Losartan Metabolite EXP3179 Activates Akt and Endothelial Nitric Oxide Synthase via Vascular Endothelial Growth Factor Receptor-2 in Endothe
http://www.100md.com
《循环学杂志》
the Cardiovascular Research Institute and Department of Medicine (T.W., J.S., H.Y., B.C.B.) and Department of Pharmacology and Physiology (V.K.S., S.-S.S.), University of Rochester, Rochester, NY.
Abstract
Background— Recent studies suggest that angiotensin type 1 receptor (AT1R) blockers have vascular protective effects beyond blood pressure lowering. Because of the importance of endothelial nitric oxide synthase (eNOS) in vascular and platelet function, we hypothesized that losartan and its metabolites would stimulate eNOS and its upstream activators Akt and phosphatidylinositol 3-kinase (PI3K).
Methods and Results— Losartan is metabolized into EXP3174 (AT1R-blocking metabolite) and EXP3179 (no AT1R-blocking properties). Treatment of endothelial cells (ECs) with losartan and both metabolites stimulated phosphorylation of Akt and eNOS in the absence of angiotensin II. However, the magnitude for EXP3179 was much greater than EXP3174, and the EC50 was significantly lower (–logEC50, 8.2±0.1 versus 5.4±0.2 mol/L), suggesting an AT1R-independent effect. Inhibiting PI3K or vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine phosphorylation abrogated EXP3179-induced eNOS phosphorylation. In endothelium of intact rat aorta, EXP3179 also stimulated Akt and eNOS phosphorylation. VEGFR2 activation was shown to be calcium and Src family kinase dependent by use of specific drug inhibitors and dominant negative kinase transfection. EXP3179 significantly inhibited tumor necrosis factor –induced apoptosis by 60% (from 30.1±5.8% to 12.2±2.0% TUNEL-positive cells), which was abolished by pretreatment with the PI3K inhibitor LY294002. Cleaved caspase-3 was suppressed by 48% with EXP3179.
Conclusions— The losartan metabolite EXP3179 stimulates eNOS phosphorylation and suppresses tumor necrosis factor –induced EC apoptosis by activating the VEGFR2/PI3K/Akt pathway.
Key Words: apoptosis endothelium nitric oxide synthase signal transduction
Introduction
Angiotensin type 1 receptor (AT1R) blockers (ARBs) are clinically effective in the management of hypertension and congestive heart failure.1 There is active debate over the relative cardiovascular benefits of drugs that inhibit the renin-angiotensin system compared with drugs from other antihypertensive classes. Currently, there is strong evidence from the HOPE trial to support the use of ACE inhibitors in patients with multiple risk factors for coronary artery disease.2 However, several other trials have raised some concerns. For example, in PROGRESS, single ACE inhibitor therapy produced no discernable reduction in the incidence of stroke.3 In VALUE, valsartan (compared with amlodipine) was associated with a higher incidence at early times in stroke and myocardial infarction.4 Recent results from LIFE, which enrolled patients with left ventricular hypertrophy, showed a significantly greater benefit of losartan compared with atenolol for stroke.5 The difference between losartan and atenolol in terms of stroke reduction was not explained by differences in blood pressure. Likely explanations for blood pressure–independent effects of ARBs include improved endothelial function and decreased coagulation and inflammation.
Recently, several articles have shown that some ARBs such as losartan and irbesartan have AT1R-independent anticoagulant and antiinflammatory effects.6,7 Krmer et al6 demonstrated that the losartan metabolite EXP3179, which has no AT1R-blocking properties, inhibited expression of cyclooxygenase-2 and suppressed thromboxane A2–induced platelet aggregation, suggesting that the AT1R-independent effects of losartan are mediated primarily by its metabolites.
Here, we show that the losartan metabolite EXP3179 promotes phosphorylation of endothelial nitric oxide synthase (eNOS) through a VEGFR2/PI3K/Akt pathway and inhibits endothelial apoptosis, suggesting another AT1R-independent beneficial effect of losartan on endothelial cells (ECs).
Methods
Materials
Antibody Sources included the following: phospho-eNOS (phospho-Ser-1177 in the human eNOS sequence, corresponding to Ser-1179 in bovine eNOS), phospho-Akt (Ser-473), Akt, cleaved caspase-3, and caspase-3 were from Cell Signaling Technologies; Flk1/KDR (vascular endothelial growth factor [VEGF] receptor 2 [VEGFR2]) and eNOS were from Santa Cruz; HA.11 was from Covance Research Products; phosphotyrosine (clone 4G10) was from Upstate Biotechnology); and phospho-IRS-1 (Tyr-612) was from Biosource. Losartan, 2-butyl-4-chloro-1{[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl}-1H-imidazole-5-carboxaldehyde (EXP3179), and 2-butyl-4-chloro-1{[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl}-1H-imidazole-5-carboxylic acid (EXP3174) were provided by Merck & Co, Inc. Irbesartan was provided by Bristol Myers Squibb. VEGF165 was purchased from R&D Systems. LY294002, VEGF receptor tyrosine kinase inhibitor (VTKi; 4-[(4'-chrolo-2'-fluoro)phenylamino]-6,7-dimethoxy-quinazoline), SU1498, PTX, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM), PP2, manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) were purchased from Calbiochem. Protein A/G PLUS-agarose was from Santa Cruz; N-acetyl cysteine (NAC), tiron, and ebselen were from Sigma. Anti-VEGF neutralizing monoclonal antibody 577B11 was a gift from Dr Bjercke. Hoechst 33342 was purchased from Molecular Probes Co. Tumor necrosis factor- (TNF) and in situ cell death detection kit were purchased from Roche Applied Science.
