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1.
Sci Rep ; 13(1): 3366, 2023 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-36849509

RESUMO

Manganese-enhanced magnetic resonance imaging can provide a surrogate measure of myocardial calcium handling. Its repeatability and reproducibility are currently unknown. Sixty-eight participants: 20 healthy volunteers, 20 with acute myocardial infarction, 18 with hypertrophic and 10 with non-ischemic dilated cardiomyopathy underwent manganese-enhanced magnetic resonance imaging. Ten healthy volunteers were re-scanned at 3 months. Native T1 values and myocardial manganese uptake were assessed for intra and inter-observer repeatability. Scan-rescan reproducibility was assessed in ten healthy volunteers. Intra-observer and inter-observer correlation was excellent in healthy volunteers for mean native T1 mapping [Lin's correlation coefficient (LCC) 0.97 and 0.97 respectively] and myocardial manganese uptake (LCC: 0.99 and 0.96 respectively). Scan-rescan correlation for native T1 and myocardial manganese uptake was also excellent. Similarly, intra-observer correlations for native T1 and myocardial manganese uptake in patients with acute myocardial infarction (LCC: 0.97 and 0.97 respectively), hypertrophic (LCC: 0.98 and 0.97 respectively) and dilated cardiomyopathy (LCC: 0.99 and 0.95 respectively) were excellent. Limits of agreement were broader in patients with dilated cardiomyopathy. Manganese-enhanced magnetic resonance imaging has high repeatability and reproducibility in healthy myocardium and high repeatability in diseased myocardium. However, further study is needed to establish robustness in pathologies with diffuse myocardial fibrosis.


Assuntos
Neoplasias da Mama , Cardiomiopatia Dilatada , Infarto do Miocárdio , Lesões Pré-Cancerosas , Humanos , Feminino , Manganês , Cardiomiopatia Dilatada/diagnóstico por imagem , Reprodutibilidade dos Testes , Infarto do Miocárdio/diagnóstico por imagem , Hipertrofia , Imageamento por Ressonância Magnética
2.
Artigo em Inglês | MEDLINE | ID: mdl-33200175

RESUMO

AIMS: The aim of this study is to quantify altered myocardial calcium handling in non-ischaemic cardiomyopathy using magnetic resonance imaging. METHODS AND RESULTS: Patients with dilated cardiomyopathy (n = 10) or hypertrophic cardiomyopathy (n = 17) underwent both gadolinium and manganese contrast-enhanced magnetic resonance imaging and were compared with healthy volunteers (n = 20). Differential manganese uptake (Ki) was assessed using a two-compartment Patlak model. Compared with healthy volunteers, reduction in T1 with manganese-enhanced magnetic resonance imaging was lower in patients with dilated cardiomyopathy [mean reduction 257 ± 45 (21%) vs. 288 ± 34 (26%) ms, P < 0.001], with higher T1 at 40 min (948 ± 57 vs. 834 ± 28 ms, P < 0.0001). In patients with hypertrophic cardiomyopathy, reductions in T1 were less than healthy volunteers [mean reduction 251 ± 86 (18%) and 277 ± 34 (23%) vs. 288 ± 34 (26%) ms, with and without fibrosis respectively, P < 0.001]. Myocardial manganese uptake was modelled, rate of uptake was reduced in both dilated and hypertrophic cardiomyopathy in comparison with healthy volunteers (mean Ki 19 ± 4, 19 ± 3, and 23 ± 4 mL/100 g/min, respectively; P = 0.0068). In patients with dilated cardiomyopathy, manganese uptake rate correlated with left ventricular ejection fraction (r2 = 0.61, P = 0.009). Rate of myocardial manganese uptake demonstrated stepwise reductions across healthy myocardium, hypertrophic cardiomyopathy without fibrosis and hypertrophic cardiomyopathy with fibrosis providing absolute discrimination between the healthy myocardium and fibrosed myocardium (mean Ki 23 ± 4, 19 ± 3, and 13 ± 4 mL/100 g/min, respectively; P < 0.0001). CONCLUSION: The rate of manganese uptake in both dilated and hypertrophic cardiomyopathy provides a measure of altered myocardial calcium handling. This holds major promise for the detection and monitoring of dysfunctional myocardium, with the potential for early intervention and prognostication.

