1 Institute for Environmental Medicine and Departments of 2 Medicine and 3 Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6068
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ABSTRACT |
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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 H2O2-generating enzyme glucose oxidase (GOX) conjugated with an antibody to platelet-endothelial cell adhesion molecule (PECAM)-1, an endothelial surface antigen. Anti-PECAM-125I-GOX conjugates specifically bind to both endothelial and PECAM-transfected cells. Approximately 70% of cell-bound anti-PECAM-125I-GOX was internalized. The cell-bound conjugate was enzymatically active and generated H2O2 from glucose. Use of the fluorescent dye dihydrorhodamine 123 revealed that 70% of H2O2 was generated intracellularly, whereas 30% of H2O2 was detected in the cell medium. Catalase added to the cells eliminated H2O2 in the medium but had little effect on the intracellular generation of H2O2 by anti-PECAM-GOX. Both H2O2 added exogenously to the cell medium (extracellular H2O2) 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 H2O2 or anti-PECAM-GOX. Catalase added to the medium protected either normal or aminotriazole-treated cells against extracellular H2O2, 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 H2O2 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.
hydrogen peroxide; drug delivery; bioconjugation; CD31; platelet-endothelial cell adhesion molecule-1
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INTRODUCTION |
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OXIDATIVE STRESS in endothelial cells plays an important role in cardiovascular and lung diseases (3, 10, 12, 21). Endothelium, particularly pulmonary endothelium, is predisposed to injury by reactive oxygen species (ROS) and their derivatives. The major factors involved in pulmonary oxidative endothelial injury include 1) exposure to high levels of environmental oxygen, ROS (e.g., ozone), and compounds that induce ROS generation in the lung (e.g., smoke) (6); 2) a pulmonary vasculature that is a target for sequestration of activated leukocytes that release ROS (3, 9, 10); and 3) pulmonary endothelial cells as well as other endothelial cells that are capable of ROS generation (5, 7, 11, 26, 32).
The effect of ROS generation is critically dependent on the available targets for the reaction, which are determined by both the reaction rate and the local concentrations of these potential reactants. Presumably, the available targets for the reaction will vary between the intracellular and extracellular environments. Because they interact with and modify different targets, it is likely that ROS generated internally and externally have different effects in cells and cause different forms of oxidative stress.
The aim of the present study was to construct a model to study an oxidative stress induced by regulated intracellular flux of a defined ROS in a cultured cell system. We hypothesized that this could be achieved by the intracellular delivery of an ROS-generating enzyme. To deliver such an enzyme in a cell-specific manner (allowing for in vivo applications), we utilized an immunotargeting strategy. In this strategy, an active compound (e.g., an ROS-generating enzyme) is conjugated with an antibody directed against a specific surface antigen on the target cell. In the present study, we used the anti-platelet-endothelial cell adhesion molecule (PECAM), a monoclonal antibody directed against PECAM-1. PECAM-1, or CD31 antigen, is a 130-kDa glycoprotein constitutively expressed on the endothelial surface (1, 20). Platelets also possess PECAM-1 but at a much lower level. In a previous study, Muzykantov et al. (17) have documented that endothelial cells internalize biotinylated PECAM antibodies conjugated with streptavidin (SA) and that the anti-PECAM-SA carrier provides intracellular delivery of the conjugated proteins to endothelial cells. Importantly, the anti-PECAM-SA carrier accumulates in the pulmonary vasculature in perfused rat lungs and in intact animals in vivo (17).
We postulated that conjugation of an ROS-generating enzyme with anti-PECAM-SA would provide specific binding of the enzyme to endothelial cells, facilitate its intracellular delivery (internalization), and cause intracellular generation of a defined ROS. To test this hypothesis, we 1) conjugated the H2O2-generating enzyme glucose oxidase (GOX) to a monoclonal anti-PECAM (anti-PECAM-GOX), 2) studied the properties of the conjugate in cultures of human endothelial cells and cells transfected with PECAM-1 antigen, and 3) characterized intracellular ROS generation and oxidative stress caused by the anti-PECAM-GOX conjugate.
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MATERIALS AND METHODS |
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Reagents. The following materials were
used in the study: IODO-GEN from Pierce (Rockford, IL);
Na125I and
51Cr from Amersham (Arlington
Heights, IL); fatty acid-free bovine serum albumin (BSA) from
Boehringer Mannheim (Indianapolis, IN); dimethyl formamide, a 30%
aqueous solution of
H2O2,
mouse IgG, biotinylated GOX (b-GOX), 3-amino-1,2,4-triazole (ATZ),
D-(+)-glucose, Triton X-100, and
components of the buffer solutions from Sigma (St. Louis, MO);
lyophilized bovine liver catalase (activity 20,000 U/mg) from Fluka
(Ronkonkoma, NY); and SA, goat antibodies against mouse IgG, and
6-biotinylaminocaproic acid
N-hydroxysuccinimide ester (BxNHS)
from Calbiochem (San Diego, CA). Protein concentration was determined
with a Bio-Rad (Hercules, CA) microassay kit. A monoclonal antibody to
human PECAM-1, MAb 62 (provided by Dr. Marian Nakada, Centocor,
Malvern, PA), is a mouse monoclonal IgG2 reacting with the first
Ig-like loop of human and rat PECAM-1 (17). Soluble purified PECAM (a
chimeric Ig-CD31 fusion protein) was generously provided by Dr. Peter
Newman (Blood Center of Southeastern Wisconsin, Milwaukee).
