RESUMO
BACKGROUND: CD40 is a receptor expressed on a wide range of cells such as leukocytes and endothelial cells (EC). As a member of the tumor necrosis factor (TNF) superfamily the activation of CD40 by CD40-ligand (CD40L) plays a crucial role for the development and progression of a variety of inflammatory processes including atherosclerosis. The aim of the present study was to investigate the effect of CD40/CD40L interaction on leukocyte adhesion to the endothelium and on endothelial cell migration. METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVEC) were stimulated with either stable transfectants of mouse myeloma cells expressing the CD40L or wild type cells (4 h). Subsequently adhesion of leukocytes expressing Sialyl Lewis X, the counterpart for E-selectin (HL60 cells), was measured under shear stress (2-2.6 dyne/cm(2)) using a flow chamber adhesion assay. Stimulation of CD40 led to a significant increase of E-selectin dependent adhesion of leukocytes to the endothelium. Incubation of cells with either the CD40L blocking antibody TRAP-1 or the E-selectin blocking antibody BBA2 during CD40 stimulation completely abolished adhesion of leukocytes to HUVEC. Similar results were found in human cardiac microvasculature endothelial cells (HCMEC). In contrast stimulation of CD40 had no effect on adhesion of L-selectin expressing NALM6-L cells. Furthermore, CD40/CD40L interaction abrogated VEGF-induced migration of HUVEC compared to non-stimulated controls. In comparison experiments, stimulation of endothelial cells with VEGF led to a significant phosphorylation of ERK1/2, Akt, and eNOS. Stimulation of endothelial CD40 had no effect on VEGF-induced phosphorylation of ERK1/2. However, VEGF-induced activation of Akt and eNOS was reduced to baseline levels when endothelial CD40 was stimulated. CONCLUSION: CD40/CD40L interaction induces E-selectin dependent adhesion of leukocytes to human endothelial cells and reduces endothelial cell migration by inhibiting the Akt/eNOS signaling pathway.
Assuntos
Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Movimento Celular , Selectina E/metabolismo , Endotélio Vascular/fisiologia , Leucócitos/fisiologia , Animais , Adesão Celular , Linhagem Celular Tumoral , Células Cultivadas , Células HL-60 , Humanos , Camundongos , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-akt/agonistas , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
BACKGROUND AND PURPOSE: Brain inflammation is a hallmark of stroke, where it has been implicated in tissue damage as well as in repair. Imaging technologies that specifically visualize these processes are highly desirable. In this study, we explored whether the inflammatory receptor CD40 can be noninvasively and specifically visualized in mice after cerebral ischemia using a fluorescent monoclonal antibody, which we labeled with the near-infrared fluorescence dye Cy5.5 (Cy5.5-CD40MAb). METHODS: Wild-type and CD40-deficient mice were subjected to transient middle cerebral artery occlusion. Mice were either intravenously injected with Cy5.5-CD40MAb or control Cy5.5-IgGMAb. Noninvasive and ex vivo near-infrared fluorescence imaging was performed after injection of the compounds. Probe distribution and specificity was further assessed with single-plane illumination microscopy, immunohistochemistry, and confocal microscopy. RESULTS: Significantly higher fluorescence intensities over the stroke-affected hemisphere, compared to the contralateral side, were only detected noninvasively in wild-type mice that received Cy5.5-CD40MAb, but not in CD40-deficient mice injected with Cy5.5-CD40MAb or in wild-type mice that were injected with Cy5.5-IgGMAb. Ex vivo near-infrared fluorescence showed an intense fluorescence within the ischemic territory only in wild-type mice injected with Cy5.5-CD40MAb. In the brains of these mice, single-plane illumination microscopy demonstrated vascular and parenchymal distribution, and confocal microscopy revealed a partial colocalization of parenchymal fluorescence from the injected Cy5.5-CD40MAb with activated microglia and blood-derived cells in the ischemic region. CONCLUSIONS: The study demonstrates that a CD40-targeted fluorescent antibody enables specific noninvasive detection of the inflammatory receptor CD40 after cerebral ischemia using optical techniques.