3.
Gene Ther ; 23(7): 592-6, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27014840

RESUMO

Intravenous delivery of adenoviruses is the optimal route for many gene therapy applications. Once in the blood, coagulation factor X (FX) binds to the adenovirus capsid and protects the virion from natural antibody and classical complement-mediated neutralisation in mice. However, to date, no studies have examined the relevance of this FX/viral immune protective mechanism in human samples. In this study, we assessed the effects of blocking FX on adenovirus type 5 (Ad5) activity in the presence of human serum. FX prevented human IgM binding directly to the virus. In individual human sera samples (n=25), approximately half of those screened inhibited adenovirus transduction only when the Ad5-FX interaction was blocked, demonstrating that FX protected the virus from neutralising components in a large proportion of human sera. In contrast, the remainder of sera tested had no inhibitory effects on Ad5 transduction and FX armament was not required for effective gene transfer. In human sera in which FX had a protective role, Ad5 induced lower levels of complement activation in the presence of FX. We therefore demonstrate for the first time the importance of Ad-FX protection in human samples and highlight subject variability and species-specific differences as key considerations for adenoviral gene therapy.


Assuntos
Adenoviridae/imunologia , Fator X/imunologia , Técnicas de Transferência de Genes , Terapia Genética/métodos , Adenoviridae/genética , Linhagem Celular Tumoral , Vetores Genéticos/sangue , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Células HEK293 , Humanos , Imunoglobulina M/imunologia , Injeções Intravenosas/métodos
4.
Clin Pharmacol Ther ; 99(5): 494-501, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26910520

RESUMO

MicroRNA (miRNA) have gained widespread attention for their role in diverse vascular processes including angiogenesis, apoptosis, proliferation, and migration. Despite great understanding of miRNA expression and function, knowledge of long noncoding RNA (lncRNA) molecular mechanisms still remains limited. The influence of miRNA on lncRNA function, and the converse, is now beginning to emerge. lncRNA may regulate miRNA function by acting as endogenous sponges to regulate gene expression and miRNA have been shown to bind and regulate lncRNA stability. A detailed understanding of the molecular and cellular effects of lncRNA-miRNA-mediated interactions in vascular pathophysiology could pave the way for new diagnostic markers and therapeutic approaches, but first there is a requirement for a more detailed understanding of the impact of such regulatory networks.


Assuntos
MicroRNAs/genética , RNA Longo não Codificante/genética , Doenças Vasculares/genética , Animais , Apoptose/genética , Biomarcadores/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Regulação da Expressão Gênica , Humanos , Neovascularização Fisiológica/genética , Doenças Vasculares/diagnóstico , Doenças Vasculares/fisiopatologia
5.
Gene Ther ; 20(12): 1158-64, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24005577

RESUMO

Ex vivo gene therapy during coronary artery bypass grafting (CABG) holds great potential to prevent excessive smooth muscle cell (SMC) proliferation, neointima formation and graft failure. The most successful preclinical strategies to date have utilised vectors based on the species C adenovirus, Ad5, which engages the Coxsackie and Adenovirus receptor (CAR) as its primary attachment receptor. Profiling receptors on human SMCs demonstrated the absence of CAR but substantial expression of the species B receptor CD46. We performed transduction experiments using Ad5 and the CD46-utilising adenovirus Ad35, and found Ad35 significantly more efficient at transducing SMCs. To evaluate whether transduction could be further augmented, we evaluated chimeric CD46-utilising Ad5/Ad35 vectors comprising the Ad5 capsid pseudotyped with the Ad35 fibre alone (Ad5/F35) or in combination with the Ad35 penton (Ad5/F35/P35). In human smooth muscle cells (hSMCs), Ad5/F35/P35 mediated significantly higher levels of transduction than either parental vector or Ad5/F35. Ex vivo transduction experiments using mouse aortas from CD46 transgenics demonstrated that Ad5/F35/P35 was significantly more efficient at transducing SMCs than the other vectors tested. Finally, ex vivo transduction and immunofluorescent colocalisation experiments using human tissue from CABG procedures confirmed the preclinical potential of Ad5/F35/P35 as an efficient vector for vascular transduction during CABG.


