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Hypoxia modulates reparative angiogenesis, which is a tightly regulated pathophysiological process. MicroRNAs (miRNAs) are important regulators of gene expression in hypoxia and angiogenesis. However, we do not yet have a clear understanding of how hypoxia-induced miRNAs fine-tune vasoreparative processes. Here, we identify miR-130a as a mediator of the hypoxic response in human primary endothelial colony-forming cells (ECFCs), a well-characterized subtype of endothelial progenitors. Under hypoxic conditions of 1% O2, miR-130a gain-of-function enhances ECFC pro-angiogenic capacity in vitro and potentiates their vasoreparative properties in vivo. Mechanistically, miR-130a orchestrates upregulation of VEGFR2, activation of STAT3, and accumulation of HIF1α via translational inhibition of Ddx6. These findings unveil a new role for miR-130a in hypoxia, whereby it activates the VEGFR2/STAT3/HIF1α axis to enhance the vasoregenerative capacity of ECFCs.
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AIMS: Cord blood-derived endothelial colony-forming cells (CB-ECFCs) are a defined progenitor population with established roles in vascular homeostasis and angiogenesis, which possess low immunogenicity and high potential for allogeneic therapy and are highly sensitive to regulation by reactive oxygen species (ROS). The aim of this study was to define the precise role of the major ROS-producing enzyme, NOX4 NADPH oxidase, in CB-ECFC vasoreparative function. METHODS AND RESULTS: In vitro CB-ECFC migration (scratch-wound assay) and tubulogenesis (tube length, branch number) was enhanced by phorbol 12-myristate 13-acetate (PMA)-induced superoxide in a NOX-dependent manner. CB-ECFCs highly-expressed NOX4, which was further induced by PMA, whilst NOX4 siRNA and plasmid overexpression reduced and potentiated in vitro function, respectively. Increased ROS generation in NOX4-overexpressing CB-ECFCs (DCF fluorescence, flow cytometry) was specifically reduced by superoxide dismutase, highlighting induction of ROS-specific signalling. Laser Doppler imaging of mouse ischaemic hindlimbs at 7 days indicated that NOX4-knockdown CB-ECFCs inhibited blood flow recovery, which was enhanced by NOX4-overexpressing CB-ECFCs. Tissue analysis at 14 days revealed consistent alterations in vascular density (lectin expression) and eNOS protein despite clearance of injected CB-ECFCs, suggesting NOX4-mediated modulation of host tissue. Indeed, proteome array analysis indicated that NOX4-knockdown CB-ECFCs largely suppressed tissue angiogenesis, whilst NOX4-overexpressing CB-ECFCs up-regulated a number of pro-angiogenic factors specifically-linked with eNOS signalling, in parallel with equivalent modulation of NOX-dependent ROS generation, suggesting that CB-ECFC NOX4 signalling may promote host vascular repair. CONCLUSION: Taken together, these findings indicate a key role for NOX4 in CB-ECFCs, thereby highlighting its potential as a target for enhancing their reparative function through therapeutic priming to support creation of a pro-reparative microenvironment and effective post-ischaemic revascularization.
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Células Progenitoras Endoteliais/transplante , Isquemia/cirurgia , Músculo Esquelético/irrigação sanguínea , NADPH Oxidase 4/metabolismo , Neovascularização Fisiológica , Animais , Movimento Celular , Células Cultivadas , Microambiente Celular , Modelos Animais de Doenças , Células Progenitoras Endoteliais/enzimologia , Sangue Fetal/citologia , Membro Posterior , Humanos , Isquemia/enzimologia , Isquemia/genética , Isquemia/fisiopatologia , Camundongos Endogâmicos NOD , NADPH Oxidase 4/genética , Espécies Reativas de Oxigênio/metabolismo , Recuperação de Função Fisiológica , Transdução de SinaisRESUMO
Canonical Gremlin1 (GREM1) signaling involves binding to and sequestering bone morphogenetic proteins (BMPs) in the extracellular matrix, preventing the activation of cognate BMP receptor. Exquisite temporospatial control of the GREM1-BMP interaction is required during development, and perturbation of this balance leads to abnormal limb formation and defective kidney development. In addition to inhibition of BMP signaling, several other noncanonical signaling modalities of GREM1 have been postulated. Some literature reports have suggested that GREM1 can bind to and activate vascular endothelial growth factor receptor-2 (VEGFR2) in endothelial cells, human kidney epithelial cells, and others. These reports suggest that the GREM1 â VEGFR2 signaling can drive angiogenesis both in vitro and in vivo We report here that, despite exhaustive attempts, we did not observe GREM1 activation of VEGFR2 in any of the cell lines reported by the above-mentioned studies. Incubation of endothelial colony-forming cells (ECFCs) or human umbilical vein endothelial cells (HUVECs) with recombinant VEGF triggered a robust increase in VEGFR2 tyrosine phosphorylation. In contrast, no VEGFR2 phosphorylation was detected when cells were incubated with recombinant GREM1 over a range of time points and concentrations. We also show that GREM1 does not interfere with VEGF-mediated VEGFR2 activation, suggesting that GREM1 does not bind with any great affinity to VEGFR2. Measurements of ECFC barrier integrity revealed that VEGF induces barrier function disruption, but recombinant human GREM1 had no effect in this assay. We believe that these results provide an important clarification of the potential interaction between GREM1 and VEGFR2 in mammalian cells.
