Role of Mitochondrial Oxidant Generation in Endothelial Cell Responses to Hypoxia
Endothelial cells increase their secretion of the cytokine interleukin-6 (IL-6) during hypoxia, which then acts in an autocrine fashion to increase the permeability of cell monolayers. These responses are attenuated by antioxidants, suggesting that reactive oxygen species (ROS) participate in signaling in hypoxic endothelium. We tested whether mitochondria are responsible for these ROS in human umbilical vein endothelial cells exposed to hypoxia. Oxidation of the probe 2′, 7′-dichlorodihydrofluorescein to fluorescent dichlorofluorescein or the probe dihydroethidium was used to assess oxidant signaling, whereas permeability was assessed by using transendothelial electrical resistance. Hypoxia elicited increases in dichlorofluorescein and dihydroethidium fluorescence that were abrogated by the mitochondrial electron transport (ET) inhibitors rotenone (2 μmol/L) and diphenyleneiodonium (5 μmol/L). The same ET inhibitors also attenuated hypoxia-induced increases in nuclear factor-κB (NF-κB) activation, although they did not abrogate NF-κB activation in response to endotoxin (lipopolysaccharide). ET inhibition also abolished the hypoxia-induced increases in IL-6 mRNA expression, hypoxia-stimulated IL-6 secretion into the media, and the hypoxia-induced increases in transendothelial electrical resistance of human umbilical vein endothelial cell monolayers. By contrast, the above responses to hypoxia were not significantly affected by treatment with the NAD(P)H oxidase inhibitor apocynin (30 μmol/L), the xanthine oxidase inhibitor allopurinol (100 μmol/L), or the NO synthase inhibitor N-nitro-l-arginine (100 μmol/L). We conclude that ROS signals originating from the mitochondrial ET chain trigger the increase in NF-κB activation, the transcriptional activation of IL-6, the secretion of IL-6 into the cell culture media, and the increases in endothelial permeability observed during hypoxia.
- reactive oxygen species
- human umbilical vein endothelial cells
- signal transduction
Increases in the expression of adhesion molecules on vascular endothelial cells occur in a variety of inflammatory states,1 along with increases in vascular permeability.2,3⇓ These responses are essential for increases in the adhesion of polymorphonuclear leukocytes and the regulated increases in the trafficking of inflammatory cells across the endothelial barrier. Hypoxic stress often precedes or accompanies tissue inflammation, and previous studies have shown that tissue hypoxia can induce manifestations of the inflammatory response. In this regard, hypoxia augments the upregulation of endothelial cell surface receptors and the regulated increases in endothelial barrier permeability.4–6⇓⇓ However, the mechanistic link between cellular hypoxia and the initiation of these responses has not been clearly established.
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Recent studies have suggested that reactive oxygen species (ROS) may be important intracellular signaling messengers linking tissue hypoxia to the subsequent inflammatory responses. For example, in mesenteric venules of rats exposed to systemic hypoxia, Wood et al7 found evidence of increased ROS generation that contributed to the stimulation of leukocyte–endothelial cell adhesion. We recently reported that ROS participate in the signaling responsible for the transient increase in endothelial monolayer permeability induced by continuous hypoxia.8 That study showed that interleukin-6 and interleukin-8 (IL-6 and IL-8, respectively) secretion from endothelial cells is increased during hypoxia and that IL-6 released to the media acts in an autocrine or paracrine fashion to initiate the changes in endothelial permeability. Because this increase in IL-6 secretion was attenuated by antioxidants, it appears likely that ROS function as intracellular messengers, triggering the secretion of this cytokine, and are indirectly responsible for the increase in permeability.
Endothelial cells could conceivably generate ROS from an NAD(P)H oxidase system, xanthine oxidase, or the mitochondrial electron transport (ET) chain. A growing body of evidence suggests that mitochondria respond to cellular hypoxia by paradoxically increasing their generation of ROS.9 Evidence of cellular ROS signaling was absent at O2 tensions >60 mm Hg but increased progressively as the Po2 decreased to 7 mm Hg.10 These ROS appear to initiate intracellular redox signaling and to contribute to a broad range of adaptive responses in hypoxic cells, including ischemic preconditioning in cardiomyocytes,11 hypoxia-inducible factor-1 activation,12,13⇓ nuclear factor-κB (NF-κB) activation, 14,15⇓ and p53 activation in a variety of cells.16
Therefore, we hypothesized that mitochondria in hypoxic endothelial cells may be responsible for the observed increase in intracellular ROS. The present study tested whether endothelial mitochondria increase ROS production in response to hypoxia and whether these ROS participate in an intracellular signaling process that leads to increased IL-6 transcription and release and to alterations in endothelial barrier permeability.