Modification of proteins and conjugation of b-GOX with anti-PECAM. Immunoglobulins were biotinylated at a 10-fold molar excess of the biotinylating reagent BxNHS as previously described (14, 18). The biotinylated proteins will be designated as b-IgG, b-MAb, and b-GOX. b-GOX was labeled with 125I with IODO-GEN-coated tubes according to the manufacturer's recommendation. To construct the trimolecular heteropolymer complex b-IgG-SA-b-GOX, we used a two-step procedure with other enzymes established in our laboratory (14, 15). Briefly, in the first step, SA and b-GOX were mixed at a molar ratio of SA to b-GOX of 5 and incubated for 1 h on ice to form a bimolecular complex, SA-b-GOX. In the second step, the SA-b-GOX complex was incubated with b-anti-PECAM or b-IgG to form the trimolecular complex b-anti-PECAM-SA-GOX or its nonimmune counterpart b-IgG-SA-GOX. In the preliminary experiments, we determined that an SA-to-b-GOX molar ratio of 5 provides optimal conditions for the further conjugation with biotinylated murine immunoglobulins. The enzymatic activity of GOX conjugated with either the immune or nonimmune carrier did not differ from that of the initial preparation of b-GOX (~100 U/mg). These conjugates will be designated as anti-PECAM-GOX and IgG-GOX, respectively.
Binding of anti-PECAM-GOX to immobilized PECAM-1 and detection of generation of H2O2. To confirm that the conjugation procedure yields a trimolecular complex (b-antibody-SA-b-GOX), we determined its binding in wells coated with a goat polyclonal antibody to murine IgG (anti-IgG). First, plastic wells of a 96-well plate were incubated overnight at 4°C with 100 µl of borate-buffered saline (BBS; pH 8.1) containing 0.1 mg/ml of anti-IgG. After being washed, the wells were incubated for 1 h at room temperature with BBS containing 2 mg/ml of BSA (BBS-BSA) to block the sites for nonspecific binding. Control wells were coated with albumin instead of anti-IgG. Anti-PECAM-GOX (10 µg/well in BBS-BSA) was incubated in the wells for 1 h at room temperature. After elimination of nonbound material, 100 µl of a 10 mM glucose solution in PBS was incubated in the wells to provide bound GOX with a substrate for H2O2 generation. At the indicated time, 100 µl of a substrate solution (10 µg/ml of peroxidase and 1 mg/ml of o-phenylenediamine in PBS) were added to the wells. After the 5-min reaction was terminated with sulfuric acid, absorbance at 495 nm was determined in an ELISA reader.
To determine the antigen-binding capacity of the conjugate, the chimeric Ig-CD31 protein was immobilized in the wells as described above for the anti-IgG. Incubation with the conjugates, elimination of nonbound material, incubation with glucose, and detection of H2O2 generated in the wells were performed as described above.Binding and internalization of
anti-PECAM-125I-GOX in cell culture.
We utilized two cell types to address the binding and uptake of
anti-PECAM: 1) human umbilical vein
endothelial cells (HUVECs) and 2)
transformed human mesothelioma REN cells transfected with human PECAM-1
cDNA (REN/PECAM cells) (1). Nontransfected REN cells were used as
PECAM-negative control cells (28). Binding and internalization of the
radiolabeled anti-PECAM-GOX conjugate in cell culture were studied as
previously described (16). Cells were subcultured in 24-well plates for
2-3 days to reach confluence. For an estimation of cellular
binding, the conjugates containing 125I-GOX were added to washed
cells (1 µg/well) in Hanks' buffer solution containing 0.2% BSA and
incubated for the indicated time at 37°C. After being washed with
buffer, the cells were lysed with 0.1% Triton X-100, and radioactivity
in the cell lysates was determined with a gamma counter. To determine
the internalization of the conjugated GOX, the cells were incubated at
37°C with conjugates containing
125I-GOX for 90 min at 37°C.
After being washed to remove unbound radioactivity, the cells were
incubated with 50 mM glycine and 100 mM NaCl, pH 2.5 (15 min at room
temperature) to release surface-associated antibody. There was no
detectable cell detachment or visible morphological changes after
treatment with glycine buffer as determined by light microscopy. After
collection of the glycine eluates, the cells were detached by
incubation with trypsin-EDTA. Surface-associated radioactivity (i.e.,
radioactivity of the glycine eluates) and cell-associated radioactivity
(i.e., radioactivity of cell lysates) were determined with a gamma
counter. The percentage of internalization was calculated as
[(total radioactivity glycine eluted) × 100]/total radioactivity.
Detection of intracellular and extracellular H2O2 generated by the cell-bound anti-PECAM-GOX in REN/PECAM cells. To detect and localize H2O2 in cell culture, we used the oxidant-sensitive fluorescent dye dihydrorhodamine (DHR) 123. DHR is not readily oxidized by the superoxide anion, but it is rapidly oxidized by peroxynitrite and the hydroxyl radical as well as by H2O2 in the presence of peroxidases (25). In our experiments, we incubated the cells with 15 µM DHR for 30 min before treatment with the conjugate. Nonbound extracellular dye was removed by washing. DHR-loaded cells were incubated with anti-PECAM-GOX (10 µg/well in Hanks' buffer containing 5 mg/ml of BSA, pH 7.4). After elimination of nonbound conjugate, the cells were further incubated for 30 min at 37°C with RPMI medium containing 10 mg/ml of glucose and inspected in a fluorescent microscope with a triple-band dichroic mirror (D/F/R-BS&M, Chroma Technology, Brattleboro, VT), with a wide-range rhodamine red filter providing good resolution from green (excitation 560 nm and emission 630 nm). To avoid potential artifacts associated with photoexposure (e.g., photobleaching), the cells were exposed to exciting light for time intervals of 10-15 s. The conditions were consistent when all images presented were taken.
