RESUMEN
In European countries, nearly 10% of all hospital admissions are related to respiratory diseases, mainly chronic life-threatening diseases such as COPD, pulmonary hypertension, IPF or lung cancer. The contribution of blood vessels and angiogenesis to lung regeneration, remodeling and disease progression has been increasingly appreciated. The vascular supply of the lung shows the peculiarity of dual perfusion of the pulmonary circulation (vasa publica), which maintains a functional blood-gas barrier, and the bronchial circulation (vasa privata), which reveals a profiled capacity for angiogenesis (namely intussusceptive and sprouting angiogenesis) and alveolar-vascular remodeling by the recruitment of endothelial precursor cells. The aim of this review is to outline the importance of vascular remodeling and angiogenesis in a variety of non-neoplastic and neoplastic acute and chronic respiratory diseases such as lung infection, COPD, lung fibrosis, pulmonary hypertension and lung cancer.
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Neovascularización Patológica , Animales , Humanos , Pulmón/irrigación sanguínea , Pulmón/patología , Neovascularización Patológica/patología , Enfermedades Respiratorias/fisiopatología , Enfermedades Respiratorias/patología , Remodelación VascularRESUMEN
High gestational weight gain (GWG) is a cardiovascular risk factor and may disturb neonatal endothelial function. Long non-coding RNAs (lncRNAs) regulate gene expression epigenetically and can modulate endothelial function. Endothelial colony forming cells (ECFCs), circulating endothelial precursors, are a recruitable source of endothelial cells and sustain endothelial function, vascular growth and repair. We here investigated whether higher GWG affects neonatal ECFC function and elucidated the role of lncRNAs herein. Wound healing of umbilical cord blood-derived ECFCs after pregnancies with GWG <13 kg versus >13 kg was determined in a scratch assay and based on monolayer impedance after electric wounding (electric cell-substrate impedance sensing, ECIS). LncRNA expression was analysed by RNA sequencing. The function of killer cell lectin-like receptor K1 antisense RNA (KLRK1-AS1) was investigated after siRNA-based knockdown. Closure of the scratch was delayed by 25% (P = 0.041) in the higher GWG group and correlated inversely with GWG (R = -0.538, P = 0.012) in all subjects (n = 22). Similarly, recovery of the monolayer barrier after electric wounding was delayed (-11% after 20 h; P = 0.014; n = 15). Several lncRNAs correlated with maternal GWG, the most significant one being KLRK1-AS1 (log2 fold change = -0.135, P < 0.001, n = 35). KLRK1-AS1 knockdown (n = 4) reduced barrier recovery after electric wounding by 21% (P = 0.029) and KLRK1-AS1 expression correlated with the time required for wound healing for both scratch (R = 0.447, P = 0.033) and impedance-based assay (R = 0.629, P = 0.014). Higher GWG reduces wound healing of neonatal ECFCs, and lower levels of the lncRNA KLRK1-AS1 may underlie this. KEY POINTS: Maternal cardiovascular risk factors such as diabetes, obesity and smoking in pregnancy disturb fetal endothelial function, and we here investigated whether also high gestational weight gain (GWG) has an impact on fetal endothelial cells. Circulating endothelial progenitor cells (endothelial colony forming cells, ECFCs) are highly abundant in the neonatal blood stream and serve as a circulating pool for vascular growth and repair. We revealed that higher GWG delays wound healing capacity of ECFCs in vitro. We identified the regulatory RNA lncRNA KLRK1-AS1 as a link between GWG and delayed ECFC wound healing. Our data show that high GWG, independent of pre-pregnancy BMI, affects neonatal ECFC function.
