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OBJECTIVE: Hypercholesterolemia is an atherosclerotic condition that is associated with impaired neovascularization in response to ischemia. This study sought to define the role of microRNAs in that pathophysiology. APPROACH AND RESULTS: Next-generation sequencing and quantitative reverse transcription polymerase chain reaction analyses identified miR-150 as a proangiogenic microRNA, which expression is significantly reduced in the ischemic muscles of hypercholesterolemic apolipoprotein E-deficient (ApoE-/-) mice, and in human umbilical vein endothelial cells exposed to oxidized low-density lipoprotein. Forced expression of miR-150 using a miR mimic could rescue oxidized low-density lipoprotein-mediated impairment of endothelial cell migration and tubule formation in vitro. In a mouse model of hindlimb ischemia, intramuscular injection of miR-150 mimic restored blood flow recuperation, vascular densities in ischemic muscles, and functional mobility in ApoE-/- mice. Treatment of ApoE-/- mice with miR-150 also increased the number and the activities of proangiogenic cells. miR-150 targets SRC kinase signaling inhibitor 1, an important regulator of Src (proto-oncogene tyrosine-protein kinase Src) activity. Here we found that hypercholesterolemia and oxidized low-density lipoprotein exposure are associated with increased SRC kinase signaling inhibitor 1 expression and decreased Src activity. However, treatment with miR-150 mimic reduces SRC kinase signaling inhibitor 1 expression and restores Src and downstream endothelial nitric oxide synthase and Akt (protein kinase B) activities both in vitro and in vivo. We also demonstrate the interrelation between miR-150 and SRC kinase signaling inhibitor 1 and their importance for endothelial cell angiogenic activities. CONCLUSIONS: Hypercholesterolemia is associated with reduced expression of miR-150, impaired Src signaling, and inefficient neovascularization in response to ischemia. Forced expression of miR-150 using a miR mimic could constitute a novel therapeutic strategy to improve ischemia-induced neovascularization in atherosclerotic conditions.
Assuntos
Aterosclerose/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Isquemia/metabolismo , MicroRNAs/metabolismo , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/fisiopatologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fenoterol , Predisposição Genética para Doença , Membro Posterior , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Isquemia/genética , Isquemia/fisiopatologia , Lipoproteínas LDL/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Transdução de Sinais , Transfecção , Quinases da Família src/metabolismoRESUMO
This study sought to determine the potential role of microRNAs (miRNAs) in the detrimental effects of cigarette smoke on angiogenesis and neovascularization. Using large-scale miRNA profiling and qRT-PCR analyses, we identified let-7f as a pro-angiogenic miRNA which expression is significantly reduced in HUVECs treated with cigarette smoke extracts (CSE), and in the ischemic muscles of mice that are exposed to cigarette smoke (MES). In a mouse model of hindlimb ischaemia, intramuscular injection of let-7f mimic restored ischaemia-induced neovascularization in MES. Doppler flow ratios and capillary density in ischemic muscles were significantly improved in MES treated with let-7f mimic. Clinically, this was associated with reduced ambulatory impairment and hindlimb ischaemic damage. Treatment with let-7f mimic could also rescue pro-angiogenic cell (PAC) number and function (attachment, proliferation, migration) in MES. ALK5 (TGF-ßR1), an important modulator of angiogenesis, is a target of let-7f. Here we show that ALK5 is increased in HUVECs exposed to CSE and in the ischaemic muscles of MES. This is associated with a downstream activation of the anti-angiogenic factors SMAD2/3 and PAI-1. Importantly, treatment with let-7f mimic reduces the expression of ALK5, SMAD2/3 and PAI-1 both in vitro and in vivo. Moreover, let-7f overexpression or ALK5 inhibition can rescue angiogenesis in HUVECs exposed to CSE. Cigarette smoke exposure is associated with reduced expression of let-7f and activation of the anti-angiogenic TGF-ß/ALK5 pathway. Overexpression of let-7f using a miRNA mimic could constitute a novel therapeutic strategy to improve ischaemia-induced neovascularization in pathological conditions.
Assuntos
Regulação da Expressão Gênica , Isquemia/patologia , MicroRNAs/metabolismo , Neovascularização Patológica/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fumar/efeitos adversos , Fator de Crescimento Transformador beta/metabolismo , Animais , Contagem de Células , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Isquemia/genética , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neovascularização Patológica/patologia , Receptor do Fator de Crescimento Transformador beta Tipo I , Transdução de SinaisRESUMO
MicroRNAs (miRs) regulate physiological and pathological processes, including ischemia-induced angiogenesis and neovascularization. They can be transferred between cells by extracellular vesicles (EVs). However, the specific miRs that are packaged in EVs released from skeletal muscles, and how this process is modulated by ischemia, remain to be determined. We used a mouse model of hindlimb ischemia and next generation sequencing (NGS) to perform a complete profiling of miR expression and determine the effect of ischemia in skeletal muscles, and in EVs of different sizes (microvesicles (MVs) and exosomes) released from these muscles. Ischemia significantly modulated miR expression in whole muscles and EVs, increasing the levels of several miRs that can have pro-angiogenic effects (angiomiRs). We found that specific angiomiRs are selectively enriched in MVs and/or exosomes in response to ischemia. In silico approaches indicate that these miRs modulate pathways that play key roles in angiogenesis and neovascularization, including HIF1/VEGF signaling, regulation of actin cytoskeleton and focal adhesion, NOTCH, PI3K/AKT, RAS/MAPK, JAK/STAT, TGFb/SMAD signaling and the NO/cGMP/PKG pathway. Thus, we show for the first time that angiomiRs are selectively enriched in MVs and exosomes released from ischemic muscles. These angiomiRs could be targeted in order to improve the angiogenic function of EVs for potential novel therapeutic applications in patients with severe ischemic vascular diseases.
