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This article reviews the current knowledge on the mechanisms of adaptive response to low doses of ionizing radiation or chemical exposure. A better knowledge of these mechanisms is needed to improve our understanding of health risks at low levels of environmental or occupational exposure and their involvement in cancer or non-cancer diseases. This response is orchestrated through a multifaceted cellular program involving the concerted action of diverse stress response pathways. These evolutionary highly conserved defense mechanisms determine the cellular response to chemical and physical aggression. They include DNA damage repair (p53, ATM, PARP pathways), antioxidant response (Nrf2 pathway), immune/inflammatory response (NF-κB pathway), cell survival/death pathway (apoptosis), endoplasmic response to stress (UPR response), and other cytoprotective processes including autophagy, cell cycle regulation, and the unfolded protein response. The coordinated action of these processes induced by low-dose radiation or chemicals produces biological effects that are currently estimated with the linear non-threshold model. These effects are controversial. They are difficult to detect because of their low magnitude, the scarcity of events in humans, and the difficulty of corroborating associations over the long term. Improving our understanding of these biological consequences should help humans and their environment by enabling better risk estimates, the revision of radiation protection standards, and possible therapeutic advances.
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Contaminantes Ambientales/toxicidad , Radiación Ionizante , Transducción de Señal/efectos de los fármacos , Antioxidantes/metabolismo , Autofagia/efectos de los fármacos , Autofagia/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Contaminantes Ambientales/química , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de Señal/efectos de la radiación , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/efectos de la radiaciónRESUMEN
The effects of low doses of ionizing radiation on atherosclerosis remain uncertain, particularly as regards the generation of pro- or anti-inflammatory responses, and the time scale at which such effects can occur following irradiation. To explore these phenomena, we exposed atheroprone ApoE(-/-) mice to a single dose of 0, 0.05, 0.5 or 1 Gy of 137Cs (γ) administered at a 10.35 mGy min-1 dose rate and evaluated short-term (1-10 days) and long-term consequences (100 days). Bone marrow-derived macrophages were derived from mice 1 day after exposure. Irradiation was associated with a significant skewing of M0 and M2 polarized macrophages towards the M2 phenotype, as demonstrated by an increased mRNA expression of Retnla, Arg1, and Chil3 in cells from mice exposed to 0.5 or 1 Gy compared with non-irradiated animals. Minimal effects were noted in M1 cells or M1 marker mRNA. Concurrently, we observed a reduced secretion of IL-1ß but enhanced IL-10 release from M0 and M2 macrophages. Effects of irradiation on circulating monocytes were most marked at day 10 post-exposure, when the 1 Gy dose was associated with enhanced numbers of both Ly6CHigh and Ly6Low cells. By day 100, levels of circulating monocytes in irradiated and non-irradiated mice were equivalent, but anti-inflammatory Ly6CLow monocytes were significantly increased in the spleen of mice exposed to 0.05 or 1 Gy. Long term exposures did not affect atherosclerotic plaque size or lipid content, as determined by Oil red O staining, whatever the dose applied. Similarly, irradiation did not affect atherosclerotic plaque collagen or smooth muscle cell content. However, we found that lesion CD68+ cell content tended to decrease with rising doses of radioactivity exposure, culminating in a significant reduction of plaque macrophage content at 1 Gy. Taken together, our results show that short- and long-term exposures to low to moderate doses of ionizing radiation drive an anti-inflammatory response, skewing bone marrow-derived macrophages towards an IL-10-secreting M2 phenotype and decreasing plaque macrophage content. These results suggest a low-grade athero-protective effect of low and moderate doses of ionizing radiation.
