial cells (Peiper et al., 1995). Previously, we reported that ACKR1 was expressed in bone marrow macrophages. ACKR1 interacts with CD82 on long-term repopulating hematopoietic stem cells (LT-HSCs) to maintain the dormancy of LT-HSCs during homeostasis (Hur et al., 2016). We also demonstrated that ACKR1 interacts with CD82 in HSCs from human umbilical cord blood (hUCB). These findings demonstrated that CD82 is a functional surface marker of LT-HSCs and this molecule maintains LT-HSC quiescence by interactions with ACKR1-expressing macrophages in mice and humans.
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Medula Óssea , Sistema do Grupo Sanguíneo Duffy , Monócitos , Animais , Camundongos , Sistema do Grupo Sanguíneo Duffy/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Macrófagos/metabolismo , Receptores de Quimiocinas/metabolismoRESUMO
The recent generation of induced pluripotent stem (iPS) cells represents a novel opportunity to complement embryonic stem (ES) cell-based approaches. iPS cells can be generated by viral transduction of specific transcription factors, but there is a potential risk of tumorigenicity by random retroviral integration. We have generated novel iPS (sFB-protein-iPS) cells from murine dermal fibroblasts (FVB-sFB) that have ES cell characteristics, using ES cell-derived cell extracts instead of performing viral transduction. Notably, only cell extracts from an ES cell line (C57-mES) on the C57/BL6 background generated iPS cells in our protocol-not an ES cell line (E14-mES) on the 129 background. Hypothesizing that determining the differences in these 2 mES cell lines will provide vital insight into the reprogramming machinery, we performed proteomic and global gene expression analysis by iTRAQ and mRNA microarray, respectively. We observed that pluripotent ES cells and ES cell extract-derived iPS cells had differential proteomes and global gene expression patterns. Notably, reprogramming-competent C57-mES cells highly expressed proteins that regulate protein synthesis and metabolism, compared with reprogramming-incompetent 129-mES cells, suggesting that there is a threshold that protein synthetic machinery must exceed to initiate reprogramming.
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Fibroblastos/química , Células-Tronco Pluripotentes Induzidas/química , Proteoma/análise , Animais , Biomarcadores/análise , Cromatografia Líquida/métodos , Análise por Conglomerados , Fibroblastos/citologia , Fibroblastos/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries/métodos , RNA Mensageiro/análise , Transdução de Sinais , Espectrometria de Massas em Tandem/métodosRESUMO
Although human induced pluripotent stem cells (iPSCs) can serve as a universal cell source for regenerative medicine, the use of iPSCs in clinical applications is limited by prohibitive costs and prolonged generation time. Moreover, allogeneic iPSC transplantation requires preclusion of mismatches between the donor and recipient human leukocyte antigen (HLA). We, therefore, generated universally compatible immune nonresponsive human iPSCs by gene editing. Transcription activator-like effector nucleases (TALENs) were designed for selective elimination of HLA DR expression. The engineered nucleases completely disrupted the expression of HLA DR on human dermal fibroblast cells (HDF) that did not express HLA DR even after stimulation with IFN-γ. Teratomas formed by HLA DR knockout iPSCs did not express HLA DR, and dendritic cells differentiated from HLA DR knockout iPSCs reduced CD4+ T cell activation. These engineered iPSCs might provide a novel translational approach to treat multiple recipients from a limited number of cell donors.
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BACKGROUND: The human skin-derived precursors (SKPs) are a good cell source for regeneration. However, the isolation of SKP from human skin is limited. To overcome this drawback, we hypothesized that the component of plant stem cells could convert human fibroblasts to SKPs. METHODS: Human dermal fibroblasts were treated with shikimic acid, a major component of Sequoiadendron giganteum callus extract. The characteristics of these reprogrammed cells were analyzed by qPCR, western blot, colony-forming assay, and immunofluorescence staining. Artificial human skin was used for CO2 laser-induced wound experiments. Human tissues were analyzed by immunohistochemistry. RESULTS: The reprogrammed cells expressed nestin (a neural precursor-specific protein), fibronectin, and vimentin and could differentiate into the ectodermal and mesodermal lineage. Nestin expression was induced by shikimic acid through the mannose receptor and subsequent MYD88 activation, leading to P38 phosphorylation and then CREB binding to the nestin gene promoter. Finally, we confirmed that shikimic acid facilitated the healing of cut injury and enhanced dermal reconstruction in a human artificial skin model. Moreover, in a clinical study with healthy volunteers, plant callus extracts increased the expression of stem cell markers in the basal layer of the epidermis and collagen deposit in the dermis. CONCLUSIONS: These results indicate that shikimic acid is an effective agent for tissue regeneration.