Cell Culture and Transfection
Bovine aortic endothelial cells (BAECs) purchased from Clonetics were cultured in medium 199 supplemented with 10% FBS (Hyclone). Confluent cells cultured in 60-mm dishes were serum starved for 18 to 24 hours and stimulated by losartan, irbesartan, EXP3179, and EXP3174. For the inhibitor studies, cells were pretreated with 50 ng/mL PTX for 24 hours and 10 μmol/L LY294002, 30 μmol/L SU1498, 10 μmol/L VTKi, 10 μg/mL 577B11, 20 μmol/L BAPTA/AM, 10 mmol/L NAC, 10 mmol/L tiron, 40 μmol/L ebselen, and 100 μmol/L MnTBAP for 30 minutes. For transfection, BAECs at >90% confluence in 6-well dishes were washed with Opti-MEM. For each dish, 5 μL Lipofectamine 2000 (Invitrogen) was mixed with Opti-MEM (250 μL), and dominant negative HA-tagged Akt1 (DN-HA-Akt, K179A, T308A, S473A=AAA-PKB)8 or pcDNA3.1 as negative control was added to the solution, mixed gently, and incubated at room temperature for 20 minutes. This mixture was added to ECs in 2 mL Opti-MEM and cultured for 3 hours. The medium was changed to medium 199 with 10% serum and cultured for 20 hours. Thereafter, cells were serum starved for 24 hours and stimulated by losartan or its metabolites. For adenovirus, BAECs at 90% confluence were infected with adenovirus-expressing kinase inactive Src (Ad.KI-Src) or LacZ (Ad.LacZ) for 1 hour at 37°C and then incubated with 5 mL M199 supplemented with 10% FBS for 48 hours as described previously.9
Intact Rat Aorta
Animal experiments were performed according to the guidelines of the National Institutes of Health and American Heart Association for the care and use of laboratory animals and were approved by the University of Rochester Animal Care Committee. Adult female Sprague-Dawley rats (9 months old) were killed, and the aortas were harvested. After equilibration at 37°C for 2 hours, aortas were treated with 10–7 mol/L EXP3179 for 30 minutes. EC and smooth muscle cell (SMC) proteins were harvested separately as previously described.10
Immunoprecipitation and Western Blot Analysis
Immunoprecipitation and Western blot experiments were performed as described previously.10,11 We performed Western blot analysis using Odyssey infrared imaging system following the manufacturer’s guidelines (Li-COR Biosciences). Odyssey blocking buffer and fluorescent secondary antibodies specific for this system were used.
[Ca2+]i Measurements in BAECs
Intracellular Ca2+ was measured as previously described.12 In brief, cells were grown on glass coverslips and loaded with 5 μmol/L fura-2/AM for 30 minutes at room temperature. After loading, the coverslip was mounted in a tissue chamber on the stage of a Nikon Diaphot inverted microscope equipped for epifluorescence (Till Imaging). The cell was sequentially excited at 340- and 380-nm wavelength light using 2 excitation monochromators, and emission fluorescence collected at 510 nm. The results are presented as the ratio of fluorescence at 340 nm (F340) to 380 nm (F380).
Apoptosis Assays by TdT-Mediated dUTP Nick-End Labeling
Measurement of TdT-mediated dUTP nick-end labeling (TUNEL) fluorescence was performed according to manufacturer’s instructions (Roche Applied Science).
Statistical Analysis
Quantitative data are reported as mean±SE. Statistical analysis was performed with ANOVA. Group comparisons were performed with Fisher’s protected least significant difference (PLSD). A confidence level of 95% was considered statistically significant.
Results
AT1R-Independent Activation of Akt and eNOS by Losartan
To evaluate the effects of losartan and its metabolites on EC function, we measured activation of eNOS and Akt. Treatment with losartan (10–7 mol/L) increased phosphorylation of Akt and eNOS (Figure 1A). Maximum Akt and eNOS phosphorylation occurred at 30 to 60 minutes (Figure 1A and 1B). The biotransformation of losartan in vivo involves cytochrome P450 oxidation of losartan first to an aldehyde intermediate (EXP3179) and then to the carboxylic acid product (EXP3174).13 EXP3174 has been shown to be pharmacologically more active than either losartan or EXP3179 with respect to binding to the AT1R.14 The 2 metabolites of losartan, EXP3174 and EXP3179, also increased Akt and eNOS phosphorylation (Figure 1C and 1D and Figure 2A). Unexpectedly, EXP3179 stimulated Akt and eNOS phosphorylation to a level equivalent to losartan and significantly greater than EXP3174, particularly at clinically relevant doses (Figure 2A and 2B).6,13 The concentration response for losartan and EXP3179 showed a significantly lower EC50 for Akt phosphorylation (–logEC50, 7.6±0.4 and 8.2±0.1 mol/L, respectively, P<0.0001) compared with EXP3174 (5.4±0.2 mol/L). The time course for phosphorylation of Akt and eNOS by physiological concentration of EXP3179 (10–7 mol/L) resembled losartan with maximum at 30 to 60 minutes (compare Figure 1C and 1D with Figure 1A and 1B), suggesting a similar mechanism of action.
Because EXP3179 binds to the AT1R with markedly lower affinity than EXP3174, the data suggested that activation of Akt and eNOS was independent of AT1R blocking. ARBs have been shown to potentiate AT2R signaling and to block AT1R signaling in the presence of angiotensin II (Ang II), leading to eNOS activation.15 To rule out this possibility, we used the competitive Ang II receptor antagonist [Sar1Ile8]Ang II that would block both AT1R and AT2R. As shown in Figure S1A (found in the online-only Data Supplement at http://circ.ahajournals.org/cgi/content/full/112/12/1798/DC1), [Sar1Ile8]Ang II did not significantly inhibit EXP3179-mediated activation of Akt. To prove that EXP3179, rather than its oxidation product EXP3174, was the dominant stimulus for Akt activation, we used sulfaphenazole, a cytochrome P450 inhibitor (especially of CYP2C9 and CYP2C10, the dominant isoforms in ECs16) that has previously been shown to inhibit EXP3179 metabolism.13 As shown in Data Supplement Figure S1B, sulfaphenazole had no significant effect on either losartan- or EXP3179-mediated Akt activation. Because sulfaphenazole did not inhibit losartan-induced Akt activation, losartan itself also stimulates Akt phosphorylation without conversion to EXP3174. Irbesartan, which has no active metabolites, failed to induce Akt and eNOS phosphorylation (Data Supplement Figure S2). These findings suggest that the AT1R-blocking properties of losartan and its metabolites is not important for activation of Akt and eNOS.
