Immunotargeting of catalase to the pulmonary endothelium alleviates oxidative stress and reduces acute lung transplantation injury

Vascular immunotargeting may facilitate the rapid and specific delivery of therapeutic agents to endothelial cells. We investigated whether targeting of an antioxidant enzyme, catalase, to the pulmonary endothelium alleviates oxidative stress in an in vivo model of lung transplantation. Intravenously injected enzymes, conjugated with an antibody to platelet-endothelial cell adhesion molecule-1, accumulate in the pulmonary vasculature and retain their activity during prolonged cold storage and transplantation. Immunotargeting of catalase to donor rats augments the antioxidant capacity of the pulmonary endothelium, reduces oxidative stress, ameliorates ischemia-reperfusion injury, prolongs the acceptable cold ischemia period of lung grafts, and improves the function of transplanted lung grafts. These findings validate the therapeutic potential of vascular immunotargeting as a drug delivery strategy to reduce endothelial injury. Potential applications of this strategy include improving the outcome of clinical lung transplantation and treating a wide variety of endothelial disorders.     

Xanthine oxidase mediates elastase-induced injury to isolated lungs and endothelium

Xanthine oxidase (XO)-generated toxic O2 metabolites appear to contribute to reperfusion injury, but the possibility that XO is involved in hyperoxic or neutrophil elastase-mediated injury has not been investigated. We found that lungs isolated from rats fed a tungsten-rich diet had negligible XO activities and after exposure to hyperoxia developed less acute edematous injury during perfusion with buffer or purified neutrophil elastase than XO-replete lungs from control rats which had been exposed to hyperoxia. In parallel, tungsten-treated XO-depleted cultured bovine pulmonary arterial endothelial cells made less superoxide anion and as monolayers leaked less 125I-labeled albumin after exposure to neutrophil elastase than XO-replete endothelial cell monolayers. Our findings suggest that XO-derived O2 metabolites contribute to acute edematous lung injury from hyperoxia directly and by enhancing susceptibility to neutrophil elastase.     

Immunotargeting of catalase to lung endothelium via anti-angiotensin-converting enzyme antibodies attenuates ischemia-reperfusion injury of the lung in vivo

Limitation of reactive oxygen species-mediated ischemia-reperfusion (I/R) injury of the lung by vascular immunotargeting of antioxidative enzymes has the potential to become a promising modality for extension of the viability of banked transplantation tissue. The preferential expression of angiotensin-converting enzyme (ACE) in pulmonary capillaries makes it an ideal target for therapy directed toward the pulmonary endothelium. Conjugates of ACE monoclonal antibody (MAb) 9B9 with catalase (9B9-CAT) have been evaluated in vivo for limitation of lung I/R injury in rats. Ischemia of the right lung was induced for 60 min followed by 120 min of reperfusion. Sham-operated animals (sham, n = 6) were compared with ischemia-reperfused untreated animals (I/R, n = 6), I/R animals treated with biotinylated catalase (CAT, n = 6), and I/R rats treated with the conjugates (9B9-CAT, n = 6). The 9B9-CAT accumulation in the pulmonary endothelium of injured lungs was elucidated immunohistochemically. Arterial oxygenation during reperfusion was significantly higher in 9B9-CAT (221 +/- 36 mmHg) and sham (215 +/- 16 mmHg; P < 0.001 for both) compared with I/R (110 +/- 10 mmHg) and CAT (114 +/- 30 mmHg). Wet-dry weight ratio of I/R (6.78 +/- 0.94%) and CAT (6.54 +/- 0.87%) was significantly higher than of sham (4.85 +/- 0.29%; P < 0.05), which did not differ from 9B9-CAT (5.58 +/- 0.80%). The significantly lower degree of lung injury in 9B9-CAT-treated animals compared with I/R rats was also shown by decreased serum levels of endothelin-1 (sham, 18 +/- 9 fmol/mg; I/R, 42 +/- 12 fmol/mg; CAT, 36 +/- 11 fmol/mg; 9B9-CAT, 26 +/- 9 fmol/mg; P < 0.01) and mRNA for inducible nitric oxide synthase (iNOS) [iNOS-GAPDH ratio: sham, 0.15 +/- 0.06 arbitrary units (a.u.); I/R, 0.33 +/- 0.08 a.u.; CAT, 0.26 +/- 0.05 a.u.; 9B9-CAT, 0.14 +/- 0.04 a.u.; P < 0.001]. These results validate immunotargeting by anti-ACE conjugates as a prospective and specific strategy to augment antioxidative defenses of the pulmonary endothelium in vivo.     

