Off-Target Vascular Effects of Cholesteryl Ester Transfer Protein

Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2016/03/03/jpet.115.230748.DC1

1521-0103/357/2/415–422$25.00 http://dx.doi.org/10.1124/jpet.115.230748 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 357:415–422, May 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Off-Target Vascular Effects of Cholesteryl Ester Transfer Protein Inhibitors Involve Redox-Sensitive and Signal Transducer and Activator of Transcription 3–Dependent Pathways s

Francisco J. Rios, Rheure A. Lopes, Karla B. Neves, Livia L. Camargo, Augusto C. Montezano, and Rhian M. Touyz Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, United Kingdom (F.J.R., R.A.L., K.B.N., L.L.C., A.C.M., R.M.T.); Ribeirao Preto Medical School (R.A.L.) and Faculty of Pharmaceutical Sciences of Ribeirao Preto (K.B.N.), University of Sao Paulo, Ribeirao Preto, Brazil

Received November 15, 2015; accepted March 2, 2016 Downloaded from

ABSTRACT Elevated blood pressure was an unexpected outcome in some vascular myosin light chain (MLC) and myosin phosphatase cholesteryl ester transfer protein (CETP) inhibitor trials, possibly target subunit 1 (MYPT1) (procontractile proteins) and stimulated due to vascular effects of these drugs. We investigated whether ROS production. CETP inhibitors increased the phosphorylation jpet.aspetjournals.org CETP inhibitors (torcetrapib, dalcetrapib, anacetrapib) influence of STAT3 (by 3- to 4-fold), a transcription factor important in cell vascular function and explored the putative underlying molecular activation. Activation of MLC was reduced by NAC, GKT137831 mechanisms. Resistance arteries and vascular smooth muscle [2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H- cells (VSMC) from rats, which lack the CETP gene, were studied. pyrazolo[4,3-c]pyridine-3,6-dione] (Nox1/4 inhibitor), and S3I- CETP inhibitors increased phenylephrine-stimulated vascular 201. The phosphorylation of STAT3 was unaffected by NAC : contraction (logEC50 6.6 6 0.1; 6.4 6 0.06, and 6.2 6 0.09 for and GKT137831. CETP inhibitors did not influence activation of torcetrapib, dalcetrapib, and anacetrapib, respectively, versus mitogen-activated proteins kinases (MAPK) or c-Src. Our data 6 control 5.9 0.05). Only torcetrapib reduced endothelium- demonstrate that CETP inhibitors influence vascular function at ASPET Journals on October 1, 2021 dependent vasorelaxation. The CETP inhibitor effects were and contraction through redox-sensitive, STAT3-dependent, ameliorated by N-acetylcysteine (NAC), a reactive oxygen and MAPK-independent processes. These phenomena do not species (ROS) scavenger, and by S3I-201 [2-hydroxy-4-[[2-(4- involve CETP because the CETP gene is absent in rodents. methylphenyl)sulfonyloxyacetyl]amino]benzoic acid], a signal Findings from our study indicate that CETP inhibitors have transducer and activator of transcription 3 (STAT3) inhibitor. vasoactive properties, which may contribute to the adverse CETP inhibitors increased the phosphorylation (2- to 3-fold) of cardiovascular effects of these drugs such as hypertension.

