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1.
Gene Ther ; 20(9): 901-12, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23535897

RESUMO

Coronary artery disease represents the leading cause of mortality in the developed world. Percutaneous coronary intervention involving stent placement remains disadvantaged by restenosis or thrombosis. Vascular gene therapy-based methods may be approached, but lack a vascular gene delivery vector. We report a safe and efficient long-term transduction of rat carotid vessels after balloon injury intervention with a translational optimized AAV2.5 vector. Compared with other known adeno-associated virus (AAV) serotypes, AAV2.5 demonstrated the highest transduction efficiency of human coronary artery vascular smooth muscle cells (VSMCs) in vitro. Local delivery of AAV2.5-driven transgenes in injured carotid arteries resulted in transduction as soon as day 2 after surgery and persisted for at least 30 days. In contrast to adenovirus 5 vector, inflammation was not detected in AAV2.5-transduced vessels. The functional effects of AAV2.5-mediated gene transfer on neointimal thickening were assessed using the sarco/endoplasmic reticulum Ca(2+) ATPase isoform 2a (SERCA2a) human gene, known to inhibit VSMC proliferation. At 30 days, human SERCA2a messenger RNA was detected in transduced arteries. Morphometric analysis revealed a significant decrease in neointimal hyperplasia in AAV2.5-SERCA2a-transduced arteries: 28.36±11.30 (n=8) vs 77.96±24.60 (n=10) µm(2), in AAV2.5-green fluorescent protein-infected, P<0.05. In conclusion, AAV2.5 vector can be considered as a promising safe and effective vector for vascular gene therapy.


Assuntos
Reestenose Coronária/terapia , Dependovirus/genética , Terapia Genética , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Artérias Carótidas/citologia , Células Cultivadas , Vasos Coronários/citologia , Dependovirus/fisiologia , Modelos Animais de Doenças , Vetores Genéticos , Humanos , Masculino , Músculo Liso Vascular/patologia , Neointima/fisiopatologia , Ratos , Ratos Sprague-Dawley , Transdução Genética
2.
Gene Ther ; 18(10): 979-85, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21512506

RESUMO

Intrapericardial drug delivery is a promising procedure, with the ability to localize therapeutics with the heart. Gelfoam particles are nontoxic, inexpensive, nonimmunogenic and biodegradable compounds that can be used to deliver therapeutic agents. We developed a new percutaneous approach method for intrapericardial injection, puncturing the pericardial sac safely under fluoroscopy and intravascular ultrasound (IVUS) guidance. In a porcine model of myocardial infarction (MI), we deployed gelfoam particles carrying either (a) autologous mesenchymal stem cells (MSCs) or (b) an adenovirus encoding enhanced green fluorescent protein (eGFP) 48 h post-MI. The presence of MSCs and viral infection at the infarct zone was confirmed by immunoflourescence and PCR. Puncture was performed successfully in 16 animals. Using IVUS, we successfully determined the size of the pericardial space before the puncture, and safely accessed that space in setting of pericardial effusion and also adhesions induced by the MI. Intrapericardial injection of gelfoam was safe and reliable. Presence of the MSCs and eGFP expression from adenovirus in the myocardium were confirmed after delivery. Our novel percutaneous approach to deliver (stem-) cells or adenovirus was safe and efficient in this pre-clinical model. IVUS-guided delivery is a minimally invasive procedure that seems to be a promising new strategy to deliver therapeutic agents locally to the heart.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Esponja de Gelatina Absorvível/administração & dosagem , Vetores Genéticos/administração & dosagem , Transplante de Células-Tronco Mesenquimais/métodos , Infarto do Miocárdio/tratamento farmacológico , Pericárdio/diagnóstico por imagem , Adenoviridae , Administração Cutânea , Animais , Primers do DNA/genética , Imunofluorescência , Fluoroscopia , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos , Ultrassonografia de Intervenção
3.
Cell Mol Life Sci ; 64(6): 661-73, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17380307

RESUMO

Regardless of erroneous claims by a minority of reports, adult cardiomyocytes are terminally differentiated cells which do not re-enter the cell-cycle under any known physiological or pathological circumstances. However, it has recently been shown that the adult heart has a robust myocardial regenerative potential, which challenges the accepted notions of cardiac cellular biology. The source of this regenerative potential is constituted by resident cardiac stem cells (CSCs). These CSCs, through both cell transplantation and in situ activation, have the capacity to regenerate significant segmental and diffuse myocyte losts, restoring anatomical integrity and ventricular function. Thus, CSC identification has started a brand new discipline of cardiac biology that could profoundly changed the outlook of cardiac physiology and the potential for treatment of cardiac failure. Nonetheless, the dawn of this new era should not be set back by premature attempts at clinical application before having accumulated the required scientifically reproducible data.


Assuntos
Miocárdio/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Fusão Celular , Linhagem da Célula , Humanos , Regeneração
4.
Biochem Soc Trans ; 31(Pt 6): 1453-5, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14641088

RESUMO

Dual-wavelength single-particle fluorescence imaging has been used to quantify the co-localization of receptors and/or ligands on cells by widefield microscopy. Methods for correction of chromatic aberration and identification of submicroscopic artefacts are presented, with data for the lipopolysaccharide/CD14 and MHC class II/CD74 systems.


Assuntos
Microscopia de Fluorescência/métodos , Receptores de Superfície Celular/metabolismo , Animais , Antígenos de Diferenciação de Linfócitos B/metabolismo , Células CHO , Cricetinae , Antígenos de Histocompatibilidade Classe II/metabolismo , Receptores de Lipopolissacarídeos/metabolismo
5.
Biochem Soc Trans ; 31(Pt 5): 1028-31, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14505473

RESUMO

SPFI (single-particle fluorescence imaging) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. The images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional Gaussian function. The spot intensities depend on whether they arise from one or more particles; this provides the basis for determining self-association of cell-surface receptors. We have used this approach to determine dimerization of MHC class II molecules and its disruption by interface peptides. We have also exploited the positional information obtained from SPFI to detect co-localization of cell-surface molecules. This involves labelling two different molecules with different coloured fluorophores and determining their positions separately by dual wavelength imaging. The images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. The technique provides quantification of the extent of co-localization and can detect whether co-localized molecules occur singly or in clusters. We have obtained preliminary data for co-localization of lipopolysaccharide and CD14 on intact cells. We also show that HLA-DR (human leukocyte antigen-DR) and CD74 are partially co-localized and that interaction between these molecules involves the peptide-binding groove of HLA-DR.


Assuntos
Membrana Celular/metabolismo , Microscopia de Fluorescência/métodos , Animais , Antígenos de Diferenciação de Linfócitos B/biossíntese , Dimerização , Antígenos HLA-DR/química , Antígenos de Histocompatibilidade Classe II/biossíntese , Humanos , Receptores de Lipopolissacarídeos/biossíntese , Distribuição Normal , Peptídeos/química
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