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1.
Eur J Nucl Med Mol Imaging ; 34(5): 715-721, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17096094

RESUMO

PURPOSE: Cell-based therapy by transplantation of progenitor cells has emerged as a promising development for organ repair, but non-invasive imaging approaches are required to monitor the fate of transplanted cells. Radioactive labelling with (111)In-oxine has been used in preclinical trials. This study aimed to validate (111)In-oxine labelling and subsequent in vivo and ex vivo detection of haematopoietic progenitor cells. METHODS: Murine haematopoietic progenitor cells (10(6), FDCPmix) were labelled with 0.1 MBq (low dose) or 1.0 MBq (high dose) (111)In-oxine and compared with unlabelled controls. Cellular retention of (111)In, viability and proliferation were determined up to 48 h after labelling. Labelled cells were injected into the cavity of the left or right cardiac ventricle in mice. Scintigraphic images were acquired 24 h later. Organ samples were harvested to determine the tissue-specific activity. RESULTS: Labelling efficiency was 75 +/- 14%. Cellular retention of incorporated (111)In after 48 h was 18 +/- 4%. Percentage viability after 48 h was 90 +/- 1% (control), 58 +/- 7% (low dose) and 48 +/- 8% (high dose) (p<0.0001). Numbers of viable cells after 48 h (normalised to 0 h) were 249 +/- 51% (control), 42 +/- 8% (low dose) and 32 +/- 5% (high dose) (p<0.0001). Cells accumulated in the spleen (86.6 +/- 27.0% ID/g), bone marrow (59.1 +/- 16.1% ID/g) and liver (30.3 +/- 9.5% ID/g) after left ventricular injection, whereas most of the cells were detected in the lungs (42.4 +/- 21.8% ID/g) after right ventricular injection. CONCLUSION: Radiolabelling of haematopoietic progenitor cells with (111)In-oxine is feasible, with high labelling efficiency but restricted stability. The integrity of labelled cells is significantly affected, with substantially reduced viability and proliferation and limited migration after systemic transfusion.


Assuntos
Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/diagnóstico por imagem , Radioisótopos de Índio , Compostos Organometálicos , Oxiquinolina/análogos & derivados , Compostos Radiofarmacêuticos , Animais , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Modelos Animais de Doenças , Feminino , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/efeitos da radiação , Células-Tronco Hematopoéticas/efeitos da radiação , Pulmão/efeitos dos fármacos , Pulmão/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Cintilografia/métodos , Fatores de Tempo
2.
Circ Res ; 94(6): 776-84, 2004 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-14963004

RESUMO

Leukocyte recruitment is crucial for the response to vascular injury in spontaneous and accelerated atherosclerosis. Whereas the mechanisms of leukocyte adhesion to endothelium or matrix-bound platelets have been characterized, less is known about the proadhesive role of smooth muscle cells (SMCs) exposed after endothelial denudation. In laminar flow assays, neointimal rat SMCs (niSMCs) supported a 2.5-fold higher arrest of monocytes and "memory" T lymphocytes than medial SMCs, which was dependent on both P-selectin and VLA-4, as demonstrated by blocking antibodies. The increase in monocyte arrest on niSMCs was triggered by the CXC chemokine GRO-alpha and fractalkine, whereas "memory" T cell arrest was mediated by stromal cell-derived factor (SDF)-1alpha. This functional phenotype was paralleled by a constitutively increased mRNA and surface expression of P-selectin and of relevant chemokines in niSMCs, as assessed by real-time PCR and flow cytometry. The increased expression of P-selectin in niSMCs versus medial SMCs was associated with enhanced NF-kappaB activity, as revealed by immunofluorescence staining for nuclear p65 in vitro. Inhibition of NF-kappaB by adenoviral IkappaBalpha in niSMCs resulted in a marked reduction of increased leukocyte arrest in flow. Furthermore, P-selectin expression by niSMCs in vivo was confirmed in a hypercholesterolemic mouse model of vascular injury by double immunofluorescence and by RT-PCR after laser microdissection. In conclusion, we have identified a NF-kappaB-mediated proinflammatory phenotype of niSMCs that is characterized by increased P-selectin and chemokine expression and thereby effectively supports leukocyte recruitment.