Introduction cardiovascular risk in patients with dyslipidemia (Okamoto et al., 2000). The cholesteryl ester-transfer protein (CETP) plays a key The Investigation of Lipid Level Management to Under- role in the metabolism of lipoproteins through the bidirec- stand Its Impact in Atherosclerotic Events (ILLUMINATE) tional transfer of neutral lipids between triglyceride-rich trial was the first designed to investigate whether CETP lipoproteins and high-density lipoproteins (HDL) (Tall, 1986; inhibition would increase HDL concentrations and reduce Barter et al., 2003). Human genetic studies demonstrated that cardiovascular events (Barter et al., 2007). The study was CETP deficiency is associated with increased HDL levels prematurely terminated, not only because torcetrapib failed to (Inazu et al., 1990), and this led to the hypothesis that achieve the clinical benefits, which was further confirmed in pharmacologic inhibition of CETP may be an effective thera- three additional large trials (ILLUSTRATE, RADIANCE 1, peutic strategy to elevate HDL cholesterol and reduce and RADIANCE 2) (Bots et al., 2007; Kastelein et al., 2007; Nissen et al., 2007), but also because there were unexpected adverse effects of elevated blood pressure, increased cardio- This work was supported by the British Heart Foundation (BHF) [Grants vascular events, and an increased death rate, despite an CH/12/4/27962 and RG/13/7/30099]. dx.doi.org/10.1124/jpet.115.230748. increase in HDL cholesterol and decrease in low-density s This article has supplemental material available at jpet.aspetjournals.org. lipoproteins. Most clinical studies using CETP inhibitors have

ABBREVIATIONS: ACh, acetylcholine; CETP, cholesteryl ester transfer protein; DMEM, Dulbecco’s modified Eagle medium; ERK, extracellular signal-regulated kinase; GKT137831, 2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6-dione; HDL, high- density lipoproteins; MAPK, mitogen-activated proteins kinase; MLC, myosin light chain; MYPT1, myosin phosphatase target subunit 1; NAC, 2 N-acetylcysteine; O2 , superoxide anion; PE, phenylephrine; ROS, reactive oxygen species; S3I-201, 2-hydroxy-4-[[2-(4-methylphenyl) sulfonyloxyacetyl]amino]benzoic acid; STAT3, signal transducer and activator of transcription 3; VSMC, vascular smooth muscle cells; WKY, Wistar Kyoto.

415 416 Rios et al. demonstrated elevated blood pressure as a major adverse cells. Torcetrapib stimulates aldosterone production in adre- event (Barter et al., 2007; Schwartz et al., 2012). The nal cells (Forrest et al., 2008; Hu et al., 2009) and adipocytes mechanisms behind this remain unclear, but the increased (Rios et al., 2015), which could contribute to hyperaldosteron- levels of plasma aldosterone that are observed in some ism and blood pressure elevation. However, treatment with patients treated with CETP inhibitors may play a role (Barter aldosterone/mineralocorticoid blockers demonstrated only et al., 2007). partial effects on torcetrapib-induced hypertension (Forrest Other small molecule CETP inhibitors have been clinically et al., 2008). Other putative mechanisms include impairment evaluated—dalcetrapib, anacetrapib, and evacetrapib—all of in endothelial function due to decreased nitric oxide synthase which increase HDL. The Dal-OUTCOMES trial of dalcetrapib activation and reduced nitric oxide production (Simic et al., was terminated in phase 3 as well; no therapeutic benefit was 2012). Increased vascular reactive oxygen species (ROS) pro- found, and a small but significant increase in systolic blood duction, which stimulates aldosterone production, may also be pressure and C-reactive protein levels was observed (Schwartz important (Simic et al., 2012; Rios et al., 2015). et al., 2012). The trial for evacetrapib was recently discontin- These studies suggest that CETP inhibitors may influence ued, but the trial for anacetrapib is ongoing (Gotto et al., 2014; endothelial function, but it is unclear whether they directly Teramoto et al., 2014; Brinton et al., 2015). impact on vascular contraction and whether the effects are Although the mechanisms underlying the adverse blood CETP dependent. Accordingly, we investigated the effects of pressure actions induced by torcetrapib and other CETP torcetrapib, dalcetrapib, and anacetrapib on vascular reactiv-

inhibitors remain elusive, in vivo and in vitro data have ity in isolated rat arteries and explored the potential molec- Downloaded from demonstrated that there may be off-target effects in different ular mechanisms underlying the vascular effects of CETP jpet.aspetjournals.org at ASPET Journals on October 1, 2021

