RESUMEN

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.

Asunto(s)

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/genética

RESUMEN

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.

Asunto(s)

Isquemia/inmunología , Monocitos/inmunología , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Receptores de Quimiocina/metabolismo , Animales , Antígenos Ly/metabolismo , Receptor 1 de Quimiocinas CX3C , Quimiocina CCL2/sangre , Quimiocina CCL5/sangre , Quimiocina CX3CL1/sangre , Quimiotaxis de Leucocito , Modelos Animales de Enfermedad , Miembro Posterior , Isquemia/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Receptores CCR2/metabolismo , Receptores CCR5/metabolismo , Receptores de Quimiocina/deficiencia , Receptores de Quimiocina/genética , Factores de Tiempo , Regulación hacia Arriba

RESUMEN

BACKGROUND: CD4+ and CD8+ T lymphocytes are key regulators of postischemic neovascularization. T-cell activation is promoted by 2 major costimulatory signalings, the B7/CD28 and CD40-CD40 ligand pathways. Interestingly, CD28 interactions with the structurally related ligands B7-1 and B7-2 are also required for the generation and homeostasis of CD4+CD25+ regulatory T cells (Treg cells), which play a critical role in the suppression of immune responses and the control of T-cell homeostasis. We hypothesized that Treg cell activation may modulate the immunoinflammatory response to ischemic injury, leading to alteration of postischemic vessel growth. METHODS AND RESULTS: Ischemia was induced by right femoral artery ligation in CD28-, B7-1/2-, or CD40-deficient mice (n=10 per group). CD40 deficiency led to a significant reduction in the postischemic inflammatory response and vessel growth. In contrast, at day 21 after ischemia, angiographic score, foot perfusion, and capillary density were increased by 2.0-, 1.2-, and 1.8-fold, respectively, in CD28-deficient mice, which showed a profound reduction in the number of Treg cells compared with controls. Similarly, disruption of B7-1/2 signaling or anti-CD25 treatment and subsequent Treg deletion significantly enhanced postischemic neovascularization. These effects were associated with enhanced accumulation of CD3-positive T cells and Mac-3-positive macrophages in the ischemic leg. Conversely, treatment of CD28(-/-) mice with the nonmitogenic anti-CD3 monoclonal antibody enhanced the number of endogenous Treg cells and led to a significant reduction of the postischemic inflammatory response and neovascularization. Finally, coadministration of Treg cells and CD28(-/-) splenocytes in Rag1(-/-) mice with hindlimb ischemia abrogated the CD28(-/-) splenocyte-induced activation of the inflammatory response and neovascularization. CONCLUSIONS: Treg cell response modulates postischemic neovascularization.

Asunto(s)

Isquemia Miocárdica/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígeno B7-1/genética , Antígeno B7-1/inmunología , Antígeno B7-2/genética , Antígeno B7-2/inmunología , Antígenos CD28/genética , Antígenos CD28/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígenos CD40/deficiencia , Antígenos CD40/genética , Antígenos CD40/inmunología , Linfocitos T CD8-positivos/inmunología , Capilares/inmunología , Citometría de Flujo , Miembro Posterior , Inmunohistoquímica , Isquemia/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/inmunología , Neovascularización Fisiológica , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

BACKGROUND: The hypoxia-inducible transcription factor (HIF) subunits are destabilized via the O(2)-dependent prolyl hydroxylase domain proteins (PHD1, PHD2, and PHD3). We investigated whether inhibition of PHDs via upregulating HIF might promote postischemic neovascularization. METHODS AND RESULTS: Mice with right femoral artery ligation were treated, by in vivo electrotransfer, with plasmids encoding for an irrelevant short hairpin RNA (shRNA) (shCON [control]) or specific shRNAs directed against HIF-1alpha (shHIF-1alpha), PHD1 (shPHD1), PHD2 (shPHD2), and PHD3 (shPHD3). The silencing of PHDs induced a specific and transient downregulation of their respective mRNA and protein levels at day 2 after ischemia and, as expected, upregulated HIF-1alpha. As a consequence, 2 key hypoxia-inducible proangiogenic actors, vascular endothelial growth factor-A and endothelial nitric oxide synthase, were upregulated at the mRNA and protein levels. In addition, monocyte chemotactic protein-1 mRNA levels and infiltration of Mac-3-positive macrophages were enhanced in ischemic leg of mice treated with shPHD2 and shPHD3. Furthermore, activation of HIF-1alpha-related pathways was associated with changes in postischemic neovascularization. At day 14, silencing of PHD2 and PHD3 increased vessel density by 2.2- and 2.6-fold, capillary density by 1.8- and 2.1-fold, and foot perfusion by 1.2- and 1.4-fold, respectively, compared with shCON (P<0.001). shPHD1 displayed a lower proangiogenic effect. Of interest, coadministration of shHIF-1alpha with shPHD3 abrogated shPHD3-related effects, suggesting that activation of endogenous HIF-1-dependent pathways mediated the proangiogenic effects of PHD silencing. CONCLUSIONS: We demonstrated that a direct inhibition of PHDs, and more particularly PHD3, promoted therapeutic revascularization. Furthermore, we showed that activation of the HIF-1 signaling pathway is required to promote this revascularization.