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Apolipoproteínas E , Radioisótopos de Cesio , Rayos gamma , Macrófagos , Placa Aterosclerótica , Animales , Macrófagos/metabolismo , Macrófagos/efectos de la radiación , Placa Aterosclerótica/patología , Placa Aterosclerótica/metabolismo , Ratones , Apolipoproteínas E/genética , Apolipoproteínas E/deficiencia , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos CD/metabolismo , Antígenos CD/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Masculino , Ratones Noqueados , Molécula CD68RESUMEN
We assessed the role of plasminogen activator inhibitor-1 (PAI-1) and matrix metalloproteinase 9 (MMP9) in wound healing process and in the bone marrow mononuclear cells (BMMNC)-related effects on physiological and pathological wound healing. A full thickness excision wound was created by removal of the skin on the midback of irradiated and nonirradiated animals. Angiogenesis and re-epithelialization were markedly increased in PAI-1-/- mice compared to wild-type (WT) animals. We revealed high MMP activity in tissue of PAI-1-/- animals. Of interest, the wound healing process was reduced in PAI-1-/-:MMP9-/- animals compared to PAI-1-/- mice, suggesting a key role of MMP9 in beneficial effect of PAI-1 deficiency on wound closure. To unravel the role of PAI-1 in BMMNC relative effects, mice were treated with or without local injection of BMMNC isolated from WT, PAI-1-/-, and PAI-1-/-: MMP9-/- animals for 14 days (10(6) cells, n = 6 per group). In WT nonirradiated mice, transplantation of BMMNC isolated from PAI-1-/- animals enhanced wound formation when compared with WT BMMNC. BMMNC differentiation into cells with endothelial phenotype was enhanced by PAI-1 deficiency. These effects were abrogated in PAI-1-/-:MMP9-/- and MMP9-/- BMMNC. In addition, using chimeric mice, we demonstrated that PAI-1 deficiency environment increased the BMMNC-GFP recruitment to the wound site, whereas this effect was abrogated when using PAI-1-/-:MMP9-/- BMMNC. PAI-1 deficiency, at least through MMP9 upregulation, enhanced wound healing and BMMNC therapeutic potential in irradiated and nonirradiated animals.
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Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Cicatrización de Heridas/fisiología , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Masculino , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Noqueados , Inhibidor 1 de Activador Plasminogénico/genética , Cicatrización de Heridas/genética , Cicatrización de Heridas/efectos de la radiaciónRESUMEN
Low dose ionizing radiation (LDIR) is known to have a protective effect on atherosclerosis in rodent studies, but how it impacts different cells types involved in lesion formation remains incompletely understood. We investigated the immunomodulatory response of different doses and dose-rates of irradiation in ApoE-/- mice. Mice were exposed to external γ rays at very low (1.4 mGy.h-1) or low (50 mGy.h-1) dose-rates, with cumulative doses spanning 50 to 1000 mGy. Flow cytometry of circulating cells revealed a significant decrease in pro-inflammatory Ly6CHi monocytes at all cumulative doses at low dose-rate, but more disparate effects at very low dose-rate with reductions in Ly6CHi cells at doses of 50, 100 and 750 mGy only. In contrast, Ly6CLo monocytes were not affected by LDIR. Similarly, proportions of CD4+ T cell subsets in the spleen did not differ between irradiated mice and non-irradiated controls, whether assessing CD25+FoxP3+ regulatory or CD69+ activated lymphocytes. In the aorta, gene expression of cytokines such as IL-1 and TGF-ß and adhesion molecules such as E-Selectin, ICAM-1, and VCAM-1 were reduced at the intermediate dose of 200 mGy. These results suggest that LDIR may reduce atherosclerotic plaque formation by selectively reducing blood pro-inflammatory monocytes and by impairing adhesion molecule expression and inflammatory processes in the vessel wall. In contrast, splenic T lymphocytes were not affected by LDIR. Furthermore, some responses to irradiation were nonlinear; reductions in aortic gene expression were significant at intermediate doses, but not at either highest or lowest doses. This work furthers our understanding of the impact of LDIR with different dose-rates on immune system response in the context of atherosclerosis.