Assuntos
Quimiotaxia de Leucócito/fisiologia , Inflamação/patologia , Integrina alfa4beta1/fisiologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , NF-kappa B/fisiologia , Selectina-P/fisiologia , Animais , Aorta Torácica/lesões , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Arteriosclerose/metabolismo , Arteriosclerose/patologia , Adesão Celular , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Quimiocina CX3CL1 , Quimiocinas CX3C/farmacologia , Constrição Patológica , Citocinas/biossíntese , Citocinas/genética , Citocinas/fisiologia , Endotélio Vascular/lesões , Regulação da Expressão Gênica , Hipercolesterolemia/metabolismo , Proteínas I-kappa B/fisiologia , Inflamação/metabolismo , Integrina alfa4beta1/biossíntese , Integrina alfa4beta1/genética , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Knockout , Monócitos/citologia , Músculo Liso Vascular/citologia , Inibidor de NF-kappaB alfa , Selectina-P/biossíntese , Selectina-P/genética , Ratos , Ratos Sprague-Dawley , Receptores de Interleucina-8B/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Recidiva , Reologia , Subpopulações de Linfócitos T/citologia
3.
Circulation ; 109(3): 380-5, 2004 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-14691040

RESUMO

BACKGROUND: Macrophage migration inhibitory factor (MIF), a cytokine that controls cell-mediated inflammatory responses, is upregulated in atherogenesis; however, its functional contribution to lesion development has not been evaluated. METHODS AND RESULTS: We studied the role of MIF on neointima lesion formation after wire-induced injury of carotid arteries in apolipoprotein E-deficient (apoE(-/-)) mice. Immunohistochemistry revealed that MIF expression was detectable in endothelial cells before injury and upregulated in smooth muscle cells (SMCs) 24 hours after endothelial denudation. Three weeks after injury, MIF was predominantly found in endothelial cells and macrophage-derived foam cells. Neutralizing MIF with a monoclonal antibody resulted in a marked reduction of neointimal macrophages and inhibited transformation of macrophages into foam cells. Conversely, the content of SMCs and of collagen in the neointima were increased, amounting to a slight but not significant reduction in neointima and media size after 3 weeks of MIF monoclonal antibody treatment. Notably, serum levels of the cytokines IL-2, IL-4, IL-6, IL-10, and tumor necrosis factor were increased in MIF monoclonal antibody-treated mice. In vitro flow assays revealed that MIF pretreatment of aortic endothelium enhanced monocyte recruitment and that the monocyte arrest induced by oxidized LDL is mediated by endothelial MIF, as shown by monoclonal antibody inhibition. CONCLUSIONS: Inhibition of MIF resulted in a shift in the cellular composition of neointimal plaques toward a stabilized phenotype with reduced macrophage/foam cell content and increased SMC content. This might be attributable to a reduction of monocyte recruitment mediated by endothelial MIF.


Assuntos
Arteriosclerose/imunologia , Arteriosclerose/patologia , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Animais , Apolipoproteínas E/genética , Doenças das Artérias Carótidas/etiologia , Doenças das Artérias Carótidas/imunologia , Doenças das Artérias Carótidas/patologia , Adesão Celular , Citocinas/sangue , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Feminino , Humanos , Fatores Inibidores da Migração de Macrófagos/farmacologia , Fatores Inibidores da Migração de Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Túnica Íntima/química , Túnica Íntima/imunologia , Túnica Íntima/patologia
4.
J Vasc Res ; 39(4): 340-52, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12187124