Fig. 1. Torcetrapib, dalcetrapib, and anacetrapib increase sensitivity to phenylephrine in small mesenteric arteries from WKY rats. Resistance mesenteric arteries were isolated from WKY rats, mounted in a wire myograph, and treated with (A) torcetrapib, (B) dalcetrapib, or (C) anacetrapib (1 mM for 1 hour). Concentration-response curves to phenylephrine (PE) or acethylcoline (ACh) were derived from endothelium-intact arteries. Results indicate the mean 6 S.E.M. of five to eight vessels of different animals. *P , 0.05 versus control vehicle group. Vascular Effects of CETP Inhibitors 417 inhibitors in cultured rat vascular smooth muscle cells (VSMC). mounted on two stainless-steel wires in standard organ chambers In particular, we focused on the redox-sensitive mitogen-activated for isometric tension recording and left to equilibrate for about protein kinase (MAPK) and signal transducer and activator of 30 minutes in Krebs–Henseleit solution. The arterial integrity was transcription 3 (STAT3) signaling pathways, which are important assessed by stimulation with 120 mM of KCl. in VSMC regulation. We studied rats as our experimental model After washing and a new stabilization period, endothelial function was assessed by stimulation with acetylcholine (ACh, 1 mM) on vessels because rodents lack the CETP gene, which makes the effects of previously contracted with phenylephrine (PE, 1 mM). Cumulative the drugs independent of the enzyme. concentration–response curves to PE or ACh (0.001–10 mM) were obtained in vessels preincubated with torcetrapib, dalcetrapib, or m Materials and Methods anacetrapib (1 M for 1 hour) or vehicle. The CETP inhibitor concentrations used in this study were based on our previous findings (Rios Animals. Male Wistar Kyoto (WKY) rats, 16 to 20 weeks old, were et al., 2015) and on dose–response experiments where we evaluated the 2 studied (n 5 8–10). Mesenteric small arteries were isolated and used effect of the drugs on superoxide anion (O2 ) production in VSMC from to study vascular function by wire myography. These arteries were WKY (Supplemental Figure 1). The concentration used in our study is also used for primary culture of VSMC. All animal procedures were similartoorlowerthantheCmax in humans treated with torcetrapib, performed in accordance with the Guide for the Care and Use of dalcetrapib, or anacetrapib (Dalvie et al., 2008; Krishna et al., 2009; Laboratory Animals as adopted and promulgated by the U.S. National Derks et al., 2010), which may have (patho)physiologic significance. Institutes of Health, and were approved the United Kingdom Home Experimental Protocols. To evaluate the effect of CETP inhibi- Office Guidance on the operation of the Animals (Scientific Proce- tors, we treated VSMC with torcetrapib, dalcetrapib, or anacetrapib (1 mM) dures) Act 1986 and institutional ethics approval. (Santa Cruz Biotechnology, Dallas, TX) for 5 minutes to 24 hours. To Downloaded from Cell Culture. VSMC derived from rat mesenteric arteries were determine the molecular mechanisms involved in cell activation by CETP isolated and characterized, as we described before (Touyz et al., 1994). inhibitors, we pretreated the cells for 30 minutes with the ROS scavenger Briefly, mesenteric arteries were cleaned of adipose and connective N-acetyl-L-cysteine (NAC; Sigma-Aldrich, St. Louis, MO), the Nox1/4 tissue, and VSMC were dissociated by the enzymatic digestion of inhibitor GKT137831 (2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5- vascular arcades. The tissue was filtered, and the cell suspension was methyl-1H-pyrazolo[4,3-c]pyridine-3,6-dione) (Genkyotex, Geneva, Switzer- centrifuged and resuspended in Dulbecco’s modified Eagle medium land), or the STAT3 inhibitor S3I-201 (2-hydroxy-4-[[2-(4-methylphenyl)

