RESUMO
BACKGROUND: Gene transfer to the vessel wall may provide new possibilities for the treatment of vascular disorders, such as postangioplasty restenosis. In this study, we analyzed the effects of adenovirus-mediated vascular endothelial growth factor (VEGF)-C gene transfer on neointima formation after endothelial denudation in rabbits. For comparison, a second group was treated with VEGF-A adenovirus and a third group with lacZ adenovirus. Clinical-grade adenoviruses were used for the study. METHODS AND RESULTS: Aortas of cholesterol-fed New Zealand White rabbits were balloon-denuded, and gene transfer was performed 3 days later. Animals were euthanized 2 and 4 weeks after the gene transfer, and intima/media ratio (I/M), histology, and cell proliferation were analyzed. Two weeks after the gene transfer, I/M in the lacZ-transfected control group was 0. 57+/-0.04. VEGF-C gene transfer reduced I/M to 0.38+/-0.02 (P:<0.05 versus lacZ group). I/M in VEGF-A-treated animals was 0.49+/-0.17 (P:=NS). The tendency that both VEGF groups had smaller I/M persisted at the 4-week time point, when the lacZ group had an I/M of 0.73+/-0.16, the VEGF-C group 0.44+/-0.14, and the VEGF-A group 0. 63+/-0.21 (P:=NS). Expression of VEGF receptors 1, 2, and 3 was detected in the vessel wall by immunocytochemistry and in situ hybridization. As an additional control, the effect of adenovirus on cell proliferation was analyzed by performing gene transfer to intact aorta without endothelial denudation. No differences were seen in smooth muscle cell proliferation or I/M between lacZ adenovirus and 0.9% saline-treated animals. CONCLUSIONS: Adenovirus-mediated VEGF-C gene transfer may be useful for the treatment of postangioplasty restenosis and vessel wall thickening after vascular manipulations.
Assuntos
Adenoviridae/genética , Angioplastia com Balão/efeitos adversos , Estenose da Valva Aórtica/prevenção & controle , Fatores de Crescimento Endotelial/farmacologia , Túnica Íntima/efeitos dos fármacos , Adenoviridae/metabolismo , Animais , Estenose da Valva Aórtica/etiologia , Estenose da Valva Aórtica/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento Endotelial/genética , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Técnicas de Transferência de Genes , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Coelhos , Receptores Proteína Tirosina Quinases/biossíntese , Receptores de Superfície Celular/biossíntese , Receptores de Fatores de Crescimento/biossíntese , Receptores de Fatores de Crescimento do Endotélio Vascular , Transfecção , Túnica Íntima/metabolismo , Túnica Íntima/patologia , Fator A de Crescimento do Endotélio Vascular , Fator C de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Receptor 3 de Fatores de Crescimento do Endotélio VascularRESUMO
Blood vessels are among the easiest targets for gene therapy. However, no data are available about the safety and feasibility of intracoronary gene transfer in humans. We studied the safety and efficacy of catheter-mediated vascular endothelial growth factor (VEGF) plasmid/liposome (P/L) gene transfer in human coronary arteries after percutaneous translumenal coronary angioplasty (PTCA) in a randomized, double-blinded, placebo-controlled study. The optimized angioplasty/gene delivery method was previously shown to lead to detectable VEGF gene expression in human peripheral arteries as analyzed from amputated leg samples. Gene transfer to coronary arteries was done with a perfusion-infusion catheter, using 1000 microg of VEGF or beta-galactosidase plasmid complexed with 1000 microl of DOTMA:DOPE liposomes. Ten patients received VEGF P/L, three patients received beta-galactosidase P/L, and two patients received Ringer lactate. Gene transfer to coronary arteries was feasible and well tolerated. Except for a slight increase in serum C-reative protein in all study groups, no adverse effects or abnormalities in laboratory parameters were detected. No VEGF plasmid or recombinant VEGF protein was present in the systemic circulation after the gene transfer. In control angiography 6 months later, no differences were detected in the degree of coronary stenosis between treatment and control groups. We conclude that catheter-mediated intracoronary gene transfer performed after angioplasty is safe and well tolerated and potentially applicable for the prevention of restenosis and myocardial ischemia.