Assuntos
Adenovírus Humanos/genética , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Transdução Genética , Adenovírus Humanos/classificação , Animais , Aorta , Células Cultivadas , Ponte de Artéria Coronária , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/patologia , Doença da Artéria Coronariana/cirurgia , Doença da Artéria Coronariana/terapia , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Terapia Genética , Vetores Genéticos , Humanos , Proteína Cofatora de Membrana/genética , Proteína Cofatora de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/virologia , Neointima/patologia , Neointima/terapia
6.
J Cardiovasc Transl Res ; 5(5): 605-17, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22855254

RESUMO

Revascularisation of ischaemic tissue remains an area of substantial unmet clinical need in cardiovascular disease. Strategies to induce therapeutic angiogenesis are therefore attractive. Our recent focus has been on human embryonic stem cell (hESC) strategies since hESC can be maintained in a pluripotent state or differentiated into any desired cell type, including endothelial cells (EC), under defined differentiation culture conditions. We recently published a protocol for non-good manufacturing practice (GMP) feeder- and serum-free hESC-EC-directed monolayer differentiation to vascular EC demonstrating the potential to generate hESC-derived EC in a GMP-compliant manner suitable for use in clinical trials. In this study we modified that laboratory protocol to GMP compliance. EC production was confirmed by flow cytometry, qRT-PCR and production of vascular structures in Matrigel®, yielding approximately 30 % mature VE-cadherin(+)/PECAM-1(+) cells using the GMP-compliant hESC line RC13. In conclusion, we have successfully demonstrated the production of vascular EC under GMP-compliant conditions suitable for clinical evaluation.


Assuntos
Biotecnologia/normas , Técnicas de Cultura de Células/normas , Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Células Endoteliais/fisiologia , Fidelidade a Diretrizes/normas , Guias como Assunto/normas , Isquemia , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Caderinas/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/transplante , Células Endoteliais/metabolismo , Células Endoteliais/transplante , Citometria de Fluxo/normas , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Isquemia/patologia , Isquemia/fisiopatologia , Isquemia/cirurgia , Neovascularização Fisiológica , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Fatores de Tempo
7.
Gene Ther ; 19(6): 630-6, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22456326

RESUMO

Autologous saphenous vein is commonly used as a conduit to bypass atherosclerotic lesions in coronary and femoral arteries. Despite the wide use of arterial conduits, which are less susceptible to complications and failure, as alternative conduits, the saphenous vein will continue to be used in coronary artery bypass grafting until acceptable alternative approaches are evaluated. Hence, preservation of vein graft patency is essential for the long-term success. Gene therapy is attractive in this setting as an ex-vivo technology to genetically manipulate the conduit before grafting. The use of safe and efficient vectors for delivery is a necessity as well as a strategy to improve patency in the long term. Here, we review the current clinical practice, the pathogenesis of bypass graft failure and adenovirus-mediated gene therapy strategies designed to improve late vein graft failure by modulation of smooth muscle cells in the vein wall.


Assuntos
Ponte de Artéria Coronária/efeitos adversos , Terapia Genética/métodos , Falha de Prótese , Grau de Desobstrução Vascular , Sobrevivência de Enxerto , Humanos , Transplante Autólogo
8.
Gene Ther ; 19(1): 109-13, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21677690

RESUMO

Coagulation factor X (FX)-binding ablated adenovirus type 5 (Ad5) vectors have been genetically engineered to ablate the interaction with FX, resulting in substantially reduced hepatocyte transduction following intravenous administration in rodents. Here, we quantify viral genomes and gene transfer mediated by Ad5 and FX-binding-ablated Ad5 vectors in non-human primates. Ad5 vectors accumulated in and mediated gene transfer predominantly to the liver, whereas FX-binding-ablated vectors primarily targeted the spleen but showed negligible liver gene transfer. In addition, we show that Ad5 binding to hepatocytes may be due to the presence of heparan sulfate proteoglycans (HSPGs) on the cell membrane. Therefore, the Ad5-FX-HSPG pathway mediating liver gene transfer in rodents is also the mechanism underlying Ad5 hepatocyte transduction in Microcebus murinus.


Assuntos
Adenoviridae/genética , Fator X/metabolismo , Técnicas de Transferência de Genes , Genoma Viral , Adenoviridae/metabolismo , Animais , Membrana Celular/metabolismo , Cheirogaleidae , Fator X/genética , Marcação de Genes/métodos , Terapia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Imuno-Histoquímica , Fígado/citologia , Fígado/metabolismo , Ligação Proteica , Baço/citologia , Baço/metabolismo
9.
Gene Ther ; 19(9): 936-46, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22071970

RESUMO

The efficient delivery of genetic material to the developing fetal brain represents a powerful research tool and a means to supply therapy in a number of neonatal lethal neurological disorders. In this study, we have delivered vectors based upon adenovirus serotype 5 (Ad5) and adeno-associated virus (AAV) pseudotypes 2/5, 2/8 and 2/9 expressing green fluorescent protein to the E16 fetal mouse brain. One month post injection, widespread caudal to rostral transduction of neural cells was observed. In discrete areas of the brain these vectors produced differential transduction patterns. AAV2/8 and 2/9 produced the most extensive gene delivery and had similar transduction profiles. All AAV pseudotypes preferentially transduced neurons whereas Ad5 transduced both neurons and glial cells. None of the vectors elicited any significant microglia-mediated immune response when compared with control uninjected mice. Whole-body imaging and immunohistological evaluation of brains 9 months post injection revealed long-term expression using these non-integrating vectors. These data will be useful in targeting genetic material to discrete or widespread areas of the fetal brain with the purpose of devising therapies for early neonatal lethal neurodegenerative disease and for studying brain development.


