RESUMEN
RATIONALE: Decades of research have examined immune-modulatory strategies to protect the heart after an acute myocardial infarction and prevent progression to heart failure but have failed to translate to clinical benefit. OBJECTIVE: To determine anti-inflammatory actions of n-apo AI (Apo AI nanoparticles) that contribute to cardiac tissue recovery after myocardial infarction. METHODS AND RESULTS: Using a preclinical mouse model of myocardial infarction, we demonstrate that a single intravenous bolus of n-apo AI (CSL111, 80 mg/kg) delivered immediately after reperfusion reduced the systemic and cardiac inflammatory response. N-apo AI treatment lowered the number of circulating leukocytes by 30±7% and their recruitment into the ischemic heart by 25±10% (all P<5.0×10-2). This was associated with a reduction in plasma levels of the clinical biomarker of cardiac injury, cardiac troponin-I, by 52±17% (P=1.01×10-2). N-apo AI reduced the cardiac expression of chemokines that attract neutrophils and monocytes by 60% to 80% and lowered surface expression of integrin CD11b on monocytes by 20±5% (all P<5.0×10-2). Fluorescently labeled n-apo AI entered the infarct and peri-infarct regions and colocalized with cardiomyocytes undergoing apoptosis and with leukocytes. We further demonstrate that n-apo AI binds to neutrophils and monocytes, with preferential binding to the proinflammatory monocyte subtype and partially via SR-BI (scavenger receptor BI). In patients with type 2 diabetes, we also observed that intravenous infusion of the same n-apo AI (CSL111, 80 mg/kg) similarly reduced the level of circulating leukocytes by 12±5% (all P<5.0×10-2). CONCLUSIONS: A single intravenous bolus of n-apo AI delivered immediately post-myocardial infarction reduced the systemic and cardiac inflammatory response through direct actions on both the ischemic myocardium and leukocytes. These data highlight the anti-inflammatory effects of n-apo AI and provide preclinical support for investigation of its use for management of acute coronary syndromes in the setting of primary percutaneous coronary interventions.
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Antiinflamatorios/administración & dosificación , Apolipoproteína A-I/administración & dosificación , Inflamación/prevención & control , Leucocitos/efectos de los fármacos , Infarto del Miocardio/tratamiento farmacológico , Nanopartículas , Administración Intravenosa , Adulto , Animales , Antígeno CD11b/metabolismo , Células Cultivadas , Quimiocinas/metabolismo , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/inmunología , Modelos Animales de Enfermedad , Esquema de Medicación , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Leucocitos/inmunología , Leucocitos/metabolismo , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Infarto del Miocardio/inmunología , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Ensayos Clínicos Controlados Aleatorios como Asunto , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/metabolismo , Troponina I/sangreAsunto(s)
Síndrome Coronario Agudo , Glucosa , Animales , Humanos , Lipoproteínas HDL , Ratones , Infarto del MiocardioRESUMEN
Protecting the heart after an acute coronary syndrome is a key therapeutic goal to support cardiac recovery and prevent progression to heart failure. A potential strategy is to target cardiac glucose metabolism at the early stages after ischemia when glycolysis is critical for myocyte survival. Building on our discovery that high-density lipoprotein (HDL) modulates skeletal muscle glucose metabolism, we now demonstrate that a single dose of reconstituted HDL (rHDL) delivered after myocardial ischemia increases cardiac glucose uptake, reduces infarct size, and improves cardiac remodeling in association with enhanced functional recovery in mice. These findings applied equally to metabolically normal and insulin-resistant mice. We further establish direct effects of HDL on cardiomyocyte glucose uptake, glycolysis, and glucose oxidation via the Akt signaling pathway within 15 min of reperfusion. These data support the use of infusible HDL preparations for management of acute coronary syndromes in the setting of primary percutaneous interventions.