Asunto(s)

Terapia Genética/métodos , Isquemia/terapia , Neovascularización Fisiológica/fisiología , Procolágeno-Prolina Dioxigenasa/genética , Transducción de Señal/fisiología , Animales , Quimiocinas/metabolismo , Arteria Femoral , Silenciador del Gen , Miembro Posterior/irrigación sanguínea , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Inflamación/metabolismo , Isquemia/metabolismo , Isquemia/fisiopatología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo III/metabolismo , Plásmidos/farmacología , Procolágeno-Prolina Dioxigenasa/metabolismo , ARN Mensajero/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

OBJECTIVE: Transplantation of adipose-derived stroma cells (ADSCs) stimulates neovascularization after experimental ischemic injury. ADSC proangiogenic potential is likely mediated by their ability to differentiate into endothelial cells and produce a wide array of angiogenic and antiapoptotic factors. Mitochondrial reactive oxygen species (ROS) have been shown to control ADSC differentiation. We therefore hypothesized that mitochondrial ROS production may change the ADSC proangiogenic properties. METHODS AND RESULTS: The use of pharmacological strategies (mitochondrial inhibitors, antimycin, and rotenone, with or without antioxidants) allowed us to specifically and precisely modulate mitochondrial ROS generation in ADSCs. We showed that transient stimulation of mitochondrial ROS generation in ADSCs before their injection in ischemic hindlimb strongly improved revascularization and the number of ADSC-derived CD31-positive cells in ischemic area. Mitochondrial ROS generation increased the secretion of the proangiogenic and antiapoptotic factors, VEGF and HGF, but did not affect ADSC ability to differentiate into endothelial cells, in vitro. Moreover, mitochondrial ROS-induced ADSC preconditioning greatly protect ADSCs against oxidative stress-induced cell death. CONCLUSIONS: Our study demonstrates that in vitro preconditioning by moderate mitochondrial ROS generation strongly increases in vivo ADSC proangiogenic properties and emphasizes the crucial role of mitochondrial ROS in ADSC fate.

Asunto(s)

Diferenciación Celular/fisiología , Células Endoteliales/citología , Células Endoteliales/fisiología , Mitocondrias/metabolismo , Neovascularización Fisiológica/fisiología , Especies Reactivas de Oxígeno/metabolismo , Adipocitos , Animales , Células Cultivadas , Masculino , Ratones , Daño por Reperfusión/fisiopatología , Células del Estroma/citología , Células del Estroma/metabolismo

RESUMEN

OBJECTIVES: Our goal was to demonstrate that microparticles (MPs) are the endogenous signal leading to neovessel formation through CD40 ligation in human atherosclerotic plaques. BACKGROUND: Vulnerable atherosclerotic plaques prone to rupture are characterized by an increased number of vasa vasorum and frequent intraplaque hemorrhage. Although inflammatory cytokines, growth factors, or CD40/CD40 ligand (CD40L) are possible candidates, the mechanism of atherosclerotic plaque neovascularization remains unknown. Atherosclerotic plaques contain large amounts of membrane-shed submicron MPs released after cell activation or apoptosis. METHODS: Microparticles were isolated from endarterectomy specimens surgically obtained from 26 patients and characterized by phosphatidylserine exposure and specific markers of cellular origin. RESULTS: Plaque MPs increased both endothelial proliferation assessed by (3)H-thymidine incorporation and cell number and stimulated in vivo angiogenesis in Matrigel (BD Biosciences, San Diego, California) assays performed in wild-type and BalbC/Nude mice, whereas circulating MPs had no effect. Microparticles from symptomatic patients expressed more CD40L and were more potent in inducing endothelial proliferation, when compared with asymptomatic plaque MPs. Most of CD40L+ MPs (93%) isolated from human plaques were of macrophage origin. Microparticle-induced endothelial proliferation was impaired by CD40L or CD40-neutralizing antibodies and abolished after endothelial CD40-ribonucleic acid silencing. In addition, the proangiogenic effect of plaque MPs was abolished in Matrigel assays performed in the presence of CD40L-neutralizing antibodies or in CD40-deficient mice. CONCLUSIONS: These results demonstrate that MPs isolated from human atherosclerotic lesions express CD40L, stimulate endothelial cell proliferation after CD40 ligation, and promote in vivo angiogenesis. Therefore, MPs could represent a major determinant of intraplaque neovascularization and plaque vulnerability.