EXP3179-Induced eNOS Phosphorylation Is Mediated by an Akt/PI3K Pathway
To elucidate the mechanism by which EXP3179 stimulated phosphorylation of Akt and eNOS, cells were pretreated with PI3K inhibitor LY294002 (10 μmol/L) for 30 minutes before exposure to EXP3179 (10–7 mol/L). LY294002 suppressed both EXP3179-induced Akt and eNOS phosphorylation in a concentration-dependent manner (Figure 3A and 3B). To show further that EXP3179 required Akt activity, we transfected cells with DN-HA-Akt. As shown in Figure 3C-D, VEGF stimulated phosphorylation of eNOS was completely inhibited by DN-HA-Akt. EXP3179-induced eNOS phosphorylation was also inhibited 100% by DN-HA-Akt (Figure 3C and 3D). These data indicate that EXP3179 mediates eNOS phosphorylation through a PI3K/Akt pathway.
VEGFR2 Phosphorylation Is Involved in Activation of the PI3K/Akt Pathway by EXP3179
To elucidate the mechanism for EXP3179-mediated PI3K/Akt/eNOS activation, we measured VEGFR2 tyrosine phosphorylation. Samples were immunoprecipitated with anti-VEGFR2 antibody, and Western blots were probed with anti-phosphotyrosine antibody (4G10) or anti-VEGFR2 antibody. As shown in Figure 4A and 4B, tyrosine phosphorylation of VEGFR2 increased at 5 minutes and peaked at 15 minutes (2.5±0.4-fold) after exposure to 10–7 mol/L EXP3179. To determine whether VEGFR2 phosphorylation is required for activation of Akt/eNOS pathway, we treated ECs with VEGFR tyrosine kinase inhibitors (SU1498 and VTKi) for 30 minutes. SU1498 and VTKi significantly inhibited phosphorylation of VEGFR2, Akt, and eNOS (Figure 5A; compare lane 2 with lanes 3 and 4). To evaluate whether secretion of VEGF by EXP3179 was responsible for VEGFR2 phosphorylation, we used the VEGF neutralization antibody 577B11. Pretreatment with 577B11 did not block EXP3179-induced phosphorylation of VEGFR2, Akt, and eNOS (Figure 5A; compare lanes 2 and 5).
To determine the mechanism by which EXP3179 stimulates VEGFR2, we examined the effects of several inhibitors on EXP3179-mediated phosphorylation of VEGFR2, Akt, and eNOS (Figure 5B). The Gi protein inhibitor PTX did not block phosphorylation of VEGFR2, Akt, and eNOS by EXP3179. In contrast, the calcium chelator BAPTA/AM and the Src kinase family inhibitor PP2 completely blocked EXP3179-induced phosphorylation (Figure 5B). To determine whether EXP3179 increased intracellular Ca2+ we used fura-2 to measure Ca2+ transients in BAECs. EXP3179 did not increase intracellular Ca2+ concentration (Data Supplement Figure S3A). To evaluate further the contribution of Src kinase to EXP3179-induced VEGFR2 phosphorylation, we infected BAECs with kinase inactive Src (Ad.KI-Src). Ad.KI-Src did not suppress EXP3179-induced Akt/eNOS phosphorylation (Data Supplement Figure S3B) at concentrations shown previously to be effective.9 The inhibition by PP2, but not by Ad.KI-Src, suggests that a Src kinase family member other than Src is probably involved in EXP3179-induced VEGFR2 transactivation. Because tyrosine kinase receptor transactivation has been reported to be reactive oxygen species dependent,17 we examined the effect of the anti-oxidant NAC on VEGFR2 phosphorylation. NAC had no effect on EXP3179-induced VEGFR2, Akt, and eNOS phosphorylation (Figure 5B). Several other antioxidants (tiron, ebselen, and MnTBAP) also had no effect on EXP3179-induced Akt phosphorylation (Data Supplement Figure S4). Finally, it has been reported that losartan may improve insulin resistance induced by Ang II by altering IRS-1 phosphorylation. Specifically, Andreozzi et al18 reported that losartan enhanced insulin-stimulated tyrosine phosphorylation of IRS-1 on Tyr612 and Tyr632 (required for interaction with the p85 subunit of PI3K). To evaluate the effect of EXP3179 on IRS-1 phosphorylation, BAECs were treated with 10–7 mol/L EXP3179 or 1 μmol/L insulin for 5 to 30 minutes. Although insulin significantly increased phosphorylation of Tyr612 at 5 minutes, there was no effect of EXP3179 at any time (Data Supplement Figure S3C). Taken together, these data show that EXP3179 stimulates VEGFR2 tyrosine phosphorylation via a pathway dependent on intracellular calcium and Src kinase family members independently of IRS-1 tyrosine phosphorylation, reactive oxygen species, increases in intracellular calcium, and PTX-sensitive G proteins.
EXP3179 Inhibits TNF-Induced Caspase-3 Activation and Prevents Endothelial Apoptosis
Akt has been shown to mediate growth factor–dependent survival signals in ECs.19 Therefore, we tested whether EXP3179 could protect ECs from apoptosis. EC apoptosis was induced by TNF (10 ng/mL) in the presence of cycloheximide (CHX; 10 μg/mL) as reported by our laboratory.20 Pretreatment with EXP3179 for 2 hours significantly suppressed TNF-induced apoptosis by 60%, comparable to the effect of VEGF (from 30.1±5.8% to 12.2±2.0% TUNEL-positive cells; Figures 6 and S5). The EXP3179 antiapoptotic effect was abolished by pretreatment with LY294002 and overexpression of DN-HA-Akt (Figure 6).
To study the EXP3179 antiapoptotic effect more fully, we measured caspase-3 activation. As shown in Figure 7A, TNF and CHX treatment increased cleaved caspase-3 that was suppressed by 48% with EXP3179 treatment. LY294002 abrogated the inhibitory effect of EXP3179 on TNF-induced caspase-3 activation (Figure 7B). Overexpression of DN-HA-Akt also abrogated the effect of EXP3179 to prevent caspase-3 activation (Figure 7C, 7D).
EXP3179 Activates Phosphorylation of eNOS and Akt in the Intact Rat Aorta
To evaluate the effects of EXP3179 on the intact vessel, we examined whether EXP3179 activated the Akt/eNOS pathway in rat aortas. After equilibration at 37°C for 2 hours, rat aortas were incubated with 10–7 mol/L EXP3179 for 30 minutes. Figure 8 shows that EXP3179 induced phosphorylation of Akt and eNOS in aortic ECs but not in SMCs (consistent with the lack of VEGFR2 in SMCs).