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Lipoproteínas HDL/uso terapéutico , Infarto del Miocardio/tratamiento farmacológico , Animales , Glucosa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Miocardio/metabolismo , Miocardio/patología , Transducción de Señal/efectos de los fármacosRESUMEN
Acute myocardial infarction (MI) is a severe ischemic disease responsible for heart failure and sudden death. Inflammatory cells orchestrate postischemic cardiac remodeling after MI. Studies using mice with defective mast/stem cell growth factor receptor c-Kit have suggested key roles for mast cells (MCs) in postischemic cardiac remodeling. Because c-Kit mutations affect multiple cell types of both immune and nonimmune origin, we addressed the impact of MCs on cardiac function after MI, using the c-Kit-independent MC-deficient (Cpa3(Cre/+)) mice. In response to MI, MC progenitors originated primarily from white adipose tissue, infiltrated the heart, and differentiated into mature MCs. MC deficiency led to reduced postischemic cardiac function and depressed cardiomyocyte contractility caused by myofilament Ca(2+) desensitization. This effect correlated with increased protein kinase A (PKA) activity and hyperphosphorylation of its targets, troponin I and myosin-binding protein C. MC-specific tryptase was identified to regulate PKA activity in cardiomyocytes via protease-activated receptor 2 proteolysis. This work reveals a novel function for cardiac MCs modulating cardiomyocyte contractility via alteration of PKA-regulated force-Ca(2+) interactions in response to MI. Identification of this MC-cardiomyocyte cross-talk provides new insights on the cellular and molecular mechanisms regulating the cardiac contractile machinery and a novel platform for therapeutically addressable regulators.
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Señalización del Calcio , Calcio/metabolismo , Mastocitos/metabolismo , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Miofibrillas/metabolismo , Animales , Carboxipeptidasas A/genética , Carboxipeptidasas A/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ratones , Ratones Noqueados , Contracción Miocárdica/genética , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocardio/patología , Miofibrillas/patología , Proteolisis , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptor PAR-2/genética , Receptor PAR-2/metabolismoRESUMEN
BACKGROUND: Cell-based therapies are being explored as a therapeutic option for patients with chronic heart failure following myocardial infarction. Extracellular vesicles (EV), including exosomes and microparticles, secreted by transplanted cells may orchestrate their paracrine therapeutic effects. We assessed whether post-infarction administration of EV released by human embryonic stem cell-derived cardiovascular progenitors (hESC-Pg) can provide equivalent benefits to administered hESC-Pg and whether hESC-Pg and EV treatments activate similar endogenous pathways. METHODS: Mice underwent surgical occlusion of their left coronary arteries. After 2-3 weeks, 95 mice included in the study were treated with hESC-Pg, EV, or Minimal Essential Medium Alpha Medium (alpha-MEM; vehicle control) delivered by percutaneous injections under echocardiographic guidance into the peri-infarct myocardium. functional and histologic end-points were blindly assessed 6 weeks later, and hearts were processed for gene profiling. Genes differentially expressed between control hearts and hESC-Pg-treated and EV-treated hearts were clustered into functionally relevant pathways. RESULTS: At 6 weeks after hESC-Pg administration, treated mice had significantly reduced left ventricular end-systolic (-4.20 ± 0.96 µl or -7.5%, p = 0.0007) and end-diastolic (-4.48 ± 1.47 µl or -4.4%, p = 0.009) volumes compared with baseline values despite the absence of any transplanted hESC-Pg or human embryonic stem cell-derived cardiomyocytes in the treated mouse hearts. Equal benefits were seen with the injection of hESC-Pg-derived EV, whereas animals injected with alpha-MEM (vehicle control) did not improve significantly. Histologic examination suggested a slight reduction in infarct size in hESC-Pg-treated animals and EV-treated animals compared with alpha-MEM-treated control animals. In the hESC-Pg-treated and EV-treated groups, heart gene profiling identified 927 genes that were similarly upregulated compared with the control group. Among the 49 enriched pathways associated with these up-regulated genes that could be related to cardiac function or regeneration, 78% were predicted to improve cardiac function through increased cell survival and/or proliferation or DNA repair as well as pathways related to decreased fibrosis and heart failure. CONCLUSIONS: In this post-infarct heart failure model, either hESC-Pg or their secreted EV enhance recovery of cardiac function and similarly affect cardiac gene expression patterns that could be related to this recovery. Although the mechanisms by which EV improve cardiac function remain to be determined, these results support the idea that a paracrine mechanism is sufficient to effect functional recovery in cell-based therapies for post-infarction-related chronic heart failure.