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Células Endoteliais da Veia Umbilical Humana/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Fosforilação , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Type 1 diabetes (T1D) is caused by autoimmune destruction of insulin-producing ß cells located in the endocrine pancreas in areas known as islets of Langerhans. The current standard-of-care for T1D is exogenous insulin replacement therapy. Recent developments in this field include the hybrid closed-loop system for regulated insulin delivery and long-acting insulins. Clinical studies on prediction and prevention of diabetes-associated complications have demonstrated the importance of early treatment and glucose control for reducing the risk of developing diabetic complications. Transplantation of primary islets offers an effective approach for treating patients with T1D. However, this strategy is hampered by challenges such as the limited availability of islets, extensive death of islet cells, and poor vascular engraftment of islets post-transplantation. Accordingly, there are considerable efforts currently underway for enhancing islet transplantation efficiency by harnessing the beneficial actions of stem cells. This review will provide an overview of currently available therapeutic options for T1D, and discuss the growing evidence that supports the use of stem cell approaches to enhance therapeutic outcomes.
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Metabolic engagement is intrinsic to immune cell function. Prostaglandin E2 (PGE2) has been shown to modulate macrophage activation, yet how PGE2 might affect metabolism is unclear. Here, we show that PGE2 caused mitochondrial membrane potential (Δψm) to dissipate in interleukin-4-activated (M(IL-4)) macrophages. Effects on Δψm were a consequence of PGE2-initiated transcriptional regulation of genes, particularly Got1, in the malate-aspartate shuttle (MAS). Reduced Δψm caused alterations in the expression of 126 voltage-regulated genes (VRGs), including those encoding resistin-like molecule α (RELMα), a key marker of M(IL-4) cells, and genes that regulate the cell cycle. The transcription factor ETS variant 1 (ETV1) played a role in the regulation of 38% of the VRGs. These results reveal ETV1 as a Δψm-sensitive transcription factor and Δψm as a mediator of mitochondrial-directed nuclear gene expression.
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Núcleo Celular/efeitos dos fármacos , Dinoprostona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Animais , Núcleo Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Interleucina-4/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/genética , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
For over a decade various cell populations have been investigated for their vasoreparative potential. Cells with the capacity to promote blood vessel regeneration are commonly known as endothelial progenitor cells (EPCs); although such a definition is currently considered too simple for the complexity of cell populations involved in the reparative angiogenic process. A subset of EPCs called endothelial colony forming cells (ECFCs) have emerged as a suitable candidate for cytotherapy, primarily due to their clonogenic progenitor characteristics, unequivocal endothelial phenotype, and inherent ability to promote vasculogenesis. ECFCs can be readily isolated from human peripheral and cord blood, expanded ex vivo and used to revascularize ischemic tissues. These cells have demonstrated efficacy in several in vivo preclinical models such as the ischemic heart, retina, brain, limb, lung and kidney. This review will summarize the current pre-clinical evidence for ECFC cytotherapy and discuss their potential for clinical application.
