RESUMO
Targeted drug delivery to the endothelium has the potential to generate localized therapeutic effects at the blood-tissue interface. For some therapeutic cargoes, it is essential to maintain contact with the bloodstream to exert protective effects. The pharmacokinetics (PK) of endothelial surface-targeted affinity ligands and biotherapeutic cargo remain a largely unexplored area, despite obvious translational implications for this strategy. To bridge this gap, we site-specifically radiolabeled mono- (scFv) and bivalent (mAb) affinity ligands specific for the endothelial cell adhesion molecules, PECAM-1 (CD31) and ICAM-1 (CD54). Radiotracing revealed similar lung biodistribution at 30 minutes post-injection (79.3% ± 4.2% vs 80.4% ± 10.6% ID/g for αICAM and 58.9% ± 3.6% ID/g vs. 47.7% ± 5.8% ID/g for αPECAM mAb vs. scFv), but marked differences in organ residence time, with antibodies demonstrating an order of magnitude greater area under the lung concentration vs. time curve (AUCinf 1698 ± 352 vs. 53.3 ± 7.9 ID/g*hrs for αICAM and 1023 ± 507 vs. 114 ± 37 ID/g*hrs for αPECAM mAb vs scFv). A physiologically based pharmacokinetic model, fit to and validated using these data, indicated contributions from both superior binding characteristics and prolonged circulation time supporting multiple binding-detachment cycles. We tested the ability of each affinity ligand to deliver a prototypical surface cargo, thrombomodulin (TM), using one-to-one protein conjugates. Bivalent mAb-TM was superior to monovalent scFv-TM in both pulmonary targeting and lung residence time (AUCinf 141 ± 3.2 vs 12.4 ± 4.2 ID/g*hrs for ICAM and 188 ± 90 vs 34.7 ± 19.9 ID/g*hrs for PECAM), despite having similar blood PK, indicating that binding strength is more important parameter than the kinetics of binding. To maximize bivalent target engagement, we synthesized an oriented, end-to-end anti-ICAM mAb-TM conjugate and found that this therapeutic had the best lung residence time (AUCinf 253 ± 18 ID/g*hrs) of all TM modalities. These observations have implications not only for the delivery of TM, but also potentially all therapeutics targeted to the endothelial surface.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Endotélio Vascular/metabolismo , Molécula 1 de Adesão Intercelular/imunologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Anticorpos de Cadeia Única/administração & dosagem , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Células Endoteliais/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Ligantes , Pulmão/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacocinética , Distribuição TecidualRESUMO
Endothelial cells actively maintain an anti-thrombotic environment; loss of this protective function may lead to thrombosis and systemic coagulopathy. The transcription factor ERG is essential to maintain endothelial homeostasis. Here, we show that inducible endothelial ERG deletion (ErgiEC-KO) in mice is associated with spontaneous thrombosis, hemorrhages and systemic coagulopathy. We find that ERG drives transcription of the anticoagulant thrombomodulin (TM), as shown by reporter assays and chromatin immunoprecipitation. TM expression is regulated by shear stress (SS) via Krüppel-like factor 2 (KLF2). In vitro, ERG regulates TM expression under low SS conditions, by facilitating KLF2 binding to the TM promoter. However, ERG is dispensable for TM expression in high SS conditions. In ErgiEC-KO mice, TM expression is decreased in liver and lung microvasculature exposed to low SS but not in blood vessels exposed to high SS. Our study identifies an endogenous, vascular bed-specific anticoagulant pathway in microvasculature exposed to low SS.
Assuntos
Regulação da Expressão Gênica , Microvasos/metabolismo , Trombomodulina/metabolismo , Trombose/metabolismo , Regulador Transcricional ERG/metabolismo , Animais , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like , Camundongos Knockout , Microvasos/citologia , Regiões Promotoras Genéticas/genética , Transdução de Sinais/genética , Estresse Mecânico , Trombomodulina/genética , Trombose/genética , Regulador Transcricional ERG/genéticaRESUMO
Targeting drugs to endothelial cells has shown the ability to improve outcomes in animal models of inflammatory, ischemic and thrombotic diseases. Previous studies have revealed that certain pairs of ligands (antibodies and antibody fragments) specific for adjacent, but distinct, epitopes on PECAM-1 enhance each other's binding, a phenomenon dubbed Collaborative Enhancement of Paired Affinity Ligands, or CEPAL. This discovery has been leveraged to enable simultaneous delivery of multiple therapeutics to the vascular endothelium. Given the known role of PECAM-1 in promoting endothelial quiescence and cell junction integrity, we sought here to determine if CEPAL might induce unintended vascular effects. Using a combination of in vitro and in vivo techniques and employing human and mouse endothelial cells under physiologic and pathologic conditions, we found only modest or non-significant effects in response to antibodies to PECAM-1, whether given solo or in pairs. In contrast, these methods detected significant elevation of endothelial permeability, pro-inflammatory vascular activation, and systemic cytokine release following antibody binding to the related endothelial junction protein, VE-Cadherin. These studies support the notion that PECAM-1-targeted CEPAL provides relatively well-tolerated endothelial drug delivery. Additionally, the analysis herein creates a template to evaluate potential toxicities of vascular-targeted nanoparticles and protein therapeutics.