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Células Progenitoras Endoteliales , Ganancia de Peso Gestacional , ARN Largo no Codificante , Embarazo , Recién Nacido , Femenino , Humanos , ARN Largo no Codificante/genética , Células Cultivadas , Cicatrización de Heridas , Subfamilia K de Receptores Similares a Lectina de Células NKRESUMEN
Preeclampsia, a pregnancy-related hypertensive disorder, is associated with endothelial dysfunction and increased cardiovascular risk of the offspring in adulthood. In preeclampsia, endothelial colony-forming cells (ECFC) are reduced in number and function. Recently, we have shown that miR-1270, which is involved in cancer in vitro proliferation, migration, and tumor progression, is downregulated in fetal ECFC from preeclamptic pregnancies. We now hypothesize that miR-1270 dysregulation contributes to vascular endothelial dysfunction occurring after preeclampsia via ATM (ataxia telangiectasia mutated) overexpression, the key kinase of DNA damage repair. Here, we show that miR-1270 silencing in normal ECFC and downregulation in preeclamptic ECFC are accompanied by an increase in the expression levels of ATM. Furthermore, ATM activation correlates with upregulated tyrosine kinase Src leading to phosphorylation and internalization of VE-cadherin (vascular endothelial-cadherin) which subsequently compromises endothelial barrier permeability and morphodynamic cell parameters. Treatment with specific ATM inhibitors reveals a novel role of ATM upstream of tyrosine kinase Src activation. Subsequently, Src phosphorylation and internalization of VE-cadherin compromise endothelial barrier permeability. Our findings suggest that downregulation of miR-1270 contributes to impaired ECFC function via the associated ATM overexpression, which further identifies ATM as a novel and critical factor for ECFC defects in preeclampsia. Our study provides new insights into the understanding of ECFC impairment associated with cardiovascular risk in preeclamptic offspring and identifies potential novel therapeutic targets.
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Proteínas de la Ataxia Telangiectasia Mutada , Células Progenitoras Endoteliales , MicroARNs , Preeclampsia , Antígenos CD , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Cadherinas/metabolismo , Regulación hacia Abajo , Células Progenitoras Endoteliales/metabolismo , Femenino , Humanos , MicroARNs/genética , Preeclampsia/genética , Preeclampsia/patología , Embarazo , Proteínas Tirosina Quinasas/metabolismoRESUMEN
Glaucoma is a severe, rapidly progressing disease that in the absence of proper treatment leads to blindness in 20% of patients. According to the World Glaucoma Association, this disease is the most socially significant in modern ophthalmology and requires searching for new and effective methods of treatment. This article presents the results of research and reviews on this issue, considers both conservative therapy and surgical methods of treatment, analyzes in detail modern methods of micro-invasive eye surgery actively used in clinical practice. The article also describes indications for a various types of interventions, as well as the effect achieved by them and the possible complications, and presents the conclusions about the possibility of using these procedures in wide clinical practice.
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Implantes de Drenaje de Glaucoma , Glaucoma , Oftalmología , Humanos , Presión Intraocular , Implantes de Drenaje de Glaucoma/efectos adversos , Glaucoma/diagnóstico , Glaucoma/cirugía , Procedimientos Quirúrgicos Oftalmológicos/efectos adversos , StentsRESUMEN
The search for a source of endothelial cells (ECs) with translational therapeutic potential remains crucial in regenerative medicine. Human blood-derived endothelial colony-forming cells (ECFCs) represent a promising source of autologous ECs due to their robust capacity to form vascular networks in vivo and their easy accessibility from peripheral blood. However, whether ECFCs have distinct characteristics with translational value compared to other ECs remains unclear. Here, we show that vascular networks generated with human ECFCs exhibited robust paracrine support for human pluripotent stem cell-derived cardiomyocytes (iCMs), significantly improving protection against drug-induced cardiac injury and enhancing engraftment at ectopic (subcutaneous) and orthotopic (cardiac) sites. In contrast, iCM support was notably absent in grafts with vessels lined by mature-ECs. This differential trophic ability was due to a unique high constitutive expression of the cardioprotective growth factor neuregulin-1 (NRG1). ECFCs, but not mature-ECs, were capable of actively releasing NRG1, which, in turn, reduced apoptosis and increased the proliferation of iCMs via the PI3K/Akt signaling pathway. Transcriptional silencing of NRG1 abrogated these cardioprotective effects. Our study suggests that ECFCs are uniquely suited to support human iCMs, making these progenitor cells ideal for cardiovascular regenerative medicine.
