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1.
J Clin Invest ; 117(6): 1527-37, 2007 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-17510705

RESUMEN

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.


Asunto(s)
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/genética
2.
Circ Res ; 103(7): 751-60, 2008 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-18723447

RESUMEN

Cell-based therapy is a promising approach designed to enhance neovascularization and function of ischemic tissues. Interaction between endothelial and smooth muscle cells regulates vessels development and remodeling and is required for the formation of a mature and functional vascular network. Therefore, we assessed whether coadministration of endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs) can increase the efficiency of cell therapy. Unilateral hindlimb ischemia was surgically induced in athymic nude mice treated with or without intravenous injection of EPCs (0.5 x 10(6)), SMPCs (0.5 x 10(6)) and EPCs+SMPCs (0.25 x 10(6)+0.25 x 10(6)). Vessel density and foot perfusion were increased in mice treated with EPCs+SMPCs compared to animals receiving EPCs alone or SMPCs alone (P<0.001). In addition, capillary and arteriolar densities were enhanced in EPC+SMPC-treated mice compared to SMPC and EPC groups (P<0.01). We next examined the role of Ang-1/Tie2 signaling in the beneficial effect of EPC and SMPC coadministration. Small interfering RNA directed against Ang-1-producing SMPCs or Tie2-expressing EPCs blocked vascular network formation in Matrigel coculture assays, reduced the rate of incorporated EPCs within vascular structure, and abrogated the efficiency of cell therapy. Production of Ang-1 by SMPCs activates Tie2-expressing EPCs, resulting in increase of EPC survival and formation of a stable vascular network. Subsequently, the efficiency of EPC- and SMPC-based cotherapy is markedly increased. Therefore, coadministration of different types of vascular progenitor cells may constitute a novel therapeutic strategy for improving the treatment of ischemic diseases.


Asunto(s)
Células Endoteliales/trasplante , Miembro Posterior/irrigación sanguínea , Isquemia/terapia , Miocitos del Músculo Liso/trasplante , Neovascularización Fisiológica , Trasplante de Células Madre , Células Madre , Angiotensina I/metabolismo , Animales , Células Endoteliales/metabolismo , Humanos , Isquemia/metabolismo , Masculino , Ratones , Ratones Desnudos , Miocitos del Músculo Liso/citología , Receptor TIE-2/metabolismo , Transducción de Señal , Células Madre/citología , Células Madre/metabolismo
3.
Arterioscler Thromb Vasc Biol ; 26(4): 773-9, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16410461

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
4.
Circ Res ; 91(5): 441-8, 2002 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-12215494

RESUMEN

Identification of factors that may stimulate ischemia-induced neovascularization without increasing atherosclerotic plaque progression is of major therapeutic importance. We hypothesized that interleukin-18 binding protein (IL-18BP), a major antiinflammatory protein with plaque-stabilizing activities, may affect the neovascularization in mice ischemic hindlimb. Ischemia was produced by artery femoral occlusion in mice that were subjected to in vivo intramuscular electrotransfer of either an empty plasmid or a murine IL-18BP plasmid. Angiographic score, capillary density (CD31 staining), and laser Doppler perfusion data at day 28 showed significant improvement in ischemic/nonischemic leg ratio by respectively 1.6-, 1.4-, and 1.5-fold in IL-18BP-treated mice compared with controls (P<0.01). This was associated with a significant 2-fold increase in both vascular endothelial growth factor (VEGF) and phospho-Akt protein content in the ischemic hindlimb of IL-18BP-treated mice (P<0.05). Similar results were obtained in IL-18-deficient mice. Because bone marrow-derived endothelial progenitor cells (BM-EPCs) are involved in postnatal vasculogenesis, EPCs were isolated and cultivated from bone marrow mononuclear cells. IL-18BP treatment led to a significant 1.8-fold increase in the percentage of BM-EPCs characterized as cells positive for both AcLDL-Dil and von Willebrand factor (P<0.001). In conclusion, IL-18BP stimulates ischemia-induced neovascularization in association with an activation of VEGF/Akt signaling and an increase in BM-EPCs mobilization and differentiation. Our findings strongly suggest a major antiangiogenic role of endogenous IL-18 in postischemic injury.