Endothelial cells were harvested from human umbilical veins (<24 hours after birth) by a modification of the method described by Jaffe et al.17 Briefly, umbilical veins were flushed with 120 mL HEPES-buffered saline (50 mmol/L) and incubated with 0.2% collagenase (Sigma Chemical Co) at 37°C for 15 minutes. This yielded human umbilical endothelial cells (HUVECs) that were grown to confluence on gelatin-coated T25 flasks (Becton Dickinson) in medium 199 supplemented with 10% human serum, 10% FCS, penicillin, streptomycin, and amphotericin B (Sigma) at pH 7.4 and 37°C. Cells were studied between the first and second passages after characterization of the endothelial phenotype via positive staining for CD31 (platelet–endothelial cell adhesion molecule-1) and factor VIII. After treatment with 0.1% collagenase and 0.25% EDTA, the cells were split 3:1 onto (1) gelatin-coated cell culture inserts (12-mm diameter, 3.0-μm pore size; Costar) for permeability analysis and Po2 determination and (2) gelatin-coated 60-mm dishes (Becton Dickinson) for the collection of supernatants for cytokine measurements. Alternatively, for the hypoxia-dichlorofluorescein (DCF) fluorescence, Northern blot, and electrophoretic mobility shift assay (EMSA) experiments, primary culture HUVECs obtained commercially (Clonetics) were grown in endothelial cell growth media supplemented with human recombinant epidermal growth factor (10 ng/mL), hydrocortisone (1.0 μg/mL), gentamicin (50 μg/mL), amphotericin B (50 ng/mL), bovine brain extract (3 mg/mL), and 2% (vol/vol) FBS in 75-cm2 polystyrene flasks (Corning). The cells were incubated at pH 7.4 and 37°C and were used after their second or third passage, as they approached 90% confluence. When pharmacological inhibitors were used during the experiment, the cells were preincubated with the agents for 2 hours before the start of the study. Agents were used at concentrations used previously or at the lowest concentration that produced the effect.
Induction of Hypoxia
Cells were subjected to hypoxia in a modular chamber (Billups-Rothenberg) that was flushed with a gas mixture (1% O2/5% CO2/94% N2) to produce the desired level of hypoxia (Po2 14±3 mm Hg) within 30 minutes. Samples of medium were analyzed for Po2 and pH at 3-hour intervals over 24 hours by use of an O2 phosphorescence quenching method (Oxyspot, Medical Systems) with a palladium-meso-tetra(4-carboxyphenyl)porphine probe.18 The pH was measured in aliquots of medium with the use of a blood gas analyzer (Radiometer). For the experiments in which DCF or dihydroethidium (DHE) fluorescence was measured, hypoxia was induced by flushing the head space of the T flasks with known gas mixtures at a flow rate of 100 mL/min. Gas mixtures and flow rates were controlled by a mass flow controller (GF-3, Cameron Instrument Co).
Measurement of Oxidant Signaling
ROS production in HUVECs was assessed by using the probe 2,7′-dichlorodihydrofluorescein-diacetate (DCFH-DA, 10 μmol/L, Molecular Probes). The diacetate form of DCFH is membrane permeable and was added to the medium immediately before the experiments. Inside the cell, esterases cleave the acetate groups, trapping the reduced form of the dye (DCFH) intracellularly.19 ROS in the cells induce the oxidation of DCFH, yielding the fluorescent product DCF.20 The fluorescence of the intracellular fluid was used as a measurement of the intracellular ROS generation. In other studies, the probe DHE (20 μmol/L, Molecular Probes) was used to detect oxidant signaling. When oxidized, this compound intercalates into DNA, resulting in an increase in quantum yield.21 After exposure to hypoxia, T flasks were immediately immersed in an ice bath, the cells were removed with a rubber scraper, and the supernatant was transferred to a 15-mL centrifuge tube. The supernatant was then centrifuged at 10 000g (1200 rpm) to separate the cells from the intracellular fluid. The supernatant was removed, and intact cells in the pellet were lysed by using RLT lysis buffer (350 μL, RNeasy Mini Kit, Quiagen) with β-mercaptoethanol (10 μL/mL). To ensure complete lysis, the mixture was aspirated through a 25-gauge needle. The solution was then diluted in 1 mL of distilled water, and the fluorescence was quantified by using a fluorescence spectrophotometer (excitation 488 nm and emission 530 nm for DCF, excitation 475 and emission 610 for DHE; Perkin-Elmer).