Asunto(s)

Aterosclerosis/metabolismo , Ligando de CD40/metabolismo , Enfermedades de las Arterias Carótidas/metabolismo , Endotelio Vascular/metabolismo , Neovascularización Patológica/metabolismo , Anciano , Apoptosis/fisiología , Aterosclerosis/patología , Enfermedades de las Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/cirugía , Proliferación Celular , Células Cultivadas , Medios de Cultivo , Endarterectomía/métodos , Endotelio Vascular/patología , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Masculino , Neovascularización Patológica/patología , Tamaño de la Partícula , Valores de Referencia , Sensibilidad y Especificidad

RESUMEN

We hypothesized that activation of angiogenesis by chronic hypoxia may affect vascular resistance and, subsequently, blood pressure levels in spontaneously hypertensive rats (SHRs). Five-week-old prehypertensive SHRs and age-matched normotensive Wistar-Kyoto (WKY) rats (n=8 per group) were maintained under normobaric normoxic or hypoxic (10% O(2)) conditions for 8 weeks. Three weeks later, the systolic blood pressure was lower by 26% in hypoxic SHRs compared to normoxic SHRs (P<0.05) and remained at the normoxic WKY level. Total peripheral vascular resistance, calculated as the mean arterial pressure/cardiac output (assessed by ultrasound imaging and Doppler), was 30% lower in hypoxic than in normoxic SHRs (P<0.001) and returned to WKY levels. Interestingly, chronic hypoxia also significantly reduced systolic blood pressure in adult 12-week-old SHRs with established hypertension; blood pressure was normalized (versus normoxic WKY rats) after 4 weeks of hypoxia. Changes in hemodynamic parameters were associated with activation of proangiogenic pathways. Protein levels of vascular endothelial growth factor (VEGF)-A in the skeletal muscles were increased by 2.2-fold in hypoxic compared to normoxic SHRs (P<0.001). At the end of the hypoxic period, capillary density in the quadriceps muscle was 1.2-fold higher in hypoxic than in normoxic SHRs (P<0.001). Myocardial capillary density and VEGF-A protein contents were also 1.2- and 2.1-fold higher in hypoxic compared to normoxic SHRs (P<0.001 and P<0.05, respectively). Moreover, treatment with neutralizing VEGF-A antibody abrogated the hypoxia-induced angiogenesis and subsequently worsened arterial hypertension. Therefore, our results suggest that chronic normobaric hypoxia (1) activates VEGF-A-induced angiogenesis and thereafter (2) prevents the occurrence of hypertension in young prehypertensive SHRs and (3) normalizes blood pressure in adult SHRs with established hypertension.

Asunto(s)

Corazón/fisiopatología , Hipoxia/fisiopatología , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Animales , Presión Sanguínea , Gasto Cardíaco , Enfermedad Crónica , Hipertensión/sangre , Hipertensión/etiología , Hipertensión/fisiopatología , Hipoxia/sangre , Hipoxia/complicaciones , Hipoxia/patología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/sangre , Resistencia Vascular

RESUMEN

We analyzed the effect of hypertension on postischemic vasculogenesis. Ischemia was induced by right femoral artery ligature in Wistar Kyoto rats (WKY) or spontaneously hypertensive rats (SHR) treated with or without angiotensin-converting enzyme inhibitor (Perindopril, 0.76 mg/kg/d) and angiotensin type 1 receptor blocker (losartan, 30 mg/kg/d). Basal postischemic neovascularization was reduced in SHR compared to WKY (P<0.05, n=8). Treatment with ACE inhibitor or angiotensin type 1 receptor blocker decreased blood pressure levels by 1.4- and 1.3-fold (P<0.001), respectively and restored vessel growth in SHR to WKY levels. Interestingly, 14 days after bone-marrow mononuclear cell (BM-MNC) transfusion, angiographic scores, capillary density, and foot perfusion were decreased by 1.4-, 1.5-, and 1.2-fold, respectively in SHR transfused with BM-MNCs isolated from SHR compared to those receiving BM-MNCs of WKY (P<0.05, n=6). Alteration in BM-MNCs proangiogenic potential was likely related to the reduction in their ability to mobilize into peripheral circulation, as revealed by the 2.9-fold decrease in number of circulating CD34+/CD117+ cells (P<0.001) and to differentiate into cells with endothelial phenotype, as revealed by the 2.1-fold reduction in percentages of DilLDL/BS-1 lectin positive cells (P<0.001). In addition, reactive oxygen species (ROS) levels were increased by 2.2-fold in SHR BM-MNCs compared to WKY BM-MNCs (P<0.01), as assessed by L-012 luminescence. Cotreatment with ACE inhibitor, angiotensin type 1 receptor blocker, or antioxidants (NAC 3 mmol/L, Apocynin 200 micromol/L) reduced ROS levels, improved the number of DilLDL/BS-1 lectin-positive cells by around 1.5-fold, and restored BM-MNCs proangiogenic effects in ischemic hindlimb. In conclusion, alteration in progenitor cell proangiogenic function may participate to the hypertension-induced impairment in postischemic revascularization.