Assuntos
Angina Pectoris/terapia , Cateterismo/métodos , Vasos Coronários/metabolismo , Fatores de Crescimento Endotelial/genética , Técnicas de Transferência de Genes , Linfocinas/genética , Isquemia Miocárdica/terapia , Adulto , Idoso , Angioplastia Coronária com Balão/métodos , Artérias/metabolismo , Método Duplo-Cego , Feminino , Humanos , Lipossomos/genética , Masculino , Pessoa de Meia-Idade , Plasmídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Gene therapy may be useful for the treatment of atherosclerosis and related diseases. Gene transfer to vascular system can be performed both via intravascular and extravascular routes. Gene transfer to other tissues, such as liver and muscle, can also be used. The first clinical trials for the induction of therapeutic angiogenesis with VEGF gene transfer are under way, and preliminary results are promising. In the prevention of restenosis genes inhibiting cellular proliferation and increasing NO production, such as NOS and VEGF, have been used. However, more basic research is needed to fully understand pathophysiological mechanisms involved in conditions related to atherosclerosis. Also, further developments in gene transfer vectors and gene delivery techniques are required for the improvement of the efficacy of gene therapy.
Assuntos
Arteriosclerose/terapia , Terapia Genética , Neovascularização Patológica/terapia , Animais , Ensaios Clínicos como Assunto , Vetores Genéticos , Humanos , RecidivaRESUMO
To study the expression of the bovine beta-lactoglobulin (BLG) gene we isolated the BLG gene from a genomic library and introduced it into murine germline. Bovine BLG gene including 2.8 kbp of 5' and 1.9 kbp of 3' flanking region was expressed efficiently and mammary gland-specifically in transgenic mice. Expression levels of BLG in milk exceeded 1 mg ml-1 in all four mouse lines analyzed. However, in two mouse lines originating from female founders BLG expression levels varied from less than 0.02 mg ml-1 up to 1 mg ml-1. In both lines originating from male founders all analyzed female mice excreted bovine BLG into their milk at a high and constant level of 1-2 mg ml-1. BLG expression was stable within individual mice in two successive lactations and the amount of BLG in the milk of mice correlated with the level of BLG mRNA in the mammary tissue. Methylation analyses of HpaII sites revealed that transgene copies were on average more methylated in mice which excreted low levels of BLG into their milk. Each mouse line had its own methylation pattern and, in addition, each mouse had more or less identical methylation patterns in mammary gland, brain and kidney DNA. Genomic sequencing of the BLG gene indicated that the promoter region (bases -162 to +391 with respect to the transcription start site) was heavily methylated except for distinct CpG sites that were only partially methylated both in transgenic mice and lactating cattle.
Assuntos
Lactoglobulinas/genética , Animais , Mama/fisiologia , Bovinos , Clonagem Molecular , Metilação de DNA , Feminino , Dosagem de Genes , Expressão Gênica/genética , Camundongos , Camundongos Transgênicos , Proteínas do Leite/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/isolamento & purificação , Análise de SequênciaRESUMO
Emerging knowledge of molecular pathology of vascular diseases provides new targets for vascular gene therapy. Sufficient expression of a gene of interest in the vessel wall can be achieved using either extravascular or intravascular gene delivery approaches (1). Plasmid DNA, transferred by an extravascular approach, gives transfection efficiency high enough to cause biological effects. Plasmids and adenoviruses can be used for both extravascular and intravascular gene therapy. The collar model allows one to use controlled gene transfer. In this chapter we describe two gene delivery methods used for extravascular and intravascular gene transfer experiments.
