RESUMO
Endothelial cells in different microvascular segments of the kidney have diverse functions and exhibit differential responsiveness to disease stimuli. The responsible molecular mechanisms are largely unknown. We previously showed that during hemorrhagic shock, VCAM-1 protein was expressed primarily in extraglomerular compartments of the kidney, while E-selectin protein was highly induced in glomeruli only (van Meurs M, Wulfert FM, Knol AJ, de Haes A, Houwertjes M, Aarts LPHJ, Molema G. Shock 29: 291-299, 2008). Here, we investigated the molecular control of expression of these endothelial cell adhesion molecules in mouse models of renal inflammation. Microvascular segment-specific responses to the induction of anti-glomerular basement membrane (anti-GBM), glomerulonephritis and systemic TNF-α treatment showed that E-selectin expression was transcriptionally regulated, with high E-selectin mRNA and protein levels preferentially expressed in the glomerular compartment. In contrast, VCAM-1 mRNA expression was increased in both arterioles and glomeruli, while VCAM-1 protein expression was limited in the glomeruli. These high VCAM-1 mRNA/low VCAM-1 protein levels were accompanied by high local microRNA (miR)-126 and Egfl7 levels, as well as higher Ets1 levels compared with arteriolar expression levels. Using miR-reporter constructs, the functional activity of miR-126 in glomerular endothelial cells could be demonstrated. Moreover, in vivo knockdown of miR-126 function unleashed VCAM-1 protein expression in the glomeruli upon inflammatory challenge. These data imply that miR-126 has a major role in the segmental, heterogenic response of renal microvascular endothelial cells to systemic inflammatory stimuli.
Assuntos
Glomerulonefrite/metabolismo , Inflamação/metabolismo , Rim/metabolismo , MicroRNAs/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Selectina E/genética , Selectina E/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Glomerulonefrite/genética , Humanos , Inflamação/genética , Glomérulos Renais/metabolismo , Camundongos , MicroRNAs/genética , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
We studied the kinetics of hepatic uptake of liposomes during serum-free recirculating perfusion of rat livers. Liposomes consisted of phosphatidylcholine, cholesterol and phosphatidylserine in a 6:4:0 or a 3:4:3 molar ratio and were radiolabelled with [3H]cholesteryl oleyl ether. The negatively charged liposomes were taken up to a 10-fold higher extent than the neutral ones. Hepatic uptake of fluorescently labelled liposomes was examined by fluorescence microscopy. The neutral liposomes displayed a typical Kupffer cell distribution pattern, in addition to weak diffuse staining of the parenchyma, while the negatively charged liposomes showed a characteristic sinusoidal lining pattern, consistent with an endothelial localization. In addition, scattered Kupffer cell staining was distinguished as well as diffuse parenchymal fluorescence. The mainly endothelial localisation of the negatively charged liposomes was confirmed by determining radioactivity in endothelial and Kupffer cells isolated following a 1-h perfusion. Perfusion in the presence of polyinosinic acid, an inhibitor of scavenger receptor activity, reduced the rate of uptake of the negatively charged liposomes twofold, indicating the involvement of this receptor in the elimination mechanism. These results are compatible with earlier in vitro studies on liposome uptake by isolated endothelial cells and Kupffer cells, which showed that in the absence of serum also endothelial cells in situ are able to take up massive amounts of negatively charged liposomes. The present results emphasize that the high in vitro endothelial cell uptake in the absence of serum from earlier observations was not an artifact induced by the cell isolation procedure.