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Insuficiencia Cardíaca , Animales , Enfermedad Crónica , Células Madre Embrionarias , Vesículas Extracelulares , Humanos , Ratones , Infarto del Miocardio , Miocardio , Miocitos CardíacosRESUMEN
Published clinical trials in patients with ischemic diseases show limited benefit of adult stem cell-based therapy, likely due to their restricted plasticity and commitment toward vascular cell lineage. We aim to uncover the potent regenerative ability of MesP1/stage-specific embryonic antigen 1 (SSEA-1)-expressing cardiovascular progenitors enriched from human embryonic stem cells (hESCs). Injection of only 10(4) hESC-derived SSEA-1(+) /MesP1(+) cells, or their progeny obtained after treatment with VEGF-A or PDGF-BB, was effective enough to enhance postischemic revascularization in immunodeficient mice with critical limb ischemia (CLI). However, the rate of incorporation of hESC-derived SSEA-1(+) /MesP1(+) cells and their derivatives in ischemic tissues was modest. Alternatively, these cells possessed a unique miR-21 signature that inhibited phosphotase and tensin homolog (PTEN) thereby activating HIF-1α and the systemic release of VEGF-A. Targeting miR-21 limited cell survival and inhibited their proangiogenic capacities both in the Matrigel model and in mice with CLI. We next assessed the impact of mR-21 in adult angiogenesis-promoting cells. We observed an impaired postischemic angiogenesis in miR-21-deficient mice. Notably, miR-21 was highly expressed in circulating and infiltrated monocytes where it targeted PTEN/HIF-1α/VEGF-A signaling and cell survival. As a result, miR-21-deficient mice displayed an impaired number of infiltrated monocytes and a defective angiogenic phenotype that could be partially restored by retransplantation of bone marrow-derived cells from wild-type littermates. hESC-derived SSEA-1(+) /MesP1(+) cells progenitor cells are powerful key integrators of therapeutic angiogenesis in ischemic milieu and miR-21 is instrumental in this process as well as in the orchestration of the biological activity of adult angiogenesis-promoting cells.
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Isquemia/terapia , MicroARNs/metabolismo , Miocardio/metabolismo , Trasplante de Células Madre , Células Madre/metabolismo , Animales , Linaje de la Célula , Supervivencia Celular/fisiología , Miembro Posterior/irrigación sanguínea , Humanos , Ratones , Neovascularización Fisiológica/genética , Transducción de Señal/fisiología , Trasplante de Células Madre/métodosRESUMEN
BACKGROUND: Interaction with heparan sulfate proteoglycans is supposed to provide chemokines with the capacity to immobilize on cell surface and extracellular matrix for accomplishing both tissue homing and signaling of attracted cells. However, the consequences of the exclusive invalidation of such interaction on the roles played by endogenous chemokines in vivo remain unascertained. METHODS AND RESULTS: We engineered a mouse carrying a Cxcl12 gene (Cxcl12(Gagtm)) mutation that precludes interactions with heparan sulfate structures while not affecting CXCR4-dependent cell signaling of CXCL12 isoforms (α, ß, γ). Cxcl12(Gagtm/Gagtm) mice develop normally, express normal levels of total and isoform-specific Cxcl12 mRNA, and show increased counting of circulating CD34(+) hematopoietic precursor cells. After induced acute ischemia, a marked impaired capacity to support revascularization was observed in Cxcl12(Gagtm/Gagtm) animals associated with a reduced number of infiltrating cells in the ischemic tissue despite the massive expression of CXCL12 isoforms. Importantly, exogenous administration of CXCL12γ, which binds heparan sulfate with the highest affinity ever reported for a cytokine, fully restores vascular growth, whereas heparan sulfate-binding CXCL12γ mutants failed to promote revascularization in Cxcl12(Gagtm/Gagtm) animals. CONCLUSION: These findings prove the role played by heparan sulfate interactions in the functions of CXCL12 in both homeostasis and physiopathological settings and document for the first time the paradigm of chemokine immobilization in vivo.
