RESUMEN
BACKGROUND: Acidic fibroblast growth factor (FGF-1) has been identified as a potent mitogen for vascular cells, inducing formation of mature blood vessels in vitro and in vivo and represents one of the most promising approaches for the treatment of ischemic cardiovascular diseases by gene therapy. Nevertheless, and most probably due to the few experimental models able to address the issue, no study has described the therapeutic effects of FGF-1 gene transfer in subjects with peripheral arterial disease (PAD) exhibiting a clinically relevant cardiovascular pathology. METHODS: In order to assess the potency of FGF-1 gene transfer for therapeutic angiogenesis in ischemic skeletal muscles displaying decreased gene expression levels and sustained impaired formation of collateral vessels and arterioles, we developed a model of PAD in hamsters with a background of hypercholesterolemia. Hamsters fed a cholesterol-rich diet and subjected to hindlimb ischemia exhibit a sustained impaired angiogenic response, as evidenced by decreased angiographic score and histological quantification of arterioles in the ischemic muscles. RESULTS: In this model, we demonstrate that NV1FGF (a human FGF-1 expression plasmid), given intramuscularly 14 days after induction of hindlimb ischemia, promoted the formation of both collateral vessels and arterioles 14 days after treatment (i.e. 28 days post-ischemia). CONCLUSIONS: Our data provide evidence that NV1FGF can reverse the cholesterol-induced impairment of revascularization in a hamster model of hindlimb ischemia by promoting the growth of both collateral vessels and arterioles in ischemic muscles exhibiting significantly decreased levels of gene expression compared with control muscles. Therefore, this study underscores the relevance of NV1FGF gene therapy to overcome perfusion defects in patients with PAD.
Asunto(s)
Factor 1 de Crecimiento de Fibroblastos/genética , Terapia Genética/métodos , Vectores Genéticos/genética , Isquemia/terapia , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Enfermedades Vasculares Periféricas/terapia , Animales , Arteriolas/crecimiento & desarrollo , Circulación Colateral/fisiología , Cricetinae , Modelos Animales de Enfermedad , Factor 1 de Crecimiento de Fibroblastos/uso terapéutico , Técnicas de Transferencia de Gen , Hipercolesterolemia/complicaciones , Enfermedades Vasculares Periféricas/complicaciones , Plásmidos/genética , Plásmidos/uso terapéuticoRESUMEN
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.
Asunto(s)
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
We used a panel of recombinant human apolipoprotein (apo) A-IV truncation mutants, in which pairs of 22-mer alpha-helices were sequentially deleted along the primary sequence, to examine the impact of protein structure and interfacial activity on the ability of apoA-IV to activate cholesterol ester transfer protein. Circular dichroism and fluorescence spectroscopy revealed that the secondary structure, conformation, and molecular stability of recombinant human apoA-IV were identical to the native protein. However, deletion of any of the alpha-helical domains in apoA-IV disrupted its tertiary structure and impaired its molecular stability. Surprisingly, determination of the water/phospholipid interfacial exclusion pressure of the apoA-IV truncation mutants revealed that, for most, deletion of amphipathic alpha-helical domains increased their affinity for phospholipid monolayers. All of the truncation mutants activated the transfer of fluorescent-labeled cholesterol esters between high and low density lipoproteins at a rate higher than native apoA-IV. There was a strong positive correlation (r = 0.790, p = 0.002) between the rate constant for cholesterol ester transfer and interfacial exclusion pressure. We conclude that molecular interfacial exclusion pressure, rather than specific helical domains, determines the degree to which apoA-IV, and likely other apolipoproteins, facilitate cholesterol ester transfer protein-mediated lipid exchange.