Northern Blot Analysis
HUVEC monolayers were grown to confluence and exposed to hypoxia (Po2 15 mm Hg for 12 hours) as described above. Cellular RNA was then harvested (RNeasy Mini Kit, Quiagen). Two identical membranes were created in which 2-μg samples of total RNA were electrophoresed and transferred to nylon membranes with the use of a semidry technique (Bio-Rad). Membranes were prehybridized (Sigma) at 42°C for 2 hours. The cDNA probe for either 28S rRNA or IL-6 mRNA was added to prewarmed hybridization buffer (Sigma) after the cDNA was randomly labeled (Rediprime II, Amersham) with [γ-32P]dCTP (Redivue, specific activity 3000 Ci/mmol, Amersham). Hybridization was continued overnight at 42°C, followed by washing (2× SSC and 0.1% SDS at room temperature twice for 15 minutes, then 0.2× SSC and 0.1% SDS at either 65°C or 50°C twice for 15 minutes). The signal was then detected by autoradiography.
Generation of cDNA Probes
A 28S rRNA cDNA oligonucleotide probe was acquired from Ambion. A probe for IL-6 mRNA was generated by using RNA isolated from HUVEC monolayers. Upstream (CACACAGACAGCCACTCACCTC) and downstream (GTGCCTGCAGCTTCGTCAGCTGG) oligonucleotide primers were purchased (GIBCO), and IL-6 cDNA was synthesized and amplified by using reverse transcriptase–polymerase chain reaction (Access RT-PCR System, Promega).
HUVEC monolayers cultured on gelatin-coated 60-mm plates were exposed to hypoxia as described above. Culture medium was collected at 3-hour intervals over 24 hours. Hypoxia-induced production of IL-6 was assessed by ELISA (R&D Systems). Each sample was measured in duplicate and is expressed as an average of these values.
Measurement of TEER as an Index of Permeability
First-passage endothelial cells were split 3:1 to 12-mm Transwell tissue culture inserts. They were rinsed with HEPES-buffered saline and fed every other day until they were confluent by visual inspection. The resistances of the monolayers were monitored daily until stable resistances were obtained (>25 Ω · cm2); the cells were then exposed to hypoxia for 3 to 24 hours. Transendothelial electrical resistance (TEER) was measured with a resistance meter together with the Endohm-12 chamber (World Precision Instruments). Measurements were taken in triplicate and reported as percentage (mean±SE) for each time point relative to the same insert at time 0.
Electrophoretic Mobility Shift Assay
Nuclear extracts were isolated by suspending the cells in buffer A (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol, 0.5 mmol/L phenylmethylsulfonyl fluoride, 1 μg/mL leupeptin, and 5 μg/mL aprotinin) for 15 minutes on ice. After adding 25 μL of 10% NP-40, the cells were subsequently centrifuged at 12 000 rpm for 30 seconds. The pellet was then resuspended in buffer B (20 mmol/L HEPES, pH 7.9, 0.4 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonyl fluoride, 1 μg/mL leupeptin, and 5 μg/mL aprotinin) for 15 minutes at 4°C. Samples were prepared and loaded onto 4% polyacrylamide gels and were run in 0.5× TBE for 4 hours at 120 V as described previously.22,23⇓
Data are reported as mean±SE and were analyzed by Student t tests or ANOVA (Minitab II) where appropriate. Significance was defined as a value of P<0.05.
