RESUMO
BACKGROUND: Proteinuria is associated with many glomerular diseases and a risk factor for the progression to renal failure. We previously showed that heparanase (HPSE) is essential for the development of proteinuria, whereas peroxisome proliferator-activated receptor ɣ (PPARɣ) agonists can ameliorate proteinuria. Since a recent study showed that PPARɣ regulates HPSE expression in liver cancer cells, we hypothesized that PPARɣ agonists exert their reno-protective effect by inhibiting glomerular HPSE expression. METHODS: Regulation of HPSE by PPARɣ was assessed in the adriamycin nephropathy rat model, and cultured glomerular endothelial cells and podocytes. Analyses included immunofluorescence staining, real-time PCR, heparanase activity assay and transendothelial albumin passage assay. Direct binding of PPARɣ to the HPSE promoter was evaluated by the luciferase reporter assay and chromatin immunoprecipitation assay. Furthermore, HPSE activity was assessed in 38 type 2 diabetes mellitus (T2DM) patients before and after 16/24 weeks treatment with the PPARɣ agonist pioglitazone. FINDINGS: Adriamycin-exposed rats developed proteinuria, an increased cortical HPSE and decreased heparan sulfate (HS) expression, which was ameliorated by treatment with pioglitazone. In line, the PPARɣ antagonist GW9662 increased cortical HPSE and decreased HS expression, accompanied with proteinuria in healthy rats, as previously shown. In vitro, GW9662 induced HPSE expression in both endothelial cells and podocytes, and increased transendothelial albumin passage in a HPSE-dependent manner. Pioglitazone normalized HPSE expression in adriamycin-injured human endothelial cells and mouse podocytes, and adriamycin-induced transendothelial albumin passage was reduced as well. Importantly, we demonstrated a regulatory effect of PPARɣ on HPSE promoter activity and direct PPARy binding to the HPSE promoter region. Plasma HPSE activity of T2DM patients treated with pioglitazone for 16/24 weeks was related to their hemoglobin A1c and showed a moderate, near significant correlation with plasma creatinine levels. INTERPRETATION: PPARɣ-mediated regulation of HPSE expression appears an additional mechanism explaining the anti-proteinuric and renoprotective effects of thiazolidinediones in clinical practice. FUNDING: This study was financially supported by the Dutch Kidney Foundation, by grants 15OI36, 13OKS023 and 15OP13. Consortium grant LSHM16058-SGF (GLYCOTREAT; a collaboration project financed by the PPP allowance made available by Top Sector Life Sciences & Health to the Dutch Kidney Foundation to stimulate public-private partnerships).
Assuntos
Diabetes Mellitus Tipo 2 , Nefropatias , Tiazolidinedionas , Ratos , Camundongos , Humanos , Animais , Pioglitazona/farmacologia , Pioglitazona/uso terapêutico , PPAR gama , Diabetes Mellitus Tipo 2/complicações , Agonistas PPAR-gama , Células Endoteliais/metabolismo , Tiazolidinedionas/farmacologia , Tiazolidinedionas/uso terapêutico , Proteinúria/tratamento farmacológico , Proteinúria/etiologia , Nefropatias/tratamento farmacológico , Doxorrubicina/efeitos adversosRESUMO
Microvascular endothelial cells in the kidney have been a neglected cell type in sepsis-induced acute kidney injury (sepsis-AKI) research; yet, they offer tremendous potential as pharmacological targets. As endothelial cells in distinct cortical microvascular segments are highly heterogeneous, this Review focuses on endothelial cells in their anatomical niche. In animal models of sepsis-AKI, reduced glomerular blood flow has been attributed to inhibition of endothelial nitric oxide synthase activation in arterioles and glomeruli, whereas decreased cortex peritubular capillary perfusion is associated with epithelial redox stress. Elevated systemic levels of vascular endothelial growth factor, reduced levels of circulating sphingosine 1-phosphate and loss of components of the glycocalyx from glomerular endothelial cells lead to increased microvascular permeability. Although coagulation disbalance occurs in all microvascular segments, the molecules involved differ between segments. Induction of the expression of adhesion molecules and leukocyte recruitment also occurs in a heterogeneous manner. Evidence of similar endothelial cell responses has been found in kidney and blood samples from patients with sepsis. Comprehensive studies are needed to investigate the relationships between segment-specific changes in the microvasculature and kidney function loss in sepsis-AKI. The application of omics technologies to kidney tissues from animals and patients will be key in identifying these relationships and in developing novel therapeutics for sepsis.
