At Least 2 Distinct Pathways Generating Reactive Oxygen Species Mediate Vascular Cell Adhesion Molecule-1 Induction by Advanced Glycation End Products
Objective— The interaction of advanced glycation end products (AGEs) with their main receptor RAGE in endothelial cells induces intracellular generation of reactive oxygen species (ROS) and the expression of vascular cell adhesion molecule (VCAM)-1. We investigated the role of distinct sources of ROS, including the mitochondrial electron transport chain, NAD(P)H oxidase, xanthine oxidase, and arachidonic acid metabolism, in AGE-induced VCAM-1 expression.
Methods and Results— The induction of ROS and VCAM-1 by AGEs in cultured human umbilical vein endothelial cells was specifically blocked by an anti-RAGE antibody. The inhibition of NAD(P)H oxidase by apocynin and diphenylene iodonium, and of the mitochondrial electron transport system at complex II by thenoyltrifluoroacetone (TTFA), significantly inhibited both AGE-induced ROS production and VCAM-1 expression, whereas these effects were potentiated by rotenone and antimycin A, specific inhibitors of mitochondrial complex I and III, respectively. The inhibition of Cu/Zn superoxide dismutase inhibited both ROS and VCAM-1 induction, indicating that H2O2 by this source is involved as a mediator of VCAM-1 expression by AGEs.
Conclusions— Altogether, these results demonstrate that ROS generated by both NAD(P)H-oxidase and the mitochondrial electron transport system are involved in AGE signaling through RAGE, and indicate potential targets for the inhibition of the atherogenic signals triggered by AGE-RAGE interaction.
- diabetes mellitus
- reactive oxygen species
- signal transduction
- vascular biology
- adhesion molecules
The formation and accumulation of advanced glycation end products (AGEs) induce vascular cellular activation and inflammation mainly through interaction of AGEs with specific receptors. The main pathological consequence of AGE interaction with the main endothelial surface receptor for AGEs (RAGE) is the induction of intracellular reactive oxygen species (ROS).1 ROS would in turn activate the redox-sensitive transcription nuclear factor NF-κB, a pleiotropic regulator of many “response-to-injury” genes, such as vascular cell adhesion molecule-1 (VCAM-1).2,3
Several reports have linked ROS with p21ras, MAP kinases, cdc42/rac, and extracellular signal regulated kinase (ERK)-1 and -2 in the signal transduction from RAGE to NF-κB–induced inflammatory molecules.1,4–8 Each of these pathways is closely linked to AGE binding to RAGE, because blockade of the receptor with either anti–RAGE IgG or excess soluble (s)RAGE prevents their activation.9 The tethering of AGEs to the cell surface by RAGE is not enough to generate ROS and cellular activation, because the RAGE carboxy-terminal cytosolic tail, containing known signaling phosphorylation sites, kinase domains, and other activation sites, is critical for RAGE-dependent cellular activation. In fact, a truncated form of RAGE, lacking only the cytosolic tail and expressed in cells, retains the binding to various ligands identically as wild-type RAGE, but does not mediate the induction of cellular activation.10
Despite such information, sources of ROS involved in the signal transduction cascade initiated by AGE–RAGE interaction remain largely unknown. Although a role for the NAD(P)H oxidase system in the generation of ROS after AGE stimulation has been recently suggested,11 the involvement of other distinct sources of ROS in this signal transduction cascade has not yet been explored. Here we show that both the mitochondrial respiratory chain and the NAD(P)H oxidase systems are implicated in AGE signaling through RAGE, highlighting the redundancy of intracellular signals elicited by AGE–RAGE interaction.
Endothelial Cell Cultures
Human umbilical vein endothelial cells (HUVEC) were obtained as previously described12 (see supplemental Methods, available online at http://atvb.ahajournals.org).