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Myeloid angiogenic cells (MACs) promote revascularization through the paracrine release of angiogenic factors and have been harnessed as therapeutic cells for many ischemic diseases. However, their proangiogenic properties have been suggested to be diminished in diabetes. This study investigates how the diabetic milieu affects the immunophenotype and function of MACs. Both MACs isolated from diabetic conditions and healthy cells exposed to a diabetic environment were used to determine the potential of MACs as a cell therapy for diabetic-related ischemia. MACs were isolated from human peripheral blood and characterized alongside proinflammatory macrophages M (LPS + IFNγ) and proangiogenic macrophages M (IL4). Functional changes in MACs in response to high-d-glucose were assessed using an in vitro 3D-tubulogenesis assay. Phenotypic changes were determined by gene and protein expression analysis. Additionally, MACs from type 1 diabetic (T1D) patients and corresponding controls were isolated and characterized. Our evidence demonstrates MACs identity as a distinct macrophage subtype that shares M2 proangiogenic characteristics, but can be distinguished by CD163hi expression. High-d-glucose treatment significantly reduced MACs proangiogenic capacity, which was associated with a significant increase in IL1ß mRNA and protein expression. Inhibition of IL1ß abrogated the antiangiogenic effect induced by high-d-glucose. IL1ß was also significantly upregulated in MACs isolated from T1D patients with microvascular complications compared to T1D patients without microvascular complications or nondiabetic volunteers. This study demonstrates that Type 1 diabetes and diabetic-like conditions impair the proangiogenic and regenerative capacity of MACs, and this response is mediated by IL-1ß. Stem Cells 2018;36:834-843.
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Interleucina-1beta/metabolismo , Células Mieloides/metabolismo , Adolescente , Adulto , Idoso , Diabetes Mellitus , Humanos , Pessoa de Meia-Idade , Adulto JovemRESUMO
Cell therapy using endothelial progenitors holds promise for vascular repair in ischemic retinopathies. Using a well-defined subpopulation of human cord blood-derived endothelial progenitors known as endothelial colony-forming cells (ECFCs), we have evaluated essential requirements for further development of this cell therapy targeting the ischemic retina, including dose response, delivery route, and toxicity. First, to evaluate therapeutic efficacy relating to cell dose, ECFCs were injected into the vitreous of mice with oxygen-induced retinopathy. Using angiography and histology, we found that intravitreal delivery of low dose (1 × 103 ) ECFCs was as effective as higher cell doses (1 × 104 , 1 × 105 ) in promoting vascular repair. Second, injection into the common carotid artery was tested as an alternative, systemic delivery route. Intracarotid ECFC delivery conferred therapeutic benefit which was comparable to intravitreal delivery using the same ECFC dose (1 × 105 ), although there were fewer human cells observed in the retinal vasculature following systemic delivery. Third, cell immunogenicity was evaluated by injecting ECFCs into the vitreous of healthy adult mice. Assessment of murine ocular tissues identified injected cells in the vitreous, while demonstrating integrity of the host retina. In addition, ECFCs did not invade into the retina, but remained in the vitreous, where they eventually underwent cell death within 3 days of delivery without evoking an inflammatory response. Human specific Alu sequences were not found in healthy mouse retinas after 3 days of ECFC delivery. These findings provide supportive preclinical evidence for the development of ECFCs as an efficacious cell product for ischemic retinopathies. Stem Cells Translational Medicine 2018;7:59-67.
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Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Endoteliais/citologia , Células Endoteliais/transplante , Sangue Fetal/citologia , Isquemia/terapia , Doenças Retinianas/terapia , Animais , Proliferação de Células/fisiologia , Células Cultivadas , Humanos , Isquemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/fisiologiaRESUMO
BACKGROUND: Endothelial colony-forming cells (ECFCs), also termed late outgrowth endothelial cells, are a well-defined circulating endothelial progenitor cell type with an established role in vascular repair. ECFCs have clear potential for cell therapy to treat ischaemic disease, although the precise mechanism(s) underlying their response to hypoxia remains ill-defined. METHODS: In this study, we isolated ECFCs from umbilical cord blood and cultured them on collagen. We defined the response of ECFCs to 1% O2 exposure at acute and chronic time points. RESULTS: In response to low oxygen, changes in ECFC cell shape, proliferation, size and cytoskeleton phenotype were detected. An increase in the number of senescent ECFCs also occurred as a result of long-term culture in 1% O2. Low oxygen exposure altered ECFC migration and tube formation in Matrigel®. Increases in angiogenic factors secreted from ECFCs exposed to hypoxia were also detected, in particular, after treatment with placental growth factor (PlGF). Exposure of cells to agents that stabilise hypoxia-inducible factors such as dimethyloxalylglycine (DMOG) also increased PlGF levels. Conditioned medium from both hypoxia-treated and DMOG-treated cells inhibited ECFC tube formation. This effect was reversed by the addition of PlGF neutralising antibody to the conditioned medium, confirming the direct role of PlGF in this effect. CONCLUSIONS: This study deepens our understanding of the response of ECFCs to hypoxia and also identifies a novel and important role for PlGF in regulating the vasculogenic potential of ECFCs.