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Vesículas Extracelulares , Isquemia , MicroRNAs , Músculo Esquelético , Neovascularização Fisiológica , Animais , Músculo Esquelético/metabolismo , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/patologia , Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , MicroRNAs/genética , Isquemia/metabolismo , Isquemia/patologia , Camundongos , Membro Posterior/irrigação sanguínea , Membro Posterior/patologia , Camundongos Endogâmicos C57BL , Transdução de Sinais , Masculino , Exossomos/metabolismo , Neovascularização Patológica/metabolismo , AngiogêneseRESUMO
BACKGROUND AND AIMS: The NO-cGMP pathway is essential for angiogenesis, vasculogenesis and post-natal neovascularization. The key enzyme responsible for the synthesis of cGMP following binding of NO is soluble guanylate cyclase (sGC). Riociguat is the first member of a novel class of compounds known as sGC stimulators. We tested the hypothesis that stimulation of sGC with riociguat might improve neovascularization in response to ischemia. METHODS: In vitro, the angiogenic effect of riociguat was tested in human umbilical vein endothelial cells (HUVECs). In vivo, neovascularization was investigated in a mouse model of limb ischemia. C57Bl/6 mice were treated by gavage with 3 mg/kg/day of riociguat for a total of 28 days. After two weeks of treatment, hindlimb ischemia was surgically induced by femoral artery removal. RESULTS: In a matrigel assay in vitro, riociguat dose-dependently stimulates tubule formation in HUVECs. Cell migration (scratch assay) is also increased in HUVECs treated with riociguat. At the molecular level, riociguat treatment leads to rapid activation of the p44/p42 MAP kinase pathway in HUVECs. Inhibition of protein kinase G (PKG) activity supresses both p44/p42 MAP kinase activation and angiogenesis in HUVECs treated with riociguat. In vivo, treatment with riociguat improves blood flow recovery after ischemia (Laser Doppler imaging), and increases capillary density in ischemic muscles (CD31 immunostaining). Clinically, this is associated with a significant decrease of ambulatory impairment and ischemic damages. Interestingly, mice treated with riociguat also show a 94% increase in the number of bone marrow-derived pro-angiogenic cells (PACs) compared to control mice. Moreover, riociguat treatment is associated with a significant improvement of PAC functions including migratory capacity, adhesion to an endothelial monolayer, and integration into endothelial tubular networks. CONCLUSIONS: The sGC stimulator riociguat promotes angiogenesis and improves neovascularization after ischemia. The mechanism involves PKG-dependent activation of p44/p42 MAP kinase pathway, together with an improvement of PAC number and functions. sGC stimulation could constitute a novel therapeutic strategy to reduce tissue ischemia in patients with severe atherosclerotic diseases.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno , Neovascularização Patológica , Humanos , Camundongos , Animais , Guanilil Ciclase Solúvel/farmacologia , Isquemia , Células Endoteliais da Veia Umbilical Humana , Neovascularização Fisiológica , Membro PosteriorRESUMO
OBJECTIVE: To investigate the effect of oxidative stress on ischemia-induced neovascularization in copper-zinc (CuZn) superoxide dismutase (SOD)-deficient mice. METHODS AND RESULTS: In the vascular wall, CuZnSOD is essential for protecting against excessive oxidative stress and maintaining endothelial function. However, its specific role for the development of new vessels in response to ischemia is unknown. After surgically induced hind limb ischemia, CuZnSOD-deficient mice showed impaired neovascularization, as assessed by blood flow recuperation (laser Doppler) and capillary density in the ischemic muscles. This was associated with increased levels of oxidative stress in ischemic tissues and peripheral blood, together with reduced plasmatic NO production. CuZnSOD-deficient mice demonstrated an important reduction in the number of endothelial progenitor cells (EPCs) in the bone marrow and spleen. Moreover, EPCs isolated from CuZnSOD-deficient mice showed increased oxidative stress levels, decreased NO production, and a reduced ability to migrate and integrate into capillary-like networks. Importantly, the functional activities of CuZnSOD-deficient EPCs were rescued after treatment with the SOD-mimetic Tempol (a membrane-permeable radical scavenger) or the NO donor sodium nitroprusside (SNP). Moreover, the neovascularization defect in CuZnSOD-deficient mice could be rescued by wild-type (but not CuZnSOD-deficient) EPC supplementation. CONCLUSIONS: Protection against oxidative stress by CuZnSOD may be essential for EPC function and reparative neovascularization after ischemia.