Assuntos
Adenoviridae/genética , Encéfalo/metabolismo , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Animais , Encéfalo/embriologia , Feminino , Proteínas de Fluorescência Verde/genética , Camundongos , Neuroglia/metabolismo , Neurônios/metabolismo , Transdução Genética
10.
Gene Ther ; 15(6): 443-51, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18004401

RESUMO

Targeted delivery of biological agents to atherosclerotic plaques may provide a novel treatment and/or useful tool for imaging of atherosclerosis in vivo. However, there are no known viral vectors that possess the desired tropism. Two plaque-targeting peptides, CAPGPSKSC (CAP) and CNHRYMQMC (CNH) were inserted into the capsid of adeno-associated virus 2 (AAV2) to assess vector retargeting. AAV2-CNH produced significantly higher levels of transduction than unmodified AAV2 in human, murine and rat endothelial cells, whereas transduction of nontarget HeLa cells was unaltered. Transduction studies and surface plasmon resonance suggest that AAV2-CNH uses membrane type 1 matrix metalloproteinase as a surface receptor. AAV2-CAP only produced higher levels of transduction in rat endothelial cells, possibly because the virus was found to be affected by proteasomal degradation. In vivo substantially higher levels of both peptide-modified AAV2 vectors was detected in the brachiocephalic artery (site of advanced atherosclerotic plaques) and aorta, whereas reduced levels were detected in all other organs examined. These results suggest that in the AAV2 platform the peptides are exposed on the capsid surface in a way that enables efficient receptor binding and so creates effective atherosclerotic plaque targeted vectors.


Assuntos
Aterosclerose/terapia , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Animais , Apolipoproteínas E/genética , Linhagem Celular , Dependovirus/metabolismo , Marcação de Genes , Engenharia Genética , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Heparina/metabolismo , Humanos , Metaloproteinase 1 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fragmentos de Peptídeos/genética , Ligação Proteica , Ratos , Ressonância de Plasmônio de Superfície , Transdução Genética/métodos , Transgenes
11.
Int J Radiat Biol ; 83(1): 13-25, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17357436

RESUMO

PURPOSE: To investigate the potential effects of stable tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) overexpression on DNA damage and cell killing following low-dose gamma-radiation and whether this up-regulation interfered with the activation of the matrix metalloproteinase -2 (MMP-2) and -9 (MMP-9) in a highly metastatic renal carcinoma cell line. MATERIALS AND METHODS: Stable transfections were carried out using the cytomegalovirus expression plasmid pRc/CMV carrying TIMP-1 cDNA and LIPOFECTAMINE reagent. TIMP-1 expression in selected clones was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. Exponentially growing Caki-1 cells were treated with sub lethal doses of ionizing radiation (0- 10Gy) either alone or following stable TIMP-1 transfection. DNA damage was assessed by the Alkaline Comet Assay and cell survival was determined by a clonogenic assay. Caki-1 cell cycle alterations following TIMP-1 transfection were assessed by fluorescence activated cell sorting (FACS) analysis of propidium iodide (PI)-stained cells. The interactions between TIMP-1 and MMP-2 and MMP-9 were analysed 24 hours post-irradiation by means of gelatin zymography. RESULTS: Three clones with varying degrees of TIMP-1 expression were selected and used for further analysis. TIMP-1 transfected Caki-1 cells displayed significantly higher mean tail moment values (p < 0.05) following irradiation at doses between 5 and 10 Gy relative to that seen with radiation alone. The TIMP-1 radiosensitizing effect was accompanied by large decreases in the survival fraction of the parental Caki-1 cell line and significant increases in the alpha-parameter of the linear-quadratic fit. These effects were directly correlated to the degree of TIMP-1 gene expression detected in the selected clones. Interestingly, elevated levels of TIMP-2 protein were detected in the three TIMP-1 clones compared to TIMP-2 levels present in Caki-l cells. The three clones also displayed marked phenotypic alterations relative to their parental cell line. Significant increases in the percentage of cells arrested in the G2/M phase of the cell cycle were detected in the three clones under normal growth conditions and reduced serum conditions (p < 0.05). When the TIMP-1 clones were assessed for their MMP-2 activity, a marked decrease in the MMP-2 mean protein levels was detected in clone T1-3 following irradiation at doses between 2 and 6 Grays (Gy) (p < 0.01) and clone T1-2 at 2- 5Gy (p < 0.05). MMP-9 activity was differentially affected by ionizing radiation in the three TIMP-1 clones. T1-3 and TI-2 displayed significantly reduced MMP-9 levels at various dose points whereas T1-1 exhibited elevated levels of MMP-9 activity at higher doses of treatment (p < 0.05). CONCLUSION: These results demonstrate a dual role for the TIMP-1 overexpression in this renal carcinoma cell line, both as radiosensitizing agents and effectors of MMP-2 and MMP-9 activity.