PECAM-directed delivery of catalase to endothelium protects against pulmonary vascular oxidative stress

Targeted delivery of drugs to vascular endothelium promises more effective and specific therapies in many disease conditions, including acute lung injury (ALI). This study evaluates the therapeutic effect of drug targeting to PECAM (platelet/endothelial cell adhesion molecule-1) in vivo in the context of pulmonary oxidative stress. Endothelial injury by reactive oxygen species (e.g., H2O2) is involved in many disease conditions, including ALI/acute respiratory distress syndrome and ischemia-reperfusion. To optimize delivery of antioxidant therapeutics, we conjugated catalase with PECAM antibodies and tested properties of anti-PECAM/catalase conjugates in cell culture and mice. Anti-PECAM/catalase, but not an IgG/catalase counterpart, bound specifically to PECAM-expressing cells, augmented their H2O2-degrading capacity, and protected them against H2O2 toxicity. Anti-PECAM/catalase, but not IgG/catalase, rapidly accumulated in the lungs after intravenous injection in mice, where it was confined to the pulmonary endothelium. To test its protective effect, we employed a murine model of oxidative lung injury induced by glucose oxidase coupled with thrombomodulin antibody (anti-TM/GOX). After intravenous injection in mice, anti-TM/GOX binds to pulmonary endothelium and produces H2O2, which causes lung injury and 100% lethality within 7 h. Coinjection of anti-PECAM/catalase protected against anti-TM/GOX-induced pulmonary oxidative stress, injury, and lethality, whereas polyethylene glycol catalase or IgG/catalase conjugates afforded only marginal protective effects. This result validates vascular immunotargeting as a prospective strategy for therapeutic interventions aimed at immediate protective effects, e.g., for augmentation of antioxidant defense in the pulmonary endothelium and treatment of ALI.     

血管内皮Nrf2-ARE通路调控的研究进展与展望

核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)是一种对细胞氧化应激十分敏感的基因转录因子,可诱导依赖抗氧化反应元件(ARE)的多种抗氧化蛋白的合成;Nrf2由Kelch样ECH相关蛋白1(Keap1)扣押于胞浆并被转运至26S蛋白酶体降解,从而维持在生理状态下Nrf2的低转录活性;创伤及氧化应激可诱导Nrf2从Keap1解离并转位至胞核,从而启动下游靶基因的转录激活.由蛋白激酶C(PKC)、丝裂原活化蛋白激酶(MAPK)、肌酸激酶2(CK2)等蛋白激酶介导的Nrf2磷酸化、亲电子物质对Keap1巯基的修饰,以及泛素-蛋白酶体系统均与Nrf2-ARE通路的转录调控有关.     

Morphologic characterization of acute injury to vascular endothelium of skin after frostbite

Cyclo-oxygenase inhibitors and free-radical scavengers protect the skin against necrosis induced by frostbite. However, the tissue component(s) that determine the evolution of skin necrosis and the mechanism of this pharmacologic protection are not precisely defined. We have studied freezing injury to rabbit ears by serial biopsies examined by light and electron microscopy. The morphologic evidence of skin injury due to freezing was localized exclusively in the endothelial cells, particularly in the arterioles. Within 1 hour, the entire microvasculature demonstrated endothelial damage. Intravascular platelet aggregation occurred just after thawing and closely paralleled the endothelial cell injury. Very few neutrophils were seen initially (at 10 minutes). By 1 hour, leukocyte aggregates were present, and they further increased at 6 hours. Swelling of the interstitium started 10 minutes after thawing, while extravasation of erythrocytes began to appear by 6 hours. Parenchymal elements of skin were relatively free of damage. In the ear cartilage, the chondrocytes showed evidence of damage immediately after freezing. The administration of superoxide dismutase (SOD) during thawing (reperfusion) did not qualitatively alter any of the initial morphologic changes induced by freezing. We conclude that the endothelial cell is the initial target of injury induced by freezing, an initial injury that is mediated by a non-free-radical-mediated mechanism. It is likely that this acute injury ultimately compromises blood flow and leads to skin necrosis.     

Cell-selective intracellular delivery of a foreign enzyme to endothelium in vivo using vascular immunotargeting.

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.     

Glyoxal causes inflammatory injury in human vascular endothelial cells

To explore mechanisms of diabetes-associated vascular endothelial cells (ECs) injury, human umbilical vein ECs were treated for 24 h with high glucose (HG; 26 mM), advanced glycation end-products (AGEs; 100 μg/ml) or their intermediate, glyoxal (GO: 50-5000 μM). HG and AGEs had no effects on ECs morphology and inflammatory states as measured by vascular cell adhesion molecule (VCAM)-1 and cyclooxygenase (COX)-2 expressions. GO (500 μM, 24 h) induced cytotoxic morphological changes and protein expression of COX-2 but not VCAM-1. GO (500 μM, 24 h) activated ERK but not JNK, p38 or NF-κB. However, ERK inhibitor PD98059 was ineffective to GO-induced COX-2. While EUK134, synthetic combined superoxide dismutase/catalase mimetic, had no effect on GO-mediated inflammation, sodium nitroprusside inhibited it. The present results indicate that glyoxal, a metabolite of glucose might be a more powerful inducer for vascular ECs inflammatory injury. Nitric oxide but not anti-oxidant is preventive against GO-mediated inflammatory injury.     