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Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Hipóxia/metabolismo , Hipóxia/patologia , Fator de Crescimento Placentário/metabolismo , Hormônios Placentários/metabolismo , Aminoácidos Dicarboxílicos/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Colágeno/metabolismo , Meios de Cultivo Condicionados/metabolismo , Combinação de Medicamentos , Células Progenitoras Endoteliais/metabolismo , Sangue Fetal/metabolismo , Sangue Fetal/fisiologia , Humanos , Laminina/metabolismo , Neovascularização Fisiológica/fisiologia , Proteoglicanas/metabolismoRESUMO
AIMS: Circulating angiogenic cells (CACs) promote revascularization of ischaemic tissues although their underlying mechanism of action and the consequences of delivering varying number of these cells for therapy remain unknown. This study investigates molecular mechanisms underpinning CAC modulation of blood vessel formation. METHODS AND RESULTS: CACs at low (2 × 105 cells/mL) and mid (2 × 106 cells/mL) cellular densities significantly enhanced endothelial cell tube formation in vitro, while high density (HD) CACs (2 × 107 cells/mL) significantly inhibited this angiogenic process. In vivo, Matrigel-based angiogenesis assays confirmed mid-density CACs as pro-angiogenic and HD CACs as anti-angiogenic. Secretome characterization of CAC-EC conditioned media identified pentraxin 3 (PTX3) as only present in the HD CAC-EC co-culture. Recombinant PTX3 inhibited endothelial tube formation in vitro and in vivo. Importantly, our data revealed that the anti-angiogenic effect observed in HD CAC-EC co-cultures was significantly abrogated when PTX3 bioactivity was blocked using neutralizing antibodies or PTX3 siRNA in endothelial cells. We show evidence for an endothelial source of PTX3, triggered by exposure to HD CACs. In addition, we confirmed that PTX3 inhibits fibroblast growth factor (FGF) 2-mediated angiogenesis, and that the PTX3 N-terminus, containing the FGF-binding site, is responsible for such anti-angiogenic effects. CONCLUSION: Endothelium, when exposed to HD CACs, releases PTX3 which markedly impairs the vascular regenerative response in an autocrine manner. Therefore, CAC density and accompanying release of angiocrine PTX3 are critical considerations when using these cells as a cell therapy for ischaemic disease.
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Proteína C-Reativa/metabolismo , Células Endoteliais/metabolismo , Células Progenitoras Endoteliais/metabolismo , Neovascularização Fisiológica , Componente Amiloide P Sérico/metabolismo , Adolescente , Adulto , Animais , Comunicação Autócrina , Proteína C-Reativa/química , Proteína C-Reativa/genética , Células Cultivadas , Técnicas de Cocultura , Citocinas/metabolismo , Modelos Animais de Doenças , Células Progenitoras Endoteliais/transplante , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Oxigênio , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Neovascularização Retiniana , Retinopatia da Prematuridade/metabolismo , Retinopatia da Prematuridade/fisiopatologia , Retinopatia da Prematuridade/cirurgia , Componente Amiloide P Sérico/química , Componente Amiloide P Sérico/genética , Transdução de Sinais , Transfecção , Adulto JovemRESUMO
Activated effector T (TE) cells augment anabolic pathways of metabolism, such as aerobic glycolysis, while memory T (TM) cells engage catabolic pathways, like fatty acid oxidation (FAO). However, signals that drive these differences remain unclear. Mitochondria are metabolic organelles that actively transform their ultrastructure. Therefore, we questioned whether mitochondrial dynamics controls T cell metabolism. We show that TE cells have punctate mitochondria, while TM cells maintain fused networks. The fusion protein Opa1 is required for TM, but not TE cells after infection, and enforcing fusion in TE cells imposes TM cell characteristics and enhances antitumor function. Our data suggest that, by altering cristae morphology, fusion in TM cells configures electron transport chain (ETC) complex associations favoring oxidative phosphorylation (OXPHOS) and FAO, while fission in TE cells leads to cristae expansion, reducing ETC efficiency and promoting aerobic glycolysis. Thus, mitochondrial remodeling is a signaling mechanism that instructs T cell metabolic programming.