(DMEM) containing heat-inactivated fetal bovine serum, HEPES, sulfonyloxyacetyl]amino]benzoic acid) (Santa Cruz Biotechnology). jpet.aspetjournals.org L-glutamine, penicillin, and streptomycin. The VSMC were cultured in NADPH Oxidase Activity and Hydrogen Peroxide Produc- DMEM containing 10% fetal bovine serum and maintained at 37°C in tion. Stimulated VSMC were washed with cold phosphate-buffered a humidified incubator (5% CO2/95% air). Early passaged cells saline and homogenized in lysis buffer (20 mM of KH2PO4,1mMof (passages 3 to 6) were used. The medium was changed to DMEM/ EGTA, 1 mg/ml of aprotinin, 1 mg/ml of leupeptin, 1 mg/ml of pepstatin, 0.5% 1 day before the experiments. and 1 mM of phenylmethylsulfonyl fluoride) and transferred to micro- Preparation and Study of Small Arteries. Third-order supe- tubes. The lucigenin-derived chemiluminescence assay was used to rior mesenteric arteries (approximately 2 mm in length) from WKY determine the NADPH oxidase activity in total protein cell homogenates. rats were dissected and placed in cold physiologic salt solution The reaction was started by the addition of NADPH (0.1 mM) to the at ASPET Journals on October 1, 2021 containing (mM): NaCl 120, NaHCO3 25, KCl 4.7, KH2PO4 1.18, sample-containing suspension (50 ml), lucigenin (5 mM), and assay MgSO4 1.18, CaCl2 2.5, EDTA 0.026, and glucose 5.5. The vessels were phosphate buffer (50 mM KH2PO4, 1 mM EGTA, 150 mM sucrose,

Fig. 2. Phenylephrine-induced contraction increased by CETP inhibitors is dependent on ROS production and STAT3 activation. Resistance mesenteric arteries were isolated from WKY rats and mounted in a wire myograph. Vessels were pretreated with (A) NAC, a ROS scavenger (10 mM), or (B) S3I-201 (SI), a STAT3 inhibitor (50 mM), for 30 minutes, followed by treatment with torcetrapib, dalcetrapib, or anacetrapib (1 mM for 1 hour). Concentration- response curves to phenylephrine (PE) derived from endothelium-intact arteries. Results indicate the mean 6 S.E.M. of five to eight vessels of different animals. *P , 0.05 versus control vehicle group; #P , 0.05 versus NAC or S3I-201 pretreated group. 418 Rios et al. pH 7.4), 250 ml final volume. Luminescence was measured every 1.8 seconds (Thr180/Tyr182), total-p38MAPK (Cell Signaling Technology, Beverly, for 3 minutes in a luminometer (Lumistar Galaxy; BMG Labtechnologies, MA), phospho-myosin phosphatase target subunit 1 (p-MYPT1) (Thr696), Ortenberg, Germany). Buffer blank was subtracted from each reading. total-MYPT1 (Santa Cruz Biotechnology), and b-actin (Sigma-Aldrich). Hydrogen peroxide was evaluated by using the Amplex Red Hydrogen As secondary antibodies, we used anti-rabbit IgG–horseradish peroxidase Peroxide/Peroxidase Assay Kit (Molecular Probes/Life Technologies, Pais- (1:2000) and anti-mouse–horseradish peroxidase (1:5000) (Jackson ley, United Kingdom) according to the manufacturer´s instructions. ImmunoResearch, West Grove, PA). Obtained values were normalized by protein concentration in the cell Protein expression was visualized using the chemiluminescent sub- lysate and are expressed as the fold change related to the vehicle control strate SuperSignal West Pico (Thermo Scientific, Rockford, IL). The group. resulting autoradiograms were analyzed using ImageJ 1.44p software Western Blot Analysis. Cells were lysed in radioimmunopreci- (Wayne Rasband, National Institutes of Health, http://imagej.nih.gov/ij). pitation assay (RIPA) buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Statistical Analysis. The results are presented as mean 6 S.E.M. Triton X-100, 0.1% SDS) supplemented with 1 mM phenylmethylsulfonyl Statistical differences between mean values were determined by one- fluoride, 1 mg/ml pepstatin A, 1 mg/ml leupeptin, 1 mg/ml aprotinin way analysis of variance followed by the Newman-Keuls test or (Sigma-Aldrich), 10 mM sodium fluorate (AnalaR Normapur; VWR Student’s t test, as appropriate, using GraphPad Prism 5.0 software International, Leuven, Belgium), and 1 mM sodium orthovanadate (Alfa (GraphPad Software, San Diego, CA). P , 0.05 was considered Aesar, Ward Hill, MA). Protein concentrations were determined using the statistically significant. DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad Laboratories). The nonspecific Results binding was blocked with nonfat dry milk. Downloaded from The membranes were incubated overnight at 4°C in constant agitation CETP-Inhibitors Increase Phenylephrine-Induced with primary antibodies specific for phospho-STAT3 (Tyr705), total- Vascular Contraction. To investigate effects of CETP inhib- STAT3, phospho-MLC (Ser19), total-MLC, phospho-extracellular signal- itors on vascular function, we treated endothelium-intact regulated kinase (p-ERK1/2) (Thr202/Tyr204), total ERK1/2, phospho-P38 mesenteric arteries from WKY rats with torcetrapib, dalcetrapib, jpet.aspetjournals.org at ASPET Journals on October 1, 2021