Discussion
The major finding of this study is that the AT1R antagonist losartan and its metabolite EXP3179 stimulate phosphorylation of eNOS by activating a VEGFR2/PI3K/Akt pathway (Data Supplement Figure S6). In rat aortic ECs, EXP3179 also induces eNOS phosphorylation via this pathway (Figure 8). Moreover, EXP3179 suppressed TNF-induced caspase-3 activation and EC apoptosis (Figure 7). The results demonstrate direct effects of losartan and EXP3179 on EC function independently of Ang II and the AT1R. The effect was clearly independent of the AT1R because EXP3179 (the non–AT1R-binding metabolite) exhibited greater effects than EXP3174 (the AT1R-binding metabolite) and [Sar1Ile8]Ang II did not inhibit EXP3179.
It has been reported that AT1R blockers improve EC dysfunction in patients with hypertension.21–23 Several groups have reported that inhibiting the AT1R promotes selective binding of Ang II to the free AT2R, leading to AT2R stimulation and potential upregulation of eNOS.15,24,25 However, the mechanism of EXP3179-induced Akt/eNOS activation reported here is probably different because Ang II was not present in the culture media. Furthermore, EXP3179 induced phosphorylation of Akt in the presence of the Ang receptor blocker [Sar1Ile8]Ang II (Data Supplement Figure S1A). In addition, Akt and eNOS phosphorylation induced by losartan and EXP3179 appears to be independent of AT1R blocking action (Figure 2) because EXP3179 does not bind the AT1R. This conclusion is supported by the fact that the affinity of EXP3174 for the AT1R is 10 times greater than losartan,13 yet losartan was 100 times more potent than EXP3174 at Akt and eNOS phosphorylation. Indeed, irbesartan had no effect on Akt and eNOS phosphorylation, also indicating that the effect of EXP3179 is independent of AT1R blocking action.
Andreozzi et al18 showed that Ang II inhibited insulin signaling (NO production and Akt phosphorylation) by increasing IRS-1 serine phosphorylation and inhibiting insulin stimulation of IRS-1 tyrosine phosphorylation necessary for interaction with PI3K. Losartan reversed this effect of Ang II by blocking the AT1R. Although these findings may be clinically important, they differ importantly from the present study. First, the effects of EXP3179 and losartan reported here are independent of AT1R binding, and all experiments were performed in the absence of Ang II. Second, there was no effect of EXP3179 on IRS-1 phosphorylation. Finally, the effect of EXP3179 and losartan requires transactivation of VEGFR2 (via a calcium and Src family kinase pathway) that is relatively unique to EC. Thus, SMCs that express the AT1R failed to show Akt activation by EXP3179 (Figure 8).
Recently, it has been reported that biological actions of all ARBs are not identical.26 For example, the ability of losartan and irbesartan to inhibit cyclooxygenase-2 or the thromboxane A2/prostaglandin endoperoxide H2 receptor is not shared by candesartan. It was also reported that telmisartan and some other ARBS27,28 may act as partial agonists of PPAR, indicating that some of the antiinflammatory and anticoagulant action of these drugs may be related to their individual chemical characteristics rather than an ARB class effect.6,7,29 However, none of these effects explain the transactivation of VEGFR2 and downstream events reported here.
The effects of losartan and EXP3179 appear to be via an EC-specific pathway requiring VEGFR2 because activation of Akt by EXP3179 was not observed in rat aortic SMCs that lack VEGR2 (Figure 8). Our data demonstrate that VEGFR2 is activated by a pathway dependent on intracellular calcium and a Src kinase family member (Data Supplement Figure S6). Importantly, signaling does not involve autocrine secretion of VEGF, a G protein–coupled receptor, reactive oxygen species, Src kinase itself, or IRS-1. Specific experimental results to support this model include the following: There was no effect of EXP3179 on intracellular Ca2+ transients after EXP3179 stimulation (Data Supplement Figure S3A); PTX did not inhibit EXP3179-induced VEGFR2 phosphorylation (Figure 5B); several antioxidants had no effect on EXP3179-induced VEGFR2, Akt, and eNOS phosphorylation (Figure 5B); adenoviral overexpression of kinase inactive Src (Ad.KI-Src) did not inhibit EXP3179-induced Akt and eNOS phosphorylation (Data Supplement Figure S3B); and EXP3179 did not stimulate tyrosine phosphorylation of IRS-1 on Tyr612 (Data Supplement Figure S3C). We did find that BAPTA/AM (intracellular calcium chelator) and PP2 (Src kinase family inhibitor) inhibited EXP3179-induced VEGFR2 autophosphorylation and Akt/eNOS phosphorylation (Figure 5B), indicating that EXP3179-induced VEGFR2 autophosphorylation required Ca2+ and Src kinase activation (transactivation). We also examined the effects of EXP3179 on other mitogen-activated protein kinases in ECs. We found that EXP3179 stimulated phosphorylation of extracellular signal-regulated kinase (ERK1/2) but not p38 and c-Jun N-terminal kinase (data not shown). ERK1/2 activation is known to promote cell survival signals in ECs as well as Akt. However, we demonstrated that LY2940002 and expression of dominant negative Akt kinase almost completely inhibited the antiapoptotic effect of EXP3179 on ECs (Figure 6). These findings indicate that the EXP3179 exerts antiapoptotic effects primarily through the PI3K/Akt pathway rather than the ERK pathway.
VEGF exhibits multiple biological activities in ECs, including promoting EC survival.30,31 VEGF effects on cell survival are mediated by a VEGFR/PI3K/Akt pathway.19,32 EXP3179 significantly suppressed EC apoptosis and caspase-3 activation via an Akt pathway, suggesting that EXP3179, in part, mimics VEGF. In summary, the present study shows that the losartan metabolite EXP3179 stimulates eNOS phosphorylation and suppresses TNF-induced EC apoptosis by activating a VEGFR2/PI3K/Akt pathway independently of AT1R-mediated signaling.
Acknowledgments
This study was supported by a grant from Merck & Co., Inc, to Dr Berk. Dr Watanabe was supported by a fellowship from Japan Heart Foundation & Bayer Yakuhin Research Grant Abroad (Tokyo, Japan).