Assuntos
Injúria Renal Aguda , Sepse , Injúria Renal Aguda/metabolismo , Animais , Modelos Animais de Doenças , Células Endoteliais , Humanos , Rim/metabolismo , Sepse/complicações , Fator A de Crescimento do Endotélio VascularRESUMO
Macrophages are key players in the pathogenesis of large-vessel vasculitis (LVV) and may serve as a target for diagnostic imaging of LVV. The radiotracer, 18F-FDG has proven to be useful in the diagnosis of giant cell arteritis (GCA), a form of LVV. Although uptake of 18F-FDG is high in activated macrophages, it is not a specific radiotracer as its uptake is high in any proliferating cell and other activated immune cells resulting in high non-specific background radioactivity especially in aging and atherosclerotic vessels which dramatically lowers the diagnostic accuracy. Evidence also exists that the sensitivity of 18F-FDG PET drops in patients upon glucocorticoid treatment. Therefore, there is a clinical need for more specific radiotracers in imaging GCA to improve diagnostic accuracy. Numerous clinically established and newly developed macrophage targeted radiotracers for oncological and inflammatory diseases can potentially be utilized for LVV imaging. These tracers are more target specific and therefore may provide lower background radioactivity, higher diagnostic accuracy and the ability to assess treatment effectiveness. However, current knowledge regarding macrophage subsets in LVV lesions is limited. Further understanding regarding macrophage subsets in vasculitis lesion is needed for better selection of tracers and new targets for tracer development. This review summarizes the development of macrophage targeted tracers in the last decade and the potential application of macrophage targeted tracers currently used in other inflammatory diseases in imaging LVV.
Assuntos
Arterite de Células Gigantes/diagnóstico , Arterite de Células Gigantes/patologia , Macrófagos/patologia , Humanos , Imagem Molecular , Tomografia por Emissão de Pósitrons , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
Dactolisib (NVP-BEZ235, also referred to as: 'BEZ235' or 'BEZ') is a dual mTOR/PI3K inhibitor that is of potential interest in the treatment of inflammatory disorders. This work focuses on formulation of BEZ-loaded polymeric nanoparticles composed of a blend of poly(D,L-lactide-co-glycolide) (PLGA) and poly(D,L-lactide-co-glycolide)-poly(ethylene glycol)-2000 (PLGA-PEG). The nanoparticles were prepared by an oil/water emulsion solvent evaporation method, and were subsequently characterized for yield, encapsulation efficiency, morphology, particle size, drug-polymer interaction and in vitro drug release profiles. A targeted formulation was developed by conjugation of a S-acetyl-thioacetyl (SATA)-modified mouse-anti human E-selectin antibody to the distal end of PLGA-PEG-SPDP containing nanoparticles. Our results show the successful preparation of spherical PLGA/PLGA-PEG nanoparticles loaded with BEZ. The particle size distribution showed a range from 250 to 360nm with a high (>75%) BEZ encapsulation efficiency. Approximately 35% of the loaded BEZ was released within 10days at 37°C in a medium containing 5% bovine serum albumin (BSA). Evaluation of efficacy of anti-E-selectin decorated BEZ-loaded nanoparticles was carried out in tumor necrosis factor-α (TNF-α) activated endothelial cells. Confocal microscopy analysis showed that cellular uptake of the targeted nanoparticles and subsequent internalization. Cell functional assays, including migration assay and phosphowestern blot analysis of the mTOR and pI3K signaling pathways, revealed that the E-selectin targeted nanoparticles loaded with BEZ had a pronounced effect on inflammation-activated endothelial cells as compared to the non-targeted BEZ-loaded nanoparticles. In conclusion, E-selectin targeted nanoparticles have a high potential in delivering the potent mTOR/pI3K inhibitor dactolisib to inflamed endothelial cells and are an interesting nanomedicine for anti-inflammatory therapy.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Endotélio Vascular/efeitos dos fármacos , Imidazóis/administração & dosagem , Nanopartículas/administração & dosagem , Inibidores de Fosfoinositídeo-3 Quinase , Polietilenoglicóis/administração & dosagem , Poliglactina 910/administração & dosagem , Quinolinas/administração & dosagem , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Imidazóis/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Nanopartículas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Polietilenoglicóis/metabolismo , Poliglactina 910/metabolismo , Quinolinas/metabolismo , Difração de Raios X/métodosRESUMO
Activated endothelial cells play a pivotal role in the pathology of inflammatory disorders and thus present a target for therapeutic intervention by drugs that intervene in inflammatory signaling cascades, such as rapamycin (mammalian target of rapamycin (mTOR) inhibitor). In this study we developed anti-E-selectin immunoliposomes for targeted delivery to E-selectin over-expressing tumor necrosis factor-α (TNF-α) activated endothelial cells. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3.;hosphocholine (DPPC), Cholesterol, and 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]-maleimide (DSPE-PEG-Mal) were loaded with rapamycin via lipid film hydration, after which they were further functionalized by coupling N-succinimidyl-S-acetylthioacetate (SATA)-modified mouse anti human E-selectin antibodies to the distal ends of the maleimidyl (Mal)-PEG groups. In cell binding assays, these immunoliposomes bound specifically to TNF-α activated endothelial cells. Upon internalization, rapamycin loaded immunoliposomes inhibited proliferation and migration of endothelial cells, as well as expression of inflammatory mediators. Our findings demonstrate that rapamycin-loaded immunoliposomes can specifically inhibit inflammatory responses in inflamed endothelial cells.