Asunto(s)

Antihipertensivos/farmacología , Hipertensión/tratamiento farmacológico , Hipertensión/fisiopatología , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/fisiología , Perindopril/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Diferenciación Celular/fisiología , Modelos Animales de Enfermedad , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Miembro Posterior/irrigación sanguínea , Hidralazina/farmacología , Hipertensión/patología , Isquemia/patología , Isquemia/fisiopatología , Ligadura , Losartán/farmacología , Masculino , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Células Madre/citología , Células Madre/fisiología

RESUMEN

Cardiovascular risk factors are associated with reduction in both the number and function of vascular progenitor cells. We hypothesized that 1) hypertension abrogates postnatal vasculogenesis, and 2) antihypertensive treatment based on the combination of perindopril (angiotensin-converting enzyme inhibitor) and indapamide (diuretic) may counteract hypertension-induced alteration in progenitor cell-related effects. Postischemic neovascularization was significantly lower in untreated spontaneously hypertensive rats (SHRs) compared with Wistar Kyoto (WKY) rats (p < 0.05). Treatment of SHRs with perindopril and the combination of perindopril/indapamide reduced the blood pressure levels and normalized vessel growth in ischemic area. Cotreatment with perindopril and indapamide increased vascular endothelial growth factor and endothelial nitric-oxide synthase protein contents, two key proangiogenic factors. It is interesting to note that 14 days after bone marrow mononuclear cell (BM-MNC) transplantation, revascularization was significantly lower in ischemic SHRs receiving BM-MNCs isolated from SHRs compared with those receiving BM-MNCs isolated from WKY rats (p < 0.05). Alteration in proangiogenic potential of SHR BM-MNCs was probably related to the reduction in their ability to differentiate into endothelial progenitor cells in vitro. Furthermore, the number of circulating endothelial progenitor cells (EPCs) was reduced by 3.1-fold in SHRs compared with WKY rats (p < 0.001). Treatments with perindopril or perindopril/indapamide restored the ability of BM-MNCs to differentiate in vitro into EPCs, increased the number of circulating EPCs, and re-established BM-MNC proangiogenic effects. Therefore, hypertension is associated with a decrease in the number of circulating progenitor cells and in the BM-MNC proangiogenic potential, probably leading to vascular complications in this setting. The combination of perindopril and indapamide counteracts hypertension-induced alterations in progenitor cell-related effects and restores blood vessel growth.

Asunto(s)

Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Antihipertensivos/uso terapéutico , Diuréticos/uso terapéutico , Hipertensión/tratamiento farmacológico , Indapamida/uso terapéutico , Neovascularización Fisiológica/efectos de los fármacos , Perindopril/uso terapéutico , Animales , Antígenos CD34/metabolismo , Quimioterapia Combinada , Arteria Femoral/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/fisiología , Hipertensión/fisiopatología , Masculino , Músculo Esquelético/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III , Proteínas Proto-Oncogénicas c-kit/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

We hypothesized that diabetes-induced oxidative stress may affect postischemic neovascularization. The response to unilateral femoral artery ligation was studied in wild-type or gp91(phox)-deficient control or type 1 diabetic mice or in animals treated with the anti-oxidant N-acetyl-l-cysteine (NAC) or with in vivo electrotransfer of a plasmid encoding dominant-negative Rac1 (50 microg) for 21 days. Postischemic neovascularization was reduced in diabetic mice in association with down-regulated vascular endothelial growth factor-A protein levels. In diabetic animals vascular endothelial growth factor levels and postischemic neovascularization were restored to nondiabetic levels by the scavenging of reactive oxygen species (ROS) by NAC administration or the inhibition of ROS generation by gp91(phox) deficiency or by administration of dominant-negative Rac1. Finally, diabetes reduced the ability of adherent bone marrow-derived mononuclear cells (BM-MNCs) to differentiate into endothelial progenitor cells. Treatment with NAC (3 mmol/L), apocynin (200 micromol/L), or the p38MAPK inhibitor LY333351 (10 micromol/L) up-regulated the number of endothelial progenitor cell colonies derived from diabetic BM-MNCs by 1.5-, 1.6-, and 1.5-fold, respectively (P < 0.05). In the ischemic hindlimb model, injection of diabetic BM-MNCs isolated from NAC-treated or gp91(phox)-deficient diabetic mice increased neovascularization by approximately 1.5-fold greater than untreated diabetic BM-MNCs (P < 0.05). Thus, inhibition of NADPH oxidase-derived ROS overproduction improves the angiogenic and vasculogenic processes and restores postischemic neovascularization in type 1 diabetic mice.

Asunto(s)

Diabetes Mellitus Experimental/inducido químicamente , Isquemia , Músculo Esquelético/irrigación sanguínea , NADPH Oxidasas/metabolismo , Neovascularización Patológica , Especies Reactivas de Oxígeno/metabolismo , Animales , Animales Recién Nacidos , Médula Ósea , Diferenciación Celular , Células Endoteliales/citología , Ratones , Monocitos/citología , NADPH Oxidasas/antagonistas & inhibidores , Subunidades de Proteína/metabolismo , Ratas , Células Madre/citología , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

BACKGROUND: Proangiogenic cell therapy based on administration of bone marrow-derived mononuclear cells (BMCs) or endothelial progenitor cells (EPCs) is now under investigation in humans for the treatment of ischemic diseases. However, mechanisms leading to the beneficial effects of BMCs and EPCs remain unclear. METHODS AND RESULTS: BMC- and CD34+-derived progenitor cells interacted with ischemic femoral arteries through SDF-1 and CXCR4 signaling and released nitric oxide (NO) via an endothelial nitric oxide synthase (eNOS)-dependent pathway. BMC-induced NO production promoted a marked vasodilation and disrupted vascular endothelial-cadherin/beta-catenin complexes, leading to increased vascular permeability. NO-dependent vasodilation and hyperpermeability were critical for BMC infiltration in ischemic tissues and their proangiogenic potential in a model of hindlimb ischemia in mice. CONCLUSIONS: Our results propose a new concept that proangiogenic progenitor cell activity does not rely only on their ability to differentiate into endothelial cells but rather on their capacity to modulate the function of preexisting vessels.

