RESUMO
Endothelial Progenitor Cells (EPCs) can actively participate in revascularization in oxygen-induced retinopathy (OIR). Yet the mechanisms responsible for their dysfunction is unclear. Nogo-A, whose function is traditionally related to the inhibition of neurite function in the central nervous system, has recently been documented to display anti-angiogenic pro-repellent properties. Based on the significant impact of EPCs in retinal vascularization, we surmised that Nogo-A affects EPC function, and proceeded to investigate the role of Nogo-A on EPC function in OIR. The expression of Nogo-A and its specific receptor NgR1 was significantly increased in isolated EPCs exposed to hyperoxia, as well as in EPCs isolated from rats subjected to OIR compared with respective controls (EPCs exposed to normoxia). EPCs exposed to hyperoxia displayed reduced migratory and tubulogenic activity, associated with the suppressed expression of prominent EPC-recruitment factors SDF-1/CXCR4. The inhibition of Nogo-A (using a Nogo-66 neutralizing antagonist peptide) or siRNA-NGR1 in hyperoxia-exposed EPCs restored SDF-1/CXCR4 expression and, in turn, rescued the curtailed neovascular functions of EPCs in hyperoxia. The in vivo intraperitoneal injection of engineered EPCs (Nogo-A-inhibited or NgR1-suppressed) in OIR rats at P5 (prior to exposure to hyperoxia) prevented retinal and choroidal vaso-obliteration upon localization adjacent to vasculature; coherently, the inhibition of Nogo-A/NgR1 in EPCs enhanced the expression of key angiogenic factors VEGF, SDF-1, PDGF, and EPO in retina; CXCR4 knock-down abrogated suppressed NgR1 pro-angiogenic effects. The findings revealed that hyperoxia-induced EPC malfunction is mediated to a significant extent by Nogo-A/NgR1 signaling via CXCR4 suppression; the inhibition of Nogo-A in EPCs restores specific angiogenic growth factors in retina and the ensuing vascularization of the retina in an OIR model.
Assuntos
Células Progenitoras Endoteliais , Hiperóxia , Doenças Retinianas , Animais , Ratos , Oxigênio/efeitos adversos , Proteínas Nogo/genética , Hiperóxia/complicaçõesRESUMO
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.
Assuntos
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
PURPOSE: To investigate whether embolization with chitosan hydrogel (CH) with or without a sclerosant (sodium tetradecyl sulphate, STS) can induce chemical endothelial ablation and prevent endothelial recanalization in a rabbit model. METHODS: Chitosan radiopaque thermogels were prepared using chitosan, ß-glycerophosphate, iopamidol, and different STS concentrations. Each auricular artery of 14 New Zealand White rabbits was cannulated and injected with 0.6 mL of chitosan (CH0; nâ=â14) on one side and either saline (nâ=â3), chitosan and 1% STS (CH1; nâ=â6), or chitosan and 3% STS (CH3; nâ=â6) in the contralateral side. Immediately after embolization and at 1, 7, 14, and 30 days, auricular artery patency and percentage of recanalization were assessed by visual inspection; microcirculation was evaluated using laser Doppler imaging (LDI). The rabbits were sacrificed at 30 days to assess endothelial ablation and inflammatory response by histological analyses. RESULTS: All arteries were catheterized and embolized with success. All saline-injected arteries rapidly recovered normal flow. The length of embolization was greater with CH3 than CH1 or CH0, regardless of the time observed (p<0.001). No difference in recanalization length was found among the gels (pâ=â0.07). Destruction of arterial wall was frequently observed independent of embolizing agent. Foreign body reaction was more frequent with CH3 as compared with CH1 and CH0 (pâ=â0.0070 and 0.0058, respectively). After 30 days, hypervascularization was observed on LDI only with CH0; it was attributed to intra- or perivascular neovessels and inflammatory response on pathological analysis. The vascular modifications appeared to be more homogenous across the length of embolization with CH3 than the other formulations. CONCLUSION: The viscosity obtained with chitosan and 3% STS permits better control during injection and longer vascular occlusion. These findings, combined with the intravascular neovascularization observed with CH0, led us to prefer the combination with STS.