Disclosure
Dr Berk has received research grants from Merck and Novartis; has served on the speakers’ bureaus of and/or received honoraria from Merck, Novartis, Pfizer, and Bristol-Myers Squibb; and has consulted for Merck, Novartis, and Bristol-Myers Squibb.
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Abstract
Background— Recent studies suggest that angiotensin type 1 receptor (AT1R) blockers have vascular protective effects beyond blood pressure lowering. Because of the importance of endothelial nitric oxide synthase (eNOS) in vascular and platelet function, we hypothesized that losartan and its metabolites would stimulate eNOS and its upstream activators Akt and phosphatidylinositol 3-kinase (PI3K).
Methods and Results— Losartan is metabolized into EXP3174 (AT1R-blocking metabolite) and EXP3179 (no AT1R-blocking properties). Treatment of endothelial cells (ECs) with losartan and both metabolites stimulated phosphorylation of Akt and eNOS in the absence of angiotensin II. However, the magnitude for EXP3179 was much greater than EXP3174, and the EC50 was significantly lower (–logEC50, 8.2±0.1 versus 5.4±0.2 mol/L), suggesting an AT1R-independent effect. Inhibiting PI3K or vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine phosphorylation abrogated EXP3179-induced eNOS phosphorylation. In endothelium of intact rat aorta, EXP3179 also stimulated Akt and eNOS phosphorylation. VEGFR2 activation was shown to be calcium and Src family kinase dependent by use of specific drug inhibitors and dominant negative kinase transfection. EXP3179 significantly inhibited tumor necrosis factor –induced apoptosis by 60% (from 30.1±5.8% to 12.2±2.0% TUNEL-positive cells), which was abolished by pretreatment with the PI3K inhibitor LY294002. Cleaved caspase-3 was suppressed by 48% with EXP3179.
Conclusions— The losartan metabolite EXP3179 stimulates eNOS phosphorylation and suppresses tumor necrosis factor –induced EC apoptosis by activating the VEGFR2/PI3K/Akt pathway.
Key Words: apoptosis endothelium nitric oxide synthase signal transduction
Introduction
Angiotensin type 1 receptor (AT1R) blockers (ARBs) are clinically effective in the management of hypertension and congestive heart failure.1 There is active debate over the relative cardiovascular benefits of drugs that inhibit the renin-angiotensin system compared with drugs from other antihypertensive classes. Currently, there is strong evidence from the HOPE trial to support the use of ACE inhibitors in patients with multiple risk factors for coronary artery disease.2 However, several other trials have raised some concerns. For example, in PROGRESS, single ACE inhibitor therapy produced no discernable reduction in the incidence of stroke.3 In VALUE, valsartan (compared with amlodipine) was associated with a higher incidence at early times in stroke and myocardial infarction.4 Recent results from LIFE, which enrolled patients with left ventricular hypertrophy, showed a significantly greater benefit of losartan compared with atenolol for stroke.5 The difference between losartan and atenolol in terms of stroke reduction was not explained by differences in blood pressure. Likely explanations for blood pressure–independent effects of ARBs include improved endothelial function and decreased coagulation and inflammation.
Recently, several articles have shown that some ARBs such as losartan and irbesartan have AT1R-independent anticoagulant and antiinflammatory effects.6,7 Krmer et al6 demonstrated that the losartan metabolite EXP3179, which has no AT1R-blocking properties, inhibited expression of cyclooxygenase-2 and suppressed thromboxane A2–induced platelet aggregation, suggesting that the AT1R-independent effects of losartan are mediated primarily by its metabolites.
Here, we show that the losartan metabolite EXP3179 promotes phosphorylation of endothelial nitric oxide synthase (eNOS) through a VEGFR2/PI3K/Akt pathway and inhibits endothelial apoptosis, suggesting another AT1R-independent beneficial effect of losartan on endothelial cells (ECs).
Methods
Materials
Antibody Sources included the following: phospho-eNOS (phospho-Ser-1177 in the human eNOS sequence, corresponding to Ser-1179 in bovine eNOS), phospho-Akt (Ser-473), Akt, cleaved caspase-3, and caspase-3 were from Cell Signaling Technologies; Flk1/KDR (vascular endothelial growth factor [VEGF] receptor 2 [VEGFR2]) and eNOS were from Santa Cruz; HA.11 was from Covance Research Products; phosphotyrosine (clone 4G10) was from Upstate Biotechnology); and phospho-IRS-1 (Tyr-612) was from Biosource. Losartan, 2-butyl-4-chloro-1{[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl}-1H-imidazole-5-carboxaldehyde (EXP3179), and 2-butyl-4-chloro-1{[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl}-1H-imidazole-5-carboxylic acid (EXP3174) were provided by Merck & Co, Inc. Irbesartan was provided by Bristol Myers Squibb. VEGF165 was purchased from R&D Systems. LY294002, VEGF receptor tyrosine kinase inhibitor (VTKi; 4-[(4'-chrolo-2'-fluoro)phenylamino]-6,7-dimethoxy-quinazoline), SU1498, PTX, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM), PP2, manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) were purchased from Calbiochem. Protein A/G PLUS-agarose was from Santa Cruz; N-acetyl cysteine (NAC), tiron, and ebselen were from Sigma. Anti-VEGF neutralizing monoclonal antibody 577B11 was a gift from Dr Bjercke. Hoechst 33342 was purchased from Molecular Probes Co. Tumor necrosis factor- (TNF) and in situ cell death detection kit were purchased from Roche Applied Science.