Asunto(s)

Permeabilidad Capilar , Isquemia/terapia , Neovascularización Fisiológica , Trasplante de Células Madre/métodos , Vasodilatación , Animales , Arterias/patología , Células de la Médula Ósea , Células Endoteliales , Miembro Posterior , Ratones , Ratones Noqueados , Ratones Desnudos , Óxido Nítrico/metabolismo

RESUMEN

BACKGROUND: We investigated the putative proangiogenic activity and molecular pathway(s) of the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) in a model of surgically induced hindlimb ischemia. METHODS AND RESULTS: Hindlimb ischemia was induced by femoral artery ligature and an osmotic minipump was implanted subcutaneously to deliver low (0.12 mg/kg per day) or high (1.2 mg/kg per day) doses of AcSDKP, for 7 or 21 days. Angiography scores, arteriole density, capillary number, and foot perfusion were increased at day 21 in the high-dose AcSDKP-treated mice (by 1.9-, 1.8-, 1.3-, and 1.6-fold, respectively) compared with control animals (P<0.05, P<0.01, P<0.01, respectively). AcSDKP treatment for 24 hours upregulated the monocyte chemoattractant protein-1 (MCP-1) mRNA and protein levels by 1.5-fold in cultured endothelial cells (P<0.01). In the ischemic hindlimb model, administration of AcSDKP also enhanced MCP-1 mRNA levels by 90-fold in ischemic leg (P<0.001) and MCP-1 plasma levels by 3-fold (P<0.001 versus untreated ischemic control mice). MCP-1 levels upregulation were associated with a 2.3-fold increase in the number of Mac3-positive cells in ischemic area of AcSDKP-treated mice (P<0.001 versus untreated animals). Interestingly, AcSDKP-induced monocyte/macrophage infiltration and postischemic neovascularization was fully blunted in MCP-1-deficient animals. CONCLUSIONS: AcSDKP stimulates postischemic neovascularization through activation of a proinflammatory MCP-1-related pathway.

Asunto(s)

Quimiocina CCL2/fisiología , Miembro Posterior/irrigación sanguínea , Isquemia/tratamiento farmacológico , Neovascularización Fisiológica/efectos de los fármacos , Oligopéptidos/administración & dosificación , Animales , Células de la Médula Ósea/patología , Diferenciación Celular , Línea Celular Transformada , Quimiocina CCL2/deficiencia , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Arteria Femoral/patología , Isquemia/fisiopatología , Ratones , Ratones Endogámicos C57BL , Monocitos/patología , Transducción de Señal/efectos de los fármacos

RESUMEN

OBJECTIVE: We analyzed the involvement of thromboxane (TX) A2/prostaglandin (PG) H2 (TP) receptor in ischemia-induced neovascularization in mice. METHODS AND RESULTS: Unilateral hindlimb ischemia was induced by right femoral artery ligature in male C57BL/6J mice (n=7 per group). Animals were then treated with or without TP receptor antagonist (S18886, 5 or 10 mg/kg per day; ramatroban, 10 mg/kg per day) or aspirin (30 mg/kg per day) in drinking water for 21 days. Hindlimb ischemia raised plasma level of TXB2, the stable metabolite of TXA2, by 4.7-fold. This increase was blocked by aspirin treatment whereas S18886 (5 or 10 mg/kg per day) had no effect. However, neither S 18886 nor aspirin affected postischemic neovascularization. We next assessed the putative involvement of TXA2 signaling in angiotensin II (Ang II) proangiogenic pathway. Ang II (0.3 mg/kg per day) enhanced TXB2 plasma levels by 2.6-fold over that of control (P<0.01). Ang II-induced TXB2 upregulation was reduced by cotreatment with Ang II type I receptor antagonist (candesartan, 20 mg/kg per day). Angiographic score, capillary number, and foot perfusion were improved by 1.7-, 1.7-, and 1.4-fold, respectively, in Ang II-treated mice compared with controls (P<0.05). Ang II proangiogenic effect was associated with a 1.6-fold increase in VEGF-A protein content (P<0.05) and a 1.4-fold increase in the number of Mac-3-positive cells (ie, macrophages) in ischemic areas (P<0.05). Interestingly, treatments with TP receptor antagonists or aspirin hampered the proangiogenic effects of Ang II. CONCLUSIONS: Endogenous activation of TXA2 receptor by eicosanoids did not modulate spontaneous neovascularization in the setting of ischemia. Conversely, TXA2 signaling is involved in Ang II-induced AT1-dependent vessel growth.