Assuntos
Ciclo Celular/efeitos da radiação , Dano ao DNA/efeitos da radiação , Inibidores Enzimáticos/farmacologia , Neoplasias Renais , Plasmídeos/efeitos da radiação , Tolerância a Radiação/fisiologia , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Linhagem Celular Tumoral/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Dano ao DNA/fisiologia , Relação Dose-Resposta à Radiação , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz , Tolerância a Radiação/genética , Tolerância a Radiação/efeitos da radiação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-1/antagonistas & inibidores , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-2/antagonistas & inibidores , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Transfecção
12.
Arterioscler Thromb Vasc Biol ; 26(2): 340-6, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16306430

RESUMO

BACKGROUND: Matrix metalloproteinase-9 (MMP-9) is involved in atherosclerosis and elevated MMP-9 activity has been found in unstable plaques, suggesting a crucial role in plaque rupture. This study aims to assess the effect of MMP-9 on plaque stability in apolipoprotein E-deficient mice at different stages of plaque progression. METHODS AND RESULTS: Atherosclerotic lesions were elicited in carotid arteries by perivascular collar placement. MMP-9 overexpression in intermediate or advanced plaques was effected by intraluminal incubation with an adenovirus (Ad.MMP-9). A subset was coincubated with Ad.TIMP-1. Mock virus served as a control. Plaques were analyzed histologically. In intermediate lesions, MMP-9 overexpression induced outward remodeling, as shown by a 30% increase in media size (p=0.03). In both intermediate and advanced lesions, prevalence of vulnerable plaque morphology tended to be increased. Half of MMP-9-treated lesions displayed intraplaque hemorrhage, whereas in controls and the Ad.MMP-9/Ad.TIMP-1 group this was 8% and 16%, respectively (p=0.007). Colocalization with neovessels may point to neo-angiogenesis as a source for intraplaque hemorrhage. CONCLUSIONS: These data show a differential effect of MMP-9 at various stages of plaque progression and suggest that lesion-targeted MMP-9 inhibition might be a valuable therapeutic modality in stabilizing advanced plaques, but not at earlier stages of lesion progression.


Assuntos
Aterosclerose/metabolismo , Aterosclerose/patologia , Hemorragia/metabolismo , Hemorragia/patologia , Metaloproteinase 9 da Matriz/genética , Adenoviridae/genética , Animais , Apolipoproteínas E/genética , Progressão da Doença , Feminino , Regulação Enzimológica da Expressão Gênica , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Ruptura , Índice de Gravidade de Doença
13.
Gene Ther ; 12(20): 1534-8, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15944729

RESUMO

Transduction of the vascular endothelium by adeno-associated virus (AAV) vectors would have broad appeal for gene therapy. However, levels of transduction by AAV serotype-2 are low, an observation linked to deficiencies in endothelial cell binding, sequestration of virions in the extracellular matrix and/or virion degradation by the proteasome. Strategies to improve transduction of endothelial cells include AAV-2 capsid targeting using small peptides isolated by phage display or the use of alternate serotypes. Previously, we have shown that AAV serotypes-3 through -6 transduce endothelial cells with poor efficiency. Recently, AAV serotypes-7 and -8 have been shown to mediate efficient transduction of the skeletal muscle and liver, respectively, although their infectivity profile for vascular cells has not been addressed. Here, we show that AAV-7 and -8 also transduce endothelial cells with poor efficiency and the levels of transgene expression are markedly enhanced by inhibition of the proteasome. In both cases proteasome blockade enhances the nuclear translocation of virions. We further show that this is vascular cell-type selective since transduction of smooth muscle cells is not sensitive to proteasome inhibition. Analysis in intact blood vessels corroborated these findings and suggests that proteasome degradation is a common limiting factor for endothelial cell transduction by AAV vectors.