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Células-Tronco Multipotentes , Ácido Chiquímico , Diferenciação Celular , Células Cultivadas , Fibroblastos , Humanos , PeleRESUMO
BACKGROUND: Little is known about endogenous inhibitors of angiogenic growth factors. In this study, we identified a novel endogenous anti-angiogenic factor expressed in pericytes and clarified its underlying mechanism and clinical significance. METHODS: Herein, we found Kai1 knockout mice showed significantly enhanced angiogenesis. Then, we investigated the anti-angiogenic roll of Kai1 in vitro and in vivo. RESULTS: KAI1 was mainly expressed in pericytes rather than in endothelial cells. It localized at the membrane surface after palmitoylation by zDHHC4 enzyme and induced LIF through the Src/p53 pathway. LIF released from pericytes in turn suppressed angiogenic factors in endothelial cells as well as in pericytes themselves, leading to inhibition of angiogenesis. Interestingly, KAI1 had another mechanism to inhibit angiogenesis: It directly bound to VEGF and PDGF and inhibited activation of their receptors. In the two different in vivo cancer models, KAI1 supplementation significantly inhibited tumor angiogenesis and growth. A peptide derived from the large extracellular loop of KAI1 has been shown to have anti-angiogenic effects to block the progression of breast cancer and retinal neovascularization in vivo. CONCLUSIONS: KAI1 from PC is a novel molecular regulator that counterbalances the effect of angiogenic factors.
Assuntos
Proteína Kangai-1/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica , Animais , Feminino , Proteína Kangai-1/genética , Masculino , Microdomínios da Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Targeting the molecular pathways underlying the cardiotoxicity associated with thoracic irradiation and doxorubicin (Dox) could reduce the morbidity and mortality associated with these anticancer treatments. Here, we find that vascular endothelial cells (ECs) with persistent DNA damage induced by irradiation and Dox treatment exhibit a fibrotic phenotype (endothelial-mesenchymal transition, EndMT) correlating with the colocalization of L1CAM and persistent DNA damage foci. We demonstrate that treatment with the anti-L1CAM antibody Ab417 decreases L1CAM overexpression and nuclear translocation and persistent DNA damage foci. We show that in whole-heart-irradiated mice, EC-specific p53 deletion increases vascular fibrosis and the colocalization of L1CAM and DNA damage foci, while Ab417 attenuates these effects. We also demonstrate that Ab417 prevents cardiac dysfunction-related decrease in fractional shortening and prolongs survival after whole-heart irradiation or Dox treatment. We show that cardiomyopathy patient-derived cardiovascular ECs with persistent DNA damage show upregulated L1CAM and EndMT, indicating clinical applicability of Ab417. We conclude that controlling vascular DNA damage by inhibiting nuclear L1CAM translocation might effectively prevent anticancer therapy-associated cardiotoxicity.
Assuntos
Anticorpos Neutralizantes/farmacologia , Cardiomiopatias/prevenção & controle , Cardiotoxicidade/prevenção & controle , Doxorrubicina/toxicidade , Raios gama/efeitos adversos , Molécula L1 de Adesão de Célula Nervosa/genética , Animais , Antibióticos Antineoplásicos/toxicidade , Cardiomiopatias/etiologia , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiotoxicidade/etiologia , Cardiotoxicidade/genética , Cardiotoxicidade/metabolismo , Estudos de Casos e Controles , Técnicas de Cocultura , Dano ao DNA , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais/efeitos da radiação , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/efeitos da radiação , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. "Serial" chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the beta-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.