Assuntos
Antibacterianos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Selectina E/administração & dosagem , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Sirolimo/administração & dosagem , Animais , Antibacterianos/imunologia , Antibacterianos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Selectina E/imunologia , Selectina E/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lipossomos , Camundongos , Sirolimo/imunologia , Sirolimo/metabolismoRESUMO
The liver as transplantation site for pancreatic islets is associated with significant loss of islets, which can be prevented by grafting in a prevascularized, subcutaneous scaffold. Supporting vascularization of a scaffold to limit the period of ischemia is challenging and was developed here by applying liposomes for controlled release of angiogenic factors. The angiogenic capacity of platelet-derived growth factor, vascular endothelial growth factor, acidic fibroblast growth factor (aFGF), and basic FGF were compared in a tube formation assay. Furthermore, the release kinetics of different liposome compositions were tested. aFGF and L-α-phosphatidylcholine/cholesterol liposomes were selected to support vascularization. Two dosages of aFGF-liposomes (0.5 and 1.0 µg aFGF per injection) were administered weekly for a month after which islets were transplanted. We observed enhanced efficacy in the immediate post-transplant period compared to the untreated scaffolds. However, on the long-term, glucose levels of the aFGF treated animals started to increase to diabetic levels. These results suggest that injections with aFGF liposomes do improve vascularization and the immediate restoration of blood glucose levels but does not facilitate the long-term survival of islets. Our data emphasize the need for long-term studies to evaluate potential beneficial and adverse effects of vascularization protocols of scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2533-2542, 2017.
Assuntos
Glicemia/metabolismo , Transplante das Ilhotas Pancreáticas , Neovascularização Fisiológica , Tela Subcutânea/irrigação sanguínea , Alicerces Teciduais/química , Animais , Preparações de Ação Retardada , Teste de Tolerância a Glucose , Células Endoteliais da Veia Umbilical Humana , Humanos , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Ilhotas Pancreáticas/patologia , Lipossomos , Masculino , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Together with mesangial cells, glomerular endothelial cells and the basement membrane, podocytes constitute the glomerular filtration barrier (GFB) of the kidney. Podocytes play a pivotal role in the progression of various kidney-related diseases such as glomerular sclerosis and glomerulonephritis that finally lead to chronic end-stage renal disease. During podocytopathies, the slit-diaphragm connecting the adjacent podocytes are detached leading to severe loss of proteins in the urine. The pathophysiology of podocytopathies makes podocytes a potential and challenging target for nanomedicine development, though there is a lack of known molecular targets for cell selective drug delivery. To identify VCAM-1 as a cell-surface receptor that is suitable for binding and internalization of nanomedicine carrier systems by podocytes, we investigated its expression in the immortalized podocyte cell lines AB8/13 and MPC-5, and in primary podocytes. Gene and protein expression analyses revealed that VCAM-1 expression is increased by podocytes upon TNFα-activation for up to 24 h. This was paralleled by anti-VCAM-1 antibody binding to the TNFα-activated cells, which can be employed as a ligand to facilitate the uptake of nanocarriers under inflammatory conditions. Hence, we next explored the possibilities of using VCAM-1 as a cell-surface receptor to deliver the potent immunosuppressant rapamycin to TNFα-activated podocytes using the lipid-based nanocarrier system Saint-O-Somes. Anti-VCAM-1-rapamycin-SAINT-O-Somes more effectively inhibited the cell migration of AB8/13 cells than free rapamycin and non-targeted rapamycin-SAINT-O-Somes indicating the potential of VCAM-1 targeted drug delivery to podocytes.