Assuntos
Anticorpos Monoclonais , Isquemia Encefálica/imunologia , Antígenos CD40/biossíntese , Inflamação/imunologia , Animais , Isquemia Encefálica/complicações , Isquemia Encefálica/patologia , Carbocianinas , Técnica Direta de Fluorescência para Anticorpo , Corantes Fluorescentes , Imuno-Histoquímica , Inflamação/etiologia , Inflamação/patologia , Camundongos , Camundongos Mutantes , Microscopia Confocal , Microscopia de Fluorescência/métodosRESUMO
OBJECTIVE: Insulin resistance and hyperinsulinemia are major causes of cardiovascular morbidity and mortality. Matrix metalloproteinases (MMPs), highly expressed in activated mononuclear cells in vulnerable atherosclerotic lesions, are the main proteolytic enzymes controlling plaque stability. The aim of this study was to investigate the regulation of monocyte MMP-9 by insulin. METHODS AND RESULTS: Stimulation of MMP-9 expression by insulin was time- and concentration-dependent in human monocytic THP-1 cells. Inhibition of insulin receptor (IR) maturation via inhibition of its activating convertase furin with the pharmacological furin-inhibitor decanoyl-RVKR-chloromethylketone, as well as blocking of IGF-1R function with a IGF-1R blocking antibody, demonstrated that insulin mediates increases in MMP-9 via IR activation. Inhibition of insulin's "metabolic" phosphatidylinositol 3-kinase signaling with wortmannin (50 nmol/L) or LY294002 (2.5 micromol/L) did not prevent insulin-dependent MMP-9 induction. In contrast inhibition of insulin's "mitogenic" Ras-Raf-mitogen-activated protein kinase-kinase pathways with PD98059 (15 micromol/L) or U0126 (2 micromol/L) inhibited insulin-induced MMP-9 gelatinolytic activity in THP-1 cells. Likewise, PD98059 inhibited insulin augmented MMP-9 levels in primary human monocytes, whereas wortmannin had no effect. CONCLUSION: This study demonstrates that insulin can induce MMP-9 via mitogenic signaling pathways in monocytes, whereas phosphatidylinositol 3-kinase-dependent signaling, typically altered in insulin resistance, is not required. Induction of MMP-9 by insulin may potentially contribute to a pro-inflammatory state and the increased cardiovascular morbidity and mortality in type 2 diabetics.
Assuntos
Hipoglicemiantes/farmacologia , Insulina/farmacologia , Metaloproteinase 9 da Matriz/metabolismo , Monócitos/enzimologia , Androstadienos/farmacologia , Anticorpos Bloqueadores/farmacologia , Butadienos/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Flavonoides/farmacologia , Humanos , Insulina/imunologia , Resistência à Insulina , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Monócitos/efeitos dos fármacos , Nitrilas/farmacologia , Fosfolipases A/antagonistas & inibidores , Receptor de Insulina/metabolismo , Estimulação Química , Fatores de Tempo , WortmaninaRESUMO
BACKGROUND: Accumulation of macrophages and their in situ expression of matrix metalloproteinases (MMPs) are important determinants of plaque stability. Activation of membrane-bound MT1-MMP, the major activator of pro-MMP-2, requires intracellular endoproteolytic cleavage of its precursor protein. This type of activation typically requires suitable furin-like proprotein convertases (PCs), specifically furin and PC5. The present study was done to investigate the function of MT1-MMP as well as furin-like PCs in mononuclear inflammatory cells. METHODS AND RESULTS: Macrophage differentiation of human monocytic THP-1 cells was accompanied by increased expression of furin, PC5, and MT1-MMP. Some pro-MMP-2 activation was found in macrophages, but pro-MMP-2 level or activation was not enhanced after stimulation with the proinflammatory mediators tumor necrosis factor-alpha or lipopolysaccharide. However, culturing of macrophages in conditioned medium from serum-starved vascular smooth muscle cells, which constitutively secrete pro-MMP-2, resulted in a strong pro-MMP-2 activation. Inhibition of furin-like PCs with the specific pharmacological inhibitor decanoyl-RVKR-chloromethylketone (dec-CMK) inhibited MT1-MMP activation in macrophages. Dec-CMK or furin-specific small interfering RNA significantly inhibited macrophage MT1-MMP-dependent activation of vascular smooth muscle cell-derived pro-MMP-2. Flow cytometry demonstrated that human circulating monocytes express furin and PC5, and MT1-MMP and immunohistochemistry revealed their colocalization in macrophages in advanced human atherosclerotic lesions. CONCLUSIONS: Furin-like PCs (furin and PC5) play a central role in a MT-MMP-MMP-2 proteolytic cascade, involving provision of macrophage MT1-MMP for the activation of pro-MMP-2 synthesized by other cells. Furin and PC5 are expressed in human peripheral blood mononuclear cells and colocalize with MT1-MMP in macrophages in the atherosclerotic plaque, supporting the hypothesis that they are potential targets in atherosclerosis.