Afterward, supernatants and cell lysates were collected, and rhodamine fluorescence (excitation at 510 nm and emission at 529 nm) was determined with a spectrofluorimeter. Background fluorescence in control wells containing DHR 123-labeled cells was 220 ± 21 arbitrary fluorescence units (AFU). Bovine liver catalase (50 µg/well) was added in some wells to degrade H2O2 in the extracellular medium.Determination of cytotoxicity of anti-PECAM-GOX or
H2O2 added to the
cells.
Cell death in culture was determined by the specific release of
51Cr. Reports from our laboratory
(19) and another group (4) document that this assay permits an accurate
and quantitative assessment of oxidant-induced cellular death (more
precisely, irreversible damage to the plasma membrane) comparable with
the results obtained with other methods of cell death detection, for example, trypan blue staining. To label the cells,
51Cr isotope (~200,000
counts · min1 · well
1)
was added 24 h before the experiment. The cells were washed with
Hanks' buffer to eliminate nonbound isotope, incubated with the
indicated amount of anti-PECAM-GOX or IgG-GOX conjugate for 1 h at
37°C in Hanks' buffer containing 5 mg/ml of BSA, and washed again
to eliminate nonbound conjugates. Afterward, cell medium (medium 199 for HUVECs or RPMI medium for REN and REN/PECAM cells) supplemented
with 10 mg/ml of glucose was added to the wells. At the indicated time,
aliquots of the supernatants were collected, and radioactivity was
determined. After 20 h of incubation of the cells in a
CO2 incubator at 37°C, total
radioactivity in the wells was determined by collecting the
supernatants and cell lysates. 51Cr release was determined as the
percent radioactivity in the supernatants.
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RESULTS |
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Antigen-binding capacity and enzymatic activity of
anti-PECAM-GOX conjugate. To demonstrate that SA
cross-links murine b-MAb (anti-PECAM) and enzymatically active b-GOX,
we first incubated anti-PECAM-GOX conjugate in wells
coated with anti-IgG. Control wells were coated with BSA. As shown in
Fig. 1A,
subsequent incubation with a glucose solution led to
H2O2
generation only in wells containing anti-PECAM-GOX bound to anti-IgG
and not in control wells. No H2O2
was detected in the wells coated with anti-IgG or BSA and incubated
with unconjugated GOX.
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To characterize the antigen-binding activity of the conjugate, we incubated the anti-PECAM-GOX or its nonimmune counterpart IgG-GOX in wells coated with either purified PECAM-1 (in the form of a PECAM-Ig fusion protein) or BSA. As shown in Fig. 1B, subsequent incubation with a glucose solution led to H2O2 generation only in the PECAM-coated wells containing anti-PECAM-GOX and not in the control wells. IgG-GOX produced no H2O2 in the wells coated with either PECAM or BSA, thus confirming the specificity of anti-PECAM-GOX binding to immobilized PECAM.
These results indicate that the conjugation procedure yields a trimolecular complex (anti-PECAM-GOX conjugate) composed of an active murine b-MAb to PECAM-1, SA cross-linker, and enzymatically active b-GOX.
Immunotargeting of anti-PECAM-GOX to PECAM-expressing
cells. To examine the specific targeting of
anti-PECAM-GOX to PECAM-expressing cells, we determined the uptake of
the conjugate by a human endothelial-like cell line (REN cells derived
from a malignant mesothelioma) that does not normally express PECAM-1
(REN/PECAM) as well as by REN cells genetically modified to
express PECAM-1 (REN/PECAM+).
To determine the binding specificity, we incubated cells with
anti-PECAM-125I-GOX or
IgG-125I-GOX. As shown in Fig.
2A,
significant binding occurred when anti-PECAM-125I-GOX was added to
REN/PECAM+ cells but not to control REN cells. IgG-125I-GOX did not bind to
either cell type. Importantly, elution of the surface-bound
125I-GOX by acidic buffer showed
that 71 ± 8% of the
anti-PECAM-125I-GOX that bound to
the REN/PECAM+ cells was internalized after 90 min of incubation at
37°C.
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To determine the enzymatic activity of the bound conjugates, REN/PECAM+
or REN/PECAM cells were incubated with anti-PECAM-GOX or Ig-GOX
conjugates, exposed to glucose for 60 min, and then lysed. Figure
2B shows that
H2O2
was detected in the lysates of REN/PECAM+ cells treated with
anti-PECAM-GOX but not with IgG-GOX. There was no significant
H2O2
production in control REN/PECAM
cells treated with either conjugate.
To assess the biological consequences of anti-PECAM-GOX targeting,
REN/PECAM+ and REN/PECAM cells were first loaded with 51Cr and then reacted with either
anti-PECAM-GOX or IgG-GOX. Analysis of
51Cr release revealed that after
exposure to glucose-containing medium, anti-PECAM-GOX caused a
significant increase in cell death in REN/PECAM+ cells but not in
control REN cells (Fig. 2C). The results of 51Cr release were
confirmed by observation of the morphological signs of cellular death,
including shape changes, vacuolization of the cytoplasm, disruption of
the cellular monolayer, and cell detachment. In contrast,
51Cr release from either cell type
treated with IgG-GOX was indistinguishable from the control level (14 ± 5%). Neither nonconjugated anti-PECAM nor b-antiPECAM-SA
conjugated with biotinylated ferritin or catalase caused cellular
damage or death in HUVECs or in REN cells transfected with PECAM (data
not shown). In addition, nonconjugated anti-PECAM did not affect the
sensitivity of REN/PECAM cells to a reagent, H2O2.
These data indicate that the cytotoxic effect of anti-PECAM-GOX is
mediated by the enzymatic activity of GOX.