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Diferenciación Celular , Células Progenitoras Endoteliales/metabolismo , Regulación de la Expresión Génica , Miocitos Cardíacos/metabolismo , Neurregulina-1/biosíntesis , Células Madre Pluripotentes/metabolismo , Células Cultivadas , Humanos , Comunicación ParacrinaRESUMEN
In regenerative medicine, autologous endothelial colony forming cells (ECFCs) bear the greatest potential to be used for surface endothelialization of tissue engineered constructs, as they are easily attainable and possess a high proliferation rate. The aim of this study was to develop a standardized pre-conditioning protocol under dynamic conditions simulating the physiology of human circulation to improve the formation of a flow resistant monolayer of ECFCs and to enhance the antithrombogenicity of the endothelial cells. The main focus of the study was to consequently compare the cellular behavior under a steady laminar flow against a pulsatile flow. Mononuclear cells were isolated out of peripheral blood (PB) buffy coats and plated on uncoated tissue culture flasks in anticipation of guidelines for Advanced Therapy Medicinal Products. ECFCs were identified by typical surface markers such as CD31, CD146 and VE-Cadherin. To explore the effects of dynamic cultivation, ECFCs and human umbilical vein endothelial cells were comparatively cultured under either laminar or pulsatile (1 Hz) flow conditions with different grades of shear stress (5 dyn/cm2versus 20 dyn/cm2). High shear stress of 20 dyn/cm2 led to a significant upregulation of the antithrombotic gene marker thrombomodulin in both cell types, but only ECFCs orientated and elongated significantly after shear stress application forming a confluent endothelial cell layer. The work therefore documents a suitable protocol to pre-condition PB-derived ECFCs for sustainable endothelialization of blood contacting surfaces and provides essential knowledge for future cultivations in bioreactor systems.
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Células Progenitoras Endoteliales/fisiología , Células Endoteliales de la Vena Umbilical Humana/fisiología , Mecanotransducción Celular , Flujo Pulsátil , Ingeniería de Tejidos , Antígenos CD/metabolismo , Reactores Biológicos , Antígeno CD146/metabolismo , Cadherinas/metabolismo , Técnicas de Cultivo de Célula/instrumentación , Forma de la Célula , Células Cultivadas , Células Progenitoras Endoteliales/metabolismo , Femenino , Glucosa/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Neovascularización Fisiológica , Fenotipo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Estrés Mecánico , Trombomodulina/genética , Trombomodulina/metabolismoRESUMEN
Endoglin (Eng) is an endothelial cell (EC) transmembrane glycoprotein involved in adhesion and angiogenesis. Eng mutations result in vessel abnormalities as observed in hereditary hemorrhagic telangiectasia of type 1. The role of Eng was investigated in endothelial functions and permeability under inflammatory conditions, focusing on the actin dynamic signaling pathway. Endothelial Colony-Forming Cells (ECFC) from human cord blood and mouse lung/aortic EC (MLEC, MAEC) from Eng+/+ and Eng+/- mice were used. ECFC silenced for Eng with Eng-siRNA and ctr-siRNA were used to test tubulogenesis and permeability +/- TNFα and +/- LIM kinase inhibitors (LIMKi). In silico modeling of TNFα-Eng interactions was carried out from PDB IDs 5HZW and 5HZV. Calcium ions (Ca2+) flux was studied by Oregon Green 488 in epifluorescence microscopy. Levels of cofilin phosphorylation and tubulin post-translational modifications were evaluated by Western blot. F-actin and actin-tubulin distribution/co-localization were evaluated in cells by confocal microscopy. Eng silencing in ECFCs resulted in a decrease of cell sprouting by 50 ± 15% (p < 0.05) and an increase in pseudo-tube width (41 ± 4.5%; p < 0.001) compared to control. Upon TNFα stimulation, ECFC Eng-siRNA displayed a significant higher permeability compared to ctr-siRNA (p < 0.01), which is associated to a higher Ca2+ mobilization (p < 0.01). Computational analysis suggested that Eng mitigated TNFα activity. F-actin polymerization was significantly increased in ECFC Eng-siRNA, MAEC+/-, and MLEC+/- compared to controls (p < 0.001, p < 0.01, and p < 0.01, respectively) as well as actin/tubulin distribution (p < 0.01). Furthermore, the inactive form of cofilin (P-cofilin at Ser3) was significantly decreased by 36.7 ± 4.8% in ECFC Eng-siRNA compared to ctr-siRNA (p < 0.001). Interestingly, LIMKi reproduced the absence of Eng on TNFα-induced ECFC-increased permeability. Our data suggest that Eng plays a critical role in the homeostasis regulation of endothelial cells under inflammatory conditions (TNFα), and loss of Eng influences ECFC-related permeability through the LIMK/cofilin/actin rearrangement-signaling pathway.