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BACKGROUND: We hypothesized that microparticles (MPs) released after ischemia are endogenous signals leading to postischemic vasculogenesis. METHODS AND RESULTS: MPs from mice ischemic hind-limb muscle were detected by electron microscopy 48 hours after unilateral femoral artery ligation as vesicles of 0.1- to 1-microm diameter. After isolation by sequential centrifugation, flow cytometry analyses showed that the annexin V(+) MP concentration was 3.5-fold higher in ischemic calves than control muscles (1392+/-406 versus 394+/-180 annexin V(+) MPs per 1 mg; P<0.001) and came mainly from endothelial cells (71% of MPs are CD(144+)). MPs isolated from ischemic muscles induced more potent in vitro bone marrow-mononuclear cell (BM-MNC) differentiation into cells with endothelial phenotype than those isolated from control muscles. MPs isolated from atherosclerotic plaques were ineffective, whereas those isolated from apoptotic or interleukin-1beta-activated endothelial cells also promoted BM-MNC differentiation. Interestingly, MPs from ischemic muscles produced more reactive oxygen species and expressed significantly higher levels of NADPH oxidase p47 (6-fold; P<0.05) and p67 subunits (16-fold; P<0.001) than controls, whereas gp91 subunit expression was unchanged. BM-MNC differentiation was reduced by 2-fold with MPs isolated from gp91-deficient animals compared with wild-type mice (P<0.05). MP effects on postischemic revascularization were then examined in an ischemic hind-limb model. MPs isolated from ischemic muscles were injected into ischemic legs in parallel with venous injection of BM-MNCs. MPs increased the proangiogenic effect of BM-MNC transplantation, and this effect was blunted by gp91 deficiency. In parallel, BM-MNC proangiogenic potential also was reduced in ABCA1 knockout mice with impaired vesiculation. CONCLUSIONS: MPs produced during tissue ischemia stimulate progenitor cell differentiation and subsequently promote postnatal neovascularization.
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Micropartículas Derivadas de Células/fisiología , Isquemia/sangre , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica/fisiología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/fisiología , Animales , Anexina A5/farmacología , Diferenciación Celular , Hipoxia de la Célula , Micropartículas Derivadas de Células/trasplante , Células Endoteliales/patología , Endotelio Vascular/patología , Inducción Enzimática , Arteria Femoral , Trasplante de Células Madre Hematopoyéticas , Humanos , Isquemia/fisiopatología , Isquemia/cirugía , Ligadura , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/biosíntesis , NADPH Oxidasas/genética , Especies Reactivas de OxígenoRESUMEN
Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development. We postulated that activation of the EphB4/ephrin-B2 system may enhance EPC proangiogenic potential. In this report, we demonstrate in a nude mouse model of hind limb ischemia that EphB4 activation with an ephrin-B2-Fc chimeric protein increases the angiogenic potential of human EPCs. This effect was abolished by EphB4 siRNA, confirming that it is mediated by EphB4. EphB4 activation enhanced P selectin glycoprotein ligand-1 (PSGL-1) expression and EPC adhesion. Inhibition of PSGL-1 by siRNA reversed the proangiogenic and adhesive effects of EphB4 activation. Moreover, neutralizing antibodies to E selectin and P selectin blocked ephrin-B2-Fc-stimulated EPC adhesion properties. Thus, activation of EphB4 enhances EPC proangiogenic capacity through induction of PSGL-1 expression and adhesion to E selectin and P selectin. Therefore, activation of EphB4 is an innovative and potentially valuable therapeutic strategy for improving the recruitment of EPCs to sites of neovascularization and thereby the efficiency of cell-based proangiogenic therapy.