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Dinâmica Mitocondrial , Linfócitos T/citologia , Linfócitos T/metabolismo , Animais , Diferenciação Celular , Transporte de Elétrons , Ácidos Graxos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Glicólise , Humanos , Memória Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Transdução de Sinais , Linfócitos T/imunologiaRESUMO
CD8α(+) and CD103(+) dendritic cells (DCs) play a central role in the development of type 1 immune responses. However, their role in type 2 immunity remains unclear. We examined this issue using Batf3(-/-) mice, in which both of these DC subsets are missing. We found that Th2 cell responses, and related events such as eosinophilia, alternative macrophage activation, and immunoglobulin class switching to IgG1, were enhanced in Batf3(-/-) mice responding to helminth parasites. This had beneficial or detrimental consequences depending on the context. For example, Batf3 deficiency converted a normally chronic intestinal infection with Heligmosomoides polygyrus into an infection that was rapidly controlled. However, liver fibrosis, an IL-13-mediated pathological consequence of wound healing in chronic schistosomiasis, was exacerbated in Batf3(-/-) mice infected with Schistosoma mansoni. Mechanistically, steady-state production of IL-12 by migratory CD103(+) DCs, independent of signals from commensals or TLR-initiated events, was necessary and sufficient to exert the suppressive effects on Th2 response development. These findings identify a previously unrecognized role for migratory CD103(+) DCs in antagonizing type 2 immune responses.
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Antígenos CD/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Helmintos/imunologia , Imunidade , Imunomodulação , Cadeias alfa de Integrinas/metabolismo , Interleucina-12/genética , Animais , Fatores de Transcrição de Zíper de Leucina Básica/deficiência , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Movimento Celular , Resistência à Doença/imunologia , Feminino , Helmintíase/genética , Helmintíase/imunologia , Helmintíase/metabolismo , Imunidade/genética , Imunização , Interleucina-12/biossíntese , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Camundongos , Camundongos Knockout , Modelos Animais , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Receptores Toll-Like/metabolismoRESUMO
The vascular complications of diabetes significantly impact the quality of life and mortality in diabetic patients. Extensive evidence from various human clinical trials has clearly established that a period of poor glycemic control early in the disease process carries negative consequences, such as an increase in the development and progression of vascular complications that becomes evident many years later. Importantly, intensive glycemic control established later in the disease process cannot reverse or slow down the onset or progression of diabetic vasculopathy. This has been named the glycemic memory phenomenon. Scientists have successfully modelled glycemic memory using various in vitro and in vivo systems. This review emphasizes that oxidative stress and accumulation of advanced glycation end products are key factors driving glycemic memory in endothelial cells. Furthermore, various epigenetic marks have been proposed to closely associate with vascular glycemic memory. In addition, we comment on the importance of endothelial progenitors and their role as endogenous vasoreparative cells that are negatively impacted by the diabetic milieu and may constitute a "carrier" of glycemic memory. Considering the potential of endothelial progenitor-based cytotherapies, future studies on their glycemic memory are warranted to develop epigenetics-based therapeutics targeting diabetic vascular complications.
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Angiopatias Diabéticas/etiologia , Células Progenitoras Endoteliais/metabolismo , Epigênese Genética , Hiperglicemia/fisiopatologia , Modelos Biológicos , Animais , Diferenciação Celular , Metilação de DNA , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/patologia , Células Progenitoras Endoteliais/patologia , Produtos Finais de Glicação Avançada/sangue , Humanos , Hiperglicemia/sangue , MicroRNAs/metabolismo , Estresse Oxidativo , Nicho de Células-TroncoRESUMO
The ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony-forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel-forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of >10(8) ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb, and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF165 as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells.