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Factores Despolimerizantes de la Actina/metabolismo , Permeabilidad de la Membrana Celular , Endoglina/metabolismo , Células Endoteliales/patología , Inflamación/patología , Quinasas Lim/metabolismo , Neovascularización Patológica/patología , Factores Despolimerizantes de la Actina/genética , Animales , Endoglina/genética , Células Endoteliales/metabolismo , Inflamación/genética , Inflamación/metabolismo , Quinasas Lim/genética , Ratones , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismoRESUMEN
BACKGROUND: Human immunodeficiency virus (HIV) may be related to cardiovascular disease through monocyte activation-associated endothelial dysfunction. METHODS: Blood samples from 15 HIV-negative participants (the uninfected group), 8 HIV-positive participants who were not receiving antiretroviral therapy (ART) (the infected, untreated group), and 15 HIV-positive participants who were receiving ART (the infected, treated group) underwent flow cytometry of endothelial colony-forming cells (ECFCs) and monocyte proportions. IncuCyte live cell imaging of 8 capillary proliferative capacity parameters were obtained from cord blood ECFCs treated with participant plasma. RESULTS: The ECFC percentage determined by flow cytometry was not different between the study groups; however, values of the majority of capillary proliferative capacity parameters (ie, cell area, network length, network branch points, number of networks, and average tube width uniformity) were significantly lower in infected, untreated participants as compared to values for uninfected participants or infected, treated participants (P < .00625 for all comparisons). CD14+CD16+ intermediate monocytes and soluble CD163 were significantly and negatively correlated with several plasma-treated, cord blood ECFC proliferative capacity parameters in the combined HIV-positive groups but not in the uninfected group. CONCLUSIONS: Cord blood ECFC proliferative capacity was significantly impaired by plasma from infected, untreated patients, compared with plasma from uninfected participants and from infected, treated participants. Several ECFC functional parameters were adversely associated with monocyte activation in the HIV-positive groups, thereby suggesting a mechanism by which HIV-related inflammation may impair vascular reparative potential and consequently increase the risk of cardiovascular disease during HIV infection.
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Endotelio/inmunología , Seronegatividad para VIH/inmunología , Seropositividad para VIH/inmunología , Monocitos , Células Madre , Adulto , Alquinos , Fármacos Anti-VIH/uso terapéutico , Benzoxazinas/uso terapéutico , Proliferación Celular , Quimiocina CCL5/sangre , Ciclopropanos , Endotelio/patología , Femenino , Sangre Fetal , Citometría de Flujo , Proteínas Ligadas a GPI/metabolismo , Seropositividad para VIH/sangre , Seropositividad para VIH/tratamiento farmacológico , Humanos , Receptores de Lipopolisacáridos/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Neovascularización Fisiológica , Plasma/inmunología , Estudios Prospectivos , Receptores de IgG/metabolismo , Células Madre/fisiología , Molécula 1 de Adhesión Celular Vascular/sangre , Receptor 1 de Factores de Crecimiento Endotelial Vascular/sangre , Receptor 2 de Factores de Crecimiento Endotelial Vascular/sangreRESUMEN
New blood vessel formation in adults was considered to result exclusively from sprouting of preexisting endothelial cells, a process referred to angiogenesis. Vasculogenesis, the formation of new blood vessels from endothelial progenitor cells, was thought to occur only during embryonic life. Discovery of adult endothelial progenitor cells (EPCs) in 1997 opened the door for cell therapy in vascular disease. Endothelial progenitor cells contribute to vascular repair and are now well established as postnatal vasculogenic cells in humans. It is now admitted that endothelial colony-forming cells (ECFCs) are the vasculogenic subtype. ECFCs could be used as a cell therapy product and also as a liquid biopsy in several vascular diseases or as vector for gene therapy. However, despite a huge interest in these cells, their tissue and molecular origin is still unclear. We recently proposed that endothelial progenitor could come from very small embryonic-like stem cells (VSELs) isolated in human from CD133 positive cells. VSELs are small dormant stem cells related to migratory primordial germ cells. They have been described in bone marrow and other organs. This chapter discusses the reported findings from in vitro data and also preclinical studies that aimed to explore stem cells at the origin of vasculogenesis in human and then explore the potential use of ECFCs to promote newly formed vessels or serve as liquid biopsy to understand vascular pathophysiology and in particular pulmonary disease and haemostasis disorders.