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Células Endoteliales/metabolismo , Células Madre Fetales/metabolismo , Glicoproteínas de Membrana/metabolismo , Neovascularización Fisiológica , Receptor EphB4/metabolismo , Animales , Secuencia de Bases , Adhesión Celular , Células Cultivadas , Cartilla de ADN/genética , Selectina E/metabolismo , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Efrina-B2/metabolismo , Efrina-B2/farmacología , Sangre Fetal/citología , Células Madre Fetales/citología , Células Madre Fetales/efectos de los fármacos , Miembro Posterior/irrigación sanguínea , Humanos , Técnicas In Vitro , Isquemia/metabolismo , Isquemia/patología , Isquemia/terapia , Masculino , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Ratones , Ratones Desnudos , Neovascularización Fisiológica/efectos de los fármacos , Selectina-P/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptor EphB4/antagonistas & inhibidores , Receptor EphB4/genéticaRESUMEN
Angiogenic cell therapy with the transplantation of endothelial progenitor cells (EPC) or bone marrow mononuclear cells (BM-MNC) receives considerable attention as an approach to revascularize ischemic tissues. Adiponectin is a circulating hormone produced by the apM1 gene in adipocytes. Adiponectin modulates lipid metabolism and obesity, and it was recently found to promote physiological angiogenesis in response to ischemia. Patients with multiple cardiovascular disease risk factors or myocardial infarction may benefit from progenitor cell therapy, but they display depressed adiponectinemia. We hypothesized that adiponectin stimulation of transplanted cells is critical for their pro-angiogenic function. We aimed to establish whether adiponectinemia in the cell donor or in the cell recipient determines the success of pro-angiogenic cell therapy. In vitro, we found that conditioned media derived from wild-type adipocytes (adipo-CM) or purified adiponectin strongly enhanced BM-MNC survival and proliferation and stimulated EPC differentiation, whereas adipo-CM from apM1-/- adipocytes was one-half less effective. On the other hand, wild-type and apM1-/- BM-MNC displayed similar resistance to apoptosis and proliferation rates. In vivo, wild-type, and apM1-/- BM-MNC induced similar angiogenic reactions in wild-type ischemic hindlimbs. In contrast, wild-type BM-MNC had much diminished effects in apM1-/- ischemic hindlimbs. We concluded that adiponectin enhances BM-MNC survival and proliferation, and adiponectinemia in the cell therapy recipient is essential for the pro-angiogenic benefits of cell therapy. These observations imply that progenitor cell transplantation might only induce angiogenesis in patients with high adiponectinemia.
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Adiponectina/fisiología , Tratamiento Basado en Trasplante de Células y Tejidos , Adipocitos/metabolismo , Adiponectina/genética , Adiponectina/farmacología , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Miembro Posterior/patología , Isquemia/metabolismo , Isquemia/terapia , Leptina/farmacología , Masculino , Ratones , Ratones Transgénicos , Trasplante de Células Madre , Células Madre/citología , Células Madre/efectos de los fármacosRESUMEN
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.
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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/metabolismoRESUMEN
A central question in radiation protection research is whether low-dose and low-dose-rate (LDR) exposures to ionizing radiation play a role in progression of cardiovascular disease. The response of endothelial cells to different LDR exposures may help estimate risk of cardiovascular disease by providing the biological mechanism involved. We investigated the effect of chronic LDR radiation on functional and molecular responses of human aorta endothelial cells (HAoECs). Human aorta endothelial cells were continuously irradiated at LDR (6 mGy/h) for 15 days and analyzed at time points when the cumulative dose reached 0.05, 0.5, 1.0, and 2.0 Gy. The same doses were administered acutely at high-dose rate (HDR; 1 Gy/min). The threshold for the loss of angiogenic capacity for both LDR and HDR radiations was between 0.5 and 1.0 Gy. At 2.0 Gy, angiogenic capacity returned to normal only for HAoEC exposed to LDR radiation, associated with increased expression of antioxidant and anti-inflammatory genes. Pre-LDR, but not pre-HDR, radiation, followed by a single acute 2.0 Gy challenge dose sustained the expression of antioxidant and anti-inflammatory genes and stimulated angiogenesis. Our results suggest that dose rate is important in cellular response and that a radioadaptive response is involved for a 2.0 Gy dose at LDR.