Asunto(s)
Glicoproteínas/fisiología , Miembro Posterior/irrigación sanguínea , Interleucina-18/fisiología , Isquemia/fisiopatología , Neovascularización Patológica/fisiopatología , Proteínas Serina-Treonina Quinasas , Angiografía , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/fisiología , Línea Celular , Factores de Crecimiento Endotelial/metabolismo , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Glicoproteínas/genética , Miembro Posterior/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Interleucina-18/genética , Interleucina-18/farmacología , Flujometría por Láser-Doppler , Linfocinas/metabolismo , Linfocinas/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Fisiológica/efectos de los fármacos , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Fosforilación , Plásmidos/administración & dosificación , Plásmidos/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular
5.
Circulation ; 108(23): 2839-42, 2003 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-14656923

RESUMEN

BACKGROUND: Bone marrow-derived mononuclear cells (BM-MNCs) enhance postischemic neovascularization, and their therapeutic use is currently under clinical investigation. We evaluated the safety of BM-MNC-based therapy in the setting of atherosclerosis. METHODS AND RESULTS: Apolipoprotein E (apoE)-knockout (KO) mice were divided into 4 groups: 20 nonischemic mice receiving intravenous injection of either saline (n=10) or 10(6) BM-MNCs from wild-type animals (n=10) and 20 mice with arterial femoral ligature receiving intravenous injection of either saline (n=10) or 10(6) BM-MNCs from wild-type animals (n=10) at the time of ischemia induction. Animals were monitored for 4 additional weeks. Atherosclerosis was evaluated in the aortic sinus. BM-MNC transplantation improved tissue neovascularization in ischemic hind limbs, as revealed by the 210% increase in angiography score (P<0.0001), the 33% increase in capillary density (P=0.01), and the 65% increase in tissue Doppler perfusion score (P=0.0002). Hindlimb ischemia without BM-MNC transplantation or BM-MNC transplantation without ischemia did not affect atherosclerotic plaque size. However, transplantation of 10(6) BM-MNCs into apoE-KO mice with hindlimb ischemia induced a significant 48% to 72% increase in lesion size compared with the other 3 groups (P=0.0025), despite similar total cholesterol levels. Transplantation of 10(5) BM-MNCs produced similar results, whereas transplantation of 10(6) apoE-KO-derived BM-MNCs had neither proangiogenic nor proatherogenic effects. There was no difference in plaque composition between groups. CONCLUSIONS: BM-MNC therapy is unlikely to affect atherosclerotic plaque stability in the short term. However, it may promote further atherosclerotic plaque progression in an ischemic setting.


Asunto(s)
Apolipoproteínas E/deficiencia , Arteriosclerosis/terapia , Trasplante de Médula Ósea/efectos adversos , Miembro Posterior/irrigación sanguínea , Isquemia/terapia , Neovascularización Fisiológica , Seno Aórtico/patología , Animales , Apolipoproteínas E/genética , Arteriosclerosis/metabolismo , Quimiocina CCL2/sangre , Colesterol/sangre , Progresión de la Enfermedad , Arteria Femoral , Ligadura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Seguridad , Insuficiencia del Tratamiento , Factor A de Crecimiento Endotelial Vascular/sangre
6.
Cardiovasc Res ; 62(1): 176-84, 2004 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-15023564