Assuntos
Lipídeos , Nanoconjugados , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Sirolimo/administração & dosagem , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Anticorpos Monoclonais , Diferenciação Celular , Linhagem Celular , Expressão Gênica , Humanos , Imunossupressores/administração & dosagem , Inflamação/metabolismo , Masculino , Camundongos , Podócitos/citologia , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/genéticaAssuntos
Neoplasias Colorretais/irrigação sanguínea , Neoplasias Hepáticas/irrigação sanguínea , Neoplasias Hepáticas/secundário , Neoplasias Primárias Múltiplas/irrigação sanguínea , Neoplasias Primárias Múltiplas/secundário , Segunda Neoplasia Primária/irrigação sanguínea , Segunda Neoplasia Primária/secundário , Neovascularização Patológica/patologia , Feminino , Humanos , MasculinoRESUMO
Circulatory shock and resuscitation are associated with systemic hemodynamic changes, which may contribute to the development of MODS (multiple organ dysfunction syndrome). In this study, we used an in vitro flow system to simulate the consecutive changes in blood flow as occurring during hemorrhagic shock and resuscitation in vivo. We examined the kinetic responses of different endothelial genes in human umbilical vein endothelial cells preconditioned to 20 dyne/cm unidirectional laminar shear stress for 48 h to flow cessation and abrupt reflow, respectively, as well as the effect of flow cessation and reflow on tumor necrosis factor-α (TNF-α)-induced endothelial proinflammatory activation. Endothelial CD31 and VE-cadherin were not affected by the changes in flow in the absence or presence of TNF-α. The messenger RNA levels of proinflammatory molecules E-selectin, VCAM-1 (vascular cell adhesion molecule 1), and IL-8 (interleukin 8) were significantly induced by flow cessation respectively acute reflow, whereas ICAM-1 (intercellular adhesion molecule 1) was downregulated on flow cessation and induced by subsequent acute reflow. Flow cessation also affected the Ang/Tie2 (Angiopoietin/Tie2 receptor tyrosine kinase) system by downregulating Tie2 and inducing its endothelial ligand Ang2, an effect that was further extended on acute reflow. Furthermore, the induction of proinflammatory adhesion molecules by TNF-α under flow cessation was significantly enhanced on subsequent acute reflow. This study demonstrated that flow alterations per se during shock and resuscitation contribute to endothelial activation and that these alterations interact with proinflammatory factors coexisting in vivo such as TNF-α. The abrupt reflow-related enhancement of cytokine-induced endothelial proinflammatory activation supports the concept that sudden regain of flow during resuscitation has an aggravating effect on endothelial activation, which may play a significant role in vascular dysfunction and consequent organ injury. This study implies that the improvement of resuscitation strategies and the pharmacological interference with proinflammatory signaling cascades at the right time of resuscitation of shock patients may be beneficial to regain and/or maintain organ function in patients after circulatory shock.
Assuntos
Citocinas/fisiologia , Células Endoteliais/imunologia , Inflamação/imunologia , Choque/imunologia , Choque/fisiopatologia , Células Cultivadas , Humanos , Fluxo Sanguíneo Regional , Ressuscitação , Choque/terapiaRESUMO
AIMS: Pulmonary arterial hypertension (PAH) is characterized by the development of unique neointimal lesions in the small pulmonary arteries, leading to increased right ventricular (RV) afterload and failure. Novel therapeutic strategies are needed that target these neointimal lesions. Recently, the transcription factor Egr-1 (early growth response protein 1) was demonstrated to be up-regulated early in experimental neointimal PAH. Its effect on disease development, however, is unknown. We aimed to uncover a novel role for Egr-1 as a molecular inductor for disease development in PAH. METHODS AND RESULTS: In experimental flow-associated PAH in rats, we investigated the effects of Egr-1 down-regulation on pulmonary vascular remodelling, including neointimal development, and disease progression. Intravenous administration of catalytic oligodeoxynucleotides (DNA enzymes, DNAzymes) resulted in down-regulation of pulmonary vascular Egr-1 expression. Compared with vehicle or scrambled DNAzymes, DNAzymes attenuated pulmonary vascular remodelling, including the development of occlusive neointimal lesions. Selective down-regulation of Egr-1 in vivo led to reduced expression of vascular PDGF-B, TGF-ß, IL-6, and p53, resulting in a reduction of vascular proliferation and increased apoptosis. DNAzyme treatment further attenuated pulmonary vascular resistance, RV systolic pressure, and RV hypertrophy. In contrast, in non-neointimal PH rodents, DNAzyme treatment had no effect on pulmonary vascular and RV remodelling. Finally, pharmacological inhibition of Egr-1 with pioglitazone, a peroxisome proliferator activated receptor-γ ligand, attenuated vascular remodelling including the development of neointimal lesions. CONCLUSIONS: These results indicate that Egr-1 governs pulmonary vascular remodelling and the development of characteristic vascular neointimal lesions in flow-associated PAH. Egr-1 is therefore a potential target for future PAH treatment.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Músculo Liso Vascular/metabolismo , Artéria Pulmonar/metabolismo , Remodelação Vascular , Animais , Regulação para Baixo , Proteína 1 de Resposta de Crescimento Precoce/genética , Hemodinâmica/efeitos dos fármacos , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/patologia , Interleucina-6/metabolismo , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/patologia , Ratos Wistar , Fator de Crescimento Transformador beta/metabolismoRESUMO
In recent years much research in RNA nanotechnology has been directed to develop an efficient and clinically suitable delivery system for short interfering RNA (siRNA). The current study describes the in vivo siRNA delivery using PEGylated antibody-targeted SAINT-based-lipoplexes (referred to as antibody-SAINTPEGarg/PEG2%), which showed superior siRNA delivery capacity and effective down-regulation of VE-cadherin gene expression in vitro in inflammation-activated primary endothelial cells of different vascular origins. PEGylation of antibody-SAINTPEGarg resulted in more desirable pharmacokinetic behavior than that of non-PEGylated antibody-SAINTPEGarg. To create specificity for inflammation-activated endothelial cells, antibodies against vascular cell adhesion molecule-1 (VCAM-1) were employed. In TNFα-challenged mice, these intravenously administered anti-VCAM-1-SAINTPEGarg/PEG2% homed to VCAM-1 protein expressing vasculature. Confocal laser scanning microscopy revealed that anti-VCAM-1-SAINTPEGarg/PEG2% co-localized with endothelial cells in lung postcapillary venules. Furthermore, they did not exert any liver and kidney toxicity. Yet, lack of in vivo gene silencing as assessed in whole lung and in laser microdissected lung microvascular segments indicates that in vivo internalization and/or intracellular trafficking of the delivery system and its cargo in the target cells are not sufficient, and needs further attention, emphasizing the essence of evaluating siRNA delivery systems in an appropriate in vivo animal model at an early stage in their development.