AGEs and CML Preparation
AGEs were prepared and characterized as described,3 and nonglycated bovine serum albumin (BSA) was used as control. A monospecific rabbit anti-human RAGE IgG antibody was used to inhibit the effects of AGEs. Nε-(carboxymethyl)lysine (CML)–BSA standard was prepared according to Reddy et al.13 For details, see the supplemental Methods.
We examined the effects of several inhibitors of mitochondrial electron transport, NAD(P)H oxidase, xanthine oxidase, and arachidonic acid metabolism on AGE-induced H2O2 production and VCAM-1 expression. All reagents here described were from Sigma, unless otherwise specified.
We used the following inhibitors of mitochondrial electron transport: rotenone (a specific inhibitor of complex I), thenoyltrifluoroacetone (TTFA, a specific inhibitor of complex II), and antimycin A (a specific inhibitor of complex III). We used 2 structurally unrelated inhibitors of NAD(P)H oxidase: apocynin and diphenylene iodonium (DPI). As inhibitors of arachidonic acid metabolism we used the false substrate 5,8,11,14-eicosatetraynoic acid (ETYA) as a combined cyclooxygenase and lipoxygenase inhibitor; nordihydroguaiaretic acid (NDGA) as an inhibitor of 5-lipoxygenase; and N-2-(cyclohexyloxy)-4-nitrophenyl-methanesulfonamide (NS-398) as a selective inhibitor of cyclooxygenase-2 (COX-2). We used the following exogenously administered antioxidants as controls: N-acetylcysteine (NAC), Cu/Zn superoxide dismutase (Cu/Zn SOD), and catalase. We also used diethyldithiocarbamate (DETC), which inactivates Cu/Zn SOD and allopurinol to inhibit xanthine oxidase. Antioxidants, enzymes, and inhibitors were added to HUVEC 30 minutes before addition of AGEs and remained present throughout AGE incubation. VCAM-1 expression was determined after overnight AGE incubation. Detection of both ROS generation and NF-κB nuclear translocation were performed after 1 hour of AGE incubation. For further details, see the supplemental Methods.
Detection of VCAM-1 Expression
VCAM-1 expression was assayed by cell surface enzyme immunoassay (EIA), as previously described.3
Detection of Intracellular ROS
Monolayers were washed and incubated with 10 μmol/L of 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate bis(acetoxymethyl)-ester C-DCF-DA (Molecular Probes Inc) for 30 minutes. Using an inverted microscope (10× objective), monolayers were epi-illuminated with a 100 watt Hg lamp and photographed with 490 nm excitation and 520 nm wavelength emission filters, respectively. Photographed images were transferred to a custom-made software for data analysis of cellular fluorescence.14 For further details, see the supplemental Methods.
Immunofluorescence Analysis for NF-κB
HUVEC were grown on precoated coverslips, fixed, rinsed, and blocked with goat serum. After overnight incubation with a primary antibody against the p65 NF-κB subunit (Santa Cruz Biotechnology Inc), cells were incubated with a biotinylated goat anti-rabbit antibody (Vector Laboratories Inc), washed, incubated with streptavidin-fluorescein isothiocyanate (FITC; Vector), and mounted in an anti-fading agent (Vectashield; Vector) on coded slides. Confocal microscopy images were transferred to a custom-made software for data analysis of nuclear fluorescence.14 For further details, see the supplemental Methods.
Electrophoretic Mobility Shift Assay
For the analysis of transcription factor activation, nuclear extracts and electrophoretic mobility shift assay (EMSA) were performed as described.15 For further details, see the supplemental Methods.
Multiple comparisons were performed by one-way ANOVA and individual differences tested by the Fisher protected least significant difference (PLSD) test after the demonstration of significant inter-group differences by ANOVA. Two-group comparisons were performed by the unpaired Student t test.