High glucose induced rat aorta vascular smooth muscle cell oxidative injury: involvement of protein tyrosine nitration

The alteration and further damage of vascular smooth muscle function have been implicated in the development of vascular complications and diabetes. Little is known about protein tyrosine nitration in vascular smooth muscle cell injury induced by high glucose. In this article, vascular smooth muscle cell was exposed to 30 and 40 mM high glucose for 72 h, and then the cell injury in vascular smooth muscle cell induced by high glucose was studied. It was found that high glucose stimulated vascular smooth muscle cell injury in a dose-dependent manner, including decreasing intracellular and extracellular glutathione contents, increasing malondialdehyde and intracellular reactive oxygen species content, increasing the production of nitric oxide (increased nitrite content in cell and medium), as well as increasing protein tyrosine nitration. By comparing protein tyrosine nitration induced by high glucose conditions and extrinsic factors (hemin–nitrite–glucose oxidase system and 3-morpholinosydnonimine), it may be speculated that protein is nitrated selectively, and specific protein tyrosine nitration is involved in diabetic vascular complications.     

An archaeal NADH oxidase causes damage to both proteins and nucleic acids under oxidative stress

NADH oxidases (NOXs) catalyze the two-electron reduction of oxygen to H₂O₂ or four-electron reduction of oxygen to H₂O. In this report, we show that an NADH oxidase from Thermococcus profundus (NOXtp) displays two forms: a native dimeric protein under physiological conditions and an oxidized hexameric form under oxidative stress. Native NOXtp displays high NADH oxidase activity, and oxidized NOXtp can accelerate the aggregation of partially unfolded proteins. The aggregates formed by NOXtp have characteristics similar to β-amyloid and Lewy bodies in neurodegenerative diseases, including an increase of β-sheet content. Oxidized NOXtp can also bind nucleic acids and cause their degradation by oxidizing NADH to produce H₂O₂. Furthermore, Escherichia coli cells expressing NOXtp are less viable than cells not expressing NOXtp after treatment with H₂O₂. As NOXtp shares similar features with eukaryotic cell death isozymes and life may have originated from hyperthermophiles, we suggest that NOXtp may be an ancestor of cell death proteins.     

Role of oxidative stress in vinorelbine-induced vascular endothelial cell injury

Vinorelbine (VNR), a vinca alkaloid anticancer drug, often causes vascular injury such as venous irritation, vascular pain, phlebitis, and necrotizing vasculitis. The purpose of this study was to identify the mechanisms that mediate the cell injury induced by VNR in porcine aorta endothelial cells (PAECs). PAECs were exposed to VNR for 10 min followed by further incubation in serum-free medium without VNR. The exposure to VNR (0.3–30 μM) decreased the cell viability concentration and time dependently. The incidence of apoptotic cells significantly increased at 12 h after transient exposure to VNR. At the same time, VNR increased the activity of caspases. Interestingly, VNR rapidly depleted intracellular glutathione (GSH) and increased intracellular reactive oxygen species (ROS) production. Moreover, VNR depolarized the mitochondrial membrane potential and decreased cellular ATP levels. These VNR-induced cell abnormalities were almost completely inhibited by GSH and N-acetylcysteine. On the other hand, l-buthionine-(S,R)-sulfoximine, a specific inhibitor of GSH synthesis, aggravated the VNR-induced loss of cell viability. These results clearly demonstrate that VNR induces oxidative stress by depleting intracellular GSH and increasing ROS production in PAECs, and oxidative stress plays an important role in the VNR-induced cell injury.     

Factors involved in cell adhesion to vascular endothelium

The adhesion of blood cells to endothelium can be studied in vitro using human endothelial cells in culture. This experimental model and radiometric techniques provide us with a simple system to quantify the adhesion of blood cells to endothelium. Normal human granulocytes isolated by density gradient adhere to normal endothelial cells in a proportion of 25%. Human promyelocytic cells (HL 60) induced by retinoic acid into mature cells adhere as well as normal granulocytes while the noninduced adhere poorly to endothelium. A small percentage of normal red cells attach to endothelial cells while red cells from patients with sickle cell anemia or diabetes mellitus have a significantly increased adhesion to endothelial cells (P greater than 0.001). This adhesion is statistically correlated with the extent and severity of vascular complications in diabetes mellitus (P less than 0.05). The addition of fibrinogen significantly increased (P less than 0.01) the adhesion of normal red cells, red cells from patients with sickle cell anemia or diabetes mellitus while gamma-globulins did not modify adhesion. Fibronectin potentiated the adhesion of normal red cells.     

