Losartan Metabolite EXP3179 Blocks NADPH Oxidase-Mediated Superoxide Production by Inhibiting Protein Kinase C
Potential Clinical Implications in Hypertension
Oxidative stress plays a critical role in the pathogenesis of hypertension. The NADPH oxidase constitutes a major source of superoxide anion in phagocytic cells, and its activation is associated with matrix metalloproteinase (MMP)-9 secretion by these cells. We investigated the effects of the angiotensin II type 1 receptor antagonist losartan and its metabolites (EXP3174 and EXP3179) on NADPH oxidase activity and MMP-9 secretion in human phagocytic cells. EXP3179, but not losartan and EXP3174, dose-dependently inhibited (P<0.05) phorbol myristate acetate and insulin-stimulated NADPH oxidase activity. EXP3179 also inhibited phorbol myristate acetate–induced NADPH oxidase in endothelial cells. In addition, EXP3179 inhibited (P<0.05) both phorbol myristate acetate–stimulated p47phox translocation from cytosol to membranes and protein kinase C activity. Affinity experiments and enzymatic assays confirmed that EXP3179 inhibited several protein kinase C isoforms. EXP3179 also inhibited (P<0.05) phorbol myristate acetate–stimulated MMP-9 secretion. In a study performed in 153 hypertensive patients, phagocytic NADPH oxidase activity was lower (P<0.05) in losartan-treated compared with untreated patients and in patients treated with other angiotensin II type 1 receptor antagonists or with angiotensin-converting enzyme inhibitors. Plasma levels of MMP-9 were lower (P<0.05) in losartan-treated hypertensives compared with the other group of patients. Thus, EXP3179 acts as a blocker of the NADPH oxidase in phagocytic cells by a potential mechanism that targets the protein kinase C signaling pathway. This effect can be involved in reduced MMP-9 secretion by these cells. It is proposed that the EXP3179 metabolite may confer to losartan the specific capacity to reduce oxidative stress mediated by phagocytic cells in hypertensive patients.
Oxidative stress induced by reactive oxygen species, including superoxide anion (O2·−), is increased in arterial hypertension.1,2⇓ The NADPH oxidase systems constitute a major source of O2·− in the vessel wall, and they are present in endothelial cells, smooth muscle cells, fibroblasts, and infiltrated monocytes/macrophages.3,4⇓ Protein kinase C (PKC)–dependent p47phox phosphorylation and translocation are major mechanisms involved in phagocytic NADPH oxidase activation.3 Phagocytic NADPH oxidase is overactivated in essential hypertension.5,6⇓ Clinical studies have demonstrated that treatment with losartan improves endothelial function and decreases blood pressure through a reduction in oxidative stress.7,8⇓ Moreover, several experimental studies have demonstrated beneficial effects of losartan treatment on vascular alterations by blocking NADPH oxidase activation.9–12⇓⇓⇓
Losartan is hepatically metabolized by the cytochrome-P450 pathway and exerts its antihypertensive actions predominantly by EXP3174, its main metabolite and the pharmacological blocker of the angiotensin II type 1 receptor (AT1R).13,14⇓ Interestingly, during losartan metabolism, the liver also produces the EXP3179 metabolite, which has no AT1R-blocking properties.14,15⇓ In recent years, several studies have reported that the EXP3179 metabolite exerts AT1R-independent actions.15–18⇓⇓⇓ Krämer et al15 demonstrated that EXP3179 mediates the anti-inflammatory properties of losartan by abolishing cyclooxygenase-2 upregulation. Furthermore, EXP3179 stimulates endothelial NO synthase and suppresses tumor necrosis factor-α–induced apoptosis.16 Schupp et al17 have demonstrated that EXP3179 acts as a peroxisome proliferator–activated receptor-γ agonist. Finally, the ability of EXP3179 to inhibit collagen-dependent platelet activation has also been described.18
Therefore, we hypothesized that EXP3179 metabolite might inhibit NADPH oxidase in phagocytic cells by AT1R-independent mechanisms. To test this hypothesis, we analyzed the ability of losartan and its metabolites, as well as other drugs interfering with the renin-angiotensin system, to block NADPH oxidase–dependent O2·− production in human phagocytic cells. Given that matrix metalloproteinase (MMP)-9 secretion by phagocytic cells is regulated by NADPH oxidase,19 we also assessed the effect of EXP3179 on this proteolytic enzyme. In addition, we evaluated NADPH oxidase–dependent O2·− production and MMP-9 levels in phagocytic cells and plasma, respectively, obtained from hypertensive patients treated with different classes of drugs interfering with the renin-angiotensin system, including losartan.