Assuntos
Anticorpos/metabolismo , Células Endoteliais/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Células Cultivadas , Citocinas/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Permeabilidade/efeitos dos fármacos , Ligação ProteicaRESUMO
Polymer-based nanogel formulations offer features attractive for drug delivery, including ease of synthesis, controllable swelling and viscoelasticity as well as drug loading and release characteristics, passive and active targeting, and the ability to formulate nanogel carriers that can respond to biological stimuli. These unique features and low toxicity make the nanogels a favorable option for vascular drug targeting. In this review, we address key chemical and biological aspects of nanogel drug carrier design. In particular, we highlight published studies of nanogel design, descriptions of nanogel functional characteristics and their behavior in biological models. These studies form a compendium of information that supports the scientific and clinical rationale for development of this carrier for targeted therapeutic interventions.
RESUMO
Tissue-type plasminogen activator (tPA) remains the sole thrombolytic approved by the FDA for the treatment of pulmonary embolism (PE). tPA has not been replaced by third generation plasminogen activators, e.g. Reteplase (Ret) and Tenecteplase (TNK) that circulate with longer life-spans and in theory should have more extended potency in vivo. One reason for this paradox is the inability to assign units of activity to plasminogen activators based on specific biologically relevant standards, which impairs objective comparison. Here, we compare clot permeation, retention and fibrinolytic activities of tPA, TNK and Ret in vitro and clot composition over time with outcome in a mouse model of disseminated pulmonary microembolism (ME). When clots were incubated in the continuous presence of drug, tPA, TNK and Ret lysed fibrin clots identically in the absence of PA inhibitor-1 (e.g. PAI-1). Ret, which has lower fibrin affinity and greater susceptibility to inhibition by PAI-1 than tPA, was less effective in lysing plasma clots, while TNK was less effective when the fibrin content of the clots was enhanced. However, when clots were afforded only brief exposure to drug, as occurs in vivo, Ret showed more extensive clot permeation, greater retention and lysis than tPA or TNK. These results were reproduced in vivo in a mouse model of ME. These studies indicate the need for more relevant tests of plasminogen activator activity in vitro and in vivo and they show that clot permeation and retention are important potential predictors of clinical utility.
Assuntos
Fibrina/química , Fibrinólise/efeitos dos fármacos , Ativadores de Plasminogênio/farmacologia , Animais , Bovinos , Difusão , Fibrina/metabolismo , Tempo de Lise do Coágulo de Fibrina , Humanos , Cinética , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativadores de Plasminogênio/química , Ativadores de Plasminogênio/metabolismo , Ativadores de Plasminogênio/uso terapêutico , Embolia Pulmonar/tratamento farmacológico , Embolia Pulmonar/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Solubilidade , Tenecteplase , Ativador de Plasminogênio Tecidual/química , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tecidual/farmacologia , Ativador de Plasminogênio Tecidual/uso terapêuticoRESUMO
The endothelium represents an important therapeutic target for containment of oxidative stress, thrombosis and inflammation involved in a plethora of acute and chronic conditions including cardiovascular and pulmonary diseases and diabetes. However, rapid blood clearance and lack of affinity to the endothelium compromise delivery to target and restrict medical utility of antioxidant enzymes (e.g., catalase) and fibrinolytics. The use of "stealth" PEG-liposomes prolongs circulation, whereas conjugation with antibodies to endothelial determinants permits targeting. Constitutive endothelial cell adhesion molecules (CAM, such as ICAM-1 and PECAM-1, which are stably expressed and functionally involved in oxidative stress and thrombosis) are candidate determinants for targeting of antioxidants and fibrinolytics. CAM antibodies and compounds conjugated with anti-CAM bind to endothelial cells and accumulate in vascularized organs (preferentially, lungs). Pathological stimuli enhance ICAM-1 expression in endothelial cells and facilitate targeting, whereas PECAM-1 expression and targeting are stable. Endothelial cells internalize 100-300 nm diameter conjugates possessing multiple copies of anti-CAM, but not monomolecular antibodies or micron conjugates. This permits size-controlled sub-cellular targeting of antioxidants into the endothelial interior and fibrinolytics to the endothelial surface. Targeting catalase to PECAM-1 or ICAM-1 protects endothelial cells against injury by oxidants in culture and alleviates vascular oxidative stress in lungs in animals. Anti-CAM/catalase conjugates are active for a few hours prior to lysosomal degradation, which can be delayed by auxiliary drugs. Conjugation of fibrinolytics to monovalent anti-ICAM permits targeting and prolonged retention on the endothelial surface. Therefore, CAM targeting of antioxidants and fibrinolytics might help to contain oxidative and thrombotic stresses, with benefits of blocking CAM. Avenues for improvement and translation of this concept into the clinical domain are discussed.