Assuntos
Dependovirus/genética , Células Endoteliais/virologia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Músculo Liso Vascular/virologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Aorta Torácica/virologia , Linhagem Celular , Células Cultivadas , DNA Viral/análise , Células Endoteliais/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Leupeptinas/uso terapêutico , Camundongos , Músculo Liso Vascular/metabolismo , Inibidores de Proteassoma , Ratos , Ratos Endogâmicos WKY , Veia Safena , Sorotipagem , Transdução Genética/métodos
14.
Gene Ther ; 12(14): 1154-7, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15829995

RESUMO

Progressive saphenous vein graft (SVG) narrowing and occlusion remains a major limitation of coronary artery bypass grafting and is an important target for gene therapy. Ex vivo adenoviral gene transfer of tissue inhibitor of metalloproteinase 3 (TIMP-3) reduces adverse SVG remodelling postarterialization, but concerns remain over the use of viral vectors in patients. Ultrasound exposure (USE) in the presence of echocontrast microbubbles (ECM) substantially enhances nonviral gene delivery. We investigated the effects of ultrasound-enhanced gene delivery (UEGD) of TIMP-3 plasmid on vascular remodelling in porcine SVG. Maximal luciferase activity (3000-fold versus naked plasmid alone) and TIMP-3 transgene expression in porcine vascular smooth muscle cells in vitro was achieved using USE at 1 MHz, 1.8 mechanical index (MI), 6% duty cycle (DC) in the presence of 50% (v/v) BR14 ECM (Bracco). These conditions were therefore utilized for subsequent studies in vivo. Yorkshire White pigs received carotid interposition SVG that were untransfected or had undergone ex vivo UEGD of lacZ (control) or TIMP-3 plasmids. At 28 d postgrafting, lumen and total vessel area were significantly greater in the TIMP-3 group (10.1+/-1.2 and 25.5+/-2.2 mm2, respectively) compared to untransfected (6.34+/-0.5 and 20.8+/-1.9 mm2) or lacZ-transfected (6.1+/-0.7 and 19.7+/-1.2 mm2) controls (P<0.01). These data indicate that nonviral TIMP-3 plasmid delivery by USE achieves significant biological effects in a clinically relevant model of SV grafting, and is the first study to demonstrate the potential for therapeutic UEGD to prevent SVG failure.


Assuntos
Ponte de Artéria Coronária , Veia Safena/transplante , Inibidor Tecidual de Metaloproteinase-3/genética , Transfecção/métodos , Ultrassom , Animais , Meios de Contraste , Oclusão de Enxerto Vascular/patologia , Oclusão de Enxerto Vascular/prevenção & controle , Plasmídeos , Veia Safena/patologia , Suínos , Inibidor Tecidual de Metaloproteinase-3/metabolismo
15.
Gene Ther ; 11(16): 1296-300, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15292916

RESUMO

Adenovirus (Ad)-mediated gene delivery is a promising approach for genetic manipulation of the vasculature and is being used in both preclinical models and clinical trials. However, safety concerns relating to infection of nontarget tissue and the poor infectivity of vascular cells compared to other cell types necessitates Ad vector refinement. Here, we combine a transductional targeting approach to improve vascular cell infectivity through RGD peptide insertion into adenovirus fibers, combined with transcriptional targeting to endothelial cells using a approximately 1 kb fragment of the fms-like tyrosine kinase receptor-1 (FLT-1) promoter. Single- and double-modified vectors were characterized in human cell lines that either support or have silenced FLT-1 expression. In rat hepatocytes and endothelial cells, the double modification substantially shifted transduction profiles toward vascular endothelial cells. Furthermore, in intact aortae derived from spontaneously hypertensive rats that display enhanced alphav integrin expression on dysfunctional endothelium, enhanced levels of transduction were observed using the double-modified vector but not in aortae derived from normotensive control rats. Our data indicate that Ad-mediated transduction can be beneficially modified in vitro and in vivo by combining fiber modification and a cell-selective promoter within a single-component vector system.