The study was performed in 2 different populations. The first population consisted of 50 apparently healthy subjects in whom the effect of different drugs that interfere with the renin-angiotensin system was analyzed on NADPH oxidase–dependent O2·− production from phagocytic cells. In addition, the effect of EXP3179 was analyzed on phorbol myristate acetate (PMA)-induced p47phox translocation, PKC activity, and MMP-9 secretion. The second population consisted of 153 patients with the diagnosis of essential hypertension. These patients were considered hypertensive if they had sitting systolic and/or diastolic blood pressures of >139 and 89 mm Hg, respectively, or if they were taking antihypertensive medication during ≥6 months before the study. Blood pressure was measured on 3 occasions using a mercury sphygmomanometer, and the mean of these 3 readings was recorded. In this study, NADPH oxidase–dependent O2·− production by phagocytic cells and plasma MMP-9 levels were evaluated in the patients classified in 4 groups: untreated patients, patients treated with losartan, patients treated with other AT1R antagonists (ARAs) different from losartan, and patients treated with angiotensin-converting enzyme inhibitors (ACEIs).
Both populations consisted of subjects who were referred to our institution for global cardiovascular risk assessment. Following institutional guidelines, the subjects were aware of the research nature of the study, and written informed consent was obtained from all of them. The study was performed in accordance with the Declaration of Helsinki, and the local committee on human research approved of the study protocol.
Determination of NADPH Oxidase–Dependent O2·− Production
Peripheral blood mononuclear cells (PBMCs) were isolated with Lymphoprep from blood samples. The methodology used to isolate mononuclear cells yielded preparations with a very high purity (≥99% were lymphocytes and monocytes). NADPH oxidase–dependent O2·− production was measured by using 5 μmol/L of lucigenin by a chemiluminescent method. Luminescence was measured in 400 000 cells every 15 to 30 s along an interval of 1 hour in a plate reader luminometer (Luminoskan Ascent, Labsystem). A buffer blank was subtracted from each reading, and the value of the area under the curve was used to quantify chemiluminescence. Data are expressed as relative light units produced per second. Although lucigenin concentration was low enough to avoid autoxidation, the measurements were validated against an independent measurement of O2·− production using O2·− dismutase–inhibitable ferricytochrome C reduction, as reported previously.6 Previous experiments performed by our group allow us to confirm that NADPH oxidase is the main source of O2·− production in response to PMA stimulation in phagocytic cells.6,19⇓
Phagocytic NADPH oxidase–dependent O2·− production in response to 3.2 μmol/L of PMA in the presence (20 minutes of preincubation) of different doses of losartan, EXP3174, and EXP3179 was evaluated. In some experiments, the effects of irbesartan, another ARA, and the effect of quinapril, an ACEI, were also evaluated. In addition, in some experiments, the effect of 100 nmol/L of human recombinant insulin on NADPH oxidase–dependent O2·− production was evaluated in the presence (20 minutes of preincubation) of different doses of losartan, EXP3174, and EXP3179. Within the study performed in the hypertensive population, PMA-stimulated O2·− production was also determined in PBMCs, following the same chemiluminescent methodology.
Finally, and to test whether EXP3179 could act as an O2·− scavenger, we determined O2·− production in a homogenate of protein from the rat kidney in the absence and presence of EXP3179. Briefly, we determined the effect of 100 μmol/L of EXP3179 on O2·− production from 100 μg of renal homogenate in the presence of 5 μmol/L of lucigenin during 5 minutes. In these experiments, we used as a positive control for the scavenging of O2·−, 100 U/mL of O2·− dismutase.