Assuntos
Antioxidantes/farmacologia , Moléculas de Adesão Celular/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Fibrinolíticos/farmacologia , Animais , Antioxidantes/efeitos adversos , Endotélio Vascular/enzimologia , Fibrinolíticos/efeitos adversos , HumanosRESUMO
BACKGROUND AND PURPOSE: To test the role of fibrinolysis in stroke, we used a mouse model in which preformed 2.5- to 3-microm-diameter fibrin microemboli are injected into the cerebral circulation. The microemboli lodge in the downstream precapillary vasculature and are susceptible to fibrinolysis. METHODS: We injected various doses of microemboli into the internal carotid artery in mice and characterized their distribution, effects on cerebral blood flow, neurological deficit, infarct area, and spontaneous dissolution. By comparing wild-type and tissue plasminogen activator (tPA) knockout (tPA-/-) mice, we analyzed the role of endogenous tPA in acute thrombotic stroke. RESULTS: Microemboli cause dose-dependent brain injury. Although moderate doses of microemboli are followed by spontaneous reperfusion, they result in reproducible injury. Gene knockout of tPA markedly delays dissolution of cerebral emboli and restoration of blood flow and aggravates ischemic thrombotic infarction in the brain. CONCLUSIONS: We describe a microembolic model of stroke, in which degree of injury can be controlled by the dose of microemboli injected. Unlike vessel occlusion models, this model can be modulated to allow spontaneous fibrinolysis. Application to tPA-/- mice supports a key role of endogenous tPA in restoring cerebral blood flow and limiting infarct size after thrombosis.
Assuntos
Modelos Animais de Doenças , Fibrinólise , Embolia Intracraniana/fisiopatologia , Ativador de Plasminogênio Tecidual/fisiologia , Animais , Dano Encefálico Crônico/etiologia , Isquemia Encefálica/etiologia , Artéria Carótida Interna , Infarto Cerebral/etiologia , Fibrina/administração & dosagem , Injeções Intra-Arteriais , Injeções Intravenosas , Embolia Intracraniana/complicações , Radioisótopos do Iodo/farmacocinética , Fluxometria por Laser-Doppler , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tamanho da Partícula , Reperfusão , Cauda/irrigação sanguínea , Distribuição Tecidual , Ativador de Plasminogênio Tecidual/deficiência , Ativador de Plasminogênio Tecidual/genéticaRESUMO
Oxidant stress may contribute to acute lung injury under some circumstances. The rapid depletion of plasma gelsolin following major trauma in patients who subsequently develop respiratory distress suggests that this actin-scavenging protein might protect against delayed pulmonary complications. The specific aim of these experiments was to explore the temporal and quantitative relationship between gelsolin levels and lung damage. Gelsolin levels were measured in three murine models of oxidant injury: immunotargeting of pulmonary endothelium with an H2O2-generating enzyme; continuous exposure to >95% O2; and single high-dose thoracic radiation. The degree of lung injury was inversely related to gelsolin levels in mice treated with glucose oxidase-conjugated antibodies against platelet endothelial cell adhesion molecule-1 (p <0.0001). By 60-72 hours of hyperoxic exposure, gelsolin levels had dropped precipitously in all mice who sustained major lung damage (p <0.0001), establishing a quantitative association between gelsolin concentration and hyperoxic lung injury (r = -0.72; 95% confidence interval: ?0.81 to ?0.59). Gelsolin levels modestly but progressively fell in irradiated mice over the 3 days following treatment (p = 0.012) despite the development of only microscopic lung damage during this timeframe. These findings are consistent with the hypothesis that gelsolin depletion is involved in the pathogenesis of acute oxidant lung injury.
Assuntos
Gelsolina/sangue , Lesão Pulmonar , Pulmão/efeitos da radiação , Oxidantes/efeitos adversos , Síndrome do Desconforto Respiratório/sangue , Síndrome do Desconforto Respiratório/etiologia , Animais , Especificidade de Anticorpos/imunologia , Líquido da Lavagem Broncoalveolar/química , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Relação Dose-Resposta à Radiação , Glucose Oxidase/administração & dosagem , Glucose Oxidase/efeitos adversos , Hiperóxia/sangue , Hiperóxia/complicações , Hiperóxia/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Oxidantes/imunologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/administração & dosagem , Molécula-1 de Adesão Celular Endotelial a Plaquetas/efeitos adversos , Proteínas/metabolismo , Doses de Radiação , Estatística como Assunto , Fatores de TempoRESUMO
A novel 85 kD glycoprotein (gp85) is a marker of the avesicular zone, a thin part of pulmonary endothelial cells separating alveolar and vascular compartments and lacking vesicles. This report presents the first evaluation whether mAb 30B3, a monoclonal antibody to gp85, can be used for targeting of drugs to the surface of lung endothelium. 125I-mAb 30B3 accumulated in isolated perfused lungs (IPL) (22.8 +/- 1.1 versus 0.5 +/- 0.1 %ID/g for 125I-IgG) and accumulated preferentially in the lungs after intravenous or intraarterial injection (10.9 +/- 0.7 and 11.0 +/- 1.5 versus 0.9 +/- 0.2 %ID/g for 125I-IgG). 125I-mAb 30B3 uptake in IPL was rapid (T1/2 15 min), saturable (Bmax appr. 10(5) molecules/cell), specific (inhibited by nonlabeled mAb 30B3) and temperature independent (26.3 +/- 2.1 versus 22.8 +/- 1.1 %ID/g at 6 degrees C versus 37 degrees C). Biotinylated mAb 30B3 permitted subsequent accumulation of perfused avidin derivative in IPL. Because these data indicated that mAb 30B3 binds to an accessible, poorly internalizable antigen in the lung, we conjugated mAb 30B3 with a plasminogen activator, 125I-tPA. After intravenous injection in rats, lung-to-blood ratio was 8.4 +/- 0.9 for mAb 30B3/125I-tPA versus 0.4 +/- 0.1 for IgG/125I-tPA, indicating that mAb 30B3 may deliver drugs, which was supposed to exert therapeutic action in the vascular lumen (e.g., antithrombotic proteins), to the surface of pulmonary endothelium.