To confirm that the cytotoxic effect of anti-PECAM-GOX was dependent on enzymatic conversion of glucose to H2O2, 51Cr-labeled cells were treated with the conjugate and then exposed to either glucose-containing or glucose-free medium. Significant 51Cr release was seen only in the presence of glucose (Fig. 2D), indicating that a lack of glucose did not cause cell death and that glucose was necessary for the toxic effects of the conjugate to occur. Therefore, anti-PECAM-GOX causes a cytotoxic effect via enzymatic conversion of glucose to H2O2, but glucose starvation is not responsible for the conjugate toxicity.
The study of transfected cells versus nontransfected cells offers a valuable system to define specificity of the targeting and the effects of anti-PECAM-GOX. However, although the REN/PECAM+ cells displayed levels of PECAM expression and localization in the plasma membrane similar to those in endothelial cells (1), they may not behave identically to endothelial cells with regard to their sensitivity to H2O2 and their ability to bind and process the conjugate. We therefore repeated these experiments with cultured HUVECs. Although the absolute values of the conjugate binding varied (depending on the HUVEC donors, passage, density, and confluence of the culture), binding of anti-PECAM-125I-GOX was markedly higher than that of IgG-125I-GOX (for example, 5.3 ± 0.2 vs. 1.3 ± 0.2 ng/well in HUVECs of fourth passage). Similar to the REN/PECAM+ cells, 60 ± 4% of anti-PECAM-125I-GOX bound to HUVECs was internalized after 90 min of incubation at 37°C. Again, like in the REN/PECAM+ cells, cell-bound anti-PECAM-GOX generated H2O2 in glucose-containing medium and caused cellular damage: 84 ± 7% of 51Cr release was detected in cells treated with anti-PECAM-GOX vs. 26 ± 3% of 51Cr release in cells treated with IgG-GOX.
Therefore, these results show that anti-PECAM-GOX specifically binds to PECAM-positive target cells, enters the cells, generates H2O2 from glucose, and kills the target cells. Because anti-PECAM-GOX had similar effects in REN/PECAM+ cells and HUVECs, we utilized the more uniform REN/PECAM+ cells in the remainder of our studies.
Intracellular generation of H2O2 by anti-PECAM-GOX. Although the experiments described in Immunotargeting of anti-PECAM-GOX to PECAM-expressing cells indicated that the majority of the cell-bound anti-PECAM-GOX was internalized and that the cell-bound conjugate generated H2O2, these studies did not exclude the theoretical possibility that only the noninternalized conjugate is active and H2O2 was produced extracellularly.
To define the site of H2O2 generation, we used the dye DHR 123 as a probe (25). DHR forms a fluorescent dye, rhodamine, when oxidized by H2O2 in the presence of peroxidases or metals. DHR is also rapidly oxidized by peroxynitrite but not by superoxide anion. Therefore, its oxidation to rhodamine is indicative of the production of ROS with a high oxidative potential. DHR is particularly useful in the monitoring of intracellular oxidant production. Both DHR and its oxidation product rhodamine are membrane permeable. However, unlike DHR, rhodamine is a charged lipophilic compound and therefore is retained in negatively charged intracellular compartments such as the mitochondrion (25). REN/PECAM+ cells loaded with DHR were incubated with anti-PECAM-GOX for 1 h at 37°C to allow for conjugate uptake by the cells. The cells were then washed and incubated in RPMI medium containing 10 mg/ml of glucose for a further 30 min in either the absence or presence of catalase (1,000 U) in the medium. In parallel wells, nonconjugated GOX (extracellular GOX; 1 µg/well) was added to REN/PECAM+ cells in glucose-containing medium to generate H2O2 extracellularly in the presence and absence of catalase in the medium. The results of this experiment were assessed qualitatively by observation with epifluorescence microscopy. Intracellular rhodamine fluorescence was observed in the wells treated with anti-PECAM-GOX (Fig. 3A). In contrast, diffuse fluorescence was seen in the medium but not in the cells treated with extracellular GOX, indicating that H2O2 was produced primarily in the medium (Fig. 3C). The addition of catalase to the medium had no detectable effect on the intracellular fluorescence in the PECAM-GOX-treated cells (Fig. 3B), whereas it diminished fluorescence in the cell medium produced by the addition of extracellular GOX (Fig. 3D). This effect of catalase confirms the intracellular location of H2O2 generated by anti-PECAM-GOX.
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Role of intracellular catalase in protection against oxidative stress caused by extracellular H2O2 or anti-PECAM-GOX. To further characterize the cytotoxic effects of extracellular H2O2 versus anti-PECAM-GOX, we determined the release of 51Cr from labeled cells as a measure of irreversible plasma membrane damage (4). Use of this technique in conjunction with ATZ treatment (a potent irreversible inhibitor of catalase) allowed us to examine the role of cellular catalase in the defense against these forms of oxidative stress.
First, we characterized the cytotoxic effect of extracellular H2O2 added to the cell medium. Figure 5A shows that 30 mM H2O2 caused a rapid and massive 51Cr release, whereas 1 mM H2O2 caused a delayed 51Cr release evident only after an overnight incubation of REN-PECAM+ cells exposed to H2O2. Reports from several groups (4, 19) documented that 1 mM H2O2 causes marked cell death in HUVECs as soon as a few hours postexposure. Thus our data imply that REN/PECAM+ cells are less sensitive to H2O2 than HUVECs. Pretreatment of the cells with ATZ accelerated the time course and increased the amplitude of 51Cr released (Fig. 5B). This result indicates that intracellular catalase plays an important role in the defense against extracellular H2O2 that diffuses through the plasma membrane into the intracellular compartment. This is illustrated more graphically by comparison of 51Cr release at 3 h in ATZ-treated versus control cells (Fig. 5C). Under these conditions, 10 mM H2O2 caused no detectable 51Cr release in control cells and a marked 51Cr release in ATZ-treated cells.
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Role of extracellular catalase in protection against oxidative
stress caused by extracellular
H2O2 or
anti-PECAM-GOX.