Assuntos
Anticorpos/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Endotélio Vascular/metabolismo , Inflamação/metabolismo , Lipídeos/química , Pulmão/irrigação sanguínea , Polietilenoglicóis/química , Compostos de Piridínio/química , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transfecção/métodos , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Anticorpos/química , Antígenos CD/genética , Caderinas/genética , Modelos Animais de Doenças , Endotélio Vascular/imunologia , Regulação da Expressão Gênica , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/prevenção & controle , Masculino , Camundongos Endogâmicos C57BL , Microscopia Confocal , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Fatores de Tempo , Distribuição Tecidual , Fator de Necrose Tumoral alfa , Molécula 1 de Adesão de Célula Vascular/imunologia , Vênulas/metabolismoRESUMO
The pivotal role of endothelial cells in the pathology of inflammatory diseases raised interest in the development of short interfering RNA (siRNA) delivery devices for selective pharmacological intervention in the inflamed endothelium. The current study demonstrates endothelial specific delivery of siRNAs and downregulation of inflammatory genes in activated endothelium in vivo by applying a novel type of targeted liposomes based on the cationic amphiphile SAINT-C18 (1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride). To create specificity for inflamed endothelial cells, these so-called SAINT-O-Somes were harnessed with antibodies against vascular cell adhesion protein 1 (VCAM-1). In TNFα challenged mice, intravenously administered anti-VCAM-1 SAINT-O-Somes exerted long circulation times and homed to VCAM-1 expressing endothelial cells in inflamed organs. The formulations were devoid of liver and kidney toxicity. Using anti-VCAM-1 SAINT-O-Somes we successfully delivered siRNA to knock down VE-cadherin mRNA in inflamed renal microvasculature, as demonstrated by using laser microdissection of different microvascular beds prior to analysis of gene expression. Using the same strategy, we demonstrated local attenuation of endothelial inflammatory response towards lipopolysaccharide in kidneys of mice treated with anti-VCAM-1 SAINT-O-Somes containing NFκB p65 specific siRNA. This study is the first demonstration of a novel, endothelial specific carrier that is suitable for selective in vivo delivery of siRNAs into inflamed microvascular segments and interference with disease associated endothelial activation.
Assuntos
Anticorpos/administração & dosagem , Antígenos CD/genética , Caderinas/genética , Compostos de Piridínio/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Fator de Transcrição RelA/genética , Molécula 1 de Adesão de Célula Vascular/imunologia , Animais , Encéfalo/metabolismo , Células Cultivadas , Regulação para Baixo , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Lipossomos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Pulmão/metabolismo , Masculino , Camundongos , Miocárdio/metabolismo , Compostos de Piridínio/farmacocinética , RNA Interferente Pequeno/farmacocinética , Distribuição Tecidual , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
The endothelium represents an attractive therapeutic target due to its pivotal role in many diseases including chronic inflammation and cancer. Small interfering RNAs (siRNAs) specifically interfere with the expression of target genes and are considered an important new class of therapeutics. However, due to their size and charge, siRNAs do not spontaneously enter unperturbed endothelial cells (EC). To overcome this problem, we developed novel lipoplexes for siRNA delivery that are based on the cationic amphiphilic lipid SAINT-C18. Antibodies recognizing disease induced cell adhesion molecules were employed to create cell specificity resulting in so-called antibody-SAINTargs. To improve particle stability, antibody-SAINTargs were further optimized for EC-specific siRNA-mediated gene silencing by addition of polyethylene glycol (PEG). Although PEGylated antibody-SAINTargs maintained specificity, they lost their siRNA delivery capacity. Coupling of antibodies to the distal end of PEG (so-called antibody-SAINTPEGargs), resulted in anti-E-selectin- and anti-vascular cell adhesion molecule (VCAM)-1-SAINTPEGarg that preserved their antigen recognition and their capability to specifically deliver siRNA into inflammation-activated primary endothelial cells. The enhanced uptake of siRNA by antibody-SAINTPEGargs was followed by improved silencing of the target gene VE-cadherin, demonstrating that antibody-SAINTPEGargs were capable of functionally delivering siRNA into primary endothelial cells originating from different vascular beds. In conclusion, the newly developed, physicochemically stable, and EC-specific siRNA carrying antibody-SAINTPEGargs selectively down-regulate target genes in primary endothelial cells that are generally difficult to transfect.