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Células Endoteliais/fisiologia , Membro Posterior/irrigação sanguínea , Isquemia/fisiopatologia , Neovascularização Fisiológica/fisiologia , Células-Tronco/fisiologia , Superóxido Dismutase/fisiologia , Animais , Células da Medula Óssea , Capilares/fisiopatologia , Modelos Animais de Doenças , Feminino , Fluxometria por Laser-Doppler , Masculino , Camundongos , Estresse OxidativoRESUMO
OBJECTIVE: Because Nox2-containing NADPH oxidase is a major source of ROS in the vasculature, we investigated its potential role for the modulation of ischemia-induced neovascularization in conditions of increased oxidative stress. METHODS AND RESULTS: To mimic a clinical situation of increased oxidative stress, mice were exposed to cigarette smoke before and after the surgical induction of hindlimb ischemia. Nox2 expression and oxidative stress in ischemic tissues were significantly increased in wild-type mice, but not in mice deficient for the Nox2-containing NADPH oxidase (Nox2(-/-)). Nox2(-/-) mice demonstrated faster blood flow recovery, increased capillary density in ischemic muscles, and improved endothelial progenitor cell functional activities compared to Nox2(+/+) mice. In addition, Nox2 deficiency was associated with increased antioxidant and nitrite concentrations in plasma, together with a preserved expression of eNOS in ischemic tissues. In vitro, Nox2(-/-) endothelial cells exhibit resistance against superoxide induction and improved VEGF-dependent angiogenic activities compared to Nox2(+/+) endothelial cells. Importantly, the beneficial effects of Nox2 deficiency on neovascularization in vitro and in vivo were lost after treatment with the NO inhibitor L-NAME. CONCLUSIONS: Nox2-containing NADPH oxidase deficiency protects against ischemia in conditions of increased oxidative stress. The mechanism involves improved neovascularization through a reduction of ROS formation, preserved activation of the VEGF/NO angiogenic pathway, and improved functional activities of endothelial progenitor cells.
Assuntos
Membro Posterior/irrigação sanguínea , Isquemia/prevenção & controle , Glicoproteínas de Membrana/fisiologia , NADPH Oxidases/fisiologia , Estresse Oxidativo , Animais , Células Endoteliais/fisiologia , Isquemia/metabolismo , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidase 2 , NADPH Oxidases/deficiência , Neovascularização Fisiológica , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fumaça/efeitos adversos , Células-Tronco/fisiologia , Nicotiana/efeitos adversosRESUMO
BACKGROUND: Classical cardiovascular risk factors (CRFs) are associated with impaired angiogenic activities of bone marrow-derived proangiogenic cells (PACs) related to peripheral artery diseases (PADs) and ischemia-induced neovascularization. MicroRNAs (miRs) are key regulators of gene expression, and they are involved in the modulation of PAC function and PAC paracrine activity. However, the effects of CRFs on the modulation of miR expression in PACs are unknown. AIMS AND METHODS: We used a model of hindlimb ischemia and next-generation sequencing to perform a complete profiling of miRs in PACs isolated from the bone marrow of mice subjected to three models of CRFs: aging, smoking (SMK) and hypercholesterolemia (HC). RESULTS: Approximately 570 miRs were detected in PACs in the different CRF models. When excluding miRs with a very low expression level (<100 RPM), 40 to 61 miRs were found to be significantly modulated by aging, SMK, or HC. In each CRF condition, we identified downregulated proangiogenic miRs and upregulated antiangiogenic miRs that could contribute to explain PAC dysfunction. Interestingly, several miRs were similarly downregulated (e.g., miR-542-3p, miR-29) or upregulated (e.g., miR-501, miR-92a) in all CRF conditions. In silico approaches including Kyoto Encyclopedia of Genes and Genomes and cluster dendogram analyses identified predictive effects of these miRs on pathways having key roles in the modulation of angiogenesis and PAC function, including vascular endothelial growth factor signaling, extracellular matrix remodeling, PI3K/AKT/MAPK signaling, transforming growth factor beta (TGFb) pathway, p53, and cell cycle progression. CONCLUSION: This study describes for the first time the effects of CRFs on the modulation of miR profile in PACs related to PAD and ischemia-induced neovascularization. We found that several angiogenesis-modulating miRs are similarly altered in different CRF conditions. Our findings constitute a solid framework for the identification of miRs that could be targeted in PACs in order to improve their angiogenic function and for the future development of novel therapies to improve neovascularization and reduce tissue damage in patients with severe PAD.
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Aging is associated with impaired neovascularization in response to ischemia. MicroRNAs are small noncoding RNAs emerging as key regulators of physiological and pathological processes. Here we investigated the potential role of microRNAs in endothelial cell senescence and age-dependent impairment of neovascularization. Next generation sequencing and qRT-PCR analyses identified miR-130a as a pro-angiogenic microRNA which expression is significantly reduced in old mouse aortic endothelial cells (ECs). Transfection of young ECs with a miR-130a inhibitor leads to accelerated senescence and reduced angiogenic functions. Conversely, forced expression of miR-130a in old ECs reduces senescence and improves angiogenesis. In a mouse model of hindlimb ischemia, intramuscular injection of miR-130a mimic in older mice restores blood flow recovery and vascular densities in ischemic muscles, improves mobility and reduces tissue damage. miR-130a directly targets antiangiogenic homeobox genes MEOX2 and HOXA5. MEOX2 and HOXA5 are significantly increased in the ischemic muscles of aging mice, but forced expression of miR-130a reduces the expression of these factors. miR-130a treatment after ischemia is also associated with increased number and improved functional activities of pro-angiogenic cells (PACs). Forced expression of miR-130a could constitute a novel strategy to improve blood flow recovery and reduce ischemia in older patients with ischemic vascular diseases.