Assuntos
Técnicas de Ablação , Quitosana/administração & dosagem , Orelha/irrigação sanguínea , Embolização Terapêutica/métodos , Células Endoteliais/efeitos dos fármacos , Soluções Esclerosantes/administração & dosagem , Tetradecilsulfato de Sódio/administração & dosagem , Técnicas de Ablação/efeitos adversos , Animais , Artérias/efeitos dos fármacos , Artérias/patologia , Artérias/fisiopatologia , Quitosana/efeitos adversos , Relação Dose-Resposta a Droga , Embolização Terapêutica/efeitos adversos , Células Endoteliais/patologia , Reação a Corpo Estranho/etiologia , Reação a Corpo Estranho/patologia , Hidrogéis , Injeções Intra-Arteriais , Fluxometria por Laser-Doppler , Microcirculação , Modelos Animais , Neovascularização Patológica , Coelhos , Fluxo Sanguíneo Regional , Soluções Esclerosantes/efeitos adversos , Tetradecilsulfato de Sódio/efeitos adversos , Fatores de Tempo , Grau de Desobstrução Vascular , ViscosidadeRESUMO
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.
Assuntos
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
BACKGROUND AND AIM: Insufficient post-ischemic neovascularization is an initial key step in the pathogenesis of Oxygen-Induced Retinopathy (OIR). During neovascularization, pro-angiogenic cells (PACs) are mobilized from the bone marrow and integrate into ischemic tissues to promote angiogenesis. However, the modulation of PAC paracrine activity during OIR and the specific mechanisms involved remain to be explored. Because Tyrosine-protein phosphatase non-receptor type 9 (PTPN9) is reported to be a negative regulator of stem cell differentiation and angiogenesis signaling, we investigated its effect on PAC activity in the context of OIR. METHODS AND RESULTS: In a rat model of OIR, higher levels of PTPN9 in the retina and in bone marrow derived PACs are associated with retinal avascular areas, lower levels of the mobilization factor SDF-1 and decreased number of CD34+/CD117+/CD133+ PACs. PACs exposed ex vivo to hyperoxia display increased PTPN9 expression, which is associated with impaired ability of PAC secretome to promote angiogenesis ex vivo (choroidal vascular sprouting) and in vitro (endothelial cell tubule formation) compared to the secretome of PACs maintained in normoxia. Suppression of PTPN9 (using siRNA) increases VEGF and SDF-1 expression to normalize PAC secretome during hyperoxia, leading to restored angiogenic ability of PAC secretome. Moreover, endothelial cells exposed to the secretome of siPTPN9-treated PACs expressed increased levels of activated form of VEGF receptor 2 (VEGFR2). In the rat model of OIR, intravitreal injection of secretome from siPTPN9-treated PACs significantly reduced retinal vaso-obliteration; this was associated with higher retinal levels of VEGF/SDF-1, and increased recruitment of PACs (CD34+ cells) to the retinal and choroidal vessels. CONCLUSION: Our results suggest that hyperoxia alters the paracrine proangiogenic activity of BM-PACs by inducing PTPN9, which can contribute to impair post-ischemic revascularization in the context of OIR. Targeting PTPN9 restores PAC angiogenic properties, and provide a new target for vessel integrity in ischemic retinopathies.
RESUMO
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
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
The coronavirus disease 2019 (COVID-19) pandemic has become the number one health problem worldwide. As of August 2020, it has affected more than 18 million humans and caused over 700,000 deaths worldwide. COVID-19 is an infectious disease that can lead to severe acute respiratory syndrome. Under certain circumstances, the viral infection leads to excessive and uncontrolled inflammatory response, which is associated with the massive release of inflammatory cytokines in pulmonary alveolar structures. This phenomenon has been referred to as the "cytokine storm," and it is closely linked to lung injury, acute respiratory syndrome and mortality. Unfortunately, there is currently no vaccine available to prevent the infection, and no effective treatment is available to reduce the mortality associated with the severe form of the disease. The cytokine storm associate with COVID-19 shows similarities with those observed in other pathologies such as sepsis, acute respiratory distress syndrome, acute lung injury and other viral infection including severe cases of influenza. However, the specific mechanisms that cause and modulate the cytokine storm in the different conditions remain to be determined. micro-RNAs are important regulators of gene expression, including key inflammatory cytokines involved in the massive recruitment of immune cells to the lungs such as IL1ß, IL6, and TNFα. In recent years, it has been shown that nutraceutical agents can modulate the expression of miRs involved in the regulation of cytokines in various inflammatory diseases. Here we review the potential role of inflammatory-regulating-miRs in the cytokine storm associated with COVID-19, and propose that nutraceutical agents may represent a supportive therapeutic approach to modulate dysregulated miRs in this condition, providing benefits in severe respiratory diseases.