Intracellular ROS Levels and Surface VCAM-1 Expression Are Increased in Endothelial Cells Exposed to AGEs
To evaluate the effect of AGEs on ROS generation, we incubated HUVEC with AGEs or native albumin at 500 μg/mL for 1 hour. Basal intracellular ROS production was significantly increased (by 47%±7%, mean±SD) by the incubation of HUVEC with AGEs (Figure IA and IB, available online at http://atvb.ahajournals.org). Exposure of HUVEC to heat-inactivated AGEs did not induce ROS production (data not shown). That these effects were mediated by RAGE was suggested by the suppression of AGE-mediated increased levels of ROS by anti-RAGE IgG, but not by nonimmune IgG (Figure IA and IB). Similarly, the exposure of HUVEC to CML (50 μg/mL) induced an increase of intracellular ROS levels that was suppressed by anti-RAGE IgG (Figure IA and IB).
We previously demonstrated that the exposure of HUVEC to AGEs results in a concentration- and RAGE-dependent increase in VCAM-1 expression.3 To understand whether this depends on extracellular ROS, we pretreated HUVEC with either Cu/Zn SOD, an enzyme that converts O2 into H2O2, or catalase alone, which scavenges H2O2, or Cu/Zn SOD plus catalase. Both Cu/Zn SOD and catalase are known to remain in the extracellular compartment, not affecting intracellular ROS.16 None of these treatments affected AGE-induced ROS levels (Figure IB) or VCAM-1 surface expression (not shown). These results suggest that AGE-induced VCAM-1 expression is not associated with an increase in extracellular ROS. Conversely, pretreatment of HUVEC for 30 minutes with the antioxidant NAC, which scavenges intracellular oxidant species, halved ROS production (Figure IB) and strongly inhibited VCAM-1 expression (down to 8.3±4.7%) induced by AGEs.
The Inhibition of Cytoplasmic Cu/Zn SOD Decreases Both AGE-Induced Intracellular ROS Generation and Surface VCAM-1 Expression
To study whether O2 or H2O2 is the effector molecule leading to VCAM-1 induction, we inactivated the cytoplasmic Cu/Zn SOD (catalyzing the conversion of O2 into H2O2) with the inhibitor DETC and determined the effect of such inhibition on ROS generation and VCAM-1 expression.
Pretreatment of HUVEC with DETC at 10 and 100 μmol/L for 30 minutes determined a clear reduction of both baseline and AGE-induced ROS levels (practically H2O2 in our assay; Figure 1A) In parallel, DETC decreased AGE-induced VCAM-1 expression (Figure 2A). From these experiments we infer that incubation of HUVEC with AGEs increases intracellular O2, which is then converted to H2O2 by Cu/Zn SOD, inducing VCAM-1 expression.
Xanthine Oxidase Inhibition Reduces AGE-Induced ROS Production, but Does Not Affect VCAM-1 Expression
The contribution of xanthine oxidase to ROS generation was explored through the administration of the xanthine oxidase inhibitor allopurinol. Treatment of HUVEC with allopurinol at 1 and 10 μmol/L significantly inhibited ROS production induced by AGEs (Figure 1B) but did not modify AGE-induced levels of VCAM-1 at all concentrations used (Figure 2B), indicating that xanthine oxidase–mediated ROS production is not involved in VCAM-1 induction.
ROS Generated via NAD(P)H-Oxidase Activity Mediate VCAM-1 Expression Induced by AGEs
To determine whether NAD(P)H oxidase activity may be induced by AGEs, we used apocynin, an inhibitor of NAD(P)H oxidase.17 Pretreatment of HUVEC with apocynin for 30 minutes inhibited AGE-induced ROS production at 1 and 2 mmol/L (Figure 1C). In line with ROS inhibition by this treatment, apocynin concentration-dependently decreased AGE-induced VCAM-1 expression (Figure 2C). Similarly, treatment with DPI, another (albeit less specific) inhibitor of NAD(P)H oxidase,18 resulted in a concentration-dependent reduction in cell surface VCAM-1 expression (Figure 2D).
