RESUMEN
We previously demonstrated that injection of IL-2-activated natural killer (NK) cells contribute to vascular remodeling via a4b7 integrin and killer cell lectin-like receptor (KLRG) 1 and promote cardiac repair following myocardial infarction (MI). The aim of the present study is to test the hypothesis that injection of recombinant human interleukin (rhIL)-2 improves angiogenesis and preserves heart function after MI. A single IV injection of rhIL-2 two days following MI improved by 27.7% the left ventricular (LV) fractional shortening of immune competent (C57Bl6) mice, but had no effect on cardiac function of immune-deficient (NOD-SCID IL2Rγnull) mice. Immunohistochemical analysis of C57Bl6 cross sections of heart revealed that collagen deposition was reduced by 23.1% and that capillary density was enhanced in the scar area and the border zone of the infarct respectively by 22.4% and 33.6% following rhIL-2 injection. In addition, rhIL-2 enhanced 1.6-fold the in vivo endothelial cell proliferation index and 1.8-fold the number of NK cell infiltrating the infarcted heart, but had no effect on the number of cardiac CD4 and CD8 cells. In vitro, rhIL-2 activated NK cells enhanced cardiac endothelial cell proliferation by 17.2%. Here we show that a single IV injection of rhIL-2 positively impacted cardiac function by improving angiogenesis through a process involving NK cells.
Asunto(s)
Pruebas de Función Cardíaca/efectos de los fármacos , Corazón/fisiopatología , Interleucina-2/farmacología , Infarto del Miocardio/fisiopatología , Neovascularización Fisiológica/efectos de los fármacos , Animales , Recuento de Células , Proliferación Celular/efectos de los fármacos , Colágeno/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Citometría de Flujo , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Interleucina-2/uso terapéutico , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Infarto del Miocardio/patología , Tamaño de los Órganos/efectos de los fármacosRESUMEN
Endothelial progenitor cells (EPCs) consist of two different subpopulations named early (eEPCs) and late EPCs (lEPCs) that are derived from CD14(+) and CD14(-) circulating cells, respectively. These cells are regularly cultured over fibronectin-coated surfaces in endothelial basal medium (EBM)-2 supplemented with insulin-like growth factor (IGF-1), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). We have developed a new and simplified method for culturing human EPCs obtained from peripheral blood and tested their ability to preserve cardiac function following infarction. We first demonstrated that eEPCs derived from human peripheral blood mononuclear cells (PBMCs) and cultured in EBM-2 medium supplemented with autologous serum (10%) over fibronectin-coated surfaces (10 µg/ml) in the presence of IGF-1 (50 ng/ml) only, have a secretome similar to eEPCs cultured under regular conditions with IGF-1, VEGF, EGF, and FGF. Our data also indicate that IGF-1 modulates PBMC secretome in a dose-dependent manner. In another series of experiments, we showed that PBMCs cultured in suspension in bags (S-PBMCs) in basal medium supplemented with fibronectin and IGF-1 secrete significant amounts of stem cell factor (SCF, 31.3 ± 3.1 pg/ml)), hepatocyte growth factor (HGF, 438.6 ± 41.4 pg/ml), soluble tumor necrosis factor receptor 1 (sTNFR1, 127.1 ± 9.9 pg/ml), VEGF (139.3 ± 9.6 pg/ml), and IGF-1 (147.2 ± 46.1 pg/ml) but very low levels of TNF-α (13.4 ± 2.5 pg/ml). S-PBMCs injected intravenously into NOD SCID mice migrated to the injured myocardium, reduced cardiac fibrosis, enhanced angiogenesis, and preserved cardiac function after myocardial infarction (MI) in a manner similar to eEPCs cultured under standard conditions. In conclusion, we show in this study a refined and optimized method for culturing eEPCs. Our data indicate that S-PBMCs are composed of several cell populations including eEPCs and that they secrete high amounts of antiapoptotic, anti-inflammatory, and proangiogenic factors capable of preserving cardiac function following MI.