Assuntos
Envelhecimento/genética , Senescência Celular/genética , Endotélio Vascular/patologia , Isquemia/patologia , MicroRNAs/metabolismo , Neovascularização Fisiológica/genética , Fatores Etários , Idoso , Animais , Aorta/citologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Senescência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Endotélio Vascular/citologia , Membro Posterior/irrigação sanguínea , Proteínas de Homeodomínio/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , MicroRNAs/agonistas , MicroRNAs/antagonistas & inibidores , Neovascularização Fisiológica/efeitos dos fármacos , Cultura Primária de Células , Fatores de Transcrição/genética , Adulto JovemRESUMO
OBJECTIVE: Adaptive thermogenesis, which is partly mediated by sympathetic input on brown adipose tissue (BAT), is a mechanism of heat production that confers protection against prolonged cold exposure. Various endogenous stimuli, for example, norepinephrine and FGF-21, can also promote the conversion of inguinal white adipocytes to beige adipocytes, which may represent a secondary cell type that contributes to adaptive thermogenesis. We previously identified an essential role of the molecular scaffold 14-3-3ζ in adipogenesis, but one of the earliest, identified functions of 14-3-3ζ is its regulatory effects on the activity of tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of norepinephrine. Herein, we examined whether 14-3-3ζ could influence adaptive thermogenesis via actions on BAT activation or the beiging of white adipocytes. METHODS: Transgenic mice over-expressing a TAP-tagged human 14-3-3ζ molecule or heterozygous mice without one allele of Ywhaz, the gene encoding 14-3-3ζ, were used to explore the contribution of 14-3-3ζ to acute (3 h) and prolonged (3 days) cold (4 °C) exposure. Metabolic caging experiments, PET-CT imaging, and laser Doppler imaging were used to determine the effect of 14-3-3ζ over-expression on thermogenic and vasoconstrictive mechanisms in response to cold. RESULTS: Transgenic over-expression of 14-3-3ζ (TAP) in male mice significantly improved tolerance to acute and prolonged cold. In response to cold, body temperatures in TAP mice did not decrease to the same extent when compared to wildtype (WT) mice, and this was associated with increased UCP1 expression in beige inguinal white tissue (iWAT) and BAT. Of note was the paradoxical finding that cold-induced changes in body temperatures of TAP mice were associated with significantly decreased energy expenditure. The marked improvements in tolerance to prolonged cold were not due to changes in sensitivity to ß-adrenergic stimulation or BAT or iWAT oxidative metabolism; instead, over-expression of 14-3-3ζ significantly decreased thermal conductance and heat loss in mice via increased peripheral vasoconstriction. CONCLUSIONS: Despite being associated with elevations in cold-induced UCP1 expression in brown or beige adipocytes, these findings suggest that 14-3-3ζ regulates an alternative, non-thermogenic mechanism via vasoconstriction to minimize heat loss during cold exposure.
Assuntos
Proteínas 14-3-3/metabolismo , Termogênese/fisiologia , Termotolerância/fisiologia , Proteínas 14-3-3/fisiologia , Adipogenia/fisiologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Marrom/fisiologia , Tecido Adiposo Branco/metabolismo , Animais , Regulação da Temperatura Corporal , Temperatura Baixa , Resposta ao Choque Frio/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Mitocondriais/metabolismo , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Termogênese/genética , Termotolerância/genética , Proteína Desacopladora 1/metabolismoRESUMO
BACKGROUND AND AIMS: MicroRNA (miR)-146 is a key regulator of inflammation, endothelial activation and atherosclerosis. This study sought to define its potential role for the modulation of ischemia-induced neovascularization in atherosclerotic conditions. METHODS: Next generation sequencing and qRT-PCR analyses were used to compare microRNA expression in the ischemic muscles of hypercholesterolemic ApoE-deficient (ApoE-/-) mice vs. wild type mice, and in HUVECs exposed or not to oxLDL. Neovascularization was investigated in a mouse model of hindlimb ischemia and the functional activities of HUVECs and pro-angiogenic cells (PACs) were assessed in vitro. RESULTS: We found that miR-146b (but not miR-146a) is significantly reduced in the ischemic muscles of ApoE-/- mice, and in HUVECs exposed to oxLDL. Inhibition of miR-146b reduces angiogenesis in vitro, whereas forced expression of miR-146b rescues oxLDL-mediated impairment of endothelial cell proliferation and tube formation. Mechanistically, miR146b directly targets tumor necrosis factor-alpha (TNFa) Receptor Associated Factor 6 (TRAF6) to inhibit inflammation. We found that hypercholesterolemia and oxLDL exposure are associated with higher levels of TRAF6, and increased expression of TNFa. However, forced expression of miR-146b in high cholesterol conditions reduces the expression of these inflammatory factors. In vivo, intramuscular injection of miR-146b mimic reduces ischemic damages and restores blood flow recuperation and capillary density in the ischemic muscles of ApoE-/- mice. Treatment with miR-146b also increases the number and functional activities of pro-angiogenic cells (PACs). CONCLUSIONS: Hypercholesterolemia is associated with reduced expression of miR-146b, which increases TRAF6-dependent inflammation and is associated with poor neovascularization in response to ischemia. Forced expression of miR-146b using a miR mimic could constitute a novel therapeutic strategy to improve ischemia-induced neovascularization in atherosclerotic conditions.