Cell Culture and Transfection
Bovine aortic endothelial cells (BAECs) purchased from Clonetics were cultured in medium 199 supplemented with 10% FBS (Hyclone). Confluent cells cultured in 60-mm dishes were serum starved for 18 to 24 hours and stimulated by losartan, irbesartan, EXP3179, and EXP3174. For the inhibitor studies, cells were pretreated with 50 ng/mL PTX for 24 hours and 10 μmol/L LY294002, 30 μmol/L SU1498, 10 μmol/L VTKi, 10 μg/mL 577B11, 20 μmol/L BAPTA/AM, 10 mmol/L NAC, 10 mmol/L tiron, 40 μmol/L ebselen, and 100 μmol/L MnTBAP for 30 minutes. For transfection, BAECs at >90% confluence in 6-well dishes were washed with Opti-MEM. For each dish, 5 μL Lipofectamine 2000 (Invitrogen) was mixed with Opti-MEM (250 μL), and dominant negative HA-tagged Akt1 (DN-HA-Akt, K179A, T308A, S473A=AAA-PKB)8 or pcDNA3.1 as negative control was added to the solution, mixed gently, and incubated at room temperature for 20 minutes. This mixture was added to ECs in 2 mL Opti-MEM and cultured for 3 hours. The medium was changed to medium 199 with 10% serum and cultured for 20 hours. Thereafter, cells were serum starved for 24 hours and stimulated by losartan or its metabolites. For adenovirus, BAECs at 90% confluence were infected with adenovirus-expressing kinase inactive Src (Ad.KI-Src) or LacZ (Ad.LacZ) for 1 hour at 37°C and then incubated with 5 mL M199 supplemented with 10% FBS for 48 hours as described previously.9
Intact Rat Aorta
Animal experiments were performed according to the guidelines of the National Institutes of Health and American Heart Association for the care and use of laboratory animals and were approved by the University of Rochester Animal Care Committee. Adult female Sprague-Dawley rats (9 months old) were killed, and the aortas were harvested. After equilibration at 37°C for 2 hours, aortas were treated with 10–7 mol/L EXP3179 for 30 minutes. EC and smooth muscle cell (SMC) proteins were harvested separately as previously described.10
Immunoprecipitation and Western Blot Analysis
Immunoprecipitation and Western blot experiments were performed as described previously.10,11 We performed Western blot analysis using Odyssey infrared imaging system following the manufacturer’s guidelines (Li-COR Biosciences). Odyssey blocking buffer and fluorescent secondary antibodies specific for this system were used.
[Ca2+]i Measurements in BAECs
Intracellular Ca2+ was measured as previously described.12 In brief, cells were grown on glass coverslips and loaded with 5 μmol/L fura-2/AM for 30 minutes at room temperature. After loading, the coverslip was mounted in a tissue chamber on the stage of a Nikon Diaphot inverted microscope equipped for epifluorescence (Till Imaging). The cell was sequentially excited at 340- and 380-nm wavelength light using 2 excitation monochromators, and emission fluorescence collected at 510 nm. The results are presented as the ratio of fluorescence at 340 nm (F340) to 380 nm (F380).
Apoptosis Assays by TdT-Mediated dUTP Nick-End Labeling
Measurement of TdT-mediated dUTP nick-end labeling (TUNEL) fluorescence was performed according to manufacturer’s instructions (Roche Applied Science).
Statistical Analysis
Quantitative data are reported as mean±SE. Statistical analysis was performed with ANOVA. Group comparisons were performed with Fisher’s protected least significant difference (PLSD). A confidence level of 95% was considered statistically significant.
Results
AT1R-Independent Activation of Akt and eNOS by Losartan
To evaluate the effects of losartan and its metabolites on EC function, we measured activation of eNOS and Akt. Treatment with losartan (10–7 mol/L) increased phosphorylation of Akt and eNOS (Figure 1A). Maximum Akt and eNOS phosphorylation occurred at 30 to 60 minutes (Figure 1A and 1B). The biotransformation of losartan in vivo involves cytochrome P450 oxidation of losartan first to an aldehyde intermediate (EXP3179) and then to the carboxylic acid product (EXP3174).13 EXP3174 has been shown to be pharmacologically more active than either losartan or EXP3179 with respect to binding to the AT1R.14 The 2 metabolites of losartan, EXP3174 and EXP3179, also increased Akt and eNOS phosphorylation (Figure 1C and 1D and Figure 2A). Unexpectedly, EXP3179 stimulated Akt and eNOS phosphorylation to a level equivalent to losartan and significantly greater than EXP3174, particularly at clinically relevant doses (Figure 2A and 2B).6,13 The concentration response for losartan and EXP3179 showed a significantly lower EC50 for Akt phosphorylation (–logEC50, 7.6±0.4 and 8.2±0.1 mol/L, respectively, P<0.0001) compared with EXP3174 (5.4±0.2 mol/L). The time course for phosphorylation of Akt and eNOS by physiological concentration of EXP3179 (10–7 mol/L) resembled losartan with maximum at 30 to 60 minutes (compare Figure 1C and 1D with Figure 1A and 1B), suggesting a similar mechanism of action.
Because EXP3179 binds to the AT1R with markedly lower affinity than EXP3174, the data suggested that activation of Akt and eNOS was independent of AT1R blocking. ARBs have been shown to potentiate AT2R signaling and to block AT1R signaling in the presence of angiotensin II (Ang II), leading to eNOS activation.15 To rule out this possibility, we used the competitive Ang II receptor antagonist [Sar1Ile8]Ang II that would block both AT1R and AT2R. As shown in Figure S1A (found in the online-only Data Supplement at http://circ.ahajournals.org/cgi/content/full/112/12/1798/DC1), [Sar1Ile8]Ang II did not significantly inhibit EXP3179-mediated activation of Akt. To prove that EXP3179, rather than its oxidation product EXP3174, was the dominant stimulus for Akt activation, we used sulfaphenazole, a cytochrome P450 inhibitor (especially of CYP2C9 and CYP2C10, the dominant isoforms in ECs16) that has previously been shown to inhibit EXP3179 metabolism.13 As shown in Data Supplement Figure S1B, sulfaphenazole had no significant effect on either losartan- or EXP3179-mediated Akt activation. Because sulfaphenazole did not inhibit losartan-induced Akt activation, losartan itself also stimulates Akt phosphorylation without conversion to EXP3174. Irbesartan, which has no active metabolites, failed to induce Akt and eNOS phosphorylation (Data Supplement Figure S2). These findings suggest that the AT1R-blocking properties of losartan and its metabolites is not important for activation of Akt and eNOS.
EXP3179-Induced eNOS Phosphorylation Is Mediated by an Akt/PI3K Pathway
To elucidate the mechanism by which EXP3179 stimulated phosphorylation of Akt and eNOS, cells were pretreated with PI3K inhibitor LY294002 (10 μmol/L) for 30 minutes before exposure to EXP3179 (10–7 mol/L). LY294002 suppressed both EXP3179-induced Akt and eNOS phosphorylation in a concentration-dependent manner (Figure 3A and 3B). To show further that EXP3179 required Akt activity, we transfected cells with DN-HA-Akt. As shown in Figure 3C-D, VEGF stimulated phosphorylation of eNOS was completely inhibited by DN-HA-Akt. EXP3179-induced eNOS phosphorylation was also inhibited 100% by DN-HA-Akt (Figure 3C and 3D). These data indicate that EXP3179 mediates eNOS phosphorylation through a PI3K/Akt pathway.