RESUMO
Gene transfer to the vessel wall using vascular endothelial growth factors (VEGFs) has shown therapeutic potential for the treatment of restenosis. In this study, we evaluated the effect of catheter-mediated adenoviral (Ad) gene transfer of the mature form of VEGF-D (VEGF-D(DeltaNDeltaC)) in balloon-denuded cholesterol-fed rabbit aorta. AdLacZ was used as a control. Transduced VEGF-D(DeltaNDeltaC) mRNA was detectable in the arterial wall with RT-PCR at 6, 14 and 28 days. Gene transfer efficiency as detected with X-gal staining 6 days after the AdLacZ transduction was 1.91 +/- 1.32% in intima. AdVEGF-D(DeltaNDeltaC) gene transfer led to 52% reduction in intima/media ratio (I/M) as compared to the AdLacZ controls at 14 days time point. At 6 days there were no differences in I/M, but the number of macrophages in the vessel wall was 85% lower in the AdVEGF-D(DeltaNDeltaC) group as compared to the controls. The therapeutic effect was no longer detectable 28 days after the gene transfer. The therapeutic effect of VEGF-D(DeltaNDeltaC) was nitric oxide (NO)-dependent as the feeding of NO synthase inhibitor, L-NAME, blocked the reduction in intimal thickening. It is concluded that AdVEGF-D(DeltaNDeltaC) gene transfer reduces intimal thickening and macrophage influx into the vessel wall in balloon-denuded rabbit aortas.
Assuntos
Adenoviridae/genética , Doenças da Aorta/terapia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Transdução Genética/métodos , Fator D de Crescimento do Endotélio Vascular/genética , Animais , Aorta , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Cateterismo , Constrição Patológica/terapia , Neovascularização Patológica , Óxido Nítrico/metabolismo , Coelhos , Recidiva , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Túnica Íntima/metabolismo , Túnica Íntima/patologia , Fator D de Crescimento do Endotélio Vascular/metabolismoRESUMO
DNA arrays are revolutionizing the analysis of gene expression and single nucleotide polymorphisms of genomic DNA. Currently, the expression of 10-15% of human genes can be analysed simultaneously in a single experiment using cDNA or oligonucleotide-based format of DNA array. Alternatively, smaller DNA arrays with a limited number of selected genes, such as cytokines, growth factors or transcription factors, can be used. In concordance with Human Genome Project, after a few years, the DNA arrays will allow the analysis of expression of the whole human genome and will have a great impact on basic research, drug development and diagnostics. It is important to characterise mechanisms of atherosclerosis-related diseases at the level of gene expression so that new therapeutic strategies can be identified. With the aid of DNA array it is possible to identify multiple, simultaneous, transcriptional events that ameliorate or contribute to atherogenesis. The results are non-physical maps of the function, hierarchy and interactions of genetic programs. In this review we focus on DNA array technology and its applications in atherosclerosis research.
Assuntos
Arteriosclerose/genética , Análise de Sequência de DNA/métodos , Biologia Computacional , Projeto Genoma Humano , Humanos , InternetRESUMO
Gene therapy for the treatment of atherosclerosis and related diseases has shown its potential in animal models and in the first human trials. Gene transfer to the vascular system can be performed both via intravascular and extravascular periadventitial routes. Intravascular gene transfer can be done with several types of catheters under fluoroscopic control. Extravascular gene transfer, on the other hand, provides a well-targeted gene delivery route available during vascular surgery. It can be done with direct injection or by using perivascular cuffs or surgical collagen sheets. Ex vivo gene delivery via transfected smooth muscle cells or endothelial cells might be useful for the production of secreted therapeutic compounds. Gene transfer to the liver has been used for the treatment of hyperlipidemia. The first clinical trials for the induction of therapeutic angiogenesis in ischemic myocardium or peripheral muscles with VEGF or FGF gene transfer are under way and preliminary results are promising. VEGF has also been used for the prevention of postangioplasty restenosis because of its capability to induce endothelial repair and production of NO and prostacyclin. However, further basic research is needed to fully understand the pathophysiological mechanisms involved in conditions related to atherosclerosis. Also, further development of gene transfer vectors and gene delivery techniques will improve the efficacy and safety of human gene therapy.
Assuntos
Arteriosclerose/terapia , Técnicas de Transferência de Genes , Terapia Genética , Animais , Arteriosclerose/etiologia , Ensaios Clínicos como Assunto , Vetores Genéticos , HumanosRESUMO
Germline mutations of the putative tumor suppressor gene APC are associated in high frequency with the familial adenomatous polyposis, predisposing the patients to colorectal neoplasia. Similarly, sequence analyses have revealed that in more than half of patients with sporadic colorectal carcinoma or adenoma, the APC gene was mutated. By employing genomic sequencing, i.e., base-specific analysis of methylated cytosines, we show here that the promoter region of the APC gene is heavily methylated at CpG sites in patients with colorectal carcinoma in comparison with normal colonic mucosa and premalignant adenomas. Our results suggest that cytosine methylation of the regulatory sequences of the APC gene could be involved in the progression of human colorectal cancer.