Assuntos
Capilares/imunologia , Endotélio Vascular/imunologia , Glicoproteínas de Membrana/farmacologia , Alvéolos Pulmonares/imunologia , Animais , Anticorpos Monoclonais/imunologia , Capilares/diagnóstico por imagem , Endotélio Vascular/diagnóstico por imagem , Masculino , Alvéolos Pulmonares/diagnóstico por imagem , Cintilografia , Ratos , Ratos Sprague-DawleyRESUMO
Vascular immunotargeting is a mean for a site-selective delivery of drugs and genes to endothelium. In this study, we compared recognition of pulmonary and systemic vessels in rats by candidate carrier monoclonal antibodies (MAbs) to endothelial antigens platelet endothelial cell adhesion molecule (PECAM)-1 (CD31), intercellular adhesion molecule (ICAM)-1 (CD54), Thy-1.1 (CD90.1), angiotensin-converting enzyme (ACE; CD143), and OX-43. Tissue immunostaining showed that endothelial cells were Thy-1.1 positive in capillaries but negative in large vessels. In the lung, anti-ACE MAb provided a positive staining in 100% capillaries vs. 5-20% capillaries in other organs. Other MAbs did not discriminate between pulmonary and systemic vessels. We determined tissue uptake after infusion of 1 microg of (125)I-labeled MAbs in isolated perfused lungs (IPL) or intravenously in intact rats. Uptake in IPL attained 46% of the injected dose (ID) of anti-Thy-1.1 and 20-25% ID of anti-ACE, anti-ICAM-1, and anti-OX-43 (vs. 0.5% ID of control IgG). However, after systemic injection at this dose, only anti-ACE MAb 9B9 displayed selective pulmonary uptake (16 vs. 1% ID/g in other organs). Anti-OX-43 displayed low pulmonary (0.5% ID/g) but significant splenic and cardiac uptake (7 and 2% ID/g). Anti-Thy-1.1 and anti-ICAM-1 displayed moderate pulmonary (4 and 6% ID/g, respectively) and high splenic and hepatic uptake (e.g., 18% ID/g of anti-Thy-1.1 in spleen). The lung-to-blood ratio was 5, 10, and 15 for anti-Thy-1.1, anti-ACE, and anti-ICAM-1, respectively. PECAM antibodies displayed low pulmonary uptake in perfusion (2% ID) and in vivo (3-4% ID/g). However, conjugation with streptavidin (SA) markedly augmented pulmonary uptake of anti-PECAM in perfusion (10-54% ID, depending on an antibody clone) and in vivo (up to 15% ID/g). Therefore, ACE-, Thy-1.1-, ICAM-1-, and SA-conjugated PECAM MAbs are candidate carriers for pulmonary targeting. ACE MAb offers a high selectivity of pulmonary targeting in vivo, likely because of a high content of ACE-positive capillaries in the lungs.
Assuntos
Anticorpos Monoclonais/metabolismo , Endotélio Vascular/metabolismo , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Especificidade de Anticorpos , Antígenos de Diferenciação/metabolismo , Capilares/citologia , Capilares/metabolismo , Endotélio Vascular/citologia , Imuno-Histoquímica , Injeções Intravenosas , Molécula 1 de Adesão Intercelular/metabolismo , Radioisótopos do Iodo , Pulmão/citologia , Masculino , Peptidil Dipeptidase A/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Ratos , Ratos Sprague-Dawley , Antígenos Thy-1/metabolismo , Distribuição TecidualRESUMO
Protection of alveolar epithelial cells (alveolocytes) and vascular endothelial cells against pulmonary oxidative stress is an important problem. An inadequate delivery to the target cells limits the protective utility of the antioxidant enzymes, superoxide dismutase (SOD) and catalase. SOD and catalase modifications, such as coupling with polyethylene glycol and encapsulation in liposomes, prolong the life span of the active enzymes in vivo. The airway administration of SOD and catalase protects alveolocytes against hyperoxic oxidative stress. Although pulmonary endothelium is poorly accessible from the airways, it is accessible from circulation. However, antioxidant enzymes and their derivatives display poor targeting to pulmonary endothelium. To improve the targeting and provide intracellular delivery to endothelium, the enzymes can be conjugated with antibodies against endothelial antigens, such as angiotensin-converting enzyme and adhesion molecules [intercellular adhesion molecule-1 (ICAM-1) or platelet-endothelial cell adhesion molecule-1 (PECAM-1)]. These immunoconjugates accumulate in the pulmonary vasculature in intact animals, enter endothelium, and augment the antioxidant defenses. The immunoconjugates directed against ICAM-1 and PECAM-1 may also provide a secondary therapeutic benefit by blocking of sequestration and infiltration of leukocytes in the lungs. Further investigations are necessary to evaluate the therapeutic effectiveness of the vascular immunotargeting of antioxidant enzymes and solve technical problems associated with production of safe, clinically useful conjugates.