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Quimiocina CXCL12/genética , Heparina/análogos & derivados , Isquemia/genética , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica/genética , Proteoglicanos/metabolismo , Animales , Quimiocina CXCL12/biosíntesis , Heparina/metabolismo , Miembro Posterior/irrigación sanguínea , Homeostasis , Isquemia/metabolismo , Ratones , Modelos Animales , Isoformas de Proteínas/genética , ARN Mensajero , Transcripción GenéticaRESUMEN
OBJECTIVE: Leukocyte infiltration in ischemic areas is a hallmark of myocardial infarction, and overwhelming infiltration of innate immune cells has been shown to promote adverse remodeling and cardiac rupture. Recruitment of inflammatory cells in the ischemic heart depends highly on the family of CC-chemokines and their receptors. Here, we hypothesized that the chemokine decoy receptor D6, which specifically binds and scavenges inflammatory CC-chemokines, might limit inflammation and adverse cardiac remodeling after infarction. METHODS AND RESULTS: D6 was expressed in human and murine infarcted myocardium. In a murine model of myocardial infarction, D6 deficiency led to increased chemokine (C-C motif) ligand 2 and chemokine (C-C motif) ligand 3 levels in the ischemic heart. D6-deficient (D6(-/-)) infarcts displayed increased infiltration of pathogenic neutrophils and Ly6Chi monocytes, associated with strong matrix metalloproteinase-9 and matrix metalloproteinase-2 activities in the ischemic heart. D6(-/-) mice were cardiac rupture prone after myocardial infarction, and functional analysis revealed that D6(-/-) hearts had features of adverse remodeling with left ventricle dilation and reduced ejection fraction. Bone marrow chimera experiments showed that leukocyte-borne D6 had no role in this setting, and that leukocyte-specific chemokine (C-C motif) receptor 2 deficiency rescued the adverse phenotype observed in D6(-/-) mice. CONCLUSIONS: We show for the first time that the chemokine decoy receptor D6 limits CC-chemokine-dependent pathogenic inflammation and is required for adequate cardiac remodeling after myocardial infarction.
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Inflamación/prevención & control , Infarto del Miocardio/inmunología , Miocardio/inmunología , Receptores CCR10/metabolismo , Receptores de Quimiocina/metabolismo , Remodelación Ventricular , Animales , Antígenos Ly/metabolismo , Trasplante de Médula Ósea , Quimiocina CCL2/metabolismo , Quimiocina CCL3/metabolismo , Quimiotaxis , Modelos Animales de Enfermedad , Genotipo , Rotura Cardíaca Posinfarto/inmunología , Rotura Cardíaca Posinfarto/patología , Humanos , Hipertrofia Ventricular Izquierda/inmunología , Hipertrofia Ventricular Izquierda/patología , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/inmunología , Infarto del Miocardio/complicaciones , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Miocardio/metabolismo , Miocardio/patología , Infiltración Neutrófila , Neutrófilos/inmunología , Fenotipo , Receptores CCR2/deficiencia , Receptores CCR2/genética , Receptores de Quimiocina/deficiencia , Receptores de Quimiocina/genética , Transducción de Señal , Volumen Sistólico , Ultrasonografía , Función Ventricular Izquierda , Receptor de Quimiocina D6RESUMEN
OBJECTIVE: Catecholamines have been shown to control bone marrow (BM)-derived cell egress, yet the cellular and molecular mechanisms involved in this effect and their subsequent participation to postischemic vessel growth are poorly understood. METHODS AND RESULTS: Tyrosine hydroxylase mRNA levels, as well as dopamine (DA) and norepinephrine (NE) contents, were increased in the ischemic BM of mice with right femoral artery ligation. Angiographic score, capillary density, and arteriole number were markedly increased by treatments with DA (IP, 50 mg/kg, 5 days) or NE (IP, 2.5 mg/kg, 5 days). Using chimeric mice lethally irradiated and transplanted with BM-derived cells from green fluorescent protein mice, we showed that DA and NE enhanced by 70% (P<0.01) and 62% (P<0.001), respectively, the number of green fluorescent protein-positive BM-derived cells in ischemic tissue and promoted their ability to differentiate into cells with endothelial and inflammatory phenotypes. Similarly, both DA and NE increased the in vitro differentiation of cultured BM-derived cells into cells with endothelial phenotype. This increase was blunted by the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester. DA and NE also upregulated the number of CD45-positive cells in blood 3 days after ischemia and that of macrophages in ischemic tissue 21 days after ischemia. Of interest, DA and NE increased BM endothelial nitric oxide synthase (eNOS) mRNA levels and were unable to promote BM-derived cell mobilization in chimeric eNOS-deficient mice lethally irradiated and transplanted with BM-derived cells from wild-type animals. Furthermore, administration of a ß2 adrenergic agonist (clenbuterol, IP, 2 mg/kg, 5 days) and that of a dopaminergic D1/D5 receptor agonist (SKF-38393, IP, 2.5 mg/kg, 5 days) also enhanced BM-derived cell mobilization and subsequently postischemic vessel growth. CONCLUSION These results unravel, for the first time, a major role for the sympathetic nervous system in BM-derived cell egress through stromal eNOS activation.