To define the role of extracellular generation of
H2O2
in the cellular toxicity of anti-PECAM-GOX conjugate,
51Cr-labeled REN/PECAM+ cells were
exposed to medium (control), anti-PECAM-GOX, or 10 mM extracellular
H2O2
in the absence and presence of extracellular catalase (Fig.
7). When the cells were examined after 9 h
of incubation in glucose-containing medium (Fig.
7A), catalase added to the medium
abrogated 51Cr release caused by
either anti-PECAM-GOX or 10 mM extracellular H2O2.
This result suggests that
H2O2
generated by the surface-bound portion of the conjugate (see Fig. 4B)
accumulated in the medium and accelerated the cytotoxic effects of
anti-PECAM-GOX. Importantly, extracellular catalase had no significant
effect on the late manifestations of cellular injury seen 20 h after
incubation with cell-bound anti-PECAM-GOX in glucose-containing medium
(Fig. 7B). This result supports the
conclusion that a major portion of
H2O2
generated by anti-PECAM-GOX is intracellular and thus is inaccessible
for the extracellular catalase. In contrast, catalase completely
abrogated the late manifestations of the cytotoxic effect of
extracellular H2O2.
Therefore, in the absence of catalase in the medium, anti-PECAM-GOX causes oxidative stress with both intracellular and extracellular components. Catalase added to the medium degrades extracellular H2O2,
diminishes an extracellular component, and delays manifestation of the
oxidative stress and cell injury.
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DISCUSSION |
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The causes, mechanisms, manifestations, and methods for therapy of oxidative stress have been the focus of enormous interest for several decades. The overwhelming antioxidant defenses or the pathological overproduction of ROS result in modification and inactivation of functionally or structurally important components in cells and in the extracellular milieu, thus leading to tissue injury, cell death, or functional abnormality.
The site of ROS formation determines the targets of oxidative injury and is critical for their action. For example, more reactive ROS (e.g., the hydroxyl radical formed from H2O2) have a very limited radius of action because they react promiscuously with target molecules in close proximity to the ROS formation site. Less reactive ROS (such as H2O2) are more diffusable, although the radius of their action is limited due to degradation by enzymes (e.g., catalase) or scavengers (e.g., GSH). Intracellularly generated ROS attack interior cellular components and the inner leaflet of the plasma membrane, whereas extracellularly generated ROS attack blood elements, glycocalix components, surface proteins, and the outer leaflet of the plasma membrane. Therefore, intracellular and extracellular ROS may cause different effects and forms of oxidative stress in the cells.
Both extracellular and intracellular ROS may cause endothelial oxidative stress in vivo. For example, activated leukocytes adhere to endothelium at the site of inflammation and release ROS. Although the ROS released from the activated leukocytes are initially extracellular, they may act as intracellular ROS after diffusion through the endothelial plasma membrane. Extracellular oxidative stress in endothelium can be studied in cell culture by direct exposure of endothelial cells to ROS (such as H2O2) or ROS-generating enzymes (such as GOX or xanthine oxidase) or by addition of activated leukocytes to the cell culture medium (4, 19, 24). It also may be modeled in vivo by systemic activation of circulating leukocytes in animals (9).
Another scenario is the oxidative stress caused by excessive ROS
generation within the endothelium. Endothelial cells can generate ROS
(e.g.,
H2O2
or O2) via enzymatic pathways
including 1) xanthine oxidase formed
from xanthine dehydrogenase on hypoxia (24, 32);
2) lipoxygenase and cyclooxygenase
activated by hormones, cytokines, or inflammatory mediators (7, 23); 3) univalent reduction of molecular
oxygen by respiratory chain enzymes in the mitochondria (22, 26);
4) uncoupled superoxide anion
generation by endothelial nitric oxide synthase (23); and
5) NADPH oxidase and/or NADH oxidase
activated on ischemia and/or by cytokines (13, 31). The first
four of these enzymatic pathways generate ROS intracellularly, whereas
the latter one is postulated to generate ROS in close proximity to the
plasma membrane. Therefore, a substantial (perhaps major) fraction of endothelium-derived ROS is generated inside the endothelium
(intracellular ROS). The amplitude of ROS generation by endothelial
cells is lower than that of leukocytes, but the duration of endothelial ROS generation may be longer.
Intracellular oxidative stress in the endothelium has been difficult to
study, although numerous approaches have been described. Chemicals such
as quinones (e.g., menadione) enter cells and cause ROS generation (2,
27). Anoxia-reoxygenation, cytokines, phorbol 12-myristate 13-acetate,
growth factors, bradykinin, and lipoproteins stimulate ROS generation
by the endothelium in culture (7, 11, 23, 29-31). These models,
however, have certain limitations. First, commonly used
(patho)physiological inducers of ROS generation, such as cytokines,
bradykinin, or hypoxia, cause complex alterations in the endothelium.
Therefore, it is difficult to dissect out the effects due directly to
ROS versus ROS-unrelated and/or secondary effects of ROS inducers.
Second, most of these inducers cause concomitant generation of several ROS (i.e., both
H2O2
and O2), thus confusing the analysis
of the role for a particular ROS. Third, there are no known methods to
provide a controlled rate of intracellular flux of ROS. Finally, none
of them is specific to the endothelium. For example, injection of
cytokines or quinone would cause ROS generation by a variety of cell
types, including leukocytes. Therefore, these previously described
strategies would not be applicable to study cell-specific endothelial
intracellular oxidative stress in vivo.