RESUMO

Proliferation, migration and invasion of smooth muscle cells (SMCs) are essential pathogenic processes in the development of a broad spectrum of cardiovascular disorders, like arteriosclerosis, restenosis after percutaneous transluminal angioplasty and stent implantation as well as transplant vessel disease. As an in vitro model mimicking these processes, the Boyden chamber was employed to characterize the diverging migratory and invasive potentials of proliferating and nonproliferating human arterial SMCs (haSMCs). Using this model, differential gene expression of both phenotypes was analyzed by a cDNA array system (Clontech human cardiovascular array). With these arrays, 558 cardiovascular-associated genes could be compared. Further, gene expression was exactly quantified by real-time RT-PCR. Protein expression was analyzed by ELISA and Western blotting. In total, 47 genes were differentially expressed more than 1.5 times. Most of the differentially regulated genes in this study were associated with the extracellular matrix (ECM) and cell motility. In detail, the respective groups were matrix-organizing proteins, ECM proteins, cell adhesion proteins, extracellular communication and cytoskeleton motility proteins. Genes known to be differentially regulated during haSMC migration and invasion, like TIMP 2, TIMP 3, and MMP 3, were confirmed by the array data. Reduced expression of several cytoskeletal proteins, like vimentin, fibronectin, cytokeratins and beta1 integrin, was shown in the invasive phenotype. Further, angio-associated protein, alpha E-catenin and atrial brain natriuretic peptide receptor were downregulated whereas TFPI 2 was strongly upregulated in invasive haSMCs. In conclusion, several relevant potential candidate genes for the quiescent and the invasive SMC phenotype were identified and genes already known to be differentially regulated by previous analysis were confirmed.


Assuntos
Expressão Gênica , Músculo Liso Vascular/metabolismo , Arteriosclerose/patologia , Divisão Celular , Movimento Celular , Células Cultivadas , Reestenose Coronária/patologia , Matriz Extracelular/metabolismo , Fibronectinas/genética , Regulação da Expressão Gênica , Humanos , Integrina beta1/genética , Queratinas/genética , Artéria Torácica Interna , Metaloproteinase 3 da Matriz/genética , Modelos Biológicos , Músculo Liso Vascular/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-3/genética , Vimentina/genética
5.
Cardiovasc Res ; 54(3): 630-9, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12031709

RESUMO

Migrating cells like coronary smooth muscle cells in restenosis change their cell shape and form cellular protrusions called filopodia. A prerequisite for filopodia formation is the rearrangement of the actin cytoskeleton. An essential role of the 78-kDa protein Moesin is described for Rho- and Rac-dependent assembly of actin filaments. In vivo Moesin is not observed in mature smooth muscle cells. The objective of this study was to demonstrate that Moesin is upregulated in migrating coronary smooth muscle cells during restenosis development. In vivo expression of Moesin was upregulated in neointimal coronary smooth muscle cells of dilated porcine coronary arteries compared to the undilated left circumflex coronary artery of the same swine. Concordant to these results Moesin expression was upregulated in migrating and invading human arterial smooth muscle cells in vitro analyzed by FACS, Western blotting and RT-PCR. In addition, the invasive potential of Moesin-positive Mel Im cells transfected with Moesin sense DNA increased by 28% as compared to mock-transfected control, whereas antisense transfected cells had a decreased invasive potential of 32%. Transfection of Moesin-negative HepG2 with Moesin sense cDNA increased the invasive potential by 43%. Finally, transfection of human arterial smooth muscle cells with Moesin sense cDNA caused an increased invasive potential of 30%. Transfection of haSMCs with antisense cDNA decreased the invasive potential by 37% in comparison to mock-transfected control. These results demonstrate for the first time an upregulation of Moesin expression in coronary smooth muscle cells of the neointima after arterial injury. The increased migrative and invasive potential of cells transfected with Moesin confirmed the functional role of Moesin in cell migration. This indicates an important role of Moesin during restenosis development.


Assuntos
Angioplastia Coronária com Balão , Estenose Coronária/metabolismo , Proteínas dos Microfilamentos/análise , Túnica Íntima/metabolismo , Actinas/análise , Animais , Biomarcadores/análise , Movimento Celular , Células Cultivadas , Estenose Coronária/patologia , Vasos Coronários , Citometria de Fluxo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Modelos Animais , Músculo Liso Vascular/patologia , Suínos , Transfecção
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