Assuntos
Hipercolesterolemia/metabolismo , Inflamação/metabolismo , MicroRNAs/genética , Neovascularização Patológica/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Animais , Velocidade do Fluxo Sanguíneo , Movimento Celular , Proliferação de Células , Membro Posterior/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Isquemia/fisiopatologia , Lipoproteínas LDL/metabolismo , Camundongos , Camundongos Knockout para ApoE , MicroRNAs/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Análise de Sequência de RNA , Células THP-1RESUMO
Moderate consumption of red wine is associated with a decreased incidence of cardiovascular diseases in populations with relatively high amount of fat in the diet. However, the mechanisms involved in this protective effect are not completely understood. Here we show that moderate consumption of red wine (equivalent to 2 glasses/day in humans) but not ethanol only, improves blood flow recovery by 32% after hindlimb ischemia in hypercholesterolemic ApoE-deficient mice. In ischemic tissues, red wine consumption reduces oxidative stress and increases capillary density by 46%. Endothelial progenitor cells (EPCs) have been shown to have an important role in postnatal neovascularization. We found that the number of EPCs is increased by 60% in ApoE mice exposed to red wine. Moreover, the migratory capacity of EPCs is significantly improved in red wine-drinking mice. The wine used in our study is a cabernet sauvignon from Languedoc-Roussillon, France, which contains a relatively high concentration (4-6 mg/L) of the polyphenolic antioxidant resveratrol. We demonstrate that resveratrol can rescue oxidized low-density lipoprotein (oxLDL)-induced impairment of in vitro angiogenic activities in human umbilical vein endothelial cells (HUVECs). Resveratrol exposure is also associated with increased activation of Akt/eNOS together with a restoration of nitric oxide production in HUVECs exposed to oxLDL. Our study suggests that moderate consumption of red wine improves ischemia-induced neovascularization in high-cholesterol conditions by increasing the number and the functional activities of EPCs and by restoring the Akt-eNOS-NO pathway.
Assuntos
Células-Tronco Adultas/patologia , Apolipoproteínas E/deficiência , Endotélio Vascular/patologia , Isquemia/complicações , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/etiologia , Óxido Nítrico/fisiologia , Vinho , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Apolipoproteínas E/genética , Movimento Celular/genética , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Membro Posterior/irrigação sanguínea , Humanos , Hipercolesterolemia/complicações , Hipercolesterolemia/genética , Hipercolesterolemia/fisiopatologia , Isquemia/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/genética , Neovascularização Patológica/fisiopatologia , Óxido Nítrico/metabolismo , Resveratrol , Estilbenos/farmacologiaRESUMO
Adverse perinatal conditions can lead to developmental programming of cardiovascular diseases. Prematurely born infants are often exposed to high oxygen levels, which in animal models has been associated with endothelial dysfunction, hypertension, and cardiac remodeling during adulthood. Here we found that adult mice that have been transiently exposed to O2 after birth show defective neovasculariation after hindlimb ischemia, as demonstrated by impaired blood flow recovery, reduced vascular density in ischemic muscles and increased tissue damages. Ischemic muscles isolated from mice exposed to O2 after birth exhibit increased oxidative stress levels and reduced expression of superoxide dismutase 1 (SOD1) and vascular endothelial growth factor (VEGF). Pro-angiogenic cells (PACs) have been shown to have an important role for postnatal neovascularisation. We found that neonatal exposure to O2 is associated with reduced number of PACs in adults. Moreover, the angiogenic activities of both PACs and mature mouse aortic endothelial cells (MAECs) are significantly impaired in mice exposed to hyperoxia after birth. Our results indicate that neonatal exposure to high oxygen levels leads to impaired ischemia-induced neovascularization during adulthood. The mechanism involves deleterious effects on oxidative stress levels and angiogenic signals in ischemic muscles, together with dysfunctional activities of PACs and mature endothelial cells.
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Membro Posterior/irrigação sanguínea , Hiperóxia/fisiopatologia , Isquemia/fisiopatologia , Neovascularização Fisiológica/fisiologia , Animais , Animais Recém-Nascidos , Adesão Celular , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos Endogâmicos C57BL , Oxigênio/efeitos adversos , Fluxo Sanguíneo Regional , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: Endothelial dysfunction is one of the earliest pathological effects of cigarette smoking. It has recently been suggested that endothelial progenitor cells (EPCs) could contribute to ongoing endothelial maintenance and repair. Accordingly, we tested the hypothesis that cigarette smoking is associated with EPC dysfunction. METHODS AND RESULTS: EPCs were isolated from the peripheral venous blood of 15 healthy smokers and 11 age-matched nonsmokers. The number of EPCs was significantly reduced in smokers versus control subjects (51.6+/-1.9 versus 120.3+/-10.0 per power field, p<0.001). Moreover, the functional activities of EPCs isolated from smokers were severely compromised. First, the proliferative and migratory response of EPCs isolated from smokers were reduced by 75% and 19%, respectively (p<0.05). Second, EPCs from smokers showed an important decreased adherence to HUVECs that had been previously activated with tumor necrosis factor-alpha (TNF-alpha) (p<0.01). Finally, the participation of EPCs to tube formation in a matrigel assay was reduced by 38% in smokers versus control subjects (p<0.001). We found that EPCs from smokers had a significant reduction in the expression of the endothelial cell-specific markers (VE-cadherin, KDR, and vWF). Moreover, ROS formation was significantly increased in EPCs from smokers, whereas the serum antioxidant and nitrite levels of smokers were reduced and correlated with impaired EPC number and functional activity. CONCLUSIONS: Cigarette smoking is associated with a reduced number of EPCs together with an important impairment of EPC differentiation and functional activities. Our results suggest that EPC dysfunction could contribute to impair blood vessel healing and growth in smokers.