Assuntos
Selectina E/química , Células Endoteliais/patologia , Endotélio Vascular/patologia , Inflamação/patologia , Lipídeos/química , Polietilenoglicóis/química , Compostos de Piridínio/química , RNA Interferente Pequeno/administração & dosagem , Molécula 1 de Adesão de Célula Vascular/química , Capilares/citologia , Células Cultivadas , Sistemas de Liberação de Medicamentos , Eletroquímica , Citometria de Fluxo , Expressão Gênica , Técnicas de Transferência de Genes , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Tamanho da Partícula , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
Activated endothelial cells play a pivotal role in the pathology of inflammatory diseases and present a rational target for therapeutic intervention by endothelial specific delivery of short interfering RNAs (siRNA). This study demonstrates the potential of the recently developed new generation of liposomes based on cationic amphiphile SAINT-C18 (1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride) for functional and selective delivery of siRNA into inflamed primary endothelial cells. To create specificity for inflamed endothelial cells, these so-called SAINT-O-Somes were harnessed with antibodies against vascular cell adhesion protein 1 (VCAM-1) or respectively E-selectin and tested in TNF-α activated primary endothelial cells from venous and aortic vascular beds. Both targeted SAINT-O-Somes carrying siRNA against the endothelial gene VE-cadherin specifically downregulated its target mRNA and protein without exerting cellular toxicity. SAINT-O-Somes formulated with siRNA formed small particles (106 nm) with a 71% siRNA encapsulation efficiency. SAINT-O-Somes were stable in the presence of serum at 37 °C, protected siRNA from degradation by serum RNases, and after i.v. injection displayed pharmacokinetic comparable to conventional long circulating liposomes. These anti-VCAM-1 and anti-E-selectin SAINT-O-Somes are thus a novel drug delivery system that can achieve specific and effective delivery of siRNA into inflamed primary endothelial cells and have physicochemical features that comply with in vivo application demands.
Assuntos
Selectina E/imunologia , Inflamação/metabolismo , RNA Interferente Pequeno/administração & dosagem , Molécula 1 de Adesão de Célula Vascular/imunologia , Animais , Anticorpos , Células Endoteliais , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipossomos/química , Masculino , Camundongos , Tamanho da Partícula , RNA Interferente Pequeno/química , Fatores de Necrose Tumoral/metabolismoRESUMO
OBJECTIVE: Resection of a primary colorectal carcinoma (CRC) can be accompanied by rapid outgrowth of liver metastases, suggesting a role for angiogenesis. The aim of this study is to investigate whether the presence of a primary CRC is associated with changes in angiogenic status and proliferation/apoptotic rate in synchronous liver metastases and/or adjacent liver parenchyma. METHODS: Gene expression and localization of CD31, HIF-1α, members of the vascular endothelial growth factor (VEGF) and Angiopoietin (Ang) system were studied using qRT-PCR and immunohistochemistry in colorectal liver metastases and nontumorous-adjacent liver parenchyma. Proliferation and apoptotic rate were quantified. Three groups of patients were included: (1) simultaneous resection of synchronous liver metastases and primary tumor (SS-group), (2) resection of synchronous liver metastases 3 to 12 months after resection of the primary tumor [late synchronous (LS-group)], and (3) resection of metachronous metastases >14 months after resection of the primary tumor (M-group). RESULTS: In all 3 groups a higher expression of the angiogenic factors was encountered in adjacent liver parenchyma as compared to the metastases. VEGFR-2 gene expression was abundant in adjacent liver parenchyma in all 3 groups. VEGF-A and VEGFR-1 were prominent in adjacent parenchyma in the SS-group. The SS-group showed the highest Ang-2/Ang-1 ratio both in the metastases and the adjacent liver. This was accompanied by a high turnover of tumor cells. CONCLUSION: In the presence of the primary tumor, the liver parenchyma adjacent to the synchronous liver metastases provides an angiogenic prosperous environment for metastatic tumor growth.