An expanded Methods section is available in the online Data Supplement at http://hyper.ahajournals.org.
EXP3179 Metabolite Blocks O2·− Production
EXP3179 dose-dependently inhibited PMA-stimulated NADPH oxidase–mediated O2·− production, reaching a significant effect at a concentration of 10 μmol/L (Figure 1). The EC50 of this metabolite for PMA-stimulated NADPH oxidase–mediated O2·− production was 30 μmol/L. In contrast, neither losartan nor EXP3174 influenced PMA-stimulated NADPH oxidase–mediated O2·− production (Figure 1). Similarly, neither quinapril nor irbesartan influenced PMA-stimulated NADPH oxidase–dependent O2·− production (Figure 2).
EXP3179 also inhibited insulin-stimulated NADPH oxidase–mediated O2·− production in a dose-dependent way, reaching a significant effect at a concentration of 50 μmol/L (Figure S1). The EC50 of this metabolite for NADPH oxidase–dependent O2·− production was 80 μmol/L. In contrast, EXP3174 did not influence insulin-stimulated NADPH oxidase–dependent O2·− production, and losartan exerted an inhibitory effect that only was statistically significant at a very high dose (500 μmol/L).
To examine whether EXP3179 inhibited O2·− production stimulated by another agonist that is known to activate NADPH oxidase through PKC, PBMCs were stimulated with 50 ng/μL of tumor necrosis factor-α. Interestingly, tumor necrosis factor-α–stimulated O2·− production was significantly reduced in the presence of 100 μmol/L of EXP3179 (Figure S2).
Finally, and to test the effect of EXP3179 on NADPH oxidase activity in vascular cells, several experiments were performed in the human endothelial cell line, Eahy926. Interestingly, 100 μmol/L of EXP3179 significantly (P<0.01) reduced PMA-induced NADPH oxidase activity (Figure S3).
To test whether EXP3179 could act as an O2·− scavenger, we determined O2·− production in a protein homogenate in the absence and presence of EXP3179. Given that O2·− levels were not diminished in the presence of EXP3179 (Figure S4), we can discard a relevant role of EXP3179 as an O2·− scavenger.
EXP3179 Metabolite Blocks p47phox Translocation
An important early step in the activation of the phagocytic NADPH oxidase is the translocation of the cytosolic subunits to membranes. Thus, after PMA stimulation, an increased signal for the p47phox protein expression in the membrane was observed (Figure 3). However, when cells were preincubated with EXP3179, the p47phox translocation induced by PMA was inhibited (P<0.05), thus suggesting that the EXP3179 metabolite may interfere with some of the mechanisms involved in this necessary step for the activation of the NADPH oxidase.
EXP3179 Metabolite Inhibits PKC
Given that PKC is a relevant activator of NADPH oxidase in phagocytic cells,3 the effect of EXP3179 on PMA-stimulated PKC activity was analyzed in PBMCs. As shown in Figure 4, PKC activity was enhanced in PMA-stimulated cells. Interestingly, EXP3179 completely inhibited (P<0.05) PKC activity stimulated by PMA in these cells. Thus, we investigated the capacity of EXP3179 to bind to human recombinant PKCα by surface plasmon resonance. As shown in Figure 5A, binding of EXP3179 to PKCα is clear and concentration dependent. We plotted the different EXP3179 concentrations against the corresponding binding responses at equilibrium. To analyze whether the physical interaction between EXP3179 and PKCα would exert a functional effect, we studied the effect of EXP3179 in enzymatic assays and we observed that EXP3179 inhibited (P<0.05) the kinase activity of recombinant PKCα. The inhibitory effect of EXP3179 on PKC activity was in the same range as those observed for bisindolyl maleimide (Figure 5B). Additional experiments demonstrated that EXP3179 also interacted with the PKCβ isoform and reduced its enzymatic activity (Figure S5).
EXP3179 Metabolite Blocks MMP-9 Secretion in Phagocytic Cells
The effect of EXP3179 on MMP-9 secretion was analyzed in human monocytes and PBMCs. EXP3179 inhibited (P<0.05) PMA-induced MMP-9 secretion in the 2 types of cellular preparations (Figure 6).