Arachidonic Acid Metabolism Does Not Mediate AGE-Induced ROS Production and VCAM-1 Expression
Arachidonic acid (AA), an important precursor of lipid mediators, is generated through the activation of a cytosolic phospholipase A2 (PLA2) in response to various stimuli.19 AA can then be oxidized through cyclooxygenases, 5-lipoxygenase, or the cytochrome P450 complex, to yield eicosanoids, in processes known to generate ROS. To establish whether AA metabolization through these pathways is necessary for VCAM-1 expression induced by AGEs, cells were treated with the false substrate ETYA (an inhibitor of all processes yielding eicosanoids), the specific 5-lipoxygenase inhibitor NDGA, and the specific COX-2 inhibitor NS-398. None of these inhibitors exerted inhibitory effects on both ROS and VCAM-1 induced by AGEs (Table I, available online at http://atvb.ahajournals.org).
ROS Generated Through the Mitochondrial Electron Transport Chain Also Mediate AGE-Induced VCAM-1 Expression
To investigate the potential contribution of mitochondria to intracellular oxidative stress and the impact of mitochondrial pathways of ROS generation on VCAM-1 expression, we studied the effects of AGEs in the absence or presence of various unrelated inhibitors of the mitochondrial respiratory chain. Rotenone, a specific inhibitor of complex I, which interferes with the electron flow from NADH dehydrogenase to the ubiquinone pool, slightly but significantly increased ROS production at 1000 nmol/L when given alone, but also consistently potentiated AGE-induced ROS generation at 100 and 1000 nmol/L (Figure 3A). In parallel, rotenone alone slightly increased baseline VCAM-1 expression at 1000 nmol/L and potentiated AGE-induced VCAM-1 expression (Figure 4A). Similarly, pretreatment of HUVEC with antimycin A, which inhibits the electron flow at complex III, alone or together with AGEs, increased ROS production (Figure 3C) and, in parallel, concentration-dependently increased VCAM-1 expression (Figure 4C). On the contrary, pretreatment of HUVEC with TTFA, a specific inhibitor of complex II, which interferes with the electron flow from succinate dehydrogenase to the ubiquinone pool, caused an attenuation of AGE-induced ROS generation at 100 and 1000 nmol/L (Figure 3B). Treatment with TTFA did not alter baseline VCAM-1 levels, whereas cotreatment with AGEs caused a significant reduction in VCAM-1 expression compared with AGE treatment alone (Figure 4B). Taken together, these results support the role of mitochondrial ROS production in AGE–RAGE signaling.
Effects of NAD(P)H Oxidase Inhibitors, Cu/Zn SOD and Mithocondrial Respiratory Chain Inhibitors, on Nuclear Translocation of p65 NF-κB Subunit Induced by AGEs
Becaue the p50-p65 heterodimer activates transcription of the VCAM-1 gene,20 we investigated whether the inhibitors of NADPH-oxidase, Cu/Zn SOD, or the mithocondrial respiratory chain proven useful, on the basis of the above-reported experiments, in reducing both ROS generation and VCAM-1 expression, also prevent ROS-dependent activation of NF-κB induced by AGEs. To this purpose, we performed immunofluorescence staining of the intracellular distribution of the p65 NF-κB subunit at confocal microscopy and gel-shift assays, the latter demonstrating the formation of nuclear complexes between NF-κB and its specific binding sites on VCAM-1 promoter.