Fig. 3. Torcetrapib, dalcetrapib, and anacetrapib induce phosphorylation of MLC, MYPT1, and STAT3 in VSMC from WKY rats. Cells were treated with torcetrapib, dalcetrapib, or anacetrapib (1 mM). Phosphorylated (A) MLC, (B) MYPT1, (C) STAT3, and (D) c-Src were evaluated by Western blot analysis after 5, 15, and 30 minutes and were normalized to total forms of MLC, MYPT1, STA3, and c-Src. Results are presented as fold change of the vehicle control group. Graphs show the mean 6 S.E.M. (n = 6), and the autoradiographs show one representative experiment. *P , 0.05 versus control vehicle group. Vascular Effects of CETP Inhibitors 419 and anacetrapib. Endothelium-intact mesenteric arteries from WKY rats treated with torcetrapib, dalcetrapib, and anacetrapib were more sensitive to contractions induced by phenylephrine compared with vehicle-treated vessels, as evidenced by the leftward shift of the concentration–response curve (Fig. 1). The logEC50 for tocetrapib, dalcetrapib, and anacetrapib was 6.6 6 0.1, 6.4 6 0.06, and 6.2 6 0.09, respectively, compared with the vehicle control (5.9 6 0.05) (P , 0.05). ACh-induced vasorelaxation was not significantly modified by dalcetrapib or anacetrapib but was significantly reduced by torcetrapib, as indicated by the slight rightward shift of the dose–response curve (Fig. 1A). Increased Phenylephrine-Induced Contraction by CETP Inhibitors Is Dependent on ROS Production and STAT3 Activation. To investigate putative molecular mechanisms underlying CETP inhibitor–induced vascular effects, we probed redox-sensitive and STAT3-dependent