NADPH Oxidase-Mediated O2·− Production and MMP-9 Levels Are Reduced in Losartan-Treated Hypertensive Patients
The demographic and clinical characteristics of the patients included in this study are summarized in the Table. No differences in blood pressure and other general clinical and biochemical parameters were found among the 3 subgroups of treated patients.
NADPH oxidase–dependent O2·− production induced by PMA was lower (P<0.05) in patients treated with losartan than in untreated hypertensives and patients treated with other ARAs or with ACEIs (Figure 7A). PMA-induced NADPH oxidase–dependent O2·− production measured in patients treated with other ARAs or with ACEIs was similar to that measured in untreated patients (Figure 7A).
Plasma levels of MMP-9 were lower (P<0.05) in patients treated with losartan than in untreated patients and patients treated with other ARAs or with ACEIs (Figure 7B). In addition, plasma levels of MMP-9 measured in patients treated with other ARAs or with ACEIs were similar to those measured in untreated patients (Figure 7B). A positive correlation (P<0.01) was observed between plasma levels of MMP-9 and NADPH oxidase–dependent O2·− production in the overall population (Figure 7C), which remained significant after controlling for age and sex (r=0.340; P<0.01).
The major findings of this study are that EXP3179 inhibits NADPH oxidase and PKC activities, as well as MMP-9 secretion, in stimulated human phagocytic cells. In addition, NADPH oxidase activity and plasma levels of MMP-9 were lower in hypertensive patients treated with losartan than in untreated hypertensive patients and hypertensive patients treated with other ARAs and with ACEIs.
It has been proposed that inhibition of phagocytic NADPH oxidase–dependent O2·− production may represent a novel pharmacological target in the treatment of cardiovascular diseases.20–22⇓⇓ In this regard, we described for the first time that EXP3179 inhibits the NADPH oxidase–dependent O2·− production in phagocytic cells. Several arguments suggest that this effect of EXP3179 is independent of the AT1R. First, the pharmacological profile of losartan metabolites indicates that the EXP3179 is not related to the antagonism of the AT1R.13,14,23⇓⇓ Second, neither losartan nor EXP3174 inhibited NADPH oxidase–dependent O2·− production in phagocytic cells. Lastly, neither irbesartan nor quinapril modified the production of O2·− by NADPH oxidase in these cells.
On cell stimulation, the NADPH oxidase cytosolic subunit p47phox becomes phosphorylated and migrates to the membrane where it binds to the cytochrome, thus activating the oxidase to produce O2·−.3 Our finding that EXP3179 completely inhibited p47phox translocation from the cytosolic fraction to the membrane fraction further supports the possibility that EXP3179 inhibits PMA-dependent O2·− production by blocking the assembly of NADPH oxidase subunits in phagocytic cells. One of the major mechanisms involved in the phosphorylation and the translocation of NADPH oxidase cytosolic subunits involves the activation of PKC.3,24⇓ Thus, our finding that EXP3179 inhibits PKC stimulation in phagocytic cells suggests that EXP3179 is inhibiting NADPH oxidase by blocking the PKC signaling pathway. In agreement with this, the surface plasmon resonance data show that EXP3179 interacts with PKCα, and the enzymatic assay data indicate that this metabolite inhibits PKCα. Collectively, these observations allow us to speculate that PKC may be the target enzyme involved in the ability of EXP3179 to inhibit NADPH oxidase–dependent O2·− production in phagocytic cells. Although several PKC isoforms have been implicated in signaling responses of phagocytic cells,25 PKCα plays a selective role in PMA-stimulated phagocytic NADPH oxidase–mediated O2·− production.26 Nevertheless, our results showing that EXP3179 also interacts with PKCβ, which is also activated by PMA in phagocytic cells,26 allow us to suggest that the inhibitory effect of EXP3179 on the PKC in phagocytic cells is not directed through the specific inhibition of only a PKC isoform. Whether the EXP3179 may also interact with other PKC isoforms need to be determined in further experiments.