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Tratamiento Basado en Trasplante de Células y Tejidos/tendencias , Células Progenitoras Endoteliales/citología , Biopsia Líquida/tendencias , Neovascularización Fisiológica , Enfermedades Vasculares/diagnóstico , Enfermedades Vasculares/terapia , Humanos , Enfermedades Vasculares/patologíaRESUMEN
Bicuspid aortic valve (BAV), the most common congenital heart defect, is associated with an increased prevalence of aortic dilation, aortic rupture and aortic valve calcification. Endothelial cells (ECs) play a major role in vessel wall integrity. Little is known regarding EC function in BAV patients due to lack of patient derived primary ECs. Endothelial colony forming cells (ECFCs) have been reported to be a valid surrogate model for several cardiovascular pathologies, thereby facilitating an in vitro system to assess patient-specific endothelial dysfunction. Therefore, the aim of this study was to investigate cellular functions in ECFCs isolated from BAV patients. Outgrowth and proliferation of ECFCs from patients with BAV (n = 34) and controls with a tricuspid aortic valve (TAV, n = 10) were determined and related to patient characteristics. Interestingly, we were only able to generate ECFCs from TAV and BAV patients without aortic dilation, and failed to isolate ECFC colonies from patients with a dilated aorta. Analyzing EC function showed that while proliferation, cell size and endothelial-to-mesenchymal transition were similar in TAV and BAV ECFCs, migration and the wound healing capacity of BAV ECFCs is significantly higher compared to TAV ECFCs. Furthermore, calcification is blunted in BAV compared to TAV ECFCs. Our results reveal ECs dysfunction in BAV patients and future research is required to unravel the underlying mechanisms and to further validate ECFCs as a patient-specific in vitro model for BAV.
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Válvula Aórtica/anomalías , Células Endoteliales/patología , Enfermedades de las Válvulas Cardíacas/patología , Adulto , Aorta/patología , Válvula Aórtica/patología , Enfermedad de la Válvula Aórtica Bicúspide , Movimiento Celular , Tamaño de la Célula , Células Cultivadas , Dilatación Patológica/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
ß-Galactosidase accumulates in the lysosomes of senescent cells of certain tissues. Cell staining with X-gal is a common procedure to detect senescent cells in culture. However, the organelle nature of the staining makes automatic count impossible, requiring time-consuming manual counting or expensive alternative techniques such as flow cytometry to effectively determine the amount of stained cells. Here we present an analysis strategy for images of X-gal stained cells which can be implemented into a macro for the ImageJ software overcoming some of the drawbacks of computational analysis of organelle staining.
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Senescencia Celular , Galactósidos/química , Indoles/química , Adulto , Células Cultivadas , HumanosRESUMEN
The prospect of using endothelial progenitors is currently hampered by their low engraftment upon transplantation. We report that mesenchymal stem/stromal cells (MSCs), independent of source and age, improve the engraftment of endothelial colony forming cells (ECFCs). MSC coculture altered ECFC appearance to an elongated mesenchymal morphology with reduced proliferation. ECFC primed via MSC contact had reduced self-renewal potential, but improved capacity to form tube structures in vitro and engraftment in vivo Primed ECFCs displayed major differences in transcriptome compared to ECFCs never exposed to MSCs, affecting genes involved in the cell cycle, up-regulating of genes influencing mesenchymal transition, adhesion, extracellular matrix. Inhibition of NOTCH signaling, a potential upstream regulator of mesenchymal transition, in large part modulated this gene expression pattern and functionally reversed the mesenchymal morphology of ECFCs. The collective results showed that primed ECFCs survive better and undergo a mesenchymal transition that is dependent on NOTCH signaling, resulting in significantly increased vasculogenic potential.-Shafiee, A., Patel, J., Wong, H. Y., Donovan, P., Hutmacher, D. W., Fisk, N. M., Khosrotehrani, K. Priming of endothelial colony-forming cells in a mesenchymal niche improves engraftment and vasculogenic potential by initiating mesenchymal transition orchestrated by NOTCH signaling.