Assuntos
Antioxidantes/administração & dosagem , Catalase/administração & dosagem , Pneumopatias/tratamento farmacológico , Pulmão/efeitos dos fármacos , Superóxido Dismutase/administração & dosagem , Animais , Antioxidantes/metabolismo , Catalase/metabolismo , Sistemas de Liberação de Medicamentos , Humanos , Pneumopatias/metabolismo , Estresse Oxidativo , Superóxido Dismutase/metabolismoRESUMO
Reactive oxygen species, such as superoxide anion (O2(-)) and H2O2, cause oxidative stress in endothelial cells, a condition implicated in the pathogenesis of many cardiovascular and pulmonary diseases. Antioxidant enzymes, superoxide dismutases (SOD, converting superoxide anion into H2O2) and catalase (converting H2O2 into water), are candidate drugs for augmentation of antioxidant defenses in endothelium. However, SOD and catalase undergo fast elimination from the bloodstream, which compromises delivery and permits rather modest, if any, protection against vascular oxidative stress. Coupling of polyethylene glycol (PEG) to the enzymes and encapsulating them in liposomes increases their bioavailability and enhances their protective effect. Chemical modifications and genetic manipulations of SOD and catalase have been proposed in order to provide more effective delivery to endothelium. For example, chimeric protein constructs consisting of SOD and heparin-binding peptides have an affinity for charged components of the endothelial glycocalix. However, the problem of developing a more effective and precise delivery of the drugs to endothelial cells persists. Endothelial surface antigens may be employed to provide targeting and subcellular addressing of drugs (vascular immunotargeting strategy). Thus, SOD and catalase conjugated to antibodies directed against the constitutively expressed endothelial antigens, angiotensin-converting enzyme (ACE) and adhesion molecules (ICAM-1 or PECAM-1), bind to endothelium in intact animals after intravascular administration, accumulate in the pulmonary vasculature, enter endothelial cells and augment their antioxidant defenses. Such immunotargeting strategies may provide secondary therapeutic benefits by inhibiting the function of target antigens. For example, blocking of ICAM-1 and PECAM-1 by carrier antibodies may attenuate inflammation and leukocyte-mediated vascular damage. Additional studies in animal models of vascular oxidative stress are necessary in order to more fully characterize potential therapeutic effects and limitations of targeting of antioxidant enzymes to endothelial cells.
Assuntos
Antioxidantes/administração & dosagem , Catalase/administração & dosagem , Endotélio Vascular/metabolismo , Superóxido Dismutase/administração & dosagem , Animais , Antioxidantes/metabolismo , Catalase/metabolismo , Humanos , Superóxido Dismutase/metabolismoRESUMO
Vascular immunotargeting, the administration of drugs conjugated with antibodies to endothelial surface antigens, has the potential for drug delivery to the endothelium. Our previous cell culture studies showed that biotinylated antibodies to PECAM-1 (a highly expressed endothelial surface antigen) coupled with streptavidin (SA, a cross-linking protein that facilitates anti-PECAM internalization and targeting) may provide a carrier for the intracellular delivery of therapeutic enzymes. This paper describes the PECAM-directed vascular immunotargeting of a reporter enzyme (beta-galactosidase, beta-Gal) in intact animals. Intravenous injection of [125I]SA-beta-Gal conjugated with either anti-PECAM or IgG led to a high 125I uptake in liver and spleen, yet beta-Gal activity in these organs rapidly declined to the background levels, suggesting rapid degradation of the conjugates. In contrast, anti-PECAM/[125I]SA-beta-Gal, but not IgG/[125I]SA-beta-Gal, accumulated in the lungs (36.0+/-1.3 vs. 3.9+/-0.6% injected dose/g) and induced a marked elevation of beta-Gal activity in the lung tissue persisting for up to 8 h after injection (10-fold elevation 4 h postinjection). Using histochemical detection, the beta-Gal activity in the lungs was detected in the endothelial cells of capillaries and large vessels. The anti-PECAM carrier also provided 125I uptake and beta-Gal activity in the renal glomeruli. Predominant intracellular localization of anti-PECAM/SA-beta-Gal was documented in the PECAM-expressing cells in culture by confocal microscopy and in the pulmonary endothelium by electron microscopy. Therefore, vascular immunotargeting is a feasible strategy for cell-selective, intracellular delivery of an active foreign enzyme to endothelial cells in vivo, and thus may be potentially useful for the treatment of acute pulmonary or vascular diseases.