Assuntos
Arteriosclerose/enzimologia , Metaloproteinase 2 da Matriz/metabolismo , Pró-Proteína Convertases/fisiologia , Arteriosclerose/etiologia , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Furina/análise , Furina/fisiologia , Humanos , Macrófagos/citologia , Macrófagos/enzimologia , Metaloproteinases da Matriz/metabolismo , Metaloproteinases da Matriz Associadas à Membrana , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Pró-Proteína Convertase 5/análise , Pró-Proteína Convertase 5/fisiologia , Pró-Proteína Convertases/análise , Precursores de Proteínas/metabolismoRESUMO
Integrins are heterodimeric alpha/beta receptors that link the cytoskeleton with the extracellular matrix, thereby regulating several cell functions important in atherosclerosis. In vitro, the subtilisin/kexin-like proprotein convertases (PCs), namely PC5 and furin, have been shown to be responsible for the endoproteolytic activation of the alpha(v) integrin subunit. Based on their cleavage activity, these PCs are potential targets in atherosclerosis. In the present study, we investigated the localization of furin and PC5 in different stages of human atherosclerosis. Immunohistochemical analysis of furin and PC5 revealed their presence in vascular smooth-muscle cells and endothelial cells in atherosclerotic and non-atherosclerotic lesions. However, in the more advanced lesions, furin and PC5 staining was significantly expressed in macrophages/foam cells. In vitro, THP-1 derived macrophages contained furin and PC5, and maturation of monocytes to macrophages was accompanied by enhanced alpha(v)beta3 cell-surface expression. Inhibition of furin/PC5 with the specific pharmacological furin-like PC-inhibitor dec-CMK inhibited alpha(v) endoproteolytic activation but did not abolish alpha(v)beta3 cell-surface expression. This indicates that furin/PC5 is required for alpha(v) endoproteolytic activation but not for alpha(v) routing and sorting to the cell surface. In conclusion, our study demonstrates that furin and PC5 are significantly expressed in mononuclear cells in advanced human atherosclerotic lesions, where they regulate alpha(v) endoproteolytic activation.
Assuntos
Arteriosclerose/enzimologia , Artéria Femoral/enzimologia , Furina/metabolismo , Proteínas de Membrana/metabolismo , Pró-Proteína Convertase 5/metabolismo , Arteriosclerose/patologia , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Endotélio Vascular/enzimologia , Endotélio Vascular/patologia , Inibidores Enzimáticos/farmacologia , Artéria Femoral/patologia , Citometria de Fluxo , Furina/antagonistas & inibidores , Humanos , Imuno-Histoquímica/métodos , Integrina alfaVbeta3/metabolismo , Macrófagos/enzimologia , Macrófagos/patologia , Proteínas de Membrana/antagonistas & inibidores , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Pró-Proteína Convertase 5/antagonistas & inibidoresRESUMO
We develop a highly specific antibody-dye conjugate for optical imaging of peripheral lymph nodes. The contrast agent consists of the monoclonal antibody recognizing endothelial ligands for the lymphocyte homing receptor L-selectin, MECA-79, and a near-infrared (near-IR) fluorescent indotricarbocyanine dye. The targeting and biodistribution behavior of MECA-79 is studied after radio-iodination and intravenous injection into mice demonstrating specific uptake in lymph nodes and accumulation in high endothelial venules (HEV). After conjugation of MECA-79 with indotricarbocyanine dye, the fluorescence imaging properties of the MECA-79 dye conjugate are examined by intravenous injection in nude mice and laser-induced fluorescence whole-body imaging in vivo. The MECA-79 antibody-dye conjugate accumulates in peripheral lymph nodes, whereas an isotype antibody-dye conjugate does not. Specific lymph node near-IR fluorescent signals become detectable within minutes after injection, and stable imaging persists for more than 24 h. The results demonstrate that vascular targeting of endothelial expression of glyocproteins is feasible to visualize the accumulation of near-IR fluorescent MECA-79 in lymph nodes, making this technology potentially useful to characterize processes of inflammation.