High glucose concentrations induce oxidative damage to mitochondrial DNA in explanted vascular smooth muscle cells

Oxidative stress is considered to be one of the mechanisms leading to atherosclerosis. It occurs in response to injury or to altered metabolic state. Alterations in cell growth (proliferation or apoptosis) can also contribute to the pathogenesis of atherosclerosis and is influenced by oxidative stress. Smooth muscle cells (SMC) from aortic explants of JCR:LA-cp homozygous cp/cp corpulent rats who are genetically predisposed to develop atherosclerosis exhibit increased SMC proliferation, which can be attenuated by exercise and food restriction. This study was conducted to characterize the effects fo oxidative stress and high glucose media on cell growth and its relationship to mitochondrial DNA integrity and gene expression in explanted aortic SMC from corpulent and lean JCR:LA-cp rats. The results show that SMC from the cp/cp rat appear to be resistant to oxidant-induced cell death and that they accumulate mitochondrial DNA mutations, probably as a result of a reduction in apoptosis. These data suggest that susceptibility to age- and glucose-related atherosclerosis may be related to alterations in redox signaling.     

Ultrastructural studies of muscle cells and vascular endothelium immediately after freeze-thaw injury

Little is known about the ultrastructural changes which occur in vascular endothelium immediately after an in vivo freezethaw insult, although most investigators will agree that tissue viability relates directly to the degree of vascular damage. In this study an electron microscopic examination of an in vivo model for frostbite injury was initiated. The horseradish peroxidase technique was utilized to follow early alterations in capillary flow and the independent effects of hypoxia, cooling to 2 °C, supercooling, and a single freeze-thaw insult were assessed. No precipitous changes in muscle cell mitochondria or capillary endothelium were detected as a result of brief hypoxia, cooling at 2 °C, or supercooling to ?13 °C. Reducing the temperature by 1 °C/min until freezing occurred, continuing to cool for 10 min postheat of fusion, and rapidly rewarming resulted in consistent mitochondrial damage in muscle cells and marked degeneration of associated capillaries. Peroxidase injected iv prior to thawing was rarely localized in the capillaries of previously frozen muscle. Since peroxidase was found in the capillaries of unfrozen legs of the same animals, it is inferred that little or no flow occurred in most capillaries postthaw. Ultrastructural integrity of capillaries immediately after thawing may be a good index for predicting tissue loss.“In conducting the research described in this report, the investigators adhered to the ‘Guide for Laboratory Animal Facilities and Care,’ as promulgated by the Committee on the Guide for Laboratory Animal Facilities and Care of the Institute of Laboratory Animal Resources, National Academy of Sciences-National Research Council.”     

The contribution of vascular endothelial xanthine dehydrogenase/oxidase to oxygen-mediated cell injury.

The conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) and the reaction of XO-derived partially reduced oxygen species (PROS) have been suggested to be important in diverse mechanisms of tissue pathophysiology, including oxygen toxicity. Bovine aortic endothelial cells expressed variable amounts of XDH and XO activity in culture. Xanthine dehydrogenase plus xanthine oxidase specific activity increased in dividing cells, peaked after achieving confluency, and decreased in postconfluent cells. Exposure of BAEC to hyperoxia (95% O2; 5% CO2) for 0-48 h caused no change in cell protein or DNA when compared to normoxic controls. Cell XDH+XO activity decreased 98% after 48 h of 95% O2 exposure and decreased 68% after 48 h normoxia. During hyperoxia, the percentage of cell XDH+XO in the XO form increased to 100%, but was unchanged in air controls. Cell catalase activity was unaffected by hyperoxia and lactate dehydrogenase activity was minimally elevated. Hyperoxia resulted in enhanced cell detachment from monolayers, which increased 112% compared to controls. Release of DNA and preincorporated [8-14C]adenine was also used to assess hyperoxic cell injury and did not significantly change in exposed cells. Pretreatment of cells with allopurinol for 1 h inhibited XDH+XO activity 100%, which could be reversed after oxidation of cell lysates with potassium ferricyanide (K3Fe(CN)6). After 48 h of culture in air with allopurinol, cell XDH+XO activity was enhanced when assayed after reversal of inhibition with K3Fe(CN)6, and cell detachment was decreased. In contrast, allopurinol treatment of cells 1 h prior to and during 48 h of hyperoxic exposure did not reduce cell damage. After K3Fe(CN)6 oxidation, XDH+XO activity was undetectable in hyperoxic cell lysates. Thus, XO-derived PROS did not contribute to cell injury or inactivation of XDH+XO during hyperoxia. It is concluded that endogenous cell XO was not a significant source of reactive oxygen species during hyperoxia and contributes only minimally to net cell production of O2- and H2O2 during normoxia.