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Diferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Células Endoteliais/citologia , Células-Tronco Pluripotentes/citologia , Animais , Proliferação de Células/genética , Células Endoteliais/metabolismo , Sangue Fetal/citologia , Humanos , Camundongos , Neuropilina-1/metabolismo , Células-Tronco/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismoAssuntos
Medicamentos sem Prescrição/efeitos adversos , Compostos Fitoquímicos/efeitos adversos , Preparações de Plantas/efeitos adversos , Psicotrópicos/efeitos adversos , Transtornos Relacionados ao Uso de Substâncias/epidemiologia , Transtornos Relacionados ao Uso de Substâncias/etiologia , Adolescente , Adulto , Feminino , Humanos , Incidência , Masculino , Fatores de Risco , Reino Unido/epidemiologia , Adulto JovemRESUMO
Alternative (M2) activation of macrophages driven via the α-chain of the receptor for interleukin 4 (IL-4Rα) is important for immunity to parasites, wound healing, the prevention of atherosclerosis and metabolic homeostasis. M2 polarization is dependent on fatty acid oxidation (FAO), but the source of the fatty acids that support this metabolic program has not been clear. We found that the uptake of triacylglycerol substrates via the scavenger receptor CD36 and their subsequent lipolysis by lysosomal acid lipase (LAL) was important for the engagement of elevated oxidative phosphorylation, enhanced spare respiratory capacity (SRC), prolonged survival and expression of genes that together define M2 activation. Inhibition of lipolysis suppressed M2 activation during infection with a parasitic helminth and blocked protective responses to this pathogen. Our findings delineate a critical role for cell-intrinsic lysosomal lipolysis in M2 activation.
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Antígenos CD36/imunologia , Ácidos Graxos/metabolismo , Interleucina-4/imunologia , Lipólise/imunologia , Lisossomos/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Fosforilação Oxidativa , Transdução de Sinais/imunologia , Esterol Esterase/imunologia , Animais , Respiração Celular , Helmintíase Animal/imunologia , Humanos , Camundongos , Consumo de Oxigênio , Receptores de Interleucina-4/imunologia , TranscriptomaRESUMO
Diabetic retinopathy (DR) is a leading cause of visual impairment worldwide. Patients with DR may irreversibly lose sight as a result of the development of diabetic macular edema (DME) and/or proliferative diabetic retinopathy (PDR); retinal blood vessel dysfunction and degeneration plays an essential role in their pathogenesis. Although new treatments have been recently introduced for DME, including intravitreal vascular endothelial growth factor inhibitors (anti-VEGFs) and steroids, a high proportion of patients (~40-50%) do not respond to these therapies. Furthermore, for people with PDR, laser photocoagulation remains a mainstay therapy despite this being an inherently destructive procedure. Endothelial progenitor cells (EPCs) are a low-frequency population of circulating cells known to be recruited to sites of vessel damage and tissue ischemia where they promote vascular healing and re-perfusion. A growing body of evidence suggests that the number and function of EPCs are altered in patients with varying degrees of diabetes duration, metabolic control, and in the presence or absence of DR. Although there are no clear-cut outcomes from these clinical studies, there is mounting evidence that some EPC sub-types may be involved in the pathogenesis of DR and may also serve as biomarkers for disease progression and stratification. Moreover, some EPC sub-types have considerable potential as therapeutic modalities for DME and PDR in the context of cell therapy. This study presents basic clinical concepts of DR and combines this with a general insight on EPCs and their relation to future directions in understanding and treating this important diabetic complication.
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Macrophage function is not restricted to the innate and adaptive immune responses, but also includes host defence, wound healing, angiogenesis and homeostatic processes. Within the spectrum of macrophage activation there are two extremes: M1 classically activated macrophages which have a pro-inflammatory phenotype, and M2 alternatively activated macrophages which are pro-angiogenic and anti-inflammatory. An important property of macrophages is their plasticity to switch from one phenotype to the other and they can be defined in their polarisation state at any point between the two extremes. In order to determine what stage of activation macrophages are in, it is essential to profile various phenotypic markers for their identification. This review describes the angiogenic role for myeloid cells: circulating monocytes, Tie-2 expressing monocytes (TEMs), myeloid-derived suppressor cells (MDSCs), tumour associated macrophages (TAMs), and neutrophils. Each cell type is discussed by phenotype, roles within angiogenesis and possible targets as a cell therapy. In addition, we also refer to our own research on myeloid angiogenic cells (MACs), outlining their ability to induce angiogenesis and their similarities to alternatively activated M2 macrophages. MACs significantly contribute to vascular repair through paracrine mechanisms as they lack the capacity to differentiate into endothelial cells. Since MACs also retain plasticity, phenotypic changes can occur according to disease states and the surrounding microenvironment. This pro-angiogenic potential of MACs could be harnessed as a novel cellular therapy for the treatment of ischaemic diseases, such as diabetic retinopathy, hind limb ischaemia and myocardial infarction; however, caution needs to be taken when MACs are delivered into an inflammatory milieu.