Asunto(s)

Angiotensina II/sangre , Isquemia/metabolismo , Neovascularización Fisiológica/fisiología , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Vasoconstrictores/sangre , Angiotensina II/farmacología , Animales , Capilares/fisiología , Miembro Posterior/irrigación sanguínea , Isquemia/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Naftalenos/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Propionatos/farmacología , Receptores de Tromboxano A2 y Prostaglandina H2/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tromboxano A2/sangre , Tromboxano B2/sangre , Vasculitis/metabolismo , Vasculitis/fisiopatología , Vasoconstrictores/farmacología

RESUMEN

Vascular endothelial growth factor (VEGF)-induced blood vessel growth is involved in both physiological and pathological angiogenesis and requires integrin-mediated signaling. We now show that an integrin-binding protein initially described in milk-fat globule, MFG-E8 (also known as lactadherin), is expressed in and around blood vessels and has a crucial role in VEGF-dependent neovascularization in the adult mouse. Using neutralizing antibodies and lactadherin-deficient animals, we show that lactadherin interacts with alphavbeta3 and alphavbeta5 integrins and alters both VEGF-dependent Akt phosphorylation and neovascularization. In the absence of VEGF, lactadherin administration induced alphavbeta3- and alphavbeta5-dependent Akt phosphorylation in endothelial cells in vitro and strongly improved postischemic neovascularization in vivo. These results show a crucial role for lactadherin in VEGF-dependent neovascularization and identify lactadherin as an important target for the modulation of neovascularization.

Asunto(s)

Inductores de la Angiogénesis/metabolismo , Antígenos de Superficie/metabolismo , Proteínas de la Leche/metabolismo , Neovascularización Fisiológica/fisiología , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Análisis de Varianza , Animales , Southern Blotting , Cruzamientos Genéticos , Femenino , Vectores Genéticos , Humanos , Integrina alfaVbeta3/metabolismo , Isquemia/metabolismo , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt

RESUMEN

Inflammatory cell infiltration is a feature of postischemic neovascularization. However, mechanisms leading to leukocyte attraction to the site of neovascularization are still undefined. We hypothesized that the CXC chemokine receptor 3 (CXCR3) may contribute to leukocyte accumulation and subsequently to blood vessel growth in the ischemic area. Ischemia induced by femoral artery ligature improved the number of CXCR3-expressing cells and the level of its ligand, CXCL10. Angiographic score, blood flow recovery measurement, and capillary density analysis showed a significant decrease of ischemic/nonischemic leg ratio in CXCR3-deficient mice when compared with controls (P<0.05), at day 21 after ischemia. Interestingly, this impairment was as important as that observed in mice deficient for the well known CC-chemokine monocyte chemoattractant protein-1 (MCP-1). At day 7 of ischemic injury, the number of CD3-positive T cells and Mac-3-positive monocytes/macrophages was 38% and 45% lower, respectively, in the ischemic leg of CXCR3-deficient mice compared with the control group (P<0.05), suggesting an important role for CXCR3 in leukocyte recruitment into the ischemic area. VEGF protein content, a classical proangiogenic factor, was also markedly reduced (80% reduction) in ischemic leg of CXCR3-deficient mice (P<0.01). Injection of bone marrow-derived mononuclear cells (BM-MNCs) isolated from wild-type animals restored the neovascularization reaction in CXCR3-deficient mice whereas BM-MNCs from CXCR3-deficient mice was ineffective. In conclusion, CXCR3 plays a key role in neovascularization and provides novel information on the mechanisms leading to leukocyte infiltration in the vessel growth area.

Asunto(s)

Miembro Posterior/irrigación sanguínea , Isquemia/fisiopatología , Neovascularización Fisiológica/fisiología , Receptores de Quimiocina/fisiología , Animales , Arteriolas/metabolismo , Trasplante de Médula Ósea , Capilares/metabolismo , Quimiocina CCL2/deficiencia , Quimiocina CCL2/fisiología , Quimiocina CXCL10 , Quimiocina CXCL9 , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/genética , Quimiotaxis de Leucocito/fisiología , Arteria Femoral , Isquemia/terapia , Flujometría por Láser-Doppler , Ligadura , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Fisiológica/genética , Receptores CXCR3 , Receptores de Quimiocina/deficiencia , Receptores de Quimiocina/genética , Subgrupos de Linfocitos T/fisiología , Factor A de Crecimiento Endotelial Vascular/análisis