Assuntos
Endotélio Vascular/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/fisiologia , beta-Galactosidase/genética , Animais , Genes Reporter , Humanos , Imunoglobulina G/metabolismo , Radioisótopos do Iodo/farmacocinética , Glomérulos Renais/enzimologia , Fígado/enzimologia , Pulmão/enzimologia , Mesotelioma , Camundongos , Camundongos Endogâmicos BALB C , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Circulação Pulmonar , Proteínas Recombinantes/metabolismo , Baço/enzimologia , Distribuição Tecidual , Transfecção , Células Tumorais Cultivadas , beta-Galactosidase/metabolismoRESUMO
The role of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in fibrinolysis remains unsettled. The contribution of uPA may depend on the vascular location, the physical properties of the clot, and its impact on tissue function. To study the contribution of urokinase within the pulmonary microvasculature, a model of pulmonary microembolism in the mouse was developed. Iodine 125 ((125)I)-labeled fibrin microparticles injected intravenously through the tail vein lodged preferentially in the lung, distributing homogeneously throughout the lobes. Clearance of (125)I-microemboli in wild type mice was rapid and essentially complete by 5 hours. In contrast, uPA(-/-) and tissue-type plasminogen activator tPA(-/-) mice, but not uPAR(-/-) mice, showed a marked impairment in pulmonary fibrinolysis throughout the experimental period. The phenotype in the uPA(-/-) mouse was rescued completely by infusion of single chain uPA (scuPA). The increment in clot lysis was 4-fold greater in uPA(-/-) mice infused with the same concentration of scuPA complexed with soluble recombinant uPAR. These data indicate that uPA contributes to endogenous fibrinolysis in the pulmonary vasculature to the same extent as tPA in this model system. Binding of scuPA to its receptor promotes fibrinolytic activity in vivo as well as in vitro. The physical properties of fibrin clots, including size, age, and cellular composition, as well as heterogeneity in endothelial cell function, may modify the participation of uPA in endogenous fibrinolysis. (Blood. 2000;96:1820-1826)
Assuntos
Fibrinólise/fisiologia , Circulação Pulmonar/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Animais , Modelos Animais de Doenças , Fibrina/análise , Fibrinogênio/administração & dosagem , Fibrinogênio/farmacocinética , Fibrinólise/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Pulmão/química , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microesferas , Tamanho da Partícula , Circulação Pulmonar/efeitos dos fármacos , Embolia Pulmonar/metabolismo , Embolia Pulmonar/fisiopatologia , Embolia Pulmonar/prevenção & controle , Receptores de Superfície Celular/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Distribuição Tecidual , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Vascular immunotargeting is a novel approach for site-selective drug delivery to endothelium. To validate the strategy, we conjugated glucose oxidase (GOX) via streptavidin with antibodies to the endothelial cell surface antigen platelet endothelial cell adhesion molecule (PECAM). Previous work documented that 1) anti-PECAM-streptavidin carrier accumulates in the lungs after intravenous injection in animals and 2) anti-PECAM-GOX binds to, enters, and kills endothelium via intracellular H(2)O(2) generation in cell culture. In the present work, we studied the targeting and effect of anti-PECAM-GOX in animals. Anti-PECAM-GOX, but not IgG-GOX, accumulated in the isolated rat lungs, produced H(2)O(2,) and caused endothelial injury manifested by a fourfold elevation of angiotensin-converting enzyme activity in the perfusate. In intact mice, anti-PECAM-GOX accumulated in the lungs (27 +/- 9 vs. 2.4 +/- 0.3% injected dose/g for IgG-GOX) and caused severe lung injury and 95% lethality within hours after intravenous injection. Endothelial disruption and blebbing, elevated lung wet-to-dry ratio, and interstitial and alveolar edema indicated that anti-PECAM-GOX damaged pulmonary endothelium. The vascular injury in the lungs was associated with positive immunostaining for iPF(2alpha)-III isoprostane, a marker for oxidative stress. In contrast, IgG-GOX caused a minor lung injury and little (5%) lethality. Anti-PECAM conjugated with inert proteins induced no death or lung injury. None of the conjugates caused major injury to other internal organs. These results indicate that an immunotargeting strategy can deliver an active enzyme to selected target cells in intact animals. Anti-PECAM-GOX provides a novel model of oxidative injury to the pulmonary endothelium in vivo.