Assuntos
Antígenos de Superfície/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Linfonodos/citologia , Linfonodos/metabolismo , Vasos Linfáticos/citologia , Vasos Linfáticos/metabolismo , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência/métodos , Animais , Anticorpos Monoclonais/efeitos dos fármacos , Anticorpos Monoclonais/farmacocinética , Meios de Contraste , Feminino , Corantes Fluorescentes , Linfonodos/irrigação sanguínea , Taxa de Depuração Metabólica , Camundongos , Camundongos Nus , Espectrofotometria Infravermelho/métodos , Distribuição TecidualRESUMO
OBJECTIVE: Recent studies indicate that regulatory T cells (Tregs) attenuate murine atherosclerosis. Since interleukin (IL)-2 induces Tregs proliferation, we tested the impact of L19-IL2, a fusion antibody specific to extra-domain B of fibronectin (ED-B) containing an active human IL-2 molecule, in experimental atherosclerosis. METHODS AND RESULTS: L19-IL2 or appropriate controls were given intravenously to 6 month old Western diet-fed apoE(-/-) mice on day 1, 3, and 5. Human IL-2 was detected on day 7 within atherosclerotic plaques of L19-IL2-treated mice, and magnetic resonance imaging of the plaques showed a significant adventitial gadolinium enhancement on day 7 and 13, suggesting microvascular leakage as a result of the pharmacodynamic activity of L19-IL2. Treatment with L19-IL2 significantly reduced the size of pre-established atherosclerotic plaques at the thoracic aorta (Sudan III stained area) and in the aortic root area (microscopic, morphometric analysis) on day 7 as compared to controls (L19, D1.3-IL2, NaCl) as well as compared to baseline (day 0). Tregs markers Foxp3 and CTLA4 were highly increased in plaques after L19-IL2 treatment compared to controls (p<0.01), whereas the macrophage marker Mac3 was significantly reduced (p<0.03). Co-treatment with IL-2-receptor blocking antibody PC61 abrogated L19-IL2-induced plaque reduction compared with IgG control (p<0.03). CONCLUSION: L19-IL2 delivers functional IL-2 to pre-established atherosclerotic plaques of WD-fed apoE(-/-) mice resulting in significant plaque size reduction mediated by local Tregs.
Assuntos
Aorta/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Fármacos Cardiovasculares/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Proteínas Recombinantes de Fusão/administração & dosagem , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Aorta/imunologia , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/imunologia , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Humanos , Injeções Intravenosas , Lipídeos/sangue , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Reguladores/imunologia , Fatores de TempoRESUMO
Vascular smooth muscle cell (VSMC) invasion is a key element in atherogenesis and restenosis, requiring integrins for adhesion/de-adhesion as well as matrix metalloproteinases (MMPs) for focalized proteolysis. Among the MMP family, pro-MMP-2 is unique in its activation, depending on the formation of a multiprotein complex with MT1-MMP/TIMP-2 at the cell surface, in which integrin alphavbeta3 participates. Integrin alphav and MT1-MMP are synthesized from precursors via furin-dependent cleavage of their pro-peptide. Furin is the prototypical proprotein convertase highly expressed in VSMCs and human atherosclerotic lesions. Its precise role in the tight network involving MMPs/integrins and their coordination and cooperation required for VSMC invasion is unknown. We demonstrate that furin-inhibition with decanoyl-RVKR-chloromethylketone inhibits VSMC invasion in a comparable degree to MMP inhibitors, which reduce the MT1-MMP-MMP-2 proteolytic cascade. Furin-inhibition did not prevent MT1-MMP/MMP-2 maturation. In contrast, it strongly reduced pro-alphav cleavage, but did not lessen its cell membrane expression. However, inhibition of pro-alphav processing via furin-inhibition strongly reduced pro-MMP-2 binding to the cell surface, thereby lessening its full maturation and diminishing the cell surface in situ proteolysis required for invasion. Thus, our data demonstrate a novel mechanism of furin-dependent alphav cleavage that enhances pro-MMP-2 binding and activation at the cell membrane in cooperation with MT1-MMP in primary VSMCs. Processing of alphav by furin contributes to the recruitment of enzymatic energy to the cell surface, thereby providing focalized proteolysis associated with VSMC invasion.