Assuntos
Neoplasias Colorretais/irrigação sanguínea , Neoplasias Hepáticas/irrigação sanguínea , Neoplasias Hepáticas/secundário , Neoplasias Primárias Múltiplas/irrigação sanguínea , Neoplasias Primárias Múltiplas/secundário , Segunda Neoplasia Primária/irrigação sanguínea , Segunda Neoplasia Primária/secundário , Neovascularização Patológica/patologia , Adulto , Idoso , Apoptose/genética , Proliferação de Células , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Fígado/irrigação sanguínea , Fígado/patologia , Neoplasias Hepáticas/cirurgia , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Neoplasias Primárias Múltiplas/genética , Neoplasias Primárias Múltiplas/patologia , Neoplasias Primárias Múltiplas/cirurgia , Segunda Neoplasia Primária/genética , Segunda Neoplasia Primária/patologia , Segunda Neoplasia Primária/cirurgia , Neovascularização Patológica/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Ribonuclease Pancreático/genética , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Increased insight in the role of endothelial cells in the pathophysiology of cancer, inflammatory and cardiovascular diseases, has drawn great interest in pharmacological interventions aiming at the endothelium in diseased sites. Their location in the body makes them suitable targets for therapeutic approaches based on targeted drug delivery. Functional heterogeneity of the microvascular bed in normal organ homeostasis has been appreciated for a long time, and more recent studies have revealed heterogeneity in endothelial reactivity to inflammatory stimuli as well. Upon stimulation, each organ displays a vascular bed specific pattern of cell adhesion molecules providing challenging opportunities to deliver drugs or small RNAs to organ specific (micro)vascular endothelial subsets. In this review we introduce general concepts of endothelial heterogeneity in relation to disease state and its consequences for targeted therapeutic interventions. Furthermore, we will describe novel approaches to interfere with endothelial cell engagement in disease with a main focus on siRNA therapeutics and currently used nonviral lipid and polymer-based siRNA delivery systems. The last part of this review addresses some technical issues that are essential in proving the concept of target mRNA knock down in a vascular bed specific manner, and the further development of effective endothelial cell specific drug delivery devices.
Assuntos
Endotélio Vascular/metabolismo , Microvasos/metabolismo , RNA Interferente Pequeno/administração & dosagem , Endotélio Vascular/citologia , Humanos , Microvasos/citologiaRESUMO
In non-phagocytic cells such as endothelial cells, processing of liposomes and subsequent release of drug content is often inefficient due to the absence of professional processing machinery, which limits pharmacological efficacy. We therefore developed a liposome based drug delivery system with superior intracellular release characteristics. The design was based on long circulating conventional liposomes that were formulated with a cationic amphiphile, 1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chlorid (SAINT-C18). These so-called SAINT-O-Somes had a diameter of 100 nm, were as stable as conventionally formulated liposomes, and showed superior release of their content at pH conditions that liposomes encounter when they are endocytosed by cells. Attachment of anti-E-selectin specific antibodies to the distal end of surface grafted poly(ethylene glycol) resulted in immuno-SAINT-O-Somes that were as efficiently taken up by inflammation activated endothelial cells as conventional anti-E-selectin specific immunoliposomes. More importantly, intracellular release of calcein encapsulated in these targeted SAINT-O-Somes was 10 fold higher as compared to the release of calcein from conventional liposomes. For intracellular delivery siRNA into activated endothelial cells, formulation with SAINT-C18 was a necessity to induce a specific down-regulation of gene expression of VE-cadherin. Additionally, targeted doxorubicin loaded SAINT-O-Somes decreased endothelial cell viability significantly more than targeted conventional doxorubicin liposomes. SAINT-O-Somes therefore represent a new class of lipid based particles with superior drug release characteristics that can be applied for the efficacious intracellular delivery of hydrophilic drugs including siRNA.
Assuntos
Antineoplásicos/administração & dosagem , Portadores de Fármacos/química , Células Endoteliais/efeitos dos fármacos , Compostos de Piridínio/química , RNA Interferente Pequeno/administração & dosagem , Tensoativos/química , Animais , Antineoplásicos/farmacocinética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Microscopia Crioeletrônica , Composição de Medicamentos , Selectina E/genética , Células Endoteliais/metabolismo , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Humanos , Lipídeos/química , Lipossomos , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , RNA Interferente Pequeno/farmacocinéticaRESUMO
Adenovirus is among the preferred vectors for gene therapy because of its superior in vivo gene-transfer efficiency. However, upon systemic administration, adenovirus is preferentially sequestered by the liver, resulting in reduced adenovirus-mediated transgene expression in targeted tissues. In the liver, Kupffer cells are responsible for adenovirus degradation and contribute to the inflammatory response. As scavenger receptors present on Kupffer cells are responsible for the elimination of blood-borne pathogens, we investigated the possible implication of these receptors in the clearance of the adenovirus vector. Polyinosinic acid [poly(I)], a scavenger receptor A ligand, was analysed for its capability to inhibit adenovirus uptake specifically in macrophages. In in vitro studies, the addition of poly(I) before virus infection resulted in a specific inhibition of adenovirus-induced gene expression in a J774 macrophage cell line and in primary Kupffer cells. In in vivo experiments, pre-administration of poly(I) caused a 10-fold transient increase in the number of adenovirus particles circulating in the blood. As a consequence, transgene expression levels measured in different tissues were enhanced (by 5- to 15-fold) compared with those in animals that did not receive poly(I). Finally, necrosis of Kupffer cells, which normally occurs as a consequence of systemic adenovirus administration, was prevented by the use of poly(I). No toxicity, as measured by liver-enzyme levels, was observed after poly(I) treatment. From our data, we conclude that poly(I) can prevent adenovirus sequestration by liver macrophages. These results imply that, by inhibiting adenovirus uptake by Kupffer cells, it is possible to reduce the dose of the viral vector to diminish the liver-toxicity effect and to improve the level of transgene expression in target tissues. In systemic gene-therapy applications, this will have great impact on the development of targeted adenoviral vectors.