Assuntos
Endotélio Vascular/enzimologia , Marcação de Genes , Glucose Oxidase/genética , Estresse Oxidativo , Circulação Pulmonar , Doenças Vasculares/induzido quimicamente , Animais , Anticorpos/genética , Anticorpos/imunologia , Anticorpos/metabolismo , Anticorpos/farmacologia , Glucose Oxidase/imunologia , Glucose Oxidase/metabolismo , Técnicas In Vitro , Pneumopatias/induzido quimicamente , Pneumopatias/patologia , Camundongos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Ratos , Distribuição Tecidual , Doenças Vasculares/patologiaRESUMO
Hydrogen peroxide formed in the lung tissue in ischemia/reperfusion or released from activated leukocytes causes oxidative injury of the vascular endothelial cells (1-3). H(2)O(2)-degrading enzyme, catalase, has been extensively explored in order to protect cells and tissues against H(2)O(2)-mediated injury (4). Catalase, however, has short lifetime in the bloodstream and provides only marginal protective effect after intravascular administration in animals (5). Catalase modification (conjugation with polyethylene glycol (6) or encapsulation in liposomes [7]), prolongs catalase lifetime in the circulation and facilitate its cellular uptake. These modifications, however, do not provide it with an affinity to the endothelial cells. In order to provide catalase with such an affinity, catalase could be chemically conjugated with a carrier antibodies recognizing the surface endothelial antigens (8). A monoclonal antibody (MAb) against such an antigen, angiotensin-converting enzyme (anti-ACE MAb 9B9, produced by Dr. Sergei M. Danilov [9]), accumulates in the pulmonary endothelium after systemic injection (10). Therefore, MAb 9B9 may serve as an affinity carrier for targeting of catalase to the pulmonary endothelial cells, for specific augmentation of their antioxidative defense.
RESUMO
Extracellular and intracellular reactive oxygen species attack different targets and may, therefore, result in different forms of oxidative stress. To specifically study an oxidative stress induced by a regulated intracellular flux of a defined reactive oxygen species in endothelium, we used immunotargeting of the H(2)O(2)-generating enzyme glucose oxidase (GOX) conjugated with an antibody to platelet-endothelial cell adhesion molecule (PECAM)-1, an endothelial surface antigen. Anti-PECAM-(125)I-GOX conjugates specifically bind to both endothelial and PECAM-transfected cells. Approximately 70% of cell-bound anti-PECAM-(125)I-GOX was internalized. The cell-bound conjugate was enzymatically active and generated H(2)O(2) from glucose. Use of the fluorescent dye dihydrorhodamine 123 revealed that 70% of H(2)O(2) was generated intracellularly, whereas 30% of H(2)O(2) was detected in the cell medium. Catalase added to the cells eliminated H(2)O(2) in the medium but had little effect on the intracellular generation of H(2)O(2) by anti-PECAM-GOX. Both H(2)O(2) added exogenously to the cell medium (extracellular H(2)O(2)) and that generated by anti-PECAM-GOX caused oxidative stress manifested by time- and dose-dependent irreversible plasma membrane damage. Inactivation of cellular catalase by aminotriazole treatment augmented damage caused by either extracellular H(2)O(2) or anti-PECAM-GOX. Catalase added to the medium protected either normal or aminotriazole-treated cells against extracellular H(2)O(2), yet failed to protect cells against injury induced by anti-PECAM-GOX. Therefore, treatment of PECAM-positive cells with anti-PECAM-GOX leads to conjugate internalization, predominantly intracellular H(2)O(2) generation and intracellular oxidative stress. These results indicate that anti-PECAM-GOX 1) provides cell-specific intracellular delivery of an active enzyme and 2) causes intracellular oxidative stress in PECAM-positive cells.
Assuntos
Endotélio Vascular/metabolismo , Peróxido de Hidrogênio/metabolismo , Membranas Intracelulares/metabolismo , Estresse Oxidativo/fisiologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos/imunologia , Catalase/fisiologia , Células Cultivadas , Endotélio Vascular/citologia , Espaço Extracelular/metabolismo , Glucose Oxidase/imunologia , Humanos , Membranas Intracelulares/enzimologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismoRESUMO
Conjugation of drugs with antibodies to surface endothelial antigens is a potential strategy for drug delivery to endothelium. We studied antibodies to platelet-endothelial adhesion molecule 1 (PECAM-1, a stably expressed endothelial antigen) as carriers for vascular immunotargeting. Although 125I-labeled anti-PECAM bound to endothelial cells in culture, the antibody was poorly internalized by the cells and accumulated poorly after intravenous administration in mice and rats. However, conjugation of biotinylated anti-PECAM (b-anti-PECAM) with streptavidin (SA) markedly stimulated uptake and internalization of anti-PECAM by endothelial cells and by cells expressing PECAM. In addition, conjugation with streptavidin markedly stimulated uptake of 125I-labeled b-anti-PECAM in perfused rat lungs and in the lungs of intact animals after either intravenous or intraarterial injection. The antioxidant enzyme catalase conjugated with b-anti-PECAM/SA bound to endothelial cells in culture, entered the cells, escaped intracellular degradation, and protected the cells against H2O2-induced injury. Anti-PECAM/SA/125I-catalase accumulated in the lungs after intravenous injection or in the perfused rat lungs and protected these lungs against H2O2-induced injury. Thus, modification of a poor carrier antibody with biotin and SA provides an approach for facilitation of antibody-mediated drug targeting. Anti-PECAM/SA is a promising candidate for vascular immunotargeting of bioactive drugs.