Effects of Hypoxia on DCF and DHE Fluorescence
Oxidant signaling was investigated in HUVECs by using DCFH-DA dye, which becomes fluorescent on oxidation to DCF in the cell. Prolonged hypoxia (Po2 15 mm Hg for 6 hours) increased intracellular DCF fluorescence (Figure 1a) compared with normoxia (control). This increase was significantly attenuated when the cells were pretreated with rotenone (2 μmol/L), an inhibitor of complex I of the mitochondrial ET chain. The effect of diphenyleneiodonium (DPI, 5 μmol/L), an inhibitor of flavoproteins that include complex I of the ET chain,24 on the oxidative response to hypoxia was also evaluated. DPI attenuated the DCF fluorescence response to hypoxia. By contrast, no attenuation was observed with apocynin (30 μmol/L), an inhibitor of the neutrophil NADPH oxidase, or with allopurinol (100 μmol/L), an inhibitor of xanthine oxidase. These results suggest that the mitochondrial ET chain predominantly accounts for the increased intracellular oxidant signaling observed during hypoxia. To clarify the possible involvement of peroxynitrite in the response to hypoxia, NO synthase was inhibited by incubation with N-nitro-l-arginine (L-NNA, 100 μmol/L). Hypoxia produced an increase in oxidant signaling, as indicated by the oxidant-sensitive intracellular probe DHE (20 μmol/L, Figure 1b). The hypoxia-induced increase in DHE oxidation was abolished by rotenone (2 μmol/L) or DPI (5 μmol/L) but not by L-NNA. The increase in DHE oxidation was consistent with the increase in DCF fluorescence seen during hypoxia. The observation that L-NNA failed to abolish the hypoxic response suggests that peroxynitrite is not involved.
Effects of Hypoxia on IL-6 mRNA Message and Secretion
Under normoxic conditions, basal IL-6 transcriptional activation was found to be minimal (Figure 2a). Hypoxia elicited a significant increase in IL-6 mRNA message, which was abrogated by the addition of the mitochondrial inhibitors rotenone and DPI to the media. By contrast, neither allopurinol nor apocynin significantly attenuated the increase in IL-6 mRNA message in HUVECs incubated under hypoxic conditions. Neither rotenone nor DPI inhibited the increase in IL-6 mRNA induced by treatment with lipopolysaccharide (LPS, Figure 2b), indicating that the cells were capable of activating IL-6 mRNA expression by a separate pathway despite the presence of ET inhibition.
Effects of Antioxidants on IL-6 Secretion During Hypoxia
Prolonged hypoxia (Po2 14±3 mm Hg, range 11 to 19 mm Hg) stimulated endothelial cells to secrete IL-6 into the culture medium. Within 18 hours, levels of IL-6 in the medium reached 140±15 pg/mL (P<0.01 compared with normoxic controls, Figure 3). To determine the requirement for mitochondrial ROS in this response, the IL-6 concentration in the media of hypoxic cells was compared with that in cells pretreated with various antioxidants. We had previously found that treatment of the endothelial cells with the antioxidant compound N-acetylcysteine (NAC) inhibited the increased secretion of IL-6 in response to hypoxia, which suggested that the observed changes in IL-6 secretion were dependent on an increase in ROS signaling.8 In the present study, we pretreated the cells with rotenone to determine whether mitochondrial ROS were required for the increase in IL-6 secretion during hypoxia. Rotenone (2 μmol/L) prevented the hypoxia-induced increases in IL-6 secretion evident at 18 hours (P<0.05, Figure 3a). DPI (5 μmol/L) produced a similar attenuation in IL-6 secretion at 18 hours (Figure 3b). We then pretreated the cells with apocynin or allopurinol to determine whether NADPH oxidase or xanthine oxidase function was required for the observed increases in IL-6 secretion during hypoxia. Neither apocynin nor allopurinol significantly affected the increased IL-6 secretion (Figure 3c), indicating that neither of these systems contributed to our earlier findings. Collectively, these results suggest that mitochondrial ROS mediate the enhanced release of IL-6 during hypoxia.
Effects of Hypoxia on Permeability
The effects of hypoxia (Po2 14±3 mm Hg) on TEER were measured in HUVEC monolayers over 24 hours. A blank Transwell insert was used as an indicator of background effects on TEER and consistently demonstrated a resistance of 6±1 Ω · cm2. TEER was measured every 3 hours, and values at each time point were reported as a percentage of the original value at 0 hours. Normoxic control monolayers maintained in a standard incubator environment (5% CO2/95% room air) showed no significant change in TEER over 24 hours (data not shown). During prolonged hypoxia, TEER changed significantly over 24 hours (Figure 4) by demonstrating an initial drop in resistance at 9 hours and a greater decrease at 18 hours (P<0.01). Thereafter, resistance began to recover until 24 hours, when it reached 91±4% of the original value. By contrast, TEER failed to change significantly over 24 hours in cells incubated at Po2 25 or 35 mm Hg (data not shown). Thus, alterations of endothelial permeability depend on the duration and severity of hypoxia.