Assuntos
Adenoviridae/imunologia , Terapia Genética/métodos , Fatores Imunológicos/farmacologia , Fígado/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Poli I/farmacologia , Receptores Depuradores/antagonistas & inibidores , Animais , Linhagem Celular , Células Cultivadas , Expressão Gênica , Testes de Função Hepática , Masculino , Camundongos , Ratos , Ensaio de Placa Viral , ViremiaRESUMO
Hepatic stellate cells (HSC) are a major target for antifibrotic therapies in the liver and in particular gene delivery to these cells would be relevant. Previously, we demonstrated that mannose 6-phosphate human serum albumin (M6P-HSA) coupled liposomes accumulate in HSC in fibrotic livers. Here we prepared a M6P-HSA modified viral vector that allows the targeted delivery of plasmid DNA to HSC. Therefore, UV inactivated hemagglutinating virus of Japan (HVJ) containing plasmid DNA was fused with M6P-HSA liposomes to yield HVJ liposomes targeted to HSC. These new particles had a diameter of approximately 200 nm, as determined by electron microscopy. In a carbon tetrachloride mouse model of liver fibrosis, M6P-HSA-HVJ-liposomes associated with HSC. In conclusion, our results demonstrate that fusion of M6P-HSA liposomes with HVJ envelopes results in HVJ particles that accumulate in HSC, allowing for new possibilities to interfere with fibrosis in the liver.
Assuntos
Vetores Genéticos/administração & dosagem , Cirrose Hepática/metabolismo , Fígado/metabolismo , Vírus Sendai/metabolismo , Proteínas do Envelope Viral/química , Animais , Linhagem Celular Tumoral , Embrião de Galinha , DNA/administração & dosagem , DNA/química , Composição de Medicamentos , Sistemas de Liberação de Medicamentos , Eletroforese em Gel de Poliacrilamida , Humanos , Imuno-Histoquímica , Lipossomos , Fígado/citologia , Cirrose Hepática/patologia , Luciferases/genética , Manosefosfatos/química , Camundongos , Camundongos Endogâmicos C57BL , Tamanho da Partícula , Plasmídeos/genética , Albumina Sérica/químicaRESUMO
In the fibrotic liver, hepatic stellate cells (HSC) produce large amounts of collagen and secrete variety of mediators that promote development of fibrosis in this organ. Therefore, these cells are considered an attractive target for antifibrotic therapies. We incorporated the bioactive lipid dilinoleoylphosphatidylcholine (DLPC) into the membrane of liposomes, and then we evaluated its effect on hepatic stellate cell activation and liver fibrosis. To target DLPC-liposomes to HSC, human serum albumin modified with mannose 6-phosphate (M6P-HSA) was coupled to the surface of these liposomes. In vitro, the effects of the carrier were determined in primary cultures of HSC, Kupffer cells, and liver endothelial cells using real-time reverse transcription-polymerase chain reaction. In vivo DLPC-liposomes were tested in bile duct-ligated rats. Targeted M6P-HSA-DLPC-liposomes and DLPC-liposomes significantly reduced gene expression levels for collagen 1alpha1, alpha-smooth muscle actin (alpha-SMA), and transforming growth factor-beta (TGF-beta) in cultured HSC. In fibrotic livers, DLPC-liposomes decreased gene expression for TGF-beta and collagen 1alpha1 as well as alpha-SMA and collagen protein expression. In contrast, M6P-HSA-DLPC-liposomes enhanced expression of profibrotic and proinflammatory genes in vivo. In cultured Kupffer and endothelial cells M6P-HSA liposomes influenced the expression of proinflammatory genes. Both types of liposomes increased hepatocyte glycogen content in fibrotic livers, indicating improved functionality of the hepatocytes. We conclude that DLPC-containing liposomes attenuate activation of cultured HSC. In fibrotic livers, M6P-HSA-mediated activation of Kupffer and endothelial cells probably counteracts this beneficial effect of DLPC-liposomes. Therefore, these bioactive drug carriers modulate the activity of all liver cells during liver fibrosis.