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Células Endoteliales/metabolismo , Células Madre Mesenquimatosas/fisiología , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Animales , Diferenciación Celular , Proliferación Celular , Técnicas de Cocultivo , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Placenta , Embarazo , Receptores Notch/genéticaRESUMEN
Cell-based approaches for bone formation require instructional cues from the surrounding environment. As an alternative to pharmacological strategies or transplanting single cell populations, one approach is to coimplant populations that can establish a new vasculature and differentiate to bone-forming osteoblasts. Mesenchymal stem/stromal cells (MSCs) possess osteogenic potential and produce numerous angiogenic growth factors. Endothelial colony-forming cells (ECFCs) are a subpopulation of endothelial progenitor cells capable of vasculogenesis in vivo and may provide endogenous cues to support MSC function. We investigated the contribution of the carrier biophysical properties to instruct entrapped human MSCs and ECFCs to simultaneously promote their osteogenic and proangiogenic potential. Compared with gels containing MSCs alone, fibrin gels engineered with increased compressive stiffness simultaneously increased the osteogenic and proangiogenic potential of entrapped cocultured cells. ECFCs produced bone morphogenetic protein-2 (BMP-2), a potent osteoinductive molecule, and increases in BMP-2 secretion correlated with gel stiffness. Coculture of MSCs with ECFCs transduced to knockdown BMP-2 production abrogated the osteogenic response to levels observed with MSCs alone. These results demonstrate that physical properties of engineered hydrogels modulate the function of cocultured cells in the absence of inductive cues, thus increasing the translational potential of coimplantation to speed bone formation and repair.
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Hidrogeles/farmacología , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Células Progenitoras Endoteliales/metabolismo , Fibrina/farmacología , Humanos , Hidrogeles/química , Células Madre Mesenquimatosas/efectos de los fármacos , Osteoblastos/efectos de los fármacosRESUMEN
Administration of human cord blood endothelial colony-forming cells (ECFCs) or their exosomes protects mice against kidney ischemia/reperfusion injury. Here we studied the microRNA (miRNA) content of ECFC exosomes and the role of miRNA transfer in kidney and endothelial cell protection. ECFC exosomes were enriched in miR-486-5p, which targets the phosphatase and tensin homolog (PTEN) and the Akt pathway. In cultured endothelial cells exposed to hypoxia, incubation with ECFC exosomes increased miR-486-5p, decreased PTEN, and stimulated Akt phosphorylation. Exposure of hypoxic endothelial cells to conditioned medium from ECFCs pretreated with anti-miR-486-5p blocked increases in miR-486-5p and phosphorylated Akt, restored expression of PTEN, and enhanced apoptosis. Coculture of endothelial cells with ECFCs enhanced endothelial miR-486-5p levels. Targeting of PTEN by miR-486-5p was observed in endothelial cells, and PTEN knockdown blocked apoptosis. In mice with ischemic kidney injury, infusion of ECFC exosomes induced potent functional and histologic protection, associated with increased kidney miR-486-5p levels, decreased PTEN, and activation of Akt. Infusion of exosomes from ECFCs transfected with anti-miR-486-5p had no protective effect. Thus, delivery of ECFC exosomes reduces ischemic kidney injury via transfer of miR-486-5p targeting PTEN. Exosomes enriched in miR-486-5p could represent a therapeutic tool in acute kidney injury.
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Lesión Renal Aguda/metabolismo , Exosomas/metabolismo , MicroARNs/metabolismo , Fosfohidrolasa PTEN/metabolismo , Daño por Reperfusión/metabolismo , Animales , Apoptosis , Células Cultivadas , Células Endoteliales/fisiología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , RatonesRESUMEN
Endothelial colony-forming cells (ECFCs) are decreased in the cord blood of preterm infants with moderate-to-severe bronchopulmonary dysplasia. We quantified ECFCs from infants with congenital diaphragmatic hernia, a neonatal disorder with severe lung hypoplasia. Unlike newborns who develop bronchopulmonary dysplasia, those with congenital diaphragmatic hernia had increased and highly-proliferative cord blood ECFCs.