Assuntos
Adenoma/genética , Neoplasias Colorretais/genética , Citosina/metabolismo , Genes APC/genética , Lesões Pré-Cancerosas/genética , Adenoma/metabolismo , Neoplasias Colorretais/metabolismo , Metilação de DNA , Mutação em Linhagem Germinativa , Humanos , Mucosa Intestinal/metabolismo , Reação em Cadeia da Polimerase , Lesões Pré-Cancerosas/metabolismo , Regiões Promotoras GenéticasRESUMO
DNA methylation has been studied intensively during the past years in order to elucidate its role in the regulation of gene expression, gene imprinting and cancer progression. Earlier studies have shown that a general genomic under-methylation is associated with chronic lymphocytic leukemia and metastatic prostate cancer. Site-specific methylation changes, as revealed by the use of methylation-sensitive restriction enzymes, have been reported to occur in the promotor region of the calcitonin gene in chronic myeloid leukemia as it progresses from the chronic phase to blast crisis, in non-Hodgkin's lymphoid neoplasms and in non-lymphocytic leukemia. We have now explored possible methylation changes associated with benign and malignant breast tumors. Two approaches were employed: (i) chemical determination of general genomic methylation status and (ii) base-specific analysis of the methylation changes in the promoter of the calcitonin gene with the aid of genomic sequencing. The results did not reveal any changes of total DNA 5-methylcytosine content in ductal carcinoma of breast in comparison with benign tumors. There was a small, yet significant, increase in 5-methylcytosine content in lobular carcinoma. Genomic sequencing of the promoter region of the calcitonin gene, however, revealed a striking hypermethylation at or around the transcription start site of the gene in ductal carcinomas. In benign tumors and lobular carcinomas, this region was either entirely unmethylated or only slightly methylated. The latter changes may reflect a regional hypermethylation of the short arm of chromosome 11, which harbors, in addition to the calcitonin gene, a number of putative or established tumor-suppressor genes. Our results demonstrate that genomic sequencing in its present form can be used for a reliable and precise DNA methylation analysis of primary human tumors.
Assuntos
Neoplasias da Mama/genética , Calcitonina/genética , Metilação de DNA , DNA de Neoplasias/química , Sequências Reguladoras de Ácido Nucleico/genética , 5-Metilcitosina , Neoplasias da Mama/química , Carcinoma Ductal de Mama/química , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/química , Carcinoma Lobular/genética , Cromatografia Líquida de Alta Pressão/métodos , Citosina/análogos & derivados , Citosina/análise , Feminino , Fibroadenoma/química , Fibroadenoma/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Análise de Sequência de DNA/métodosRESUMO
BACKGROUND: Periadventitial gene therapy is a promising alternative for the treatment of stenosis, vessel wall thickening and other complications in vascular surgery. METHODS: We compared lacZ gene transfer efficiency of DOTMA: DOPE (1:1 w/w) plasmid/liposome complexes and adenoviruses in pig carotid arteries using perivascular delivery with either a collagen collar or a wrap of collagen sheet. Safety of the gene transfer was studied by clinical chemistry, tissue pathology and PCR analysis of lung, liver, kidney, spleen, skeletal muscle and gonads. RESULTS: Gene transfer efficiency using the periadventitial collar was fourfold higher than using the collagen wrap with adenovirus at 7 days (10.22 +/- 2.96 vs 2.78 +/- 1.28 positive cells/mm2; p = 0.18) and 4.3-fold at 14 days (13.46 +/- 3.49 vs 3.11 +/- 0.88 positive cells/mm2; p = 0.03). Gene transfer efficiency at 7 days with adenovirus was fivefold higher than with the plasmid/liposome complexes both using the collar (10.22 +/- 2.96 vs 2.07 +/- 0.95 positive cells/mm2; p = 0.01) and the collagen wrap (2.78 +/- 1.28 vs 0.45 +/- 0.35 positive cells/mm2; p = 0.03). No lacZ activity was detected in plasmid/liposome transfected arteries at 14 days. In spite of the local gene delivery methods a moderate systemic distribution of the transgene was detected in the major organs by PCR analysis. CONCLUSIONS: This study shows that: (i) adenovirus delivered with the periadventitial collar or the collagen wrap is well tolerated and may become an efficient new tool in vascular gene therapy, and (ii) gene transfer vector delivered in the periadventitial collar reaches the target tissue more efficiently than the vector in the collagen wrap.