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Células de la Médula Ósea/enzimología , Médula Ósea/enzimología , Diferenciación Celular , Movimiento Celular , Células Endoteliales/metabolismo , Isquemia/enzimología , Músculo Esquelético/irrigación sanguínea , Óxido Nítrico Sintasa de Tipo III/metabolismo , Sistema Nervioso Simpático/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/inervación , Células de la Médula Ósea/efectos de los fármacos , Trasplante de Médula Ósea , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Dopamina/metabolismo , Agonistas de Dopamina/farmacología , Células Endoteliales/efectos de los fármacos , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Arteria Femoral/cirugía , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Miembro Posterior , Isquemia/fisiopatología , Ligadura , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neovascularización Fisiológica , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/genética , Norepinefrina/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal , Células del Estroma/enzimología , Sistema Nervioso Simpático/efectos de los fármacos , Sistema Nervioso Simpático/fisiopatología , Factores de Tiempo , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: C/EBP homologous protein-10 (CHOP-10) is a novel developmentally regulated nuclear protein that emerges as a critical transcriptional integrator among pathways regulating differentiation, proliferation, and survival. In the present study, we analyzed the role of CHOP-10 in postnatal neovascularization. METHODS AND RESULTS: Ischemia was induced by right femoral artery ligation in wild-type and CHOP-10(-/-) mice. In capillary structure of skeletal muscle, CHOP-10 mRNA and protein levels were upregulated by ischemia and diabetes mellitus. Angiographic score, capillary density, and foot perfusion were increased in CHOP-10(-/-) mice compared with wild-type mice. This effect was associated with a reduction in apoptosis and an upregulation of endothelial nitric oxide synthase (eNOS) levels in ischemic legs of CHOP-10(-/-) mice compared with wild-type mice. In agreement with these results, eNOS mRNA and protein levels were significantly upregulated in CHOP-10 short interfering RNA-transfected human endothelial cells, whereas overexpression of CHOP-10 inhibited basal transcriptional activation of the eNOS promoter. Using a chromatin immunoprecipitation assay, we also showed that CHOP-10 was bound to the eNOS promoter. Interestingly, enhanced postischemic neovascularization in CHOP-10(-/-) mice was fully blunted in CHOP-10/eNOS double-knockout animals. Finally, we showed that induction of diabetes mellitus is associated with a marked upregulation of CHOP-10 that substantially inhibited postischemic neovascularization. CONCLUSIONS: This study identifies CHOP-10 as an important transcription factor modulating vessel formation and maturation.
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Regulación Enzimológica de la Expresión Génica , Neovascularización Patológica/enzimología , Óxido Nítrico Sintasa de Tipo III/genética , Factor de Transcripción CHOP/genética , Animales , Animales Recién Nacidos , Células Cultivadas , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/genética , Arteria Femoral/enzimología , Arteria Femoral/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica/genética , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Unión Proteica/genética , Factor de Transcripción CHOP/biosíntesis , Factor de Transcripción CHOP/deficiencia , Activación Transcripcional/genética , Regulación hacia Arriba/genéticaRESUMEN
AIMS: Monocyte systemic levels are known to be a major determinant of ischaemic tissue revascularization, but the mechanisms mediating mobilization of different monocyte subsets-Ly6C(hi) and Ly6C(lo)-to the blood and their respective role in post-ischaemic neovascularization are not clearly understood. Here, we hypothesized that distinct chemokine/chemokine receptor pathways, namely CCL2/CCR2, CX3CL1/CX3CR1, and CCL5/CCR5, differentially control monocyte subset systemic levels, and might thus impact post-ischaemic vessel growth. METHODS AND RESULTS: In a model of murine hindlimb ischaemia, both Ly6C(hi) and Ly6C(lo) monocyte circulating levels were increased after femoral artery ligation. CCL2/CCR2 activation enhanced blood Ly6C(hi) and Ly6C(lo) monocyte counts, although the opposite effect was seen in mice with CCL2 or CCR2 deficiency. CX3CL1/CX3CR1 strongly impacted Ly6C(lo) monocyte levels, whereas CCL5/CCR5 had no role. Only CCL2/CCR2 signalling influenced neovascularization, which was increased in mice overexpressing CCL2, whereas it markedly decreased in CCL2-/- mice. Moreover, adoptive transfer of Ly6C(hi)-but not Ly6C(lo)-monocytes enhanced vessel growth and blood flow recovery. CONCLUSION: Altogether, our data demonstrate that regulation of proangiogenic Ly6C(hi) monocytes systemic levels by CCL2/CCR2 controls post-ischaemic vessel growth, whereas Ly6C(lo) monocytes have no major role in this setting.