pathways by pre-exposing vessels to NAC, a ROS scavenger, Downloaded from and S3I-201, a STAT3 inhibitor. As shown in Fig. 2, the PE- induced contraction in vessels stimulated by CETP inhibitors was normalized by NAC (Fig. 2A) and S31-201 (Fig. 2B) pretreatment. CETP Inhibitors Induce Phosphorylation of MLC, MYPT1, and STAT3 in Rat VSMC. Vascular smooth jpet.aspetjournals.org muscle contraction is dependent on phosphorylation of Ser19 and Thr18 of MLC kinase, and sustained by phosphorylation of MYPT1 (Thr696), which inhibits MLC phosphatase (Webb, 2003). Both MLC and MYPT1 were significantly phosphorylated in VSMCs stimulated with torcetrapib, dalcetrapib, and anacetrapib (Fig. 3, A and B) versus control conditions (P , 0.05). STAT3 phosphorylation is associated with vascular reactivity (Johnson et al., 2013). We found that at ASPET Journals on October 1, 2021 torcetrapib, dalcetrapib, and anacetrapib induced signifi- , cant phosphorylation of STAT3 (P 0.05) (Fig. 3C). No Fig. 4. Phosphorylation of ERK1/2 and p38 are not affected by torcetrapib, differences were observed for c-Src phosphorylation (Fig. 3D) dalcetrapib, or anacetrapib in VSMC from WKY rats. Cells were or for ERK1/2 and p38MAPK activation (Fig. 4, A and B), treated with 1 mM of torcetrapib, dalcetrapib, or anacetrapib. Phosphor- which are upstream signaling proteins involved in VSMC ylated ERK1/2 (A) and p38 (B) were evaluated by Western blot analysis after 5 minutes and normalized to total ERK1/2 or p38. Results are activation. CETP inhibitors had no effect on proliferating presented as fold change of the vehicle control group. Graphs show cell nuclear antigen (PCNA) expression, an index of cell mean 6 S.E.M. (n = 6), and the autoradiographs show one representa- growth (Supplemental Figure 2). tive experiment. CETP Inhibitors Induce ROS Production in Rat VMSC. Because ROS play an important role in the regulation inhibitor S31-201 before stimulation with CETP inhibitors. As of VSMC function, we questioned whether the effects of CETP shown in Fig. 6C, the MLC phosphorylation induced by inhibitors observed in our study are mediated through ROS- torcetrapib and dalcetrapib was inhibited by S3I-201 without dependent processes. All three drugs increased generation of 2 an effect on p-MYPT1 (Supplemental Figure 4). O2 and hydrogen peroxide (H2O2) by 2- to 4-fold in VSMC (Fig. 5, Supplemental Figure 1). The kinetics for ROS generation differed between the CETP inhibitors. Torcetrapib Discussion and dalcetrapib induced a rapid response within 5 to 10 2 minutes, with dalcetrapib having a biphasic effect on O2 The major findings from our present study demonstrate that 2 production (Fig. 5, A and B). Maximal O2 and H2O2 effects torcetrapib, dalcetrapib, and anacetrapib have vasoconstric- of anacetrapib were evident at 60 and 10 minutes, respectively tor actions and that phenylephrine-induced contractile re- (Fig. 5C). sponses in small mesenteric arteries from rats are enhanced To determine whether ROS play a role in the effects ob- by these agents. Molecular mechanisms underlying vascular served with CETP inhibitors, VSMC were pretreated with NAC actions of CETP inhibitors involve ROS- and STAT3-dependent (a ROS scavenger) or GKT137831 (an inhibitor of Nox1 and processes. These vascular effects are independent of CETP Nox4, major sources of ROS in VSMCs). Both NAC and inhibition because rats lack the CETP gene and thus do not GKT137831 inhibited the phosphorylation of MLC induced by express the enzyme. torcetrapib, dalcetrapib, and anacetrapib (Fig. 6A). No signif- Findings from clinical trials have shown that elevated blood icant effects were observed for the expression of pMYTP1 or pressure is an adverse off-target effect of CETP inhibitors. The pSTAT3 (Supplemental Figure 3 and Fig. 6B). To assess the prohypertensive and CETP-independent actions of these role of STAT3, VSMCs were pretreated with the STAT3 agents are further supported by two preclinical studies where 420 Rios et al.

Fig. 5. Torcetrapib, dalcetrapib, and anacetrpib induce ROS generation in VSMC from WKY rats. Cells were treated with 1 mM of torcetrapib (A), dalcetrapib (B), or anacetrapib (C). ROS pro-

2 Downloaded from duction (O2 and H2O2) was evaluated in the cell lysate by lucigenin chemiluminescence assay and Amplex Red assay, respectively, after 5, 10, 30, 60, and 120 minutes. Values are normalized by total protein concentration of the cell lysate. Data are presented as fold change of the control vehicle values and are expressed as mean 6 S.E.M. (n =6). *P , 0.05 versus control vehicle group. jpet.aspetjournals.org at ASPET Journals on October 1, 2021

torcetrapib induced a significant increase in blood pressure ex vivo effects of CETP inhibitors on small vessel function at a in mice and rats, which lack