Assuntos
Artérias Carótidas/fisiologia , Colágeno/farmacologia , Marcação de Genes , Técnicas de Transferência de Genes , Implantes Absorvíveis , Adenoviridae/genética , Animais , Artérias Carótidas/anatomia & histologia , Estudos de Avaliação como Assunto , Genes Reporter/genética , Óperon Lac/genética , Lipossomos/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SuínosRESUMO
The short arm of the chromosome 11, known to harbour a number of putative and established tumour-suppressor genes, is frequently hypermethylated in various human neoplasms. We subjected the promoter regions of two genes residing at 11p, namely the tumour-suppressor gene WT1 (Wilms' tumour gene) (11p13) and the calcitonin gene (11p15.5), to methylation analysis in human sporadic colorectal cancer using genomic sequencing. Both genes showed significant hypermethylation of CpG sites within their promoter regions in adenomas and carcinomas compared with normal colonic mucosa. Although the WT1 promoter region was significantly hypermethylated, two CpG sites located in Sp1 motifs were unmethylated in the majority of cases (68-74% of carcinomas). The expression of WT1 gene, as revealed by in situ hybridization, showed no differences between normal colonic mucosa and malignant carcinoma. Together with earlier observations, our present results support the view that the short arm of human chromosome 11 is subjected to widespread regional hypermethylation in various human malignancies.
Assuntos
Calcitonina/genética , Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genética , Adenoma/metabolismo , Cromossomos Humanos Par 11 , Colo/metabolismo , Neoplasias Colorretais/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Genes do Tumor de Wilms/genética , Humanos , Hibridização In Situ , Mucosa Intestinal/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Fatores de Transcrição/metabolismo , Proteínas WT1RESUMO
Extracellular superoxide dismutase (EC-SOD) protects arteries against deleterious effects of superoxide anions and the development of atherosclerosis. In this study, we cloned and characterized rabbit ec-sod gene. We identified 6 rabbit C-elements and 5 CpG clusters in the cloned sequence. One of the CpG clusters is located on the coding sequence. Because CpG clusters are potential sites for methylation and may explain the occurrence of mutations, methylation status of each of the CpG dimers located in the coding sequence CpG cluster was characterized using direct genomic sequencing. Unexpectedly, a marked reduction in the amount of methylated CpG dinucleotides in ec-sod gene was detected in atherosclerotic aortas as compared with normal aortic intima-media. Although alterations in DNA methylation are well characterized in malignant tumors, the presence of methylation changes in atherosclerosis has not been studied even though both diseases are characterized by excess cellular proliferation and alterations in gene expression. Further analysis of the whole genomic methylation by high-pressure liquid chromatography in normal and atherosclerotic aortas revealed a tendency for a decreased 5-methylcytosine (5-mC) content in atherosclerotic aortas as compared with normal arteries. Hypomethylation in atherosclerotic aortas occurred at the same level as has been reported from malignant tumors. Although a causal relationship between the methylation level and expression of EC-SOD cannot be proven, our results show that ec-sod hypomethylation is associated with the development of atherosclerosis and suggest that it may affect structure and function of ec-sod and other genes possibly involved in the development of atherosclerotic lesions.