Role of Mitochondria in Altered Endothelial Permeability
We previously reported that the antioxidant NAC blocks the increase in endothelial permeability observed during hypoxia, suggesting that ROS mediate this response.8 To clarify the source of this oxidant signal, the TEER of HUVEC monolayers was compared with the TEER of identical cells pretreated with rotenone (2 μmol/L) or DPI (5 μmol/L). Rotenone and DPI prevented hypoxia-induced increases in TEER (Figure 4a). DPI treatment also abrogated the hypoxia-induced decreases in TEER at 18 hours (Figure 4b). No increase in cell death was detected after treatment with these inhibitors for 24 hours (data not shown). The increase in permeability during hypoxia was not affected by pretreatment with inhibitors of NADPH oxidase (Figure 4c) or xanthine oxidase (Figure 4d). These findings suggest that the mitochondrial ET chain acts as a source of ROS that mediate the increased endothelial permeability observed during hypoxia.
EMSA for NF-κB
To demonstrate that ROS generated by the mitochondria during hypoxia were primarily responsible for the increase in IL-6 mRNA observed in our experiments, we conducted EMSAs for NF-κB, one of the chief regulatory transcription factors for IL-6.25 After 2 hours, an increase in the NF-κB p65/p50 heterodimer was observed in hypoxic cells compared with normoxic control cells. This response was abrogated in hypoxic HUVECs pretreated with rotenone or DPI (Figure 5a). However, pretreatment with allopurinol or apocynin did not alter the hypoxic activation of NF-κB (Figure 5a and data not shown, respectively). Neither DPI nor rotenone altered the activation of NF-κB by LPS. Also, these inhibitors had no effect on normoxic cells (Figure 5b). To clarify the possible involvement of peroxynitrite in the response to hypoxia, NF-κB activation was assessed in the presence of L-NNA (100 μmol/L). Treatment with L-NNA had no effect on NF-κB activation during either normoxia or hypoxia (Figure 5c). Collectively, these results suggest that an ROS signal from the mitochondria affects IL-6 transcription through an NF-κB–mediated pathway.
Microvascular hypoxia can develop if local tissue blood flow is decreased, if tissue metabolic activity is stimulated, or if arterial Po2 is decreased by lung gas exchange failure or by travel to high altitude. Previous studies indicate that endothelial cells can detect hypoxia. Moreover, they respond by initiating the transcription of specific genes and by activating functional responses that include increases in permeability. For example, Graham et al26 found that endothelial (HUVEC) cells increased the mRNA expression of urokinase-type plasminogen activator during incubation under 1% O2 or in response to CoCl2 (100 mmol/L). Collard et al27 found that HUVECs incubated for 48 hours under 1% O2 exhibited an increase in mRNA message and protein expression of CD35, a receptor for complement C3b. In bovine retinal endothelial cells, Oh et al28 found that hypoxia (1% O2) increased the mRNA expression of angiopoietin-2, whereas in HUVECs, Karakurum et al29 demonstrated that hypoxia (Po2 14 to 18 mm Hg) elicited an increase in mRNA expression for IL-8 and for macrophage chemotactic protein-1. In that study, the increases in IL-8 protein secretion were shown to mediate the increase in chemotactic activity for polymorphonuclear cells.29 Other studies have demonstrated O2-dependent responses in platelet-derived growth factor B-chain gene expression,30 endothelin gene expression,31 and interleukin-1 gene expression.32 Thus, endothelial cells activate numerous responses to hypoxia, yet the underlying mechanism of O2 sensing that is responsible for the activation of these responses has not been identified.
Some of the transcriptional responses to hypoxia may involve NF-κB, which becomes activated during hypoxia.15 For example, Zhang et al33 found increases in NF-κB activation in association with increases in calpain mRNA expression during hypoxia in pulmonary artery endothelial cells. More recent evidence suggests that NF-κB activation during hypoxia requires ROS14 signals generated from the mitochondrial ET chain. Our previous study8 demonstrated that the IL-6 secretion response and the changes in permeability of HUVEC monolayers during hypoxia were virtually abolished by the antioxidant NAC and the glutathione peroxidase mimetic compound ebselen. In addition, these compounds abrogated the DCF fluorescence response to hypoxia, which suggests that the ROS signals detected by the DCF probe were required for the functional responses. The present study extends those findings by demonstrating that mitochondrial ET is required for the ROS response to hypoxia, which in turn triggers the activation of NF-κB and the mRNA expression of IL-6. Collectively, these studies implicate the mitochondria as the O2 sensor underlying the responses to hypoxia in endothelial cells.