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Existing and future nuclear fusion technologies involve the production and use of large quantities of tritium, a highly volatile, but low toxicity beta-emitting isotope of hydrogen. Tritium has received international attention because of public and scientific concerns over its release to the environment and the potential health impact of its internalization. This article provides a brief summary of the current state of knowledge of both the biological and regulatory aspects of tritium exposure; it also explores the gaps in this knowledge and provides recommendations on the best ways forward for improving our understanding of the health effects of low-level exposure to it. Linking health effects specifically to tritium exposure is challenging in epidemiological studies due to high uncertainty in tritium dosimetry and often suboptimal cohort sizes. We therefore argued that limits for tritium in drinking water should be based on evidence derived from controlled in vivo animal tritium toxicity studies that use realistically low levels of tritium. This article presents one such mouse study, undertaken within an international collaboration, and discusses the implications of its main findings, such as the similarity of the biokinetics of tritiated water (HTO) and organically bound tritium (OBT) and the higher biological effectiveness of OBT. This discussion is consistent with the position expressed in this article that in vivo animal tritium toxicity studies carried out within large, multi-partner collaborations allow evaluation of a great variety of health-related endpoints and essential to the development of international consensus on the regulation of tritium levels in the environment. Environ. Mol. Mutagen. 59:586-594, 2018. © 2018 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.
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Agua Potable/efectos adversos , Tritio/efectos adversos , Aminoácidos/análisis , Aminoácidos/farmacocinética , Animales , Sitios de Unión , Consenso , Agua Potable/análisis , Rayos gamma/efectos adversos , Dosimetría in Vivo , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Monitoreo de Radiación , Riesgo , Distribución Tisular , Tritio/análisis , Tritio/farmacocinética , Tritio/toxicidad , Organización Mundial de la SaludRESUMEN
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.
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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/metabolismoRESUMEN
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.
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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 fármacosRESUMEN
Vascular endothelial growth factors (VEGFs) and their receptors have emerged as central regulators of the angiogenic process. However, involvement of VEGF-B, one of these factors, in angiogenesis remains obscure. Mice received subcutaneous injection of Matrigel alone or Matrigel with human recombinant protein rhVEGF-B167 or with rhVEGF-A165. After 14 days, cell ingrowth in the Matrigel plug was increased by 2.0- and 2.5-fold in rhVEGF-B167-treated and rhVEGF-A165-treated mice, respectively (P<0.01), in association with a raise in phospho-Akt/Akt (1.8-fold, P<0.01) and endothelial NO synthase (eNOS) (1.80- and 1.60-fold, respectively; P<0.05) protein levels measured by Western blot. VEGF-B-induced cell ingrowth was impaired by treatment with NOS inhibitor (NG-nitro-l-arginine methyl ester; L-NAME, 10 mg/kg per day). Treatment with neutralizing antibody directed against the VEGF-B receptor VEGF-R1 (anti-VEGFR1, 10 microg) completely abrogated VEGF-B-related effects. Proangiogenic effect of VEGF-B was confirmed in a mouse model of surgically induced hindlimb ischemia. Plasmids containing human form of VEGF-A (phVEGF-A165) or VEGF-B (phVEGF-B167 or phVEGF-B186) were administered by in vivo electrotransfer. Angiographic score at day 28 showed significant improvement in ischemic/nonischemic leg ratio by 1.4- and 1.5-fold in mice treated with phVEGF-B167 and phVEGF-B186, respectively (P<0.05). Laser Doppler perfusion data also evidenced a 1.5-fold increase in phVEGF-B167-treated and phVEGF-B186-treated mice (P<0.05). Such an effect was associated with an upregulation of phospho-Akt/Akt and eNOS protein levels in the ischemic legs and was hampered by treatment with anti-VEGFR1. This study demonstrates for the first time that VEGF-B, in part through its receptor VEGF-R1, promotes angiogenesis in association with an activation of Akt and eNOS-related pathways.
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Factores de Crecimiento Endotelial/fisiología , Neovascularización Fisiológica/fisiología , Proteínas Serina-Treonina Quinasas , Animales , Arteriolas/efectos de los fármacos , Bioensayo , Capilares/efectos de los fármacos , Recuento de Células , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Colágeno/metabolismo , Combinación de Medicamentos , Factores de Crecimiento Endotelial/farmacología , Femenino , Miembro Posterior/irrigación sanguínea , Miembro Posterior/fisiopatología , Isquemia/fisiopatología , Laminina/metabolismo , Masculino , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica/efectos de los fármacos , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Fosforilación , Proteoglicanos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factor B de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
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.