Asunto(s)
Células Sanguíneas/citología , Técnicas de Cultivo de Célula/métodos , Células Endoteliales/citología , Células Endoteliales/trasplante , Isquemia/terapia , Enfermedades Vasculares/terapia , Inductores de la Angiogénesis/metabolismo , Animales , Apoptosis/efectos de los fármacos , Células Sanguíneas/efectos de los fármacos , Células Sanguíneas/metabolismo , Adhesión Celular/efectos de los fármacos , Técnicas de Cultivo de Célula/economía , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Fibronectinas/farmacología , Pruebas de Función Cardíaca/efectos de los fármacos , Humanos , Mediadores de Inflamación/metabolismo , Inyecciones Intravenosas , Factor I del Crecimiento Similar a la Insulina/farmacología , Isquemia/complicaciones , Isquemia/fisiopatología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Infarto del Miocardio/complicaciones , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Infarto del Miocardio/terapia , Trasplante de Células Madre , Enfermedades Vasculares/complicaciones , Enfermedades Vasculares/fisiopatologíaRESUMEN
In this study, we have investigated the hypothesis that previously reported beneficial effect of peripheral blood mononuclear cells cultured under angiogenic conditions on cardiovascular function following ischemia is not limited to EPCs but also to monocytes contained therein. We first purified and analyzed the phenotype and secretome of human and murine blood monocytes cultured under angiogenic conditions (named MDs for monocyte derivatives) and tested their effect in a mouse model of myocardial infarction (MI). FACS analysis of MDs shows that these cells express mature endothelial cell markers and that their proliferative capacity is virtually absent, consistent with their end-differentiated monocytic ontogeny. MDs secreted significant levels of HGF, IGF-1, MCP-1, and sTNFR-1 relative to their monocyte precursors. MDs were unable to form vascular networks in vitro when cultured on matrix coated flasks. Treatment of murine HL-1 cardiomyocyte cell line with MD-conditioned medium reduced their death induced by TNF-alpha, staurosporine, and oxidative stress, and this effect was dependent upon MD-derived sTNFR-1, HGF, and IGF-1. We further demonstrate that MD secretome promoted endothelial cell proliferation and capacity to form vessels in vitro and this was dependent upon MD-derived MCP-1, HGF, and IGF-1. Echocardiography analysis showed that MD myocardial implantation improved left ventricle fractional shortening of mouse hearts following MI and was associated with reduced myocardial fibrosis and enhancement of angiogenesis. Transplanted MDs and their secretome participate in preserving functional myocardium after ischemic insult and attenuate pathological remodeling.
Asunto(s)
Monocitos/metabolismo , Células Musculares/citología , Infarto del Miocardio/terapia , Neovascularización Fisiológica , Animales , Apoptosis , Células Cultivadas , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/citología , Células Endoteliales/metabolismo , Femenino , Citometría de Flujo , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Endogámicos C57BL , Monocitos/trasplante , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Estaurosporina/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Remodelación VentricularRESUMEN
Haemophilia B is characterized by a deficiency of the gamma-carboxylated protein, factor IX (FIX). As a first step to optimize a gene therapy strategy to treat haemophilia B, we employed a previously described approach (Biochemistry 2000;39: 14322) of altering the propeptide of vitamin K-dependent proteins in vitro, to improve the carboxylation efficiency of FIX. Both native FIX and FIX with a prothrombin propeptide (proPT-FIX) produced recombinant FIX in vitro following transfection of their cDNAs into human embryonic kidney (HEK) 293 cells. Using hydroxyapatite chromatography to separate carboxylated from uncarboxylated FIX, we are able to show that >90% of FIX is gamma-carboxylated and that substituting the propeptide of prothrombin into FIX does not further increase the relative amounts of carboxylated material. These results demonstrate that the nature of the propeptide, per se is not the sole determinant of optimal carboxylation of FIX in our expression system in HEK 293 cells.
Asunto(s)
Factor IX/genética , Vitamina K/fisiología , Western Blotting/métodos , Ligasas de Carbono-Carbono , Línea Celular , Cromatografía Liquida/métodos , ADN Complementario/genética , Factor IX/biosíntesis , Factor IX/aislamiento & purificación , Humanos , Protrombina/genética , Proteínas Recombinantes/biosíntesis , TransfecciónRESUMEN
Haemophilia B, or factor IX (FIX) deficiency, represents 15% of the hereditary haemophilias. The serious morbidity from the transmission of infectious agents in plasma-derived material has mandated a need for the production of recombinant product. The rate-limiting step for the production of recombinant FIX is gamma-carboxylation, a post-translational modification carried out only in mammalian cells. To test the carboxylation efficiency of recombinantly produced FIX in vitro and to improve the isolation of the pure active product, we produced FIX in a transfected human cell line (293 human embryonic kidney cells) and isolated material by immunoaffinity chromatography followed by hydroxyapatite chromatography. Unexpectedly, during hydroxyapatite chromatography, we discovered that purified FIX was contaminated by a heretofore unknown protein. Further analysis by mass spectrometry (MS) sequencing revealed this protein to be galectin-3-binding protein (G3BP). The above results raise an important note of caution regarding the production of recombinant FIX and, indeed, other proteins produced recombinantly in mammalian cells.