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Células Endoteliales , Sangre Fetal/citología , Hernias Diafragmáticas Congénitas , Células Madre , Proliferación Celular , Células Cultivadas , Células Endoteliales/fisiología , Femenino , Hernia Diafragmática/sangre , Humanos , Recién Nacido , Recuento de Leucocitos , Leucocitos Mononucleares/fisiología , Masculino , Células Madre/fisiologíaRESUMEN
Nestin, an intermediate filament protein expressed by progenitor cells, is associated with tissue regeneration. Although nestin expression has been reported in poorly differentiated and newly formed blood vessels, its role in endothelial cells remains unclear. In this study, we investigated the involvement of nestin in the angiogenic properties of endothelial colony-forming cells (ECFCs) derived from human umbilical cord blood. Our results demonstrate that ECFCs express high levels of nestin, and that its inhibition by small interfering RNAs decreased ECFC proliferation, migration in response to SDF-1 and VEGF-A, tubulogenesis, and adhesion on collagen. These effects are associated with modulation of focal adhesion kinase phosphorylation. Furthermore, nestin silencing resulted in reduced revascularization in a mouse hindlimb ischemia model. In conclusion, these findings provide evidence that nestin more than being a structural protein, is an active player in ECFC angiogenic properties.
RESUMEN
BACKGROUND: The involvement of thrombin receptor PAR1 in blood vessel development has been largely demonstrated in knockout mice; however, its implication in adult mouse angiogenesis seems very moderate. OBJECTIVES: We aimed to explore the potential relationships between PAR1, stemness, and angiogenic properties of human endothelial colony-forming cells (ECFCs). METHODS AND RESULTS: PAR1 activation on ECFCs using the selective PAR1-activating peptide induced a significant decrease in CD133 expression (RTQ-PCR analysis). In line, silencing of PAR1 gene expression with siRNA increased CD133 mRNA as well as intracellular CD133 protein expression. To confirm the link between CD133 and PAR1, we explored the association between PAR1 and CD133 levels in fast and slow fibroblasts prone to reprogramming. An imbalance between PAR1 and CD133 levels was evidenced, with a decreased expression of PAR1 in fast reprogramming fibroblasts expressing a high CD133 level. Regarding in vitro ECFC angiogenic properties, PAR1 silencing with specific siRNA induced cell proliferation evidenced by the overexpression of Ki67. However, it did not impact migration properties nor ECFC adhesion on smooth muscle cells or human arterial endothelial cells. In a mouse model of hind-limb ischemia, PAR1 silencing in ECFCs significantly increased postischemic revascularization compared to siCtrl-ECFCs along with a significant increase in cutaneous blood flows (P < .0001), microvessel density (P = .02), myofiber regeneration (P < .0001), and human endothelial cell incorporation in muscle (P < .0001). CONCLUSION: In conclusion, our work describes for the first time a link between PAR1, stemness, and vasculogenesis in human ECFCs.
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Células Endoteliales , Receptor PAR-1 , Humanos , Células Cultivadas , Células Endoteliales/metabolismo , Neovascularización Fisiológica , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Cerebral ischemia is a serious disease characterized by brain tissue ischemia and hypoxic necrosis caused by the blockage of blood vessels within the central nervous system. Although stem cell therapy is a promising approach for treating ischemic stroke, the inflammatory, oxidative, and hypoxic environment generated by cerebral ischemia greatly reduces the survival and therapeutic effects of transplanted stem cells. Endothelial colony-forming cells (ECFCs) are a class of precursor cells with strong proliferative potential that can migrate and differentiate directly into mature vascular endothelial cells. Consequently, ECFCs can exert significant therapeutic and reparative effects in diseases associated with vascular injury. Monocyte chemoattractant protein-induced protein 1 (MCPIP-1) exerts multiple biological effects; however, no studies have yet reported its role in the angiogenic function of ECFCs. In this study, we performed Proteome Profiler™ Human Angiogenesis Antibody arrays and tandem mass tag protein profiling to investigate the effect of MCPIP-1 on ECFCs. We demonstrated that MCPIP-1 knockdown enhanced the proliferation, migration, and in vivo and in vitro angiogenic capacity of ECFCs by upregulating the transferrin receptor-activated AKT/m-TOR signaling pathway to promote cellular trophic factor secretion. Furthermore, we found that the lateral ventricular transplantation of ECFCs with lentiviral MCPIP-1 knockdown into mice with middle cerebral artery occlusion increased serum vacular endothelial growth factor(VEGF), angiopoietin-1, and HIF-1a levels, enhanced neovascularization and neurogenesis in the ischemic penumbra, reduced the size of cerebral infarcts, and promoted neurological recovery. Together, these findings suggest new avenues for enhancing the therapeutic efficacy of ECFCs.