RESUMEN

OBJECTIVE: We analyzed the beneficial therapeutic effect of angiotensin converting enzyme inhibitor (ACEI) on both retinal and hind limb neovascularization in diabetic mice. METHODS AND RESULTS: Diabetic mice (streptozotocin, 40 mg/kg) were treated with or without ACEI (Perindopril, 3 mg/kg per day) or AT1 receptor blocker (Candesartan, 20 mg/kg) for 4 months. Hind limb ischemia was then induced by right femoral artery ligature for 1 additional month. In the ischemic leg, angiographic score, capillary density, and foot perfusion were increased by 2.7, 2.0-fold, and 1.6-fold, respectively, in ACEI-treated diabetic mice compared with untreated diabetic animals (P<0.01). ACEI also raised vascular endothelial growth factor (VEGF) protein level by 1.4-fold in ischemic diabetic leg. This ACEI pro-angiogenic effect was totally blunted in diabetic bradykinin B2 receptor-deficient animals, suggesting that it was mediated by the bradykinin pathway. In the diabetic retina, angiotensinogen and ACE mRNA levels were increased by 2.8-fold and 4.1-fold, respectively (P<0.01 versus nondiabetic mice), highlighting a local activation of renin-angiotensin system. Diabetes also raised VEGF protein level by 1.5-fold (P<0.05 versus nondiabetic mice). Treatments with ACEI and AT1 receptor blocker hampered diabetes-induced VEGF upregulation and retinal neovascularization. CONCLUSIONS: ACE inhibition improved neovascularization in the diabetic ischemic leg through activation of bradykinin signaling, whereas it reduced vessel growth in the diabetic retina through inhibition of overacting Ang II pathway.

Asunto(s)

Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Diabetes Mellitus Tipo 1/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Angiografía/métodos , Animales , Peso Corporal , Bradiquinina/metabolismo , Capilares/metabolismo , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/inducido químicamente , Miembro Posterior/irrigación sanguínea , Miembro Posterior/efectos de los fármacos , Isquemia/metabolismo , Flujometría por Láser-Doppler/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Peptidil-Dipeptidasa A/metabolismo , Peptidil-Dipeptidasa A/fisiología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Mensajero/metabolismo , Retina/química , Retina/metabolismo , Retina/patología , Vasos Retinianos/efectos de los fármacos , Estreptozocina

RESUMEN

BACKGROUND: We analyzed the role of aldosterone in ischemia-induced neovascularization and the involvement of angiotensin II (Ang II) signaling in this effect. METHODS AND RESULTS: Ischemia was induced by right femoral artery ligature in mice treated or not with aldosterone (4.5 microg/day), aldosterone plus spironolactone (aldosterone receptor blocker; 20 mg/kg per day), or aldosterone plus valsartan (angiotensin type 1 [AT1] receptor blocker; 20 mg/kg per day). After 21 days, neovascularization was evaluated by microangiography, capillary density measurement, and laser-Doppler perfusion imaging. Protein level of vascular endothelial growth factor (VEGF) was determined by Western blot analysis in hindlimbs. mRNA levels of renin-angiotensin system components were also assessed by semiquantitative reverse transcription-polymerase chain reaction. Angiographic score, capillary number, and foot perfusion were improved in ischemic/nonischemic leg ratio by 1.4-, 1.5-, and 1.4-fold, respectively, in aldosterone-treated mice compared with controls (P<0.05). Aldosterone proangiogenic effect was associated with 2.3-fold increase in VEGF protein content (P<0.05). Treatments with spironolactone or with neutralizing VEGF antibody hampered the proangiogenic effect of aldosterone (P<0.05 versus aldosterone-treated mice). Interestingly, AT1 receptor blockade completely abrogated the aldosterone proangiogenic effect, emphasizing the involvement of Ang II-related pathway in aldosterone-induced vessel growth. In this view, angiotensinogen mRNA content was 2.2-fold increased in aldosterone-treated mice in reference to controls (P<0.05), whereas that of renin, angiotensin-converting enzyme, and AT1 receptor subtype was unaffected. Aldosterone treatment also decreased AT2 mRNA content by 2-fold (P<0.05 versus controls), suggesting that aldosterone may switch the Ang II pathway toward activation of vessel growth. CONCLUSIONS: This study shows for the first time that aldosterone increases neovascularization in the setting of ischemia through activation of Ang II signaling.

Asunto(s)

Aldosterona/farmacología , Inductores de la Angiogénesis/farmacología , Angiotensina II/metabolismo , Isquemia/tratamiento farmacológico , Neovascularización Fisiológica , Aldosterona/uso terapéutico , Inductores de la Angiogénesis/uso terapéutico , Animales , Vasos Sanguíneos/efectos de los fármacos , Isquemia/diagnóstico por imagen , Isquemia/metabolismo , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Radiografía , Flujo Sanguíneo Regional/efectos de los fármacos , Sistema Renina-Angiotensina , Transducción de Señal , Piel/irrigación sanguínea , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

The aim of the study was to investigate in vivo arterial reactivity in the mouse hind limb using Orthogonal Polarisation Spectral (OPS) imaging, which delivers high-contrast images of vascular beds by visualizing red blood cells. After minimal skin invasion of anesthetized mice, the OPS probe was placed on the hind limb continuously superfused with physiological saline solution. Then, the response of the saphenous artery (average luminal diameter 127 +/- 3 microm; n = 15) to topical application of increasing concentrations of acetylcholine or phenylephrine was examined. Mean carotid arterial blood pressure was unaffected during the experiment. The basal diameter decreased by 70% during exposure to phenylephrine (pD2: 5.65 +/- 0.08; n = 9), while acetylcholine augmented basal diameter up to 199% (pD2: 6.55 +/- 0.12; n = 6). Application of sodium nitroprusside did not further increase arterial diameter following acetylcholine exposure. After washing out, arterial luminal diameters returned to initial values. Second exposure to vasoactive agents demonstrated that changes in diameter were reproducible with time and not different between left and right saphenous arteries. Thus, OPS imaging is an in vivo dye-free, simple and minimally invasive approach, which provides unique information regarding the behavior of vascular network within conditions of cellular and physiological homeostasis.