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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 , EstreptozocinaRESUMEN
After Chernobyl and Fukushima Daï Chi, two major nuclear accidents, large amounts of radionuclides were released in the environment, mostly caesium 137 (137Cs). Populations living in contaminated territories are chronically exposed to radionuclides by ingestion of contaminated food. However, questions still remain regarding the effects of low dose ionizing radiation exposure on the development and progression of cardiovascular diseases. We therefore investigated the effects of a chronic internal exposure to 137Cs on atherosclerosis in predisposed ApoE-/- mice. Mice were exposed daily to 0, 4, 20 or 100 kBq/l 137Cs in drinking water, corresponding to range of concentrations found in contaminated territories, for 6 or 9 months. We evaluated plaque size and phenotype, inflammatory profile, and oxidative stress status in different experimental groups. Results did not show any differences in atherosclerosis progression between mice exposed to 137Cs and unexposed controls. However, 137Cs exposed mice developed more stable plaques with decreased macrophage content, associated with reduced aortic expression of pro-inflammatory factors (CRP, TNFα, MCP-1, IFNγ) and adhesion molecules (ICAM-1, VCAM-1 and E-selectin). Lesions of mice exposed to 137Cs were also characterized by enhanced collagen and smooth muscle cell content, concurrent with reduced matrix metalloproteinase MMP8 and MMP13 expression. These results suggest that low dose chronic exposure of 137Cs in ApoE-/- mice enhances atherosclerotic lesion stability by inhibiting pro-inflammatory cytokine and MMP production, resulting in collagen-rich plaques with greater smooth muscle cell and less macrophage content.
Asunto(s)
Apolipoproteínas E/genética , Aterosclerosis/patología , Rayos gamma , Animales , Aorta/metabolismo , Aorta/patología , Aorta/efectos de la radiación , Apolipoproteínas E/deficiencia , Apolipoproteínas E/metabolismo , Aterosclerosis/metabolismo , Moléculas de Adhesión Celular/metabolismo , Radioisótopos de Cesio/química , Colesterol/sangre , Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Lipoproteínas HDL/sangre , Lipoproteínas LDL/sangre , Macrófagos/citología , Macrófagos/inmunología , Masculino , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Estrés Oxidativo/efectos de la radiaciónRESUMEN
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/metabolismoRESUMEN
We hypothesized that formation of advanced glycation end products (AGEs) associated with diabetes reduces matrix degradation by metalloproteinases (MMPs) and contributes to the impairment of ischemia-induced angiogenesis. Mice were treated or not with streptozotocin (40 mg/kg) and streptozotocin plus aminoguanidine (AGEs formation blocker, 50 mg/kg). After 8 weeks of treatment, hindlimb ischemia was induced by right femoral artery ligature. Plasma AGE levels were strongly elevated in diabetic mice when compared with control mice (579 +/- 21 versus 47 +/- 4 pmol/ml, respectively; P < 0.01). Treatment with aminoguanidine reduced AGE plasma levels when compared with untreated diabetic mice (P < 0.001). After 28 days of ischemia, ischemic/nonischemic leg angiographic score, capillary density, and laser Doppler skin-perfusion ratios were 1.4-, 1.5-, and 1.4-fold decreased in diabetic mice in reference to controls (P < 0.01). Treatment with aminoguanidine completely normalized ischemia-induced angiogenesis in diabetic mice. We next analyzed the role of proteolysis in AGE formation-induced hampered neovascularization process. After 3 days of ischemia, MMP-2 activity and MMP-3 and MMP-13 protein levels were increased in untreated and aminoguanidine-treated diabetic mice when compared with controls (P < 0.05). Despite this activation of the MMP pathway, collagenolysis was decreased in untreated diabetic mice. Conversely, treatment of diabetic mice with aminoguanidine restored collagenolysis toward levels found in control mice. In conclusion, blockade of AGE formation by aminoguanidine normalizes impaired ischemia-induced angiogenesis in diabetic mice. This effect is probably mediated by restoration of matrix degradation processes that are disturbed as a result of AGE accumulation.