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Isquemia Encefálica , Células Endoteliales , Neovascularización Fisiológica , Animales , Humanos , Ratones , Isquemia Encefálica/metabolismo , Células Cultivadas , Células Endoteliales/metabolismo , Isquemia/metabolismo , Isquemia/terapia , Neovascularización Fisiológica/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
BACKGROUND: Assessment of endothelial colony-forming cell (ECFC) number and vasculogenic properties is crucial for exploring vascular diseases and regeneration strategies. A previous survey of the Scientific and Standardization Committee on Vascular Biology of the International Society on Thrombosis and Haemostasis clarified key methodological points but highlighted a lack of standardization associated with ECFC culture. OBJECTIVES: The aim of this study was to provide expert consensus guidance on ECFC isolation and culture. METHODS: We surveyed 21 experts from 10 different countries using a questionnaire proposed during the 2019 International Society on Thrombosis and Haemostasis Congress in Melbourne (Australia) to attain a consensus on ECFC isolation and culture. RESULTS: We report here the consolidated results of the questionnaire. There was agreement on several general statements, mainly the technical aspects of ECFC isolation and cell culture. In contrast, on the points concerning the definition of a colony of ECFCs, the quantification of ECFCs, and the estimation of their age (in days or number of passages), the expert opinions were widely dispersed. CONCLUSION: Our survey clearly indicates an unmet need for rigorous standardization, multicenter comparison of results, and validation of ECFC isolation and culture procedures for clinical laboratory practice and robustness of results. To this end, we propose a standardized protocol for the isolation and expansion of ECFCs from umbilical cord and adult peripheral blood.
Asunto(s)
Técnicas de Cultivo de Célula , Células Endoteliales , Adulto , Humanos , Biología , Australia , Células Cultivadas , Neovascularización FisiológicaRESUMEN
BACKGROUND: Fetal growth restriction (FGR) is associated with deficits in the developing brain, including neurovascular unit (NVU) dysfunction. Endothelial colony forming cells (ECFC) can mediate improved vascular stability, and have demonstrated potential to enhance vascular development and protection. This investigation examined whether ECFCs from human umbilical cord blood (UCB) enhanced NVU development in FGR and appropriate for gestational age (AGA) fetal sheep. METHODS: Twin-bearing ewes had surgery performed at 88-90 days' gestation, inducing FGR in one fetus. At 113 days, ECFCs (1 × 107 cells) cultured from human UCB were administered intravenously to fetal sheep in utero. At 127 days, ewes and their fetuses were euthanised, fetal brains collected, and NVU components analysed by immunohistochemistry. RESULTS: Twenty-four fetal lambs, arranged in four groups: AGA (n = 7), FGR (n = 5), AGA + ECFC (n = 6), and FGR + ECFC (n = 6), were included in analyses. FGR resulted in lower body weight than AGA (P = 0.002) with higher brain/body weight ratio (P = 0.003). ECFC treatment was associated with increased vascular density throughout the brain in both AGA + ECFC and FGR + ECFC groups, as well as increased vascular-astrocyte coverage and VEGF expression in the cortex (P = 0.003, P = 0.0006, respectively) and in the subcortical white matter (P = 0.01, P = 0.0002, respectively) when compared with the untreated groups. CONCLUSIONS: ECFC administration enhanced development of NVU components in both the AGA and FGR fetal brain. Further investigation is required to assess how to optimise the enhanced angiogenic capabilities of ECFCs to provide a therapeutic strategy to protect the developing NVU against vulnerabilities associated with FGR.