Assuntos
Endotélio Vascular/metabolismo , Imunotoxinas/farmacocinética , Pulmão/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Estreptavidina/farmacocinética , Animais , Anticorpos/metabolismo , Transporte Biológico/efeitos dos fármacos , Biotinilação , Células Cultivadas , Endotélio Vascular/ultraestrutura , Ferritinas/metabolismo , Humanos , Radioisótopos do Iodo/farmacocinética , Cinética , Masculino , Camundongos , Coelhos , Ratos , Ratos Sprague-Dawley , Estreptavidina/farmacologia , Distribuição Tecidual , Veias UmbilicaisRESUMO
The pulmonary endothelium is susceptible to oxidative insults. Catalase conjugated with monoclonal antibodies (MAbs) against endothelial surface antigens, angiotensin-converting enzyme (MAb 9B9) or intercellular adhesion molecule-1 (MAb 1A29), accumulates in the lungs after systemic injection in rats (V. Muzykantov, E. Atochina, H. Ischiropoulos, S. Danilov, and A. Fisher. Proc. Natl. Acad. Sci. USA 93: 5213-5218, 1996). The present study characterizes the augmentation of antioxidant defense by these antibody-catalase conjugates in isolated rat lungs perfused for 1 h with catalase conjugated with either MAb 9B9, MAb 1A29, or control mouse IgG. Approximately 20% of the injected dose of Ab-125I-catalase accumulated in the perfused rat lungs (vs. <5% for IgG-125I-catalase). After elimination of nonbound material, the lungs were perfused further for 1 h with 5 mM hydrogen peroxide (H2O2). H2O2 induced an elevation in tracheal and pulmonary arterial pressures (126 +/- 7 and 132 +/- 5%, respectively, of the control level), lung wet-to-dry weight ratio (7.1 +/- 0.4 vs. 6.0 +/- 0.01 in the control lungs), and ACE release into the perfusate (436 +/- 20 vs. 75 +/- 7 mU in the control perfusates). Both MAb 9B9-catalase and MAb 1A29-catalase significantly attenuated the H2O2-induced elevation in 1) angiotensin-converting enzyme release to the perfusate (215 +/- 14 and 217 +/- 38 mU, respectively), 2) lung wet-to-dry ratio (6.25 +/- 0.1 and 6.3 +/- 0.3, respectively), 3) tracheal pressure (94 +/- 4 and 101 +/- 4%, respectively, of the control level), and 4) pulmonary arterial pressure (103 +/- 3 and 104 +/- 7%, respectively, of the control level). Nonconjugated catalase, nonconjugated antibodies, nonspecific IgG, and IgG-catalase conjugate had no protective effect, thus confirming the specificity of the effect of MAb-catalase. These results support a strategy of catalase immunotargeting for protection against pulmonary oxidative injury.
Assuntos
Catalase/metabolismo , Endotélio Vascular/fisiologia , Imunotoxinas/farmacologia , Molécula 1 de Adesão Intercelular/fisiologia , Pulmão/fisiologia , Estresse Oxidativo , Peptidil Dipeptidase A/fisiologia , Circulação Pulmonar/fisiologia , Animais , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Biotinilação , Catalase/farmacocinética , Endotélio Vascular/citologia , Peróxido de Hidrogênio/metabolismo , Imunoglobulina G/farmacologia , Imunotoxinas/farmacocinética , Molécula 1 de Adesão Intercelular/imunologia , Radioisótopos do Iodo , Pulmão/citologia , Masculino , Camundongos , Peptidil Dipeptidase A/imunologia , Ratos , Ratos Sprague-DawleyRESUMO
Previous work has suggested that not all immunoreactive angiotensin-converting enzyme (ACE) in tissues or cells is in a biologically active state. We have explored this possibility in cultured human umbilical vein endothelial cells (HUVEC), one of the most widely studied in vitro endothelial cell systems. Our approach included characterization of the effect of increasing passage number on ACE activity and expression of immunoreactive ACE at the single cell level, the subcellular compartmentalization of active ACE, and the effect of phorbol ester (PMA) treatment. We found that both ACE activity and expression of ACE antigen were downregulated by cultivation (30% of ACE-positive cells at seventh passage vs. 90% in primary culture). ACE downregulation is specific (number of CD31-positive cells did not change with cultivation) and correlated with downregulation of factor VIII-antigen. The percentage of ACE-positive cells in permeabilized HUVEC at third passage was almost twice that in nonpermeabilized HUVEC (90% vs. 50%), indicating that HUVEC contain intracellular immunoreactive ACE. ACE activity, however, was similar when measured in intact cells and in cell lysates. Moreover, diazonium salt of sulfanilic acid (DASA), a membrane-impermeable ACE inhibitor, inhibited ACE activity in intact cells and in cell lysates at the same extent, thus implying that intracellular ACE is inactive. PMA (100 nM) treatment increased the percentage of ACE-positive cells at third passage from 57 to 96%. ACE activity was increased 3-fold in cell and 1.5-fold in the culture medium of PMA-treated cells. Analysis of ACE activity in intact monolayers and cell lysates of control and PMA-treated cells revealed that all enzymatically active ACE in PMA-treated cells is localized on the plasma membrane and acts as an ectoenzyme. We conclude that expression of ACE by HUVEC is downregulated by repeated passage in culture but can be restored by PMA treatment. In addition, ACE expression is heterogeneous between neighboring cells, and total immunoreactive ACE protein associated with HUVEC includes an inactive pool of the enzyme.