Assuntos
Aterosclerose/patologia , Endotélio Vascular/patologia , Fumar/patologia , Células-Tronco/patologia , Adulto , Antioxidantes/metabolismo , Aterosclerose/sangue , Biomarcadores , Adesão Celular , Contagem de Células , Divisão Celular , Movimento Celular , Colágeno , Combinação de Medicamentos , Endotélio Vascular/metabolismo , Feminino , Humanos , Laminina , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Estresse Oxidativo , Proteoglicanas , Espécies Reativas de Oxigênio/metabolismo , Fumar/sangue , Células-Tronco/metabolismoRESUMO
The protein tyrosine phosphatase PTPRJ/DEP-1 has been implicated in negative growth regulation in endothelial cells, where its expression varies at transitions between proliferation and contact inhibition. However, in the same cells, DEP-1 has also been implicated in VEGF-dependent Src activation, permeability, and capillary formation, suggesting a positive role in regulating these functions. To resolve this dichotomy in vivo, we investigated postnatal angiogenesis and vascular permeability in a DEP-1-deficient mouse. In this study, we report that DEP-1 is required for Src activation and phosphorylation of its endothelial cell-specific substrate, VE-cadherin, after systemic injection of VEGF. Accordingly, VEGF-induced vascular leakage was abrogated in the DEP-1-deficient mice. Furthermore, capillary formation was impaired in murine aortic tissue rings or Matrigel plugs infused with VEGF. In the absence of DEP-1, angiogenesis triggered by ischemia or during tumor formation was defective, which in the latter case was associated with reduced tumor cell proliferation and increased apoptosis. Macrophage infiltration was also impaired, reflecting reduced vascular permeability in the tumors or a possible cell autonomous effect of DEP-1. Consequently, the formation of spontaneous and experimental lung metastases was strongly decreased in DEP-1-deficient mice. In clinical specimens of cancer, less vascularized tumors exhibited lower microvascular expression of DEP-1. Altogether, our results established DEP-1 as an essential driver of VEGF-dependent permeability, angiogenesis, and metastasis, suggesting a novel therapeutic route to cancer treatment. Cancer Res; 76(17); 5080-91. ©2016 AACR.
Assuntos
Permeabilidade Capilar/fisiologia , Neoplasias Experimentais/patologia , Neovascularização Patológica/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Progressão da Doença , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Experimentais/metabolismoRESUMO
BACKGROUND: Renin is the rate limiting step for the activation of the renin-angiotensin-aldosterone system, which is linked to the development of endothelial dysfunction, hypertension and atherosclerosis. However, the specific role of renin during physiological responses to tissue ischemia is currently unknown. Aliskiren is the only direct renin inhibitor that is clinically used as an orally active antihypertensive drug. Here we tested the hypothesis that aliskiren might improve neovascularization in response to ischemia. METHODS AND RESULTS: At a dose that did not modulate blood pressure (10 mg/kg), aliskiren led to improved blood flow recovery after hindlimb ischemia in C57BL/6 mice (Doppler flow ratios 0.71 ± 0.07 vs. 0.55 ± 0.03; P < 0.05). In ischemic muscles, treatment with aliskiren was associated with a significant increase of vascular density, reduced oxidative stress levels and increased expression of VEGF and eNOS. Aliskiren treatment also significantly increased the number of bone marrow-derived endothelial progenitor cells (EPCs) after hindlimb ischemia. Moreover, the angiogenic properties of EPCs (migration, adhesion, integration into tubules) were significantly improved in mice treated with aliskiren. In vitro, aliskiren improves cellular migration and tubule formation in HUVECs. This is associated with an increased expression of nitric oxide (NO), and a significant reduction of oxidative stress levels. Importantly, the angiogenic properties of aliskiren in vitro and in vivo are completely abolished following treatment with the NOS inhibitor l-NAME. CONCLUSION: Direct renin inhibition with aliskiren leads to improved ischemia-induced neovascularization that is not dependant on blood pressure lowering. The mechanism involves beneficial effects of aliskiren on oxidative stress and NO angiogenic pathway, together with an increase in the number and the functional activities of EPCs.