Assuntos
Arteriosclerose/genética , Metilação de DNA , Espaço Extracelular/genética , Superóxido Dismutase/genética , Sequência de Aminoácidos/genética , Animais , Aorta/fisiopatologia , Sequência de Bases/genética , Northern Blotting , Células Cultivadas , Ilhas de CpG/genética , Análise Mutacional de DNA , Dados de Sequência Molecular , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , CoelhosRESUMO
Different lipids and cationic polymers were tested in vitro for their ability to transfect rabbit aortic smooth muscle cells and human endothelial cells with lacZ marker gene. Toxicity of the complexes was evaluated with MTT assay. Selected plasmid-polymer complexes with different charge ratios were then tested for in vivo gene transfer efficiency using adventitial gene transfer by placing a silastic gene delivery reservoir (collar) around the carotid artery. Transfection efficiency was determined by X-gal staining 3 days after the gene transfer. Based on in vitro experiments, fractured polyamidoamine dendrimers and polyethylenimines (PEI) were selected for in vivo experiments. Fractured dendrimers (generation 6, +/- charge ratio of 3) had the highest in vivo gene transfer efficiency (4.4% +/- 1.7). PEI with molecular size of 25 kDa (+/- charge ratio 4) was also effective (2.8% +/- 1.8) in this model. PEI of 800 kDa showed a constant but modest gene transfer efficiency (1.8% +/- 0.1) with all charge ratios. A low level gene transfer was also detected with naked DNA (0.5% +/- 0.3). No signs of inflammation were seen in any of the study groups. We show here that in vitro cell culture experiments can be used to identify efficient in vivo gene transfer methods for arterial gene therapy, but the charge ratios for each complex must be optimized in vivo. It is concluded that fractured dendrimer and PEI are efficient gene delivery vehicles and can be used for arterial gene therapy via adventitial gene delivery route.
Assuntos
Artérias Carótidas , Vetores Genéticos , Transfecção/métodos , beta-Galactosidase/genética , Animais , Artérias Carótidas/anatomia & histologia , Cátions , Células Cultivadas , Endotélio Vascular , Masculino , Músculo Liso Vascular , Plasmídeos , Polímeros , CoelhosRESUMO
Recombinant Autographa californica multiple nuclear polyhedrosis viruses (AcMNPV) have recently been shown to transduce mammalian cells in vitro. Since baculoviruses offer many advantages over viruses currently used in gene therapy, we have tested them for in vivo gene transfer by constructing a baculovirus bearing a nuclear targeted beta-galactosidase marker gene (LacZ) under a CMV promoter. Both rabbit aortic smooth muscle cells (RAASMC) and human ECV-304 cells were susceptible to LacZ-baculovirus transduction. Transgene expression was evaluated in vivo by applying 1 x 10(9) p.f.u. of LacZ-baculoviruses or LacZ-adenoviruses in a silastic collar placed around rabbit carotid arteries in the absence of contact with blood components. As a result, baculoviruses led to transgene expression in adventitial cells in rabbit carotid arteries with efficiency comparable to adenoviruses. The beta-galactosidase gene expression was transient staying at a high level for 1 week but disappearing at the 14 day time-point. The arterial structure and endothelium remained intact in the baculovirus-transduced arteries, but macrophage-specific immunostaining detected signs of inflammation comparable to adenoviruses. Baculoviruses are thus able to mediate transient gene transfer in vivo and may become useful tools for gene therapy.
Assuntos
Baculoviridae/genética , Artérias Carótidas , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Transfecção/métodos , Animais , Expressão Gênica , Humanos , Masculino , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/imunologia , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta-Galactosidase/genéticaRESUMO
Expression of transgene other than in the target tissue may cause side effects and safety problems in gene therapy. We analyzed biodistribution of transgene expression after intravascular and periadventitial gene delivery methods using the first generation nuclear-targeted lacZ adenovirus. RT-PCR and X-Gal stainings were used to study transgene expression 14 days after the gene transfer. After intravascular catheter-mediated gene transfer to rabbit aorta mimicking angioplasty procedure, the target vessel showed 1.1% +/- 0. 5 gene transfer efficiency. Other tissues showed varying lacZ gene expression indicating a systemic leakage of the vector with the highest transfection efficiency in hepatocytes (0.7% +/- 0.5). X-Gal staining of blood cells 24 h after the intravascular gene transfer indicated that a significant portion (1.8% +/- 0.8) of circulating monocytes was transfected. X-Gal-positive cells were also found in testis. After periadventitial gene transfer using a closed silicon capsule placed around the artery, 0.1% +/- 0.1 lacZ-positive cells were detected in the artery wall. Positive cells were also found in the liver and testis (<0.01%), indicating that the virus escapes even from the periadventitial space, although less extensively than during the intravascular application. We conclude that catheter-mediated intravascular and, to a lesser extent, periadventitial gene transfer lead to leakage of adenovirus to systemic circulation, followed by expression of the transgene in several tissues. Possible consequences of the ectopic expression of the transgene should be evaluated in gene therapy trials even if local gene delivery methods are used.