Given that EXP 3179 presents various potentially vasoprotective effects,15–18⇓⇓⇓ it is possible to speculate that the pleiotropic actions of EXP3179 occur by a mechanism involving the inhibition of PKC in other vascular cell types. PKC signaling pathways mediate vascular effects, such as the cyclooxygenase-2 expression,27 endothelial NO synthase stimulation,28 regulation of peroxisome proliferator–activated receptor,29 and platelet activation.30 Additional studies will be needed to establish the relationship between EXP3179 and PKC in these vascular cell types. Interestingly, our findings show that EXP3179 inhibits the NADPH oxidase activity in endothelial cells. In fact, the activation of the NADPH oxidase in vascular cells has been shown to also be mediated through PKC-dependent pathways.31
Our group has demonstrated recently that increased synthesis and secretion of MMP-9 may be consequences of NADPH oxidase–dependent O2·− production in phagocytic cells.19 In this context, we show that PMA-stimulated MMP-9 secretion by phagocytic cells is inhibited in the presence of EXP3179. The potential relevance of this finding is related to both the fact that MMP-9 has been found to be colocalized with NADPH oxidase components in macrophage-rich areas in endarterectomies from atherosclerotic patients19 and the findings that support a role for MMP-9 in the development of the plaque.32,33⇓
The clinical meaning of the in vitro observations here reported is underlined by our observations in hypertensive patients. We show, for the first time, that patients treated with losartan exhibit lower values of NADPH oxidase–dependent O2·− production than untreated hypertensives. Because no changes in this parameter were observed either in patients treated with other ARAs or in patients treated with ACEIs, we can speculate that the observed effect in losartan-treated patients is independent of angiotensin II and the AT1R, thus reinforcing the potential contribution of EXP3179 to the above findings.
Finally, we found that hypertensive patients treated with losartan exhibited lower plasma levels of MMP-9 than untreated hypertensives and hypertensive patients treated with other ARAs or ACEIs. In addition, we found a significant positive bivariate correlation between phagocytic NADPH oxidase–dependent O2·− production and plasma levels of MMP-9 in the whole population of hypertensive patients. Nevertheless, we cannot discard the contribution of other vascular cells, including endothelial cells and smooth muscle cells, on the generation of MMP-9.34,35⇓ Collectively, these findings support the possibility that reduced MMP-9 might be an additional beneficial effect of losartan in hypertension, likely attributed to the ability of its EXP3179 metabolite to inhibit phagocytic NADPH oxidase–dependent O2·− production. This specific property of losartan may be involved in its capacity to inhibit experimental atherosclerosis,36 as well as to prevent ischemic atherosclerotic events in hypertensive patients.37
Albeit preliminary, these data suggest that EXP3179 acts as an inhibitor of NADPH oxidase, namely in phagocytic cells, by a potential mechanism that targets the PKC signaling pathway. Furthermore, the observed effects of EXP3179 on MMP-9 secretion from phagocytic cells might be downstream effectors of PKC inhibition. Thus, it is proposed that EXP3179 can be a specific mediator of the ability of losartan to reduce cell-mediated oxidative stress in hypertension.
We thank Merck & Co, Inc, for kindly providing losartan, EXP3174, and EXP3179. We gratefully acknowledge the technical assistance of Raquel Ros, Idoia Rodríguez, and Ana Montoya.
Sources of Funding
This project was funded through the agreement between the Foundation for Applied Medical Research and “Unión Temporal de Empresas Project Centro para la Investigación Médica Aplicada”; by a medical school grant from Merck & Co, Inc; by the Red Temática de Investigación Cooperativa en Enfermedades Cardiovasculares from the Instituto de Salud Carlos III, Ministry of Health, Spain (grant RD06/0014/0008); by the European Union (InGenious HyperCare, grant LSHM-CT-2006-037093); by the Department of Health of the Government of Navarra (grant 2538/2008); and by the Ministry of Science and Culture, Spain (grant SAF2007-62553).
- Received January 16, 2009.
- Revision received February 3, 2009.
- Accepted July 16, 2009.
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- ↵Cathcart MK. Regulation of superoxide anion production by NADPH oxidase in monocytes/macrophages: contributions to atherosclerosis. Arterioscler Thromb Vasc Biol. 2004; 24: 23–28.
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