Assuntos
Infecções por Adenoviridae/genética , Adenoviridae/genética , Marcação de Genes/métodos , Animais , Aorta/fisiopatologia , Linhagem Celular , Células Cultivadas , Células Endoteliais/fisiologia , Terapia Genética/métodos , Vetores Genéticos/genética , Hepatócitos/fisiologia , Humanos , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptores Proteína Tirosina Quinases , Receptores Virais/genética , Transcrição Gênica/genética , Transdução Genética/métodos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética
16.
Curr Gene Ther ; 4(1): 89-113, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15032617

RESUMO

It has become apparent that the clinical success anticipated in the field of gene therapy has been limited by progress in several of the fundamental areas of genetics, molecular and cellular biology relevant to its application. Whilst a great deal of effort has been made in the evaluation of transgenes, it is only more recently with the advance of vector systems that attention has begun to be focused upon the means and control of transgene expression. Until recently, the majority of constructs have employed ubiquitous viral promoters to drive expression from simple gene expression cassettes using viral promoters and lacking introns, 3' untranslated regions (UTRs), locus control regions (LCR's), matrix attachment regions (MAR's) and other such genetic components. It has consequently emerged that these elements may have a key role in determining the levels and longevity of gene expression attainable in vivo, irrespective of the vector system utilised. The majority of gene therapy applications would also benefit from the specific optimisation of 'tailor-made' expression cassettes to optimise their therapeutic efficacy. In conjunction with modification of vector tropism and strategies to limit their immunogenicity, this should create vectors suitable for the clinical application of gene therapy. This review aims to highlight some of the principle considerations of gene expression in vivo, and the means by which it may most effectively be achieved, whether this is via the minimal modification of an existing eukaryotic promoter or by the more extensive design of a novel promoter and associated elements.


Assuntos
Regulação da Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos/química , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Animais , Células Eucarióticas/metabolismo , Vetores Genéticos/genética , Humanos , Neoplasias/terapia , Fatores de Transcrição/metabolismo , Vírus/genética
17.
Gene Ther ; 9(2): 110-7, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11857069

RESUMO

Gene transfer may be appropriate for therapeutic protocols targeted at the vascular endothelium. Endothelial dysfunction is the principal phenotype associated with atherosclerosis and hypertension. Oxidative stress has been implicated in the development of endothelial dysfunction. We have explored the ability of overexpressing anti-oxidant genes (superoxide dismutases; SODs) in vitro and in vivo to assess their potential for reversing endothelial dysfunction in a rat model, the stroke-prone spontaneously hypertensive rat (SHRSP). Western blotting and immunofluorescence assays in vitro showed efficient overexpression of MnSOD and ECSOD with respect to localisation to the mitochondria and extracellular surface, respectively. Transgene functional activity was quantified with SOD activity assays. MnSOD and ECSOD overexpression in intact SHRSP vessels in vivo led to endothelial and adventitial overexpression. Pharmacological assessment of transduced vessels following in vivo delivery by basal NO availability quantification demonstrated that the "null" adenovirus and MnSOD adenovirus did not significantly increase NO availability. However, AdECSOD-treated carotid arteries showed a significant increase in NO availability (1.91 +/- 0.04 versus 0.75 +/- 0.08 g/g, n = 6, P = 0.029). In summary, efficient overexpression of ECSOD, but not MnSOD in vivo, results in improved endothelial function in a rat model of hypertension and has important implications for the development of endothelial-based vascular gene therapy.


Assuntos
Endotélio Vascular/fisiopatologia , Sequestradores de Radicais Livres/metabolismo , Terapia Genética/métodos , Hipertensão/terapia , Superóxido Dismutase/metabolismo , Adenoviridae/genética , Animais , Western Blotting , Células Cultivadas , Técnicas de Transferência de Genes , Vetores Genéticos/uso terapêutico , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Masculino , Ratos , Ratos Endogâmicos SHR , Superóxido Dismutase/genética
18.
Mol Ther ; 4(6): 534-42, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11735337

RESUMO

Adenovirus type 5 (Ad) based vectors transduce vascular endothelial cells (EC) and have been widely used for vascular gene transfer. However, many cell types express the Ad receptor (cox-sackievirus adenovirus receptor; CAR), preventing selective EC infection and precluding clinical use. We previously isolated the human EC-binding peptides SIGYPLP and LSNFHSS by phage display and demonstrated by means of a bispecific antibody that SIGYPLP directs efficient, high-level, EC-selective Ad-mediated gene transfer. We now generate genetically modified Ad fiber proteins with selective EC tropism by engineering these peptides into the HI loop of the Ad fiber. SIGYPLP, but not LSNFHSS, enhanced EC selectivity, demonstrating maintenance of peptide-cell binding fidelity upon incorporation into virions. Combining fiber mutations that block CAR binding (detargeting) with SIGYPLP insertion (retargeting) generated a novel Ad vector, AdKO1SIG, in a single component system. AdKO1SIG demonstrated efficient and selective tropism for EC compared with control Ad vectors. This is the first demonstration of genetic incorporation of a novel, mammalian, cell-selective ligand that retains its targeting fidelity in the Ad fiber HI loop, in combination with point mutations that abolish fiber-CAR interaction. This study demonstrates the potential for improving the cell-selectivity and safety of adenoviral vectors.