RESUMEN

OBJECTIVES: Recent studies have provided increasing evidence that postnatal neovascularization does not rely exclusively on sprouting of preexisting vessels, but also involves bone marrow-derived circulating endothelial precursors (BM-EPCs). Animal studies revealed that neovascularization of ischemic tissue can be enhanced by BM-EPCs transplantation. But a possible limitation to the use of vascular precursors for therapeutic angiogenesis is the relatively low number of these cells. In this study, we demonstrate that ex vivo expanded differentiated endothelial cells (ECs) and smooth muscle cells (SMCs), may home to the tumor vasculature allowing targeting of transgene expression to the neoangiogenic site. METHODS: Mononuclear cells (MNCs) or CD34+ -enriched cells were purified from cord blood. We have defined culture conditions in which we observed two types of clones easily differentiated according to their morphology: cobblestone or spindle-shaped. Phenotypic characterization was assessed by immunocytochemistry, flow cytometry analysis and polymerase reaction with reverse transcription. Formation of capillary-like network in vitro was studied in three-dimensional collagen culture. And recruitment of these cells to a tumoral neoangiogenic site was assessed into tumor-bearing Severe Combined Immunodeficient (SCID) mouse model. RESULTS: The cobblestone cells uniformly positive for CD31, VE-cadherin, vWF, VEGF R1 and R2, ecNOS and incorporating acetylated LDL were ECs. Spindle-shaped cells expressed alpha-smooth muscle actin (alpha-SMA), Smooth Muscle Heavy Chain (SMHC), SM22 and calponin. They also displayed a carbachol-induced contractility in a medium containing IGF1. So we concluded that spindle-shaped cells were SMCs. ECs and SMCs interacted with each other to form a capillary like network in three-dimensional type I collagen culture. Moreover, these ex vivo differentiated cells are able to home to the tumor vasculature. CONCLUSION: We provide evidence that progenitors for ECs and SMCs circulate in human cord blood and differentiate into functional ECs and SMCs. These differentiated cells could provide a biomaterial for vascular cell therapy, because of their homing capacity to the neovascularization site.


Asunto(s)
Células Endoteliales/citología , Sangre Fetal , Músculo Liso/citología , Células Madre/citología , Animales , Western Blotting/métodos , Línea Celular Tumoral , Movimiento Celular , Citometría de Flujo , Humanos , Ratones , Ratones SCID , Trasplante de Neoplasias , Neovascularización Patológica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor 1 de Factores de Crecimiento Endotelial Vascular/análisis
7.
Exp Cell Res ; 313(7): 1337-46, 2007 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-17362928

RESUMEN

We described the ex vivo production of mature and functional human smooth muscle cells (SMCs) derived from skeletal myoblasts. Initially, myoblasts expressed all myogenic cell-related markers such as Myf5, MyoD and Myogenin and differentiate into myotubes. After culture in a medium containing vascular endothelial growth factor (VEGF), these cells were shown to have adopted a differentiated SMC identity as demonstrated by alphaSMA, SM22alpha, calponin and smooth muscle-myosin heavy chain expression. Moreover, the cells cultured in the presence of VEGF did not express MyoD anymore and were unable to fuse in multinucleated myotubes. We demonstrated that myoblasts-derived SMCs (MDSMCs) interacted with endothelial cells to form, in vitro, a capillary-like network in three-dimensional collagen culture and, in vivo, a functional vascular structure in a Matrigel implant in nonobese diabetic-severe combined immunodeficient mice. Based on the easily available tissue source and their differentiation into functional SMCs, these data argue that skeletal myoblasts might represent an important tool for SMCs-based cell therapy.


Asunto(s)
Diferenciación Celular , Colágeno/farmacología , Laminina/farmacología , Mioblastos Esqueléticos/fisiología , Miocitos del Músculo Liso/fisiología , Proteoglicanos/farmacología , Técnicas de Cultivo de Tejidos , Animales , Biomarcadores/análisis , Células Cultivadas , Combinación de Medicamentos , Células Epiteliales/fisiología , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fibras Musculares Esqueléticas/fisiología , Mioblastos Esqueléticos/efectos de los fármacos , Mioblastos Esqueléticos/metabolismo , Mioblastos Esqueléticos/trasplante , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/trasplante , Neovascularización Fisiológica , Trasplante Heterólogo , Factor A de Crecimiento Endotelial Vascular/farmacología
8.
Am J Pathol ; 164(2): 457-66, 2004 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-14742252

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
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