RESUMO
We have developed an ex vivo gene transfer technique to rabbit arterial wall using autologous smooth muscle cells (SMCs). SMCs were harvested from rabbit ear artery, transduced in vitro with vesicular stomatitis virus G-glycoprotein pseudotyped retrovirus or feline immunodeficiency virus (FIV) and returned to the adventitial surface of the carotid artery using a periadventitial silicone collar or collagen sheet placed around the artery. Beta-galactosidase (lacZ) and human apolipoprotein E3 (apoE3) cDNAs were used as transgenes. After retrovirus-mediated gene transfer of lacZ the selected cells implanted with high efficiency and expressed lacZ marker gene at a very high level 7 and 14 days after the operation. The level of lacZ expression decreased thereafter but was still detectable 12 weeks after the gene transfer, and was exclusively localized to the site of cell implantation inside the collar. Utilizing FIV vector expressing apoE3, low levels of apoE were measured from serum collected from a low-density lipoprotein receptor deficient Watanabe heritable hyperlipidemic rabbits 1 month after the gene transfer. The physiological effect of apoE expression was detected as transiently elevated serum cholesterol levels. The results indicate that the model can be used for high efficiency local gene transfer in arteries, e.g. during vascular surgery. The model is also valuable for studying expression, stability and safety of new gene transfer vectors and their expression products in vivo.
Assuntos
Vetores Genéticos , Vírus da Imunodeficiência Felina , Miócitos de Músculo Liso/fisiologia , Transdução Genética , Animais , Apolipoproteína E3 , Apolipoproteínas E/genética , Artérias Carótidas/citologia , Artérias Carótidas/fisiologia , Gatos , Terapia Genética/métodos , Humanos , Glicoproteínas de Membrana/genética , Miócitos de Músculo Liso/transplante , Coelhos , Transdução Genética/métodos , Transplante Autólogo , Doenças Vasculares/terapia , Procedimentos Cirúrgicos Vasculares , Proteínas do Envelope Viral/genética , beta-Galactosidase/genéticaRESUMO
In this study we have attached cyclic targeting peptides by way of a poly-lysine spacer on the surface of an adenovirus using a transglutaminase enzymatic reaction to enhance transduction efficiency and to modify tissue tropism in vivo. Nuclear targeted lacZ- and TIMP-1-encoding adenoviruses were coupled to a peptide-motif (HWGF) that can bind to matrix metalloproteinase (MMP)-2 and MMP-9. Modified viruses were used to evaluate gene transfer efficiency, biodistribution, and the effect on neointima formation following balloon denudation injury. In vitro, both rabbit aortic smooth muscle cells and human endothelial hybridoma cells demonstrated significantly increased reporter gene expression with HWGF-modified adenoviruses (AdlacZ(HWGF)) compared with control (AdlacZ) or mismatch peptide-modified (AdlacZ(MM)) adenoviruses. However, in human hepatocellular Hep-G2 cells, both AdlacZ(HWGF) and AdlacZ(MM) produced significantly lower transgene expression compared with the respective control viruses. In vivo, local intravascular catheter-mediated gene transfer of a HWGF-targeted TIMP-1-encoding adenovirus (AdTIMP-1(HWGF)) significantly reduced intimal thickening in a rabbit aortic balloon denudation model (P < 0.05) compared with the control adenovirus. X-Gal staining and biodistribution analyses with TaqMan RT-PCR revealed that the cyclic peptides altered vector tropism and, in particular, reduced transduction of the liver. We found that the HWGF peptide modification increased transduction efficiency of the adenovirus-mediated gene transfer in smooth muscle cells and endothelial cells in in vitro and enhanced gene transfer to the arterial wall in vivo; that peptide modification of adenoviruses beneficially modulated tissue tropism in vivo; and that efficient TIMP-1 gene transfer reduced intimal thickening in an established restenosis model in rabbits.
Assuntos
Adenoviridae/genética , Reestenose Coronária/terapia , Vetores Genéticos , Inibidor Tecidual de Metaloproteinase-1/genética , Transdução Genética/métodos , Motivos de Aminoácidos/genética , Animais , Reestenose Coronária/genética , Reestenose Coronária/patologia , Endotélio Vascular/fisiologia , Técnicas de Transferência de Genes , Humanos , Técnicas In Vitro , Músculo Liso Vascular/fisiologia , Especificidade de Órgãos , Peptídeos Cíclicos/genética , Reação em Cadeia da Polimerase , Coelhos , Inibidor Tecidual de Metaloproteinase-1/administração & dosagemRESUMO
Arterial smooth muscle cell (SMC) migration and proliferation are central features in atherogenesis. Altered gene expression and cell proliferation in atherosclerotic lesions have some similar characteristics with certain solid tumors and thus might have similar mechanisms that lead to SMC proliferation. Among cancer cells common features are genome-wide hypomethylation which correlates with transformation and tumor progression, and coincident overexpression of methyltransferase (MTase). The purpose of the present study was to analyze whether alterations in DNA methylation and MTase expression are present in atherosclerotic lesions. A significant reduction in genomic 5-methylcytosine content was detected in advanced human atherosclerotic lesions and in lesions of ApoE knock-out mice. SMC were shown to develop hypomethylation in vitro during transformation from a contractile to synthetic phenotype. Balloon denudation of New Zealand White rabbit aorta caused proliferation of intimal SMC with concomitant genomic hypomethylation in the thickened intima. By using in situ hybridization the overall transcriptional activity was found to be increased in clusters of lesion SMC. Marked heterogeneity was seen in MTase mRNA expression in various types of atherosclerotic lesions among intimal and medial SMC. These findings show that (1) genomic hypomethylation occurs during atherogenesis in human, mouse and rabbit lesions and that it correlates with increased transcriptional activity; (2) MTase is expressed in atherosclerotic lesions; and (3) hypomethylation is present in advanced lesions at the same level as in malignant tumors and may affect cellular proliferation and gene expression in atherosclerotic lesions.
Assuntos
Arteriosclerose/genética , Arteriosclerose/metabolismo , Metilação de DNA , Metilases de Modificação do DNA/biossíntese , Metilases de Modificação do DNA/genética , DNA/genética , DNA/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Aorta/metabolismo , Aorta/patologia , Movimento Celular/genética , Criança , Modelos Animais de Doenças , Feminino , Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Modelos Cardiovasculares , Miócitos de Músculo Liso/metabolismo , Proteínas Proto-Oncogênicas c-sis/biossíntese , Proteínas Proto-Oncogênicas c-sis/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Coelhos , Túnica Íntima/metabolismoRESUMO
Real-time PCR is a powerful method for the quantification of gene expression in biological samples. This method uses TaqMan chemistry based on the 5' -exonuclease activity of the AmpliTaq Gold DNA polymerase which releases fluorescence from hybridized probes during synthesis of each new PCR product. Many gene therapy studies use lacZ, encoding Escherichia coli beta-galactosidase, as a marker gene. Our results demonstrate that E. coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ gene expression and decreases sensitivity of the method below the level required for biodistribution and long-term gene expression studies. In biodistribution analyses the contamination can lead to false-negative results by masking low-level lacZ expression in target and ectopic tissues, and false-positive results if sufficient controls are not used. We conclude that, to get reliable TaqMan results with lacZ, adequate controls should be included in each run to rule out contamination from AmpliTaq Gold polymerase.