Assuntos
Adenoviridae/fisiologia , Endotélio Vascular/virologia , Terapia Genética/métodos , Fragmentos de Peptídeos/genética , Receptores Virais/metabolismo , Sítios de Ligação , Ligação Competitiva , Células Cultivadas , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Primers do DNA/química , Citometria de Fluxo , Marcação de Genes/métodos , Vetores Genéticos , Proteínas de Fluorescência Verde , Humanos , Immunoblotting , Proteínas Luminescentes/metabolismo , Plasmídeos/genética , Receptores Virais/genética , Linfócitos T/metabolismo , Transdução Genética , Transfecção
19.
Mol Ther ; 4(3): 174-81, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11545607

RESUMO

Gene therapy vectors based on adeno-associated virus-2 (AAV2) offer considerable promise for human gene therapy. Applications for AAV vectors are limited to tissues efficiently transduced by the vector due to its natural tropism, which is predominantly skeletal muscle, neurons, and hepatocytes. Tropism modification to elevate efficiency and/or selectivity to individual cell types would enhance the scope of AAV for disease therapies. The vascular endothelium is implicitly important in cardiovascular diseases and cancer, but is relatively poorly transduced by AAV vectors. We therefore genetically incorporated the peptide SIGYPLP, which targets endothelial cells (EC), into position I-587 of AAV capsids. SIGYPLP-modified AAV (AAVsig) showed enhanced transduction of human EC compared with AAV with a wild-type capsid (AAVwt), a phenotype independent of heparan sulphate proteoglycan (HSPG) binding. In contrast, AAVsig did not enhance transduction of primary human vascular smooth muscle cells or human hepatocytes, principal targets for AAV vectors in local or systemic gene delivery applications, respectively. Furthermore, infection of EC in the presence of bafilomycin A(2) indicated that intracellular trafficking of AAV particles was altered by targeting AAV by means of SIGYPLP. AAV vectors with enhanced tropism for EC will be useful for diverse gene therapeutics targeted at the vasculature.


Assuntos
Dependovirus/genética , Endotélio Vascular/metabolismo , Macrolídeos , Transdução Genética/métodos , Sequência de Aminoácidos , Antibacterianos/farmacologia , Transporte Biológico/efeitos dos fármacos , Capsídeo/química , Capsídeo/genética , Capsídeo/metabolismo , Células Cultivadas , Dependovirus/efeitos dos fármacos , Dependovirus/metabolismo , Dependovirus/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/virologia , Expressão Gênica/efeitos dos fármacos , Engenharia Genética , Terapia Genética/métodos , Células HeLa , Proteoglicanas de Heparan Sulfato/antagonistas & inibidores , Proteoglicanas de Heparan Sulfato/metabolismo , Heparina/farmacologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/virologia , Mutação , Especificidade de Órgãos , Receptores Virais/antagonistas & inibidores , Receptores Virais/metabolismo
20.
Nat Biotechnol ; 19(9): 838-42, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11533642

RESUMO

The promise of gene therapy for health care will not be realized until gene delivery systems are capable of achieving efficient, cell-specific gene delivery in vivo. Here we describe an adenoviral system for achieving cell-specific transgene expression in pulmonary endothelium. The combination of transductional targeting to a pulmonary endothelial marker (angiotensin-converting enzyme, ACE) and an endothelial-specific promoter (for vascular endothelial growth factor receptor type 1, flt-1) resulted in a synergistic, 300,000-fold improvement in the selectivity of transgene expression for lung versus the usual site of vector sequestration, the liver. This combined approach should be useful for the design of other gene delivery systems.


Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Transcrição Gênica , Transdução Genética , Transgenes , Adenoviridae/genética , Animais , Linhagem Celular , Fatores de Crescimento Endotelial/genética , Endotélio/metabolismo , Endotélio Vascular/metabolismo , Vetores Genéticos , Imuno-Histoquímica , Fígado/metabolismo , Luciferases/metabolismo , Pulmão/metabolismo , Linfocinas/genética , Masculino , Camundongos , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular
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