Compared with native albumin, stimulation of HUVECs with AGEs determined the translocation of the p65 NF-κB subunit from the cytosol to the nucleus. NAC and DETC completely prevented AGE-induced p65 nuclear translocation (Figure 5A and 5B) and markedly decreased the intensity of the shifted band produced by nuclear extracts treated with AGEs (Figure 5C). Pretreatment with the inhibitor of NAD(P)H oxidase apocynin (2 mmol/L) significantly attenuated both nuclear immunofluorescence staining for p65 and the intensity of the shifted band produced by AGEs (Figure 5). On the contrary, the mitochondrial inhibitor TTFA (1000 nmol/L) did not modify AGE-induced NF-κB activation (Figure 5). The two other mitochondrial inhibitors rotenone and antimycin A actually stimulated p65 translocation in the presence or in the absence of AGEs (Figure 5B), indicating that oxidant stress in mitochondria can promote the extra-mitochondrial activation of NF-κB and therefore affect nuclear gene expression.
ROS are known to play an important role in the regulation of leukocyte adhesion to the vascular endothelium.21 Previous studies have indicated that an initial event in AGE–RAGE interaction is the generation of enhanced cellular oxidant stress, in turn determining increased production of VCAM-11,2 and increased monocyte adhesion. However, the intracellular sources of these agonist-induced ROS are not clear.22,23
Here we show that generation of ROS in HUVEC by selective intracellular sources is involved in the AGE–RAGE signaling pathways leading to the expression of VCAM-1. Here we first demonstrate that interaction of AGEs with their primary receptor increases intracellular ROS. That these effects are mediated by RAGE is suggested by the suppression of AGE-mediated ROS production in the presence of an anti-RAGE IgG, but not by nonimmune IgG. We next analyzed the role of selective intracellular sources of ROS by assessing ROS generation, NF-κB activation, and VCAM-1 expression under treatment of a variety of inhibitors of various enzymatic systems involved in ROS generation. The inhibition of xanthine oxidase clearly decreases the level of ROS in AGE-treated cells, but this decrease was not functionally coupled to a decreased expression of VCAM-1. This contrasts with the findings of a separate experiment (not shown) demonstrating that the same treatment leads to a net decrease in the expression of another endothelial–leukocyte adhesion molecule, intercellular adhesion molecule (ICAM)-1. These contrasting results on two distinct adhesion molecules highlight that specific sources of intracellular ROS are differently involved in the expression of different genes, and that the signal transduction pathways underlying the expression of adhesion molecules is not completely overlapping. Cyclooxygenase and 5-lipoxygenase metabolization of arachidonic acid is another well known source of ROS.24,25 We addressed the issue of the involvement of this pathway of ROS generation in AGE-induced VCAM-1 expression by using several inhibitors of arachidonic acid metabolization. All inhibitors used to this purpose did not affect both the AGE-induced VCAM-1 and ROS production in our experiments, thus ruling out that arachidonate pathway is involved as a source of ROS relevant for VCAM-1 induction by AGEs.
We confirm that NAD(P)H oxidase is an enzymatic source of ROS triggered by AGEs and is involved in AGE signal transduction.11 A major new finding of our study, however, is that also another discrete source of intracellular ROS participates in the oxidative burst induced by AGEs. Mitochondrial dysfunction, leading to energy impairment and/or oxidative stress, is associated with a number of disease states such as ischemia/reperfusion injury, aging, and neurodegenerative diseases.26 Here we show that the mitochondrial respiratory chain is also involved in AGE-RAGE-induced signal transduction and VCAM-1 expression.
The possibility of a mitochondrial involvement in the production of ROS is supported by data describing their induction by several stimuli.27–29 Generation of ROS in mitochondria is involved in endothelial cell activation by angiotensin II30 and hyperglycemia, in the latter case at the level of mitochondrial complex II.31 That mitochondrial redox status may influence the expression of adhesion molecules is suggested by the demonstration that tumor necrosis factor (TNF)-α–induced expression of VCAM-1 and E-selectin is markedly enhanced when the mitochondrial pool of reduced glutathione is depleted.22 On this basis we hypothesized that a flow of information exists from the mitochondria to the nucleus as a consequence of changes occurring in mitochondria after stimulation with AGEs.