VEGFR2 Phosphorylation Is Involved in Activation of the PI3K/Akt Pathway by EXP3179
To elucidate the mechanism for EXP3179-mediated PI3K/Akt/eNOS activation, we measured VEGFR2 tyrosine phosphorylation. Samples were immunoprecipitated with anti-VEGFR2 antibody, and Western blots were probed with anti-phosphotyrosine antibody (4G10) or anti-VEGFR2 antibody. As shown in Figure 4A and 4B, tyrosine phosphorylation of VEGFR2 increased at 5 minutes and peaked at 15 minutes (2.5±0.4-fold) after exposure to 10–7 mol/L EXP3179. To determine whether VEGFR2 phosphorylation is required for activation of Akt/eNOS pathway, we treated ECs with VEGFR tyrosine kinase inhibitors (SU1498 and VTKi) for 30 minutes. SU1498 and VTKi significantly inhibited phosphorylation of VEGFR2, Akt, and eNOS (Figure 5A; compare lane 2 with lanes 3 and 4). To evaluate whether secretion of VEGF by EXP3179 was responsible for VEGFR2 phosphorylation, we used the VEGF neutralization antibody 577B11. Pretreatment with 577B11 did not block EXP3179-induced phosphorylation of VEGFR2, Akt, and eNOS (Figure 5A; compare lanes 2 and 5).
To determine the mechanism by which EXP3179 stimulates VEGFR2, we examined the effects of several inhibitors on EXP3179-mediated phosphorylation of VEGFR2, Akt, and eNOS (Figure 5B). The Gi protein inhibitor PTX did not block phosphorylation of VEGFR2, Akt, and eNOS by EXP3179. In contrast, the calcium chelator BAPTA/AM and the Src kinase family inhibitor PP2 completely blocked EXP3179-induced phosphorylation (Figure 5B). To determine whether EXP3179 increased intracellular Ca2+ we used fura-2 to measure Ca2+ transients in BAECs. EXP3179 did not increase intracellular Ca2+ concentration (Data Supplement Figure S3A). To evaluate further the contribution of Src kinase to EXP3179-induced VEGFR2 phosphorylation, we infected BAECs with kinase inactive Src (Ad.KI-Src). Ad.KI-Src did not suppress EXP3179-induced Akt/eNOS phosphorylation (Data Supplement Figure S3B) at concentrations shown previously to be effective.9 The inhibition by PP2, but not by Ad.KI-Src, suggests that a Src kinase family member other than Src is probably involved in EXP3179-induced VEGFR2 transactivation. Because tyrosine kinase receptor transactivation has been reported to be reactive oxygen species dependent,17 we examined the effect of the anti-oxidant NAC on VEGFR2 phosphorylation. NAC had no effect on EXP3179-induced VEGFR2, Akt, and eNOS phosphorylation (Figure 5B). Several other antioxidants (tiron, ebselen, and MnTBAP) also had no effect on EXP3179-induced Akt phosphorylation (Data Supplement Figure S4). Finally, it has been reported that losartan may improve insulin resistance induced by Ang II by altering IRS-1 phosphorylation. Specifically, Andreozzi et al18 reported that losartan enhanced insulin-stimulated tyrosine phosphorylation of IRS-1 on Tyr612 and Tyr632 (required for interaction with the p85 subunit of PI3K). To evaluate the effect of EXP3179 on IRS-1 phosphorylation, BAECs were treated with 10–7 mol/L EXP3179 or 1 μmol/L insulin for 5 to 30 minutes. Although insulin significantly increased phosphorylation of Tyr612 at 5 minutes, there was no effect of EXP3179 at any time (Data Supplement Figure S3C). Taken together, these data show that EXP3179 stimulates VEGFR2 tyrosine phosphorylation via a pathway dependent on intracellular calcium and Src kinase family members independently of IRS-1 tyrosine phosphorylation, reactive oxygen species, increases in intracellular calcium, and PTX-sensitive G proteins.
EXP3179 Inhibits TNF-Induced Caspase-3 Activation and Prevents Endothelial Apoptosis
Akt has been shown to mediate growth factor–dependent survival signals in ECs.19 Therefore, we tested whether EXP3179 could protect ECs from apoptosis. EC apoptosis was induced by TNF (10 ng/mL) in the presence of cycloheximide (CHX; 10 μg/mL) as reported by our laboratory.20 Pretreatment with EXP3179 for 2 hours significantly suppressed TNF-induced apoptosis by 60%, comparable to the effect of VEGF (from 30.1±5.8% to 12.2±2.0% TUNEL-positive cells; Figures 6 and S5). The EXP3179 antiapoptotic effect was abolished by pretreatment with LY294002 and overexpression of DN-HA-Akt (Figure 6).
To study the EXP3179 antiapoptotic effect more fully, we measured caspase-3 activation. As shown in Figure 7A, TNF and CHX treatment increased cleaved caspase-3 that was suppressed by 48% with EXP3179 treatment. LY294002 abrogated the inhibitory effect of EXP3179 on TNF-induced caspase-3 activation (Figure 7B). Overexpression of DN-HA-Akt also abrogated the effect of EXP3179 to prevent caspase-3 activation (Figure 7C, 7D).
EXP3179 Activates Phosphorylation of eNOS and Akt in the Intact Rat Aorta
To evaluate the effects of EXP3179 on the intact vessel, we examined whether EXP3179 activated the Akt/eNOS pathway in rat aortas. After equilibration at 37°C for 2 hours, rat aortas were incubated with 10–7 mol/L EXP3179 for 30 minutes. Figure 8 shows that EXP3179 induced phosphorylation of Akt and eNOS in aortic ECs but not in SMCs (consistent with the lack of VEGFR2 in SMCs).