Assuntos
Células Endoteliais/metabolismo , Lipossomos/administração & dosagem , Lipossomos/química , Fígado/citologia , Fosfatidilserinas/administração & dosagem , Animais , Transporte Biológico/efeitos dos fármacos , Células Endoteliais/citologia , Células de Kupffer/citologia , Células de Kupffer/metabolismo , Lipossomos/farmacocinética , Microscopia de Fluorescência , Perfusão , Fosfatidilserinas/análise , Poli I/farmacologia , Ratos , SoroRESUMO
Adsorption of serum proteins to the liposomal surface plays a critical role in liposome clearance from the blood. The aim of this study was to investigate the role of liposome-adsorbed serum proteins in the interaction of liposomes with hepatocytes. We analyzed the serum proteins adsorbing to the surface of differently composed small unilamellar liposomes during incubation with human or rat serum, and found that one protein, with a molecular weight of around 55 kDa, adsorbed in a large amount to negatively charged liposomes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). The binding was dependent on the liposomal charge density. The approximately 55-kDa protein was identified as beta2-glycoprotein I (beta2GPI) by Western blotting. Despite the high affinity of beta2GPI for strongly negatively charged liposomes, in vitro uptake and binding experiments with isolated rat hepatocytes, Kupffer cells or liver endothelial cells, and with HepG2 cells showed no enhancing effect of this protein on the association of negatively charged liposomes with any of these cells. On the contrary, an inhibitory effect was observed. We conclude that despite abundant adsorption to negatively charged liposomes, beta2GP1 inhibits, rather than enhances, liposome uptake by liver cells.
Assuntos
Glicoproteínas/metabolismo , Hepatócitos/metabolismo , Lipossomos/metabolismo , Lipossomos/farmacocinética , Adsorção , Animais , Proteínas Sanguíneas/metabolismo , Western Blotting , Linhagem Celular , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Células Endoteliais/metabolismo , Humanos , Células de Kupffer/metabolismo , Lipossomos/química , Fígado/citologia , Masculino , Peso Molecular , Ratos , Ratos Endogâmicos , Propriedades de Superfície , beta 2-Glicoproteína IRESUMO
In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor κB signal transduction could silence the proinflammatory activation status of endothelial cells. For this, an adenovirus encoding dominant-negative IκB (dnIκB) as a therapeutic transgene was employed. Selectivity for the endothelial cells was achieved by introduction of antibodies specific for inflammatory endothelial adhesion molecules E-selectin or VCAM-1 chemically linked to the virus via polyethylene glycol. In vitro, the retargeted adenoviruses selectively infected cytokine-activated endothelial cells to express functional transgene. The comparison of transductional capacity of both retargeted viruses revealed that E-selectin based transgene delivery exerted superior pharmacological effects. Targeted delivery mediated dnIκB transgene expression in endothelial cells inhibited the induced expression of several inflammatory genes, including adhesion molecules, cytokines, and chemokines. In vivo, in mice suffering from glomerulonephritis, E-selectin-retargeted adenovirus selectively homed in the kidney to microvascular glomerular endothelium. Subsequent downregulation of endothelial adhesion molecule expression 2 days after induction of inflammation demonstrated the pharmacological potential of this gene therapy approach. The data justify further studies towards therapeutic virus design and optimization of treatment schedules to investigate their capacity to interfere with inflammatory disease progression.
Assuntos
Adenoviridae/genética , Expressão Gênica , Glomerulonefrite , Proteínas I-kappa B/genética , NF-kappa B/antagonistas & inibidores , Transgenes , Animais , Ligação Competitiva , Técnicas de Cultura de Células , Modelos Animais de Doenças , Selectina E/genética , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Feminino , Glomerulonefrite/genética , Glomerulonefrite/terapia , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Transdução de Sinais/genética , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
We investigated the potential role of scavenger receptor B-I (SR-BI) in the selective removal of liposomal markers from blood by hepatocytes. Liposomes were labeled with [(3)H]cholesteryloleyl-ether ([(3)H]COE), 1,2-di[1-(14)C]palmitoyl-phosphatidylcholine ([(14)C]PC), and N-(lissamine rhodamine-B sulfonyl)-phosphatidylethanolamine (N-Rh-PE). The radiolabels were eliminated at identical rates from plasma, while N-Rh-PE was cleared twice as fast. Involvement of SR-BI in the selective removal of N-Rh-PE from liposomes was studied in transfected Chinese hamster ovary cells over-expressing SR-BI. Uptake of N-Rh-PE from liposomes containing phosphatidylserine was higher than [(3)H]COE, and was further enhanced by apolipoprotein A-I, confirming involvement of SR-BI in the selective uptake of liposomal N-Rh-PE by cells.