RESUMO
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.
RESUMO
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: Data related to long-term safety of intracoronary (IC) injection of CD133+ bone marrow stem cells (BMSC) following an acute myocardial infarction (MI) are still lacking. METHODS: COMPARE-AMI is a double-blind, placebo-controlled phase II clinical trial evaluating the safety and efficacy of IC injection of CD133+ enriched hematopoietic BMSC in patients with ST-elevation myocardial infarction (STEMI) and persistent left ventricular (LV) dysfunction following successful primary percutaneous coronary intervention (PCI). Herein, we report outcomes up to ten years of follow-up. RESULTS: Between November 2007 and July 2012, we enrolled 38 patients in our study. Males were 89% and the median age was 50.5â¯years. Baseline left ventricular ejection fraction (LVEF) was 40.0%, and 90% of lesions were located in the left anterior descending (LAD) artery. The median follow-up time was 8.5â¯years IQR [7.9, 10.0]. Using Kaplan-Meier methods, MACE-free survival up to 10â¯years was 77.3% overall. IC injection of CD133+ BMSC was associated with a similar event-free survival rate compared to placebo (87.8% vs. 66.3%, pâ¯=â¯.37). Two cancer cases in each group were recorded. No malignant arrhythmias were observed. CONCLUSIONS: IC injection of CD133+ BMSC is safe up to 10â¯years of follow-up. The long-term efficacy needs to be confirmed by a larger randomized trial.
Assuntos
Intervenção Coronária Percutânea , Disfunção Ventricular Esquerda , Antígeno AC133 , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Intervenção Coronária Percutânea/efeitos adversos , Volume Sistólico , Resultado do Tratamento , Função Ventricular EsquerdaRESUMO
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
BACKGROUND: Preterm birth is linked to cardiovascular risks and diseases. Endothelial progenitor cells play a critical role in vascular development and repair. Cord blood endothelial progenitor cells of preterm-born infants, especially endothelial colony-forming cells (ECFC), show enhanced susceptibility to prematurity-related pro-oxidant stress. Whether ECFC dysfunction is present in adulthood following preterm birth is unknown. METHODS AND RESULTS: This cross-sectional observational study includes 55 preterm-born (≤29 gestational weeks) young adults (18-29 years old, 38% male) and 55 sex- and age-matched full-term controls. ECFC were isolated from peripheral blood; cell proliferative and vascular cord formation capacities were assessed in vitro. Daytime systolic blood pressure was higher, whereas glucose tolerance and body mass index were lower in preterm-born subjects. ECFC colonies grew in culture for 62% of full-term- and 58% of preterm-born participants. Preterm-born participants have formed ECFC colonies later in culture and have reduced proliferation compared with controls. Only in preterm-born individuals, we observed that the later the ECFC colony grows in culture, the worse was overall ECFC function. In addition, in preterms, elevated systolic blood pressure significantly correlated with reduced ECFC proliferation (rS=-0.463; P=0.030) and numbers of branches formed on matrigel (rS=-0.443; P=0.039). In preterm-born subjects, bronchopulmonary dysplasia was associated with impaired ECFC function, whereas exposure to antenatal steroids related to better ECFC function. CONCLUSIONS: This study is the first to examine ECFC in preterm-born adults and to demonstrate ECFC dysfunction compared with full-term controls. In the preterm-born group, ECFC dysfunction was associated with bronchopulmonary dysplasia, the major prematurity-related neonatal morbidity, and with increased systolic blood pressure into adulthood.
Assuntos
Doenças Cardiovasculares/sangue , Células Progenitoras Endoteliais/patologia , Recém-Nascido Prematuro , Adolescente , Adulto , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/epidemiologia , Contagem de Células , Proliferação de Células , Células Cultivadas , Estudos Transversais , Ecocardiografia , Feminino , Seguimentos , Idade Gestacional , Humanos , Incidência , Recém-Nascido , Masculino , Quebeque/epidemiologia , Estudos Retrospectivos , Fatores de Risco , Adulto JovemRESUMO
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.