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Macrófagos/imunologia , Monócitos/imunologia , Células Mieloides/imunologia , Neovascularização Patológica/imunologia , Diferenciação Celular/imunologia , Humanos , Imunoterapia/métodos , Inflamação/imunologia , Ativação de Macrófagos/imunologia , Neutrófilos/imunologia , Receptor TIE-2/biossíntese , Receptor TIE-2/metabolismoRESUMO
Elevated levels of circulating proinflammatory cytokines are associated with obesity and increased risk of type 2 diabetes, but the mechanism is unknown. We tested whether proinflammatory cytokines IL-1B+IL-6 at low picogram per milliliter concentrations (consistent with serum levels) could directly trigger pancreatic islet dysfunction. Overnight exposure to IL-1B+IL-6 in islets isolated from normal mice and humans disrupted glucose-stimulated intracellular calcium responses; cytokine-induced effects were more severe among islets from prediabetic db/db mice that otherwise showed no signs of dysfunction. IL-1B+IL-6 exposure reduced endoplasmic reticulum (ER) calcium storage, activated ER stress responses (Nos2, Bip, Atf4, and Ddit3 [CHOP]), impaired glucose-stimulated insulin secretion, and increased cell death only in islets from prediabetic db/db mice. Furthermore, we found increased serum levels of IL-1B and IL-6 in diabetes-prone mice at an age before hyperglycemia was exhibited, suggesting that low-grade systemic inflammation develops early in the disease process. In addition, we implanted normal outbred and inbred mice with subcutaneous osmotic mini-pumps containing IL-1B+IL-6 to mimic the serum increases found in prediabetic db/db mice. Both IL-1B and IL-6 were elevated in serum from cytokine-pump mice, but glucose tolerance and blood glucose levels did not differ from controls. However, when compared with controls, isolated islets from cytokine-pump mice showed deficiencies in calcium handling and insulin secretion that were similar to observations with islets exposed to cytokines in vitro. These findings provide proof of principle that low-grade systemic inflammation is present early in the development of type 2 diabetes and can trigger ER stress-mediated islet dysfunction that can lead to islet failure.
Assuntos
Estresse do Retículo Endoplasmático , Insulina/metabolismo , Interleucina-1beta/sangue , Interleucina-6/sangue , Ilhotas Pancreáticas/metabolismo , Estado Pré-Diabético/sangue , Animais , Animais não Endogâmicos , Apoptose/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Implantes de Medicamento , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glucose/metabolismo , Humanos , Infusões Subcutâneas , Insulina/sangue , Secreção de Insulina , Interleucina-1beta/administração & dosagem , Interleucina-1beta/efeitos adversos , Interleucina-6/administração & dosagem , Interleucina-6/efeitos adversos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Estado Pré-Diabético/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , Organismos Livres de Patógenos Específicos , Técnicas de Cultura de TecidosRESUMO
Harnessing outgrowth endothelial cells (OECs) for vasoreparative therapy and tissue engineering requires efficient ex vivo expansion. How such expansion impacts on OEC function is largely unknown. In this study, we show that OECs become permanently cell-cycle arrested after ex vivo expansion, which is associated with enlarged cell size, ß-galactosidase activity, DNA damage, tumor suppressor pathway activation, and significant transcriptome changes. These senescence hallmarks were coupled with low telomerase activity and telomere shortening, indicating replicative senescence. OEC senescence limited their regenerative potential by impairing vasoreparative properties in vitro and in vivo. Integrated transcriptome-proteome analysis identified inflammatory signaling pathways as major mechanistic components of the OEC senescence program. In particular, IL8 was an important facilitator of this senescence; depletion of IL8 in OECs significantly extended ex vivo lifespan, delayed replicative senescence, and enhanced function. While the ability to expand OEC numbers prior to autologous or allogeneic therapy remains a useful property, their replicative senescence and associated impairment of vasorepair needs to be considered. This study also suggests that modulation of the senescence-associated secretory phenotype could be used to optimize OEC therapy.