Assuntos
Aorta/metabolismo , Vetores Genéticos/genética , Transfecção/métodos , Adenoviridae/genética , Animais , Expressão Gênica , Técnicas de Transferência de Genes , Histocitoquímica , Técnicas In Vitro , Óperon Lac/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Coloração e Rotulagem , Distribuição Tecidual , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The class A scavenger receptors (SR-As) are trimeric, integral membrane glycoproteins that exhibit unusually broad ligand-binding properties. A number of studies have suggested that these receptors may play an important role in host defense and in many macrophage-associated pathological processes, including atherosclerosis and Alzheimer's disease. The study of the expression and function of these receptors in human disease has been hampered by the lack of suitable antibodies recognizing human SR-A. This has generated questions regarding the nature of receptors responsible for scavenger receptor activity detected in a variety of cell types, including monocytes, macrophages, smooth muscle cells, and endothelial cells. To address these questions, we have produced high-titer antisera recognizing human SR-A by using mice deficient for SR-A (SR-A -/-). We show that SR-A -/- mice produce a significantly higher-titer immune response than do wild-type (SR-A +/+) littermates, with antisera of the former having a broad species reactivity and recognizing SR-A from humans, mice, and rabbits. The antisera recognize both type I and II SR-A in a wide range of immunological techniques. Using these antisera we show that the expression of SR-A protein is induced during monocyte to macrophage differentiation and that SR-A mediates 80% of the uptake of acetylated low density lipoprotein by human monocyte-derived macrophages. We also establish that human SR-A is expressed by tissue macrophages in liver and lung and by macrophage-derived foam cells within aortic atherosclerotic lesions, with little detectable expression by smooth muscle cells or aortic endothelium.
Assuntos
Aorta/química , Aorta/patologia , Arteriosclerose/patologia , Receptores Imunológicos/análise , Receptores Imunológicos/genética , Actinas/análise , Actinas/imunologia , Animais , Anticorpos , Aorta/lesões , Doenças da Aorta/genética , Doenças da Aorta/patologia , Arteriosclerose/genética , Células CHO , Cateterismo , Moléculas de Adesão Celular/análise , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Células Cultivadas , Cricetinae , Endotélio Vascular/química , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Citometria de Fluxo , Expressão Gênica/fisiologia , Humanos , Macrófagos/química , Macrófagos/fisiologia , Camundongos , Camundongos Knockout , Músculo Liso Vascular/química , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Coelhos , Receptores Imunológicos/imunologia , Receptores Depuradores , Receptores Depuradores Classe A , TransfecçãoRESUMO
Liver-directed gene therapy is a promising alternative for the treatment of various liver diseases. Pseudotyped (VSV-G) retroviruses can be produced in high titres which is essential to overcome the problem of low gene transfer efficiency detected previously with first generation Moloney murine (MMLV) retroviruses and plasmid vectors. We compared the lacZ gene transfer efficiency of MMLV retroviruses and VSV-G retroviruses in Watanabe heritable hyperlipidaemic rabbit liver using an intraportal administration route. Hepatocyte proliferation was stimulated by a partial (10%) liver resection and a thymidine kinase-ganciclovir treatment. We also studied the safety of the gene transfer by clinical chemistry, tissue pathology and PCR analysis of lung, kidney, spleen and gonads. Gene transfer efficiency with the VSV-G retrovirus was significantly higher than with the traditional MMLV-based retrovirus (9.5+/-5.26 vs 0.21+/-0.10 positive hepatocytes mm(-2), P<0.05). After a 12-month follow-up period no lacZ expression was detected in liver samples. No transgene was detected in plasma or in lung, kidney, spleen and gonads by PCR analysis 7 days after gene transfer. Transient increases were found in plasma c-reactive protein, aspartyl aminotransferase and alanine aminotransferase levels shortly after the operation with both types of retroviruses. VSV-G retrovirus was well tolerated and may become an efficient new tool in liver gene therapy. The absence of transgene in systemic circulation or in extrahepatic tissues including gonads is an important safety feature required for in vivo gene therapy.