Discussion
The major finding of this study is that the AT1R antagonist losartan and its metabolite EXP3179 stimulate phosphorylation of eNOS by activating a VEGFR2/PI3K/Akt pathway (Data Supplement Figure S6). In rat aortic ECs, EXP3179 also induces eNOS phosphorylation via this pathway (Figure 8). Moreover, EXP3179 suppressed TNF-induced caspase-3 activation and EC apoptosis (Figure 7). The results demonstrate direct effects of losartan and EXP3179 on EC function independently of Ang II and the AT1R. The effect was clearly independent of the AT1R because EXP3179 (the non–AT1R-binding metabolite) exhibited greater effects than EXP3174 (the AT1R-binding metabolite) and [Sar1Ile8]Ang II did not inhibit EXP3179.
It has been reported that AT1R blockers improve EC dysfunction in patients with hypertension.21–23 Several groups have reported that inhibiting the AT1R promotes selective binding of Ang II to the free AT2R, leading to AT2R stimulation and potential upregulation of eNOS.15,24,25 However, the mechanism of EXP3179-induced Akt/eNOS activation reported here is probably different because Ang II was not present in the culture media. Furthermore, EXP3179 induced phosphorylation of Akt in the presence of the Ang receptor blocker [Sar1Ile8]Ang II (Data Supplement Figure S1A). In addition, Akt and eNOS phosphorylation induced by losartan and EXP3179 appears to be independent of AT1R blocking action (Figure 2) because EXP3179 does not bind the AT1R. This conclusion is supported by the fact that the affinity of EXP3174 for the AT1R is 10 times greater than losartan,13 yet losartan was 100 times more potent than EXP3174 at Akt and eNOS phosphorylation. Indeed, irbesartan had no effect on Akt and eNOS phosphorylation, also indicating that the effect of EXP3179 is independent of AT1R blocking action.
Andreozzi et al18 showed that Ang II inhibited insulin signaling (NO production and Akt phosphorylation) by increasing IRS-1 serine phosphorylation and inhibiting insulin stimulation of IRS-1 tyrosine phosphorylation necessary for interaction with PI3K. Losartan reversed this effect of Ang II by blocking the AT1R. Although these findings may be clinically important, they differ importantly from the present study. First, the effects of EXP3179 and losartan reported here are independent of AT1R binding, and all experiments were performed in the absence of Ang II. Second, there was no effect of EXP3179 on IRS-1 phosphorylation. Finally, the effect of EXP3179 and losartan requires transactivation of VEGFR2 (via a calcium and Src family kinase pathway) that is relatively unique to EC. Thus, SMCs that express the AT1R failed to show Akt activation by EXP3179 (Figure 8).
Recently, it has been reported that biological actions of all ARBs are not identical.26 For example, the ability of losartan and irbesartan to inhibit cyclooxygenase-2 or the thromboxane A2/prostaglandin endoperoxide H2 receptor is not shared by candesartan. It was also reported that telmisartan and some other ARBS27,28 may act as partial agonists of PPAR, indicating that some of the antiinflammatory and anticoagulant action of these drugs may be related to their individual chemical characteristics rather than an ARB class effect.6,7,29 However, none of these effects explain the transactivation of VEGFR2 and downstream events reported here.
The effects of losartan and EXP3179 appear to be via an EC-specific pathway requiring VEGFR2 because activation of Akt by EXP3179 was not observed in rat aortic SMCs that lack VEGR2 (Figure 8). Our data demonstrate that VEGFR2 is activated by a pathway dependent on intracellular calcium and a Src kinase family member (Data Supplement Figure S6). Importantly, signaling does not involve autocrine secretion of VEGF, a G protein–coupled receptor, reactive oxygen species, Src kinase itself, or IRS-1. Specific experimental results to support this model include the following: There was no effect of EXP3179 on intracellular Ca2+ transients after EXP3179 stimulation (Data Supplement Figure S3A); PTX did not inhibit EXP3179-induced VEGFR2 phosphorylation (Figure 5B); several antioxidants had no effect on EXP3179-induced VEGFR2, Akt, and eNOS phosphorylation (Figure 5B); adenoviral overexpression of kinase inactive Src (Ad.KI-Src) did not inhibit EXP3179-induced Akt and eNOS phosphorylation (Data Supplement Figure S3B); and EXP3179 did not stimulate tyrosine phosphorylation of IRS-1 on Tyr612 (Data Supplement Figure S3C). We did find that BAPTA/AM (intracellular calcium chelator) and PP2 (Src kinase family inhibitor) inhibited EXP3179-induced VEGFR2 autophosphorylation and Akt/eNOS phosphorylation (Figure 5B), indicating that EXP3179-induced VEGFR2 autophosphorylation required Ca2+ and Src kinase activation (transactivation). We also examined the effects of EXP3179 on other mitogen-activated protein kinases in ECs. We found that EXP3179 stimulated phosphorylation of extracellular signal-regulated kinase (ERK1/2) but not p38 and c-Jun N-terminal kinase (data not shown). ERK1/2 activation is known to promote cell survival signals in ECs as well as Akt. However, we demonstrated that LY2940002 and expression of dominant negative Akt kinase almost completely inhibited the antiapoptotic effect of EXP3179 on ECs (Figure 6). These findings indicate that the EXP3179 exerts antiapoptotic effects primarily through the PI3K/Akt pathway rather than the ERK pathway.
VEGF exhibits multiple biological activities in ECs, including promoting EC survival.30,31 VEGF effects on cell survival are mediated by a VEGFR/PI3K/Akt pathway.19,32 EXP3179 significantly suppressed EC apoptosis and caspase-3 activation via an Akt pathway, suggesting that EXP3179, in part, mimics VEGF. In summary, the present study shows that the losartan metabolite EXP3179 stimulates eNOS phosphorylation and suppresses TNF-induced EC apoptosis by activating a VEGFR2/PI3K/Akt pathway independently of AT1R-mediated signaling.
Acknowledgments
This study was supported by a grant from Merck & Co., Inc, to Dr Berk. Dr Watanabe was supported by a fellowship from Japan Heart Foundation & Bayer Yakuhin Research Grant Abroad (Tokyo, Japan).
Disclosure
Dr Berk has received research grants from Merck and Novartis; has served on the speakers’ bureaus of and/or received honoraria from Merck, Novartis, Pfizer, and Bristol-Myers Squibb; and has consulted for Merck, Novartis, and Bristol-Myers Squibb.
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