Assuntos
Amidas/química , Pressão Sanguínea/efeitos dos fármacos , Fumaratos/química , Isquemia/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Renina/antagonistas & inibidores , Animais , Anti-Hipertensivos/química , Células da Medula Óssea/citologia , Adesão Celular , Movimento Celular , Células Endoteliais/citologia , Células Progenitoras Endoteliais/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , NG-Nitroarginina Metil Éster/química , Óxido Nítrico/química , Estresse Oxidativo , Oxigênio/química , Espécies Reativas de Oxigênio , Sistema Renina-Angiotensina/efeitos dos fármacos , Superóxidos/químicaRESUMO
BACKGROUND: Psychological stress (PS) has been associated with the development of cardiovascular diseases and adverse long-term outcomes after ischemic events. However, the precise mechanisms involved are not completely understood. Here we investigated the effect of PS on ischemia-induced neovascularization, and the potential therapeutic effect of fluoxetine in this condition. METHODS AND RESULTS: Balb/c mice were subjected or not to chronic restraint stress. After 3 weeks, hindlimb ischemia was surgically induced by femoral artery removal. We found that blood flow recovery was significantly impaired in mice exposed to PS compared to controls (Doppler flow ratio (DFR) 0.61 ± 0.07 vs. 0.80 ± 0.07, p < 0.05). At the microvascular level, capillary density was significantly reduced in ischemic muscles of mice exposed to PS (38 ± 1 vs. 74 ± 3 capillaries per field, p < 0.001). This correlated with increased oxidative stress levels and reduced expression of VEGF and VEGF signalling molecules (p44/p42 MAPK, Akt) in ischemic muscles. We found that the number of pro-angiogenic cells (PACs) was significantly reduced in mice exposed to PS. In addition, oxidative stress levels (DCF-DA, DHE) were increased in PACs isolated from mice exposed to PS, and this was associated with impaired PAC functional activities (migration, adhesion, and integration into tubules). Importantly, treatment of mice exposed to PS with the selective serotonin reuptake inhibitor (SSRI) fluoxetine improved all the angiogenic parameters, and completely rescued PS-induced impairment of neovascularization. CONCLUSION: PS impairs ischemia-induced neovascularization. Potential mechanisms involved include reduced activation of the VEGF pathway in ischemic tissues, increased oxidative stress levels and reduced number and functional activities of PACs. Our results suggest that fluoxetine may represent a novel therapeutic strategy to improve neovascularization and reduce ischemia in patients suffering from cardiovascular diseases and exposed to PS.
Assuntos
Fluoxetina/uso terapêutico , Isquemia/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Estresse Psicológico , Animais , Antidepressivos de Segunda Geração/uso terapêutico , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Peso Corporal , Movimento Celular , Colágeno/química , Combinação de Medicamentos , Células Endoteliais/metabolismo , Membro Posterior/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana , Humanos , Isquemia/psicologia , Laminina/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estresse Oxidativo , Proteoglicanas/química , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Elsibucol is a metabolically stable derivative of probucol with antioxidant, anti-inflammatory and antiproliferative properties. Here we investigated the effect of elsibucol on the development of atherosclerosis following arterial injury in hypercholesterolemic rabbits. METHODS AND RESULTS: New Zealand White rabbits were fed a high cholesterol diet that was supplemented or not with 0.5% elsibucol, 1% elsibucol or 1% probucol. An angioplasty of the iliac artery was performed after 3 weeks of diet. We found that treatment with elsibucol significantly decreases blood total cholesterol, LDLc and triglyceride levels. This is associated with a significant 46% reduction of neointimal hyperplasia following arterial injury. Interestingly, the effect of elsibucol on cholesterol levels and neointimal formation appears to be more pronounced than that of probucol. In vitro, elsibucol reduces vascular smooth muscle cell proliferation without affecting cell viability. In vivo, treatment with elsibucol is associated with a significant reduction of cellular proliferation (PCNA immunostaining), oxidative stress (nitrotyrosine immunostaining), VCAM-1 expression and macrophage infiltration in injured arteries. Despite its potent effect on neointimal hyperplasia, elsibucol does not prevent endothelial healing (Evans blue staining) following arterial injury. CONCLUSIONS: In hypercholesterolemic animals, elsibucol inhibits atherosclerosis and preserves endothelial healing following arterial injury. The mechanisms involved include lowering of blood cholesterol levels together with a reduction of oxidative stress and inflammation in injured arteries.
Assuntos
Artérias/patologia , Aterosclerose/tratamento farmacológico , Butiratos/uso terapêutico , Colesterol/sangue , Inflamação/sangue , Estresse Oxidativo , Fenóis/uso terapêutico , Doenças Vasculares/tratamento farmacológico , Animais , Antioxidantes/química , Artérias Carótidas/patologia , Proliferação de Células , Sobrevivência Celular , LDL-Colesterol/sangue , Endotélio Vascular/patologia , Hipercolesterolemia/tratamento farmacológico , Artéria Ilíaca/patologia , Imuno-Histoquímica , Masculino , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Neointima/patologia , CoelhosRESUMO
BACKGROUND: Fish oil consumption has been associated with a reduced incidence of cardiovascular diseases. However, the precise mechanisms involved are not completely understood. Here we tested the hypothesis that a fish oil-enriched diet improves neovascularization in response to ischemia. METHODS AND RESULTS: C57Bl/6 mice were fed a diet containing either 20% fish oil, rich in long-chain n-3 polyunsaturated fatty acids (PUFAs), or 20% corn oil, rich in n-6 PUFAs. After 4 weeks, hindlimb ischemia was surgically induced by femoral artery removal. We found that blood flow recovery was significantly improved in mice fed a fish oil diet compared to those fed a corn oil diet (Doppler flow ratio (DFR) at day 21 after surgery 78 ± 5 vs. 56 ± 4; p < 0.01). Clinically, this was associated with a significant reduction of ambulatory impairment and ischemic damage in the fish oil group. At the microvascular level, capillary density was significantly improved in ischemic muscles of mice fed a fish oil diet. This correlated with increased expression of VEGF and eNOS in ischemic muscles, and higher NO concentration in the plasma. Endothelial progenitor cells (EPCs) have been shown to have an important role for postnatal neovascularization. We found that the number of EPCs was significantly increased in mice fed a fish oil diet. In addition, oxidative stress levels (DCF-DA, DHE) were reduced in EPCs isolated from mice exposed to fish oil, and this was associated with improved EPC functional activities (migration and integration into tubules). In vitro, treatment of EPCs with fish oil resulted in a significant increase of cellular migration. In addition, the secretion of angiogenic growth factors including IL6 and leptin was significantly increased in EPCs exposed to fish oil. CONCLUSION: Fish oil-enriched diet is associated with improved neovascularization in response to ischemia. Potential mechanisms involved include activation of VEGF/NO pathway in ischemic tissues together with an increase in the number and the functional activities of EPCs.