Asunto(s)

Acetilcolina/farmacología , Cardiotónicos/farmacología , Músculo Liso Vascular/efectos de los fármacos , Fenilefrina/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía de Polarización

RESUMEN

BACKGROUND: Adipose tissue development and remodeling are closely associated with the growth of vascular network. We hypothesized that adipose tissue may contain progenitor cells with angiogenic potential and that therapy based on adipose tissue-derived progenitor cells administration may constitute a promising cell therapy in patients with ischemic disease. METHODS AND RESULTS: In mice, cultured stromal-vascular fraction (SVF) cells from adipose tissue have a great proangiogenic potential, comparable to that of bone marrow mononuclear cells in the mouse ischemic hindlimb model. Similarly, cultured human SVF cells differentiate into endothelial cells, incorporate into vessels, and promote both postischemic neovascularization in nude mice and vessel-like structure formation in Matrigel plug. In vitro, these cells represent a homogeneous population of CD34- and CD13-positive cells, which can spontaneously express the endothelial cell markers CD31 and von Willebrand factor when cultured in semisolid medium. Interestingly, dedifferentiated mature human adipocytes have the potential to rapidly acquire the endothelial phenotype in vitro and to promote neovascularization in ischemic tissue and vessel-like structure formation in Matrigel plug, suggesting that cells of endothelial and adipocyte phenotypes may have a common precursor. CONCLUSIONS: This study demonstrates, for the first time, that adipocytes and endothelial cells have a common progenitor. Such adipose lineage cells participate in vascular-like structure formation in Matrigel plug and enhance the neovascularization reaction in ischemic tissue. These results also highlight the concept that adipose lineage cells represent a suitable new cell source for therapeutic angiogenesis in ischemic disease.

Asunto(s)

Tejido Adiposo/citología , Endotelio Vascular/citología , Isquemia/terapia , Neovascularización Fisiológica , Trasplante de Células Madre , Adipocitos/citología , Animales , Biomarcadores/análisis , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Humanos , Isquemia/diagnóstico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Músculo Esquelético/irrigación sanguínea , Fenotipo , Células Madre/citología , Células Madre/metabolismo , Células del Estroma/metabolismo , Células del Estroma/trasplante

RESUMEN

Mechanisms that hinder ischemia-induced neovascularization in diabetes remain poorly understood. We hypothesized that endogenous bone marrow mononuclear cell (BM-MNC) dysfunction may contribute to the abrogated postischemic revascularization reaction associated with diabetes. We first analyzed the effect of diabetes (streptozotocin, 40 mg/kg) on BM-MNC pro-angiogenic potential in a model of surgically induced hindlimb ischemia. In nondiabetic animals, transplantation of BM-MNCs isolated from nondiabetic animals raised the ischemic/nonischemic angiographic score, capillary number, and blood flow recovery by 1.8-, 2.7-, and 2.2-fold, respectively, over that of PBS-injected nondiabetic animals (P < 0.05). Administration of diabetic BM-MNCs also improved the neovascularization reaction in ischemic hindlimbs of nondiabetic mice but to a lesser extent from that observed with nondiabetic BM-MNC transplantation. In diabetic mice, injection of nondiabetic BM-MNCs was still more efficient than that of diabetic BM-MNCs. Such BM-MNC dysfunction was associated with the impairment of diabetic BM-MNC capacity to differentiate into endothelial progenitor cells (EPCs) in vitro and to participate in vascular-like structure formation in a subcutaneous Matrigel plug. Placenta growth factor (PlGF) administration improved by sixfold the number of EPCs differentiated from diabetic BM-MNCs in vitro and enhanced ischemic/nonischemic angiographic score, capillary number and blood flow recovery by 1.9-, 1.5- and 1.6-fold, respectively, over that of untreated diabetic animals (P < 0.01). Endogenous BM-MNC pro-angiogenic potential was affected in diabetes. Therapeutic strategy based on PlGF administration restored such defects and improved postischemic neovascularization in diabetic mice.

Asunto(s)

Diabetes Mellitus Experimental/fisiopatología , Isquemia/patología , Neovascularización Fisiológica , Proteínas Gestacionales/farmacología , Angiografía , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Trasplante de Médula Ósea , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Miembro Posterior/irrigación sanguínea , Miembro Posterior/patología , Miembro Posterior/cirugía , Isquemia/tratamiento farmacológico , Isquemia/cirugía , Flujometría por Láser-Doppler , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/patología , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Factor de Crecimiento Placentario