Assuntos
Antivirais/farmacologia , Proteínas de Ligação ao GTP/genética , Ganciclovir/farmacologia , Técnicas de Transferência de Genes , Fígado/metabolismo , Retroviridae/genética , Timidina Quinase/farmacologia , Vírus da Estomatite Vesicular Indiana/genética , Animais , Feminino , Óperon Lac/genética , Fígado/efeitos dos fármacos , Masculino , Plasmídeos/genética , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Distribuição TecidualRESUMO
We examined the feasibility of gene transfer to rabbit placenta using adenoviruses, plasmid/liposomes and plasmid/polyethyleneimine (PEI) complexes. Pregnant New Zealand White rabbits (n = 17) were anesthetized and local gene transfer was done via a catheter inserted in uterine arteries under direct angiographic control. Either nuclear targeted LacZ adenoviruses (1.0 x 10(10) p.f.u.), nuclear targeted LacZ plasmid (500 microg)/liposome (DOTMA:DOPE 1:1) complexes or nuclear targeted LacZ plasmid (250 microg)/PEI (25 kDa) complexes (charge ratio +/-4) were used. Animals were killed 3 days later and detection of the transgene expression was done by X-gal staining and RT-PCR. Adenovirus-mediated gene transfer resulted in a high transfection efficiency (34 +/- 10%) in placental trophoplastic cells. Very little, if any, transfection was seen in fetal membranes. Plasmid/liposomes and plasmid/PEI complexes led to a very low (<0.01%) transfection efficiency in trophoblastic cells, but some transfection was seen in fetal membranes. A total of 25 fetuses were analyzed for the presence of transgene at the time of death. In most fetuses expression of the LacZ gene was below the sensitivity of the X-gal staining, but expression was detected by PCR in 50%, 50% and 42% of the analyzed fetuses after adenoviral, plasmid/PEI and plasmid/liposome gene transfer, respectively. No major inflammatory changes were present in the transfected placentas as analyzed by general histology and macrophage- and T cell-specific immunostainings. We conclude that catheter-mediated intravascular gene transfer with adenoviruses can be used for the transfection of placental trophoplastic cells, but plasmid complexes are inefficient for this purpose. However, selective angiographically guided gene transfer also led to leakage of the vector to fetuses. Therefore, if gene therapy is developed for the treatment of placental disorders, the gene-vector combination should not be harmful to the fetus and the expression of the transgene should only occur in placenta.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Placenta/metabolismo , Transfecção/métodos , Útero/irrigação sanguínea , Animais , Artérias , Feminino , Feto/metabolismo , Expressão Gênica , Lipossomos , Plasmídeos , Polietilenoimina , Gravidez , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We studied the expression of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), an enzyme capable of hydrolyzing platelet-activating factor (PAF), PAF-like phospholipids, and polar-modified phosphatidylcholines, in human and rabbit atherosclerotic lesions. Oxidative modification of low-density lipoprotein, which plays an important role in atherogenesis, generates biologically active PAF-like modified phospholipid derivatives with polar fatty acid chains. PAF is known to have a potent proinflammatory activity and is inactivated by its hydrolysis. On the other hand, lysophosphatidylcholine and oxidized fatty acids released from oxidized low-density lipoprotein as a result of Lp-PLA(2) activity are thought to be involved in the progression of atherosclerosis. Using combined in situ hybridization and immunocytochemistry, we detected Lp-PLA(2) mRNA and protein in macrophages in both human and rabbit atherosclerotic lesions. Reverse transcriptase-polymerase chain reaction analysis indicated an increased expression of Lp-PLA(2) mRNA in human atherosclerotic lesions. In addition, approximately 6-fold higher Lp-PLA(2) activity was detected in atherosclerotic aortas of Watanabe heritable hyperlipidemic rabbits compared with normal aortas from control rabbits. It is concluded that (1) macrophages in both human and rabbit atherosclerotic lesions express Lp-PLA(2), which could cleave any oxidatively modified phosphatidylcholine present in the lesion area, and (2) modulation of Lp-PLA(2) activity could lead to antiatherogenic effects in the vessel wall.