Assuntos
Indutores da Angiogênese/farmacologia , Endotélio Vascular/efeitos dos fármacos , Óleos de Peixe/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Isquemia/prevenção & controle , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Peso Corporal , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Óleo de Milho/farmacologia , Modelos Animais de Doenças , Endotélio Vascular/fisiologia , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Membro Posterior/irrigação sanguínea , Isquemia/dietoterapia , Isquemia/fisiopatologia , Lipídeos/sangue , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/fisiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Aging is associated with increased oxidative stress levels and impaired neovascularization following ischemia. Because Nox2-containing NADPH oxidase is a major source of ROS in the vasculature, we investigated its potential role for the modulation of ischemia-induced neovascularization in the context of aging. METHODS AND RESULTS: Hindlimb ischemia was surgically induced by femoral artery removal in young (2 months) and old (10 months) Nox2-deficient (Nox2(-/-)) and wild type mice. We found that Nox2 expression is increased by aging in ischemic muscles of wild type mice. This is associated with a significant reduction of blood flow recovery after ischemia in old compared to young mice at day 21 after surgery (Doppler flow ratios: 0.51 ± 0.05 vs. 0.72 ± 0.05; p < 0.05). We also demonstrate that capillary and arteriolar densities are significantly reduced in ischemic muscles of old animals, while oxidative stress levels are increased (nitrotyrosine immunostaining). Importantly, Nox2 deficiency reduces oxidative stress levels in ischemic tissues and restores blood flow recuperation and vascular densities in old animals. Endothelial progenitor cells (EPCs) have an important role for postnatal neovascularization. Here we show that the functional activities of EPCs (migration, adhesion to mature endothelial cells) are significantly impaired in old compared to young mice. However, Nox2 deficiency rescues EPC functional activities in old animals. We also demonstrate an age-dependent pathological increase of oxidative stress levels in EPCs (DHE, DCF-DA) that is not present in Nox2-deficient animals. CONCLUSION: Nox2-containing NADPH oxidase deficiency protects against age-dependent impairment of neovascularization. Potential mechanisms include reduced ROS generation in ischemic tissues and preserved angiogenic activities of EPCs.
Assuntos
Envelhecimento/metabolismo , Células Endoteliais/enzimologia , Isquemia/enzimologia , Glicoproteínas de Membrana/deficiência , Músculo Esquelético/irrigação sanguínea , NADPH Oxidases/deficiência , Neovascularização Fisiológica , Células-Tronco/enzimologia , Doenças Vasculares/prevenção & controle , Fatores Etários , Envelhecimento/genética , Envelhecimento/patologia , Animais , Velocidade do Fluxo Sanguíneo , Adesão Celular , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/patologia , Membro Posterior , Isquemia/genética , Isquemia/patologia , Isquemia/fisiopatologia , Fluxometria por Laser-Doppler , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2 , NADPH Oxidases/genética , Estresse Oxidativo , Recuperação de Função Fisiológica , Fluxo Sanguíneo Regional , Células-Tronco/patologia , Superóxidos/metabolismo , Fatores de Tempo , Tirosina/análogos & derivados , Tirosina/metabolismo , Doenças Vasculares/enzimologia , Doenças Vasculares/genética , Doenças Vasculares/patologia , Doenças Vasculares/fisiopatologiaRESUMO
BACKGROUND: Hypercholesterolemia has been associated with impaired angiogenesis and reduced blood flow recuperation after ischemia. However, the precise mechanisms involved are unknown. Here we investigated the role of Nox2-derived reactive oxygen species (ROS) in the modulation of neovascularization by hypercholesterolemia. METHODS AND RESULTS: Mice deficient for the Nox2-containing NADPH oxidase (Nox2(-/-)) and control mice (Nox2(+/+)) were put on a high cholesterol diet (HCD) for a total of 15 weeks. After three months, hindlimb ischemia was surgically induced by femoral artery removal. Nox2 expression and oxidative stress levels in ischemic tissues were significantly increased by HCD in control mice, but not in Nox2(-/-) mice. Nox2(-/-) mice were also protected against hypercholesterolemia-induced impairment of neovascularization, as demonstrated by faster blood flow recovery after ischemia and increased capillary density in ischemic muscles. Nox2 deficiency was associated with preserved activity of eNOS in ischemic tissues, and improved activity of endothelial progenitor cells (EPCs). In vitro, HUVECs treated with the NADPH oxidase inhibitor apocynin or endothelial cells isolated from the aorta of Nox2(-/-) mice exhibited reduced ROS formation following exposure to oxLDL. This was associated with improved nitric oxide (NO) bioavailability and protection against oxLDL-induced inhibition of angiogenic activities. CONCLUSIONS: Nox2-containing NADPH oxidase deficiency protects against hypercholesterolemia-induced impairment of neovascularization. The potential mechanisms involved include reduced ROS formation, preserved activation of angiogenic signals, and improved functional activities of EPCs and mature endothelial cells.