Assuntos
Membrana Celular/metabolismo , Movimento Celular , Integrinas/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Movimento Celular/efeitos dos fármacos , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Concanavalina A/farmacologia , Combinação de Medicamentos , Precursores Enzimáticos/metabolismo , Citometria de Fluxo , Furina/metabolismo , Gelatina/metabolismo , Humanos , Laminina/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteoglicanas/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Serum concentrations of soluble L-selectin by far exceed those of other soluble adhesion molecules, and serum soluble L-selectin concentrations are remarkably stable upon prolonged storage. We present evidence for Ca(2+)-dependent binding interactions between human serum amyloid P (SAP), a proteolysis-resistant pentraxin glycoprotein, and L-selectin, as shown by surface plasmon resonance measurements, protein band shift assays in a native PAGE system, and after SDS-PAGE and membrane transfer. Monoclonal antibodies to L-selectin strongly reduced binding of biotinylated SAP to L-selectin-IgG chimeras immobilized on microtiter plates. As binding was reduced by prior glycopeptidase F treatment of L-selectin but not of SAP, it appears to be based on SAP lectin domain interactions with N-linked L-selectin carbohydrates. In freshly prepared human lymphocytes, SAP incubation induced expression of a beta2 integrin neoepitope associated with high-affinity binding. This was partially blocked by pre-incubation with Fab fragments of two anti-L-selectin antibodies. In flow chamber experiments, SAP inhibited the adherence of human neutrophils to activated endothelium under shear stress. Thus, SAP binds to human L-selectin and affects L-selectin-dependent leukocyte-endothelial interactions.
Assuntos
Anticorpos Monoclonais/química , Cálcio/química , Selectina L/química , Componente Amiloide P Sérico/química , Anticorpos Monoclonais/imunologia , Cálcio/metabolismo , Carboidratos/química , Carboidratos/imunologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/imunologia , Humanos , Selectina L/imunologia , Linfócitos/citologia , Linfócitos/imunologia , Neutrófilos/imunologia , Ligação Proteica/imunologia , Componente Amiloide P Sérico/imunologia , Componente Amiloide P Sérico/farmacologia , Estresse Mecânico , Ressonância de Plasmônio de Superfície/métodos , Microglobulina beta-2/imunologiaRESUMO
Soluble l-selectin (sCD62L) plasma concentrations at diagnosis and outcome were investigated in 193 children at first relapse of acute lymphoblastic leukaemia (ALL) after treatment according to the Berlin-Frankfurt-Münster relapsed ALL multicentre trials, ALL-REZ BFM 95 and 96. sCD62L was low (< fifth paediatric reference percentile) in 63 (33%) and high (> 95th percentile) in 36 (19%) children, and was independent of remission duration, sex, BCR-ABL fusion or extramedullary disease. High sCD62L was associated with circulating blasts and T-cell phenotype. More initial adverse events occurred in children with high and low levels of sCD62L (23 out of 99) than in those with normal levels (9 out of 94, P = 0.018). Among 75 worst-prognosis patients (risk groups S3/S4, isolated bone marrow relapse occurring less than 6 months after elective cessation of front-line therapy, or T-cell phenotype with bone marrow involvement), 27 had low sCD62L and decreased event-free survival (EFS) probability (PEFS5 = 0.09 at 5 years) and duration (219 d) compared with normal sCD62L (29 out of 75, PEFS5 = 0.24, 640 d, P = 0.01). Low (44 out of 118), normal (72 out of 118), and high (19 out of 118) sCD62L non-S3/S4 patients fared similarly (average PEFS5 = 0.45, 1369 d; P = 0.5). Low sCD62L may be a marker of malignant blasts replacing normal sCD62L-producing haematopoietic cells. In children with first relapse of ALL and worst prognosis, plasma sCD62L may be useful for risk-adapted stratification.