RESUMO
Coronary artery disease is a major cause of death and disability worldwide. New therapies are needed for patients who do not benefit or are not suitable for current treatments. Angiogenic gene therapy using vascular endothelial growth factors (VEGFs) has shown potential in preclinical trials. However, undesired side effects, such as increased permeability, limit their therapeutic potential. The aim of this study was to investigate if adenoviral gene transfer of a VEGF receptor 2 (VEGFR-2) ligand Gremlin, given simultaneously with VEGF-A, could modulate VEGFR-2-mediated increase in permeability without impairing the angiogenic effect of VEGF-A gene therapy. Gene transfers were done in pigs (n = 22) using endocardial injections with an endovascular injection catheter. Animals were divided in three groups receiving adenoviral (Ad) VEGF-A (n = 10), Gremlin (n = 6), or VEGF-A+Gremlin (n = 6) gene therapy. Animals were sacrificed and samples collected 6 days later for histological, safety, and permeability analyses. The mean capillary area was significantly increased in both treatment groups with AdVEGF-A when compared with the AdGremlin group. Also, the capillary area was significantly larger in AdVEGF-A group without AdGremlin. No significant differences in tissue permeability were observed using modified Miles assay between AdVEGF-A and AdVEGF-A+AdGremlin groups. However, cardiac tamponade and sudden cardiac deaths were observed only in the AdVEGF-A group. AdVEGF-A induces strong angiogenesis in porcine myocardium. Our results suggest that AdGremlin can limit the side effects of AdVEGF-A therapy, even though no direct effect on tissue permeability could be demonstrated. This could enable the use of larger AdVEGF-A doses to increase the treatment area and angiogenic effects without adverse side effects.
Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Miocárdio/metabolismo , Neovascularização Fisiológica/genética , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Permeabilidade Capilar/genética , Doença da Artéria Coronariana/etiologia , Doença da Artéria Coronariana/patologia , Doença da Artéria Coronariana/terapia , Modelos Animais de Doenças , Expressão Gênica , Vetores Genéticos/administração & dosagem , Testes de Função Cardíaca , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Suínos , Transdução Genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Endothelial cells (ECs) play a key role in tissue homeostasis, in several pathological conditions, and specifically in the control of vascular functions. ECs are frequently used as in vitro model systems for cardiovascular studies and vascular biology. The porcine model is commonly used in human clinical cardiovascular studies. Currently, however, there is no robust protocol for the isolation of porcine heart ECs. We have developed a fast isolation protocol, which is cost effective, takes only 1-2 h, and produces EC purity of over 97%. This protocol is optimized for porcine hearts but can be adapted for use with other large animals. METHODS: Heart is washed by flushing with PBS, whereafter endothelial cells are detached by collagenase incubation and the cells can then be collected immediately after the incubation and plated within an hour after the heart is isolated from a pig. RESULTS: The swiftness of the protocol limits changes in the phenotype and RNA expression profile of the cells. Cells were identified as ECs with CD31 (PECAM-1) antibody immunostaining. Functionality of ECs were ensured with in vitro angiogenesis assay. The purity of the ECs was verified by using fluorescence assisted cell sorting (FACS) with the CD31 antibody. CONCLUSION: We developed a new, fast, and cost-effective isolation method for pig heart ECs. Successful isolation of pure ECs is a prerequisite for several cardiovascular and vascular biology studies.