In the present study, we have developed a novel approach that allows intracellular generation of a defined ROS, H2O2, within endothelial cells by using intracellular immunotargeting of GOX. Our results indicate that GOX conjugated with a monoclonal antibody to the endothelial cell surface antigen PECAM-1 binds selectively to endothelial cells, enters the cells, generates H2O2 intracellularly, and induces an oxidative stress, which leads to irreversible damage to the endothelial plasma membrane. Intracellular catalase plays an important role in the cellular defense against anti-PECAM-GOX. It also protects cells against extracellular H2O2, presumably via degradation of H2O2 diffusing through the plasma membrane into the intracellular compartment. Oxidative stress induced by anti-PECAM-GOX markedly differs from that induced by extracellular H2O2: extracellular catalase fails to protect cells against anti-PECAM-GOX, whereas it effectively protects either normal or ATZ-treated cells against extracellular H2O2.
Failure of extracellular catalase to provide protection against anti-PECAM-GOX, taken together with fluorescent visualization of the intracellular H2O2 generated by anti-PECAM-GOX, provides strong evidence that the internalized conjugate is enzymatically active. This is the first direct experimental evidence of the intracellular delivery of an active ROS-generating enzyme. This result, therefore, provides important support for the strategy of intracellular delivery of enzymes (and perhaps other compounds and drugs) by immunotargeting (15-17).
Although anti-PECAM-GOX was readily internalized, ~30% of the cell-bound conjugate remained on the cell surface and produced H2O2 in the extracellular compartment. Analysis of DHR fluorescence showed that ~30% of the total H2O2 generated by the cell-bound anti-PECAM-GOX was also located in the extracellular compartment. Thus production of H2O2 by the surface-associated conjugate and/or diffusion of H2O2 from the intracellular to the extracellular compartment occurs in our model. In the absence of extracellular catalase, both processes lead to accumulation of extracellular H2O2. Because extracellular H2O2 is not decomposed by the cellular antioxidant defense (unless it diffuses back to the intracellular compartments), its concentration may reach toxic levels. Thus extracellularly accumulated H2O2 contributes to the oxidative stress, augments the toxic effect of intracellular H2O2, and aggravates cellular injury caused by anti-PECAM-GOX.
In the presence of extracellular catalase, the manifestation of the conjugate cytotoxicity is delayed because anti-PECAM-GOX requires longer time to generate enough purely intracellular H2O2 to cause cell death. In addition, lower doses of H2O2 require a longer time to reveal the toxicity (Fig. 5). We conclude that extracellular catalase allowed us to model a pure intracellular oxidative stress caused by anti-PECAM-GOX. Under physiological conditions, the endothelial extracellular luminal compartment is blood, a tissue possessing an extremely high antioxidant potential. Thus cell culture experiments with anti-PECAM-GOX in the presence of catalase in the cell medium adequately models the situation in vivo. Such an adequate design is not possible in cell culture experiments with exposure of cells to extracellular ROS because antioxidants in the cell medium degrade these readily accessible ROS and completely abrogate oxidative stress. Therefore, intracellular immunotargeting of ROS-generating enzymes may permit a better approximation to pathophysiological conditions in cell culture experiments.
In addition to providing a way to study a defined intracellular
oxidative enzyme in endothelial cell culture, two key features of
immunotargeting (specific recognition of the endothelial cells and
intracellular delivery of ROS-generating enzymes to the vascular endothelium) allow application of this strategy in intact animals. We
have recently found that an intravenous injection of anti-PECAM-GOX, but not of IgG-GOX, in mice causes oxidative vascular injury in the
pulmonary endothelium (M. Christofidou-Solomidou, G. Pietra, E. Argiris, D. Harshaw, G. FitzGerald, S. Albelda, and V. Muzykantov, unpublished data). Therefore, immunotargeting offers a new modality for
the investigation of the intracellular oxidative stress in endothelial
cells in vivo. Immunotargeting of xanthine oxidase (that generates both
O2 and
H2O2)
or other ROS-generating enzymes may help to define the contribution of
a specific ROS in the stress and will provide further flexibility to
the models.
Like other approaches, an immunotargeting strategy has some potential limitations. First, intracellular generation of ROS without concomitant cellular alterations caused by ROS inducers (e.g., cytokines) represents a significant departure from pathophysiological settings. We view this as an inevitable "cost" for a well-defined experimental system allowing us to isolate a role for a specific intracellular ROS.
Although GOX represents a relatively simple system for ROS generation, glucose and H2O2 are not the only components of the reaction. GOX consumes oxygen along with glucose and coproduces uric acid with H2O2. Uric acid is a weak antioxidant; hence it unlikely contributes to the cytotoxic effect of the conjugate. More importantly, 1) GOX may cause hypoxia via oxygen consumption and 2) the tissue level of oxygen may modulate enzymatic activity of anti-PECAM-GOX. Both factors may potentially confuse the results of experiments in cell culture or in vivo. At the present time, in collaboration with Dr. Donald Buerk (University of Pennsylvania, Philadelphia), we are directly characterizing the levels of H2O2 and oxygen in glucose-GOX mixtures utilizing selective electrodes. The goal of this ongoing study is to quantify the efflux of H2O2 in the cells as well as the consumption of oxygen and regulation of enzymatic activity of GOX (e.g., inhibition by the products). Preliminary results of our measurements indicate that 1 µg of GOX causes no more than a 10% reduction of oxygen level in the presence of 10 mg/ml of glucose. Apparently, oxygen diffusion compensates for the further decline in oxygen level and diminishes the "hypoxic" effect of anti-PECAM-GOX. The amount of cell-associated anti-PECAM-GOX in our experiments does not exceed 0.5-0.7 µg/well. We conclude, therefore, that oxygen consumption plays only a minor, if any, role in anti-PECAM-GOX action. Also, results of our in vivo study (Cristofidou-Solomidou et al., unpublished data) directly indicate that anti-PECAM-GOX does cause an oxidative lung injury in the pulmonary vasculature. Most likely, continued ventilation and oxygen transport/diffusion compensate for a slow consumption of oxygen by anti-PECAM-GOX.