Our experiments show that the treatment of endothelial cells with inhibitors of the ET chain (rotenone and DPI) blocked the increases in intracellular oxidant signaling during hypoxia and prevented the increases in the DNA binding of NF-κB along with the subsequent IL-6 transcription and secretion. Mitochondrial inhibitors also prevented the increased endothelial barrier permeability seen during prolonged hypoxic treatment of cell monolayers. One possible interpretation is that ET blockade abolished these responses by limiting mitochondrial ATP production. However, several pieces of evidence suggest that this is not the case. Most cells obtain the majority of their ATP from mitochondrial oxidative phosphorylation, which becomes limited by the oxygen supply when the Po2 falls below 5 to 7 mm Hg.34,35⇓ Interestingly, endothelial cells appear to be capable of sustaining normal responses even during sustained anoxia.36 In control experiments, we found that ET inhibitors failed to abolish the activation of NF-κB by LPS. LPS has been shown to activate NF-κB by ROS-dependent and ROS-independent pathways.14 Therefore, the observation that LPS could still trigger NF-κB activation and IL-6 expression during ET inhibition indicates that a loss of mitochondrial ATP cannot explain the loss of the hypoxic response. In further support of this conclusion, the endothelial monolayers were capable of sustaining a high electrical resistance for 24 hours in the presence of rotenone or DPI. If ET inhibition had caused cellular damage, we would have expected rotenone or DPI to cause decreases in TEER. Finally, cell death was not increased in the HUVECs treated with rotenone or DPI for 24 hours, as assessed with the use of propidium iodide. These observations support the conclusion that ET inhibition blocked the hypoxic responses by abolishing the mitochondrial signals that require a functional ET chain.
Collectively, our results suggest that ROS generation occurs at an early step in the signaling pathway activated during hypoxia. However, the mitochondrial ET chain represents only one of many potential sources of intracellular ROS in hypoxic cells. Therefore, to identify whether other oxidase systems produce ROS that contribute to the increases in IL-6 expression and the changes in endothelial permeability, we inhibited 2 alternative pathways of ROS generation by using apocynin to block the NADPH oxidase system and allopurinol to inhibit xanthine oxidase. Neither inhibitor prevented the increases in intracellular oxidant signaling, the IL-6 response, or the changes in endothelial permeability, supporting the conclusion that stimulation of the mitochondrial ROS generation is primarily responsible for triggering the hypoxic responses. Likewise, inhibition of NO synthase failed to abolish the responses to hypoxia, suggesting that peroxynitrite is not responsible for these results.
That the mitochondria act as a source of intracellular ROS responsible for changes in endothelial permeability is an interesting discovery for several reasons. First, the mitochondrial response to hypoxia may represent one of the initial steps in the cascade of events leading to increased endothelial permeability and, ultimately, contributing to the development of organ dysfunction in pathophysiological conditions. Second, these findings suggest that low levels of intracellular ROS serve as intracellular signals capable of mediating adaptive responses to cellular hypoxia. The sequence of events suggested by our data involves the following: hypoxia→mitochondrial ET chain→increased ROS→NF-κB activation/DNA binding→IL-6 mRNA message→IL-6 secretion and increased endothelial permeability. Although ROS previously have been thought to function as cytotoxic molecules in host defense or as byproducts of other biologic reactions, the present study suggests that ROS function as signaling messengers in hypoxic endothelium.
Pathophysiological conditions resulting in uncontrolled increases in endothelial permeability, such as sepsis and adult respiratory distress syndrome, must begin at a cellular or subcellular level. Recent strategies to attack these processes on a molecular level, such as monoclonal antibodies to tumor necrosis factor and receptor antagonists for interleukin-1, have not succeeded in the clinical arena,37,38⇓ but these strategies may fail because they are not acting at a step early enough in these processes. Our results suggest that the mitochondrial production of ROS represents an early signaling step in the proinflammatory effects of hypoxia on integrin expression and increased permeability, making this a potentially interesting target for future therapeutic intervention.
This study was supported in part by National Heart, Lung, and Blood Institute grants HL-32646, HL-35440, and HL-66315.
Received December 20, 2001; revision accepted January 22, 2002.
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