Our results show that treatment with mitochondrial respiratory chain inhibitors affecting intracellular ROS cause directionally similar changes in VCAM-1 expression. Antimycin A and rotenone increase intracellular ROS levels as well as VCAM-1 expression. Conversely, AGE-induced signaling to VCAM-1 expression is sensitive to inhibition of mitochondrial complex II with TTFA. The downstream signaling mechanism by which AGEs mediate mitochondrial ROS and VCAM-1 induction it is not known. Several lines of evidence suggest that mitochondria may be involved in ceramide-induced H2O2 production.28 Ceramide has emerged as a mediator of diverse molecular events after stimulation with various agents such as TNF-α32 and, more recently, AGEs.33 Whether the increase in mitochondrial ROS induced by AGEs in HUVEC occurs through the ceramide-dependent signaling pathway, however, remains to be elucidated.
ROS have been largely described as activators of redox-sensitive transcription factors, such as NF-κB and AP-1,27,34 both also activated by AGEs and involved in VCAM-1 gene expression.2,35 Here we found that AGE-induced NF-κB activation was repressed not only by the antioxidants NAC and DETC, but also, to a lower extent, by the NAD(P)H oxidase inhibitor apocynin. By altering the generation of ROS through inhibition of the mitochondrial electron transport chain with rotenone and antimycin A, at the level of complex I and III respectively, we found an activation of NF-κB, as assessed by immunofluorescence analysis of the p65 subunit nuclear translocation. These results, in agreement with another report,36 suggest that oxidative stress within mitochondria can promote extra-mitochondrial activation of NF-κB. This effect was potentiated by the cotreatment with AGEs. One of our inhibitors, TTFA, inhibited both ROS production and VCAM-1 expression induced by AGEs, but had no effect on AGE-induced NF-κB activation (Figure 5). This result is, however, compatible with the possibility that TTFA inhibits AGE-mediated VCAM-1 expression through a redox-sensitive mechanism independent from the better characterized NF-κB transcription factor.
A number of questions about the involvement of ROS in AGE-induced VCAM-1 expression, also deriving from the experiments reported here, remain to be elucidated. Because a virtually complete inhibition of AGE-induced ROS generation by DETC (at 100 μmol/L) or TTFA (at 1000 nmol/L) is paralleled by <50% inhibitory effect on VCAM-1 expression, one can reasonably argue that a parallel ROS-independent pathway for AGE-induced VCAM-1 expression is likely to exist. This is compatible with the suggestion that a number of parallel pathways can be simultaneously activated by the AGE–RAGE interaction.5 Also, the notion that a suppression of ROS expression by the xanthine oxidase inhibitor allopurinol in our system is not accompanied by any change in VCAM-1 expression requires further investigation. One may speculate that the gross semiquantitative estimate of ROS levels by our system may not accurately reflect the level of ROS generation in discrete subcellular pools that are more directly linked to NF-κB activation and VCAM-1 expression, and that this correspondence may fail to occur in specific cases, such as the generation of ROS by xanthine oxidase. One has to realize, however, that no technique is currently available to pinpoint the levels of ROS generation in those hypothetical pools, and that the approach with inhibitors, extensively used in the present study, is currently the best possible approximation to the study of the causal link between AGE-induced ROS generation and VCAM-1 expression.
Overall, the data herein reported provide indications that multiple (at least 2) intracellular sources are involved in ROS generation after endothelial cell activation with AGEs. The identification of these intracellular targets for AGE-initiated signal transduction advances our knowledge on the intimate mechanism of vascular disease mediated by AGEs, such as diabetic vasculopathy, and might prove useful for the pharmacological manipulation of the intracellular redox balance in endothelial cells in diabetes and other disease processes involving AGEs.
This work was partially supported by funding of the Italian Ministero dell’Università e della Ricerca (ex-60%) of the Center of Excellence on Aging Project (to R.D.C.).
- Received January 19, 2004.
- Accepted April 8, 2005.
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