The amplitude of ROS generation by the intracellularly delivered enzymes may exceed that occurring at physiological and even pathological conditions in vivo. This represents a potential problem because the supraphysiological levels of H2O2 in model studies require careful interpretation. The actual levels of ROS generation by the endothelium in various pathological settings are still to be determined. Utilization of radiolabeled GOX or xanthine oxidase may help to quantitate the amount of ROS-generating enzymes delivered to the target cells (either in cell culture or in vivo) and may thus provide a correlation between the effects of ROS-generating enzymes and the rate of ROS flux.
The issue of dose effects may be particularly interesting when one begins to differentiate the cytotoxic effects of intracellular H2O2 from lower doses that may affect signaling pathways. H2O2 is becoming increasingly recognized as a signal transduction molecule with widespread effects on the cytoskeleton, arachidonic acid metabolism, antioxidant defenses, apoptosis, and other physiological parameters of endothelial cells. Delivery of lower amounts of the conjugates coupled with careful measurements of H2O2 production may provide a very useful system for studying signaling events.
Finally, we can envision endothelial GOX immunotargeting as a potential therapeutic agent. For example, by replacing the anti-PECAM carrier with an antibody recognizing antigens upregulated in the tumor vasculature (i.e., the vascular endothelial cell growth factor receptor), one could potentially generate an agent useful for the eradication of tumors. This approach could complement antitumor therapy based on the immunotargeting of toxins or procoagulants to the tumor vascular endothelium (8).
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ACKNOWLEDGEMENTS |
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We thank Drs. A. Fisher, H. Ischiropoulos, G. FitzGerald, and E. Manevich (University of Pennsylvania, Philadelphia) for numerous discussions, reading of the manuscript, helpful comments, and suggestions. We are grateful to Dr. D. Buerk for experiments with an oxygen electode.
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FOOTNOTES |
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V. R. Muzykantov was supported by American Heart Association Established Investigator Grant 9640204N and National Heart, Lung, and Blood Institute (NHLBI) Specialized Center of Research in Acute Lung Injury Grant HL-60290 (Project 4). A. J. Gow was supported by a grant from the ALS Association and NHLBI National Research Service Award HL-07748.
Present address of A. J. Gow: Medical Research Bldg. Rm. 317, Duke Univ. Medical Center, Box 2612, Durham, NC 27713.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: V. R. Muzykantov, IFEM, Univ. of Pennsylvania Medical Center, 1 John Morgan Bldg., 36th St. and Hamilton Walk, Philadelphia, PA 19104-6068 (E-mail: muzykant{at}mail.med.upenn.edu).
Received 13 November 1998; accepted in final form 22 March 1999.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Albelda, S. M.,
W. A. Muller,
C. A. Buck,
and
P. J. Newman.
Molecular and cellular properties of PECAM-1 (endoCAM/CD31): a novel vascular cell-cell adhesion molecule.
J. Cell Biol.
114:
1059-1068,
1991[Abstract].
2.
Arai, T.,
S. A. Kelley,
M. L. Brengman,
M. Takano,
E. Smith,
P. J. Goldsmith-Clermont,
and
G. B. Bulkley.
Ambient but not incremental oxidant generation effects intracellular adhesion molecule 1 induction by tumour necrosis factor alpha in endothelium.
Biochem. J.
331:
853-861,
1998[Medline].
3.
Brigham, K.,
and
B. Meyrick.
Endotoxin and lung injury.
Am. Rev. Respir. Dis.
133:
913-927,
1986[Medline].
4.
Chopra, J.,
J. Joist,
and
R. Webster.
Loss of 51chromium, lactate dehydrogenase and 111indium as indicators of endothelial cell injury.
Lab. Invest.
57:
578-584,
1987[Medline].
5.
Fisher, A. B.,
C. Dodia,
Z. Tan,
I. Ayene,
and
R. G. Eckenhoff.
Oxygen-dependent lipid peroxidation during lung ischemia.
J. Clin. Invest.
88:
674-679,
1991[Medline].
6.
Freeman, B.,
and
J. Crapo.
Hyperoxia increases oxygen radical production in rat lungs and lung mitochondria.
J. Biol. Chem.
256:
10986-10992,
1981
7.
Holland, J. A.,
K. A. Pritchard,
M. A. Pappolla,
M. S. Wolin,
N. J. Rogers,
and
M. B. Stemerman.
Bradykinin induces superoxide anion release from human endothelial cells.
J. Cell. Physiol.
143:
21-25,
1990[Medline].
8.
Huang, X.,
G. Molema,
S. King,
L. Watkins,
T. Edgington,
and
P. E. Thorpe.
Tumor infarction by antibody-directed targeting of tissue factor to tumor vasculature.
Science
275:
547-550,
1997
9.
Leff, J.,
J. Bayer,
M. Bodman,
J. Kirkman,
P. Stanley,
L. Patton,
C. Beehler,
J. McCord,
and
J. Repine.
Interleukin-1-induced lung neutrophil accumulation and oxygen metabolite-induced lung leak in rats.
Am. J. Physiol.
266 (Lung Cell. Mol. Physiol. 10):
L2-L8,
1994
10.
Louie, S.,
B. Halliwell,
and
C. Cross.
Adult respiratory distress syndrome: a radical perspective.
Adv. Pharmacol.
38:
457-490,
1997[Medline].
11.
Matsubara, T.,
and
M. Ziff.
Increased superoxide anion release from human endothelial cells in response to cytokines.
J. Immunol.
137:
3295-3298,
1986
12.
McCord, J. M.
Human disease, free radicals and oxidant/antioxidant balance.
Clin. Biochem.
26:
351-357,
1993[Medline].
13.
Mohazzab, K.,
P. Kaminski,
and
M. Wolin.
NADH oxidoreducatase is a major source of superoxide anion in bovine coronary artery endothelium.
Am. J. Physiol.
266 (Heart Circ. Physiol. 35):
H2568-H2572,
1994
14.
Muzykantov, V. R.
Conjugation of catalase to a carrier antibody via streptavidin-biotin cross-linker.
Biotechnol. Appl. Biochem.
26:
103-109,
1997[Medline].
15.
Muzykantov, V.,
E. Atochina,
H. Ischiropoulos,
S. Danilov,
and
A. Fisher.
Immunotargeting of antioxidant enzymes to the pulmonary endothelium.
Proc. Natl. Acad. Sci. USA
93:
5213-5218,
1996
16.
Muzykantov, V.,
E. Atochina,
A. Kuo,
E. Barnathan,
K. Notarfrancesco,
H. Shuman,
C. Dodia,
and
A. Fisher.
Endothelial cells internalize monoclonal antibody to angiotensin-converting enzyme.
Am. J. Physiol.
270 (Lung Cell. Mol. Physiol. 14):
L704-L713,
1996
17.
Muzykantov, V.,
M. Christofidou-Solomidou,
I. Balyasnikova,
D. Harshaw,
L. Schultz,
A. Fisher,
and
S. Albelda.
Streptavidin facilitates internalization and pulmonary targeting of anti-endothelial antibody (PECAM-1): a strategy for intraendothelial drug delivery.
Proc. Natl. Acad. Sci. USA
96:
2379-2384,
1999
18.
Muzykantov, V.,
V. Gavriluk,
A. Reinecke,
E. Atochina,
A. Kuo,
E. Barnathan,
and
A. Fisher.
The functional effects of biotinylation of anti-ACE monoclonal antibody in terms of targeting in vivo.
Anal. Biochem.
226:
279-287,
1995[Medline].
19.
Muzykantov, V.,
D. Sakharov,
S. Domogatsky,
N. Goncharov,
and
S. Danilov.
Directed targeting of immunoerythrocytes provides local protection of human endothelial cells from damage by hydrogen peroxide.
Am. J. Pathol.
128:
226-234,
1987.
20.
Newman, P. J.
The biology of PECAM-1.
J. Clin. Invest.
99:
3-7,
1997
21.
Ohara, Y.,
T. E. Peterson,
and
D. G. Harrison.
Hypercholesterolemia increases endothelial superoxide anion production.
J. Clin. Invest.
91:
2546-2551,
1993[Medline].
22.
Panus, P. C.,
R. Radi,
P. Chimney,
R. H. Lillard,
and
B. A. Freeman.
Detection of H2O2 released from vascular endothelial cells.
Free Radic. Biol. Med.
14:
217-223,
1993[Medline].
23.
Pritchard, K. A., Jr.,
L. Groszek,
D. M. Smalley,
W. C. Sessa,
M. Wu,
P. Villalon,
M. S. Wolin,
and
M. B. Stemerman.
Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion.
Circ. Res.
77:
510-518,
1995
24.
Rinaldo, J. E.,
and
H. Gorry.
Protection by deferoxamine from endothelial injury: a possible link with inhibition of intracellular xanthine oxidase.
Am. J. Respir. Cell Mol. Biol.
3:
525-533,
1990[Medline].
25.
Royal, J. A.,
and
H. Ischiropoulos.
Evaluation of 2,7-dichlorofluorescein and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells.
Arch. Biochem. Biophys.
302:
348-355,
1993[Medline].
26.
Sanders, S. P.,
J. L. Zweier,
P. Kuppusamy,
S. J. Harrison,
D. J. P. Basset,
E. W. Gabrielson,
and
J. T. Sylvester.
Hyperoxic sheep pulmonary microvascular endothelial cells generate free radicals via mitochondrial electron transport.
J. Clin. Invest.
91:
46-52,
1993[Medline].
27.
Shi, M. M.,
T. Iwamoto,
and
H. J. Forman.
-Glutamylcysteine synthetase and GSH increase in quinone-induced oxidative stress in BPAEC.
Am. J. Physiol.
267 (Lung Cell. Mol. Physiol. 11):
L414-L421,
1994
28.
Smythe, W. R.,
H. C. Hwang,
K. M. Amin,
S. L. Eck,
B. L. Davidson,
J. M. Wilson,
L. R. Kaiser,
and
S. M. Albelda.
Use of recombinant adenovirus to transfer the herpes simplex virus thymidine kinase (HSVTk) gene to thoracic neoplasms: an effective in vitro drug sensitization system.
Cancer Res.
54:
2055-2059,
1994[Abstract].
29.
Terada, L.
Hypoxia-reoxygenation increases O2 efflux which injures endothelial cell by an extracellular mechanism.
Am. J. Physiol.
270 (Heart Circ. Physiol. 39):
H945-H950,
1996
30.
Thannickal, V. J.,
P. M. Hassoun,
A. C. White,
and
B. L. Farndurg.
Enhanced rate H2O2 of release from bovine pulmonary artery endothelial cells induced by TGF-1.
Am. J. Physiol.
265 (Lung Cell. Mol. Physiol. 9):
L622-L626,
1993
31.
Zulueta, J. J.,
R. Sawhney,
F. S. Yu,
C. C. Cote,
and
P. M. Hassoun.
Intracellular generation of reactive oxygen species in endothelial cells exposed to anoxia-reoxygenation.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L897-L902,
1997
32.
Zweier, J. L.,
P. Kuppusamy,
and
G. A. Lutty.
Measurement of endothelial cell free radical generation: evidence for a central mechanism of free radical injury in postischemic tissues.
Proc. Natl. Acad. Sci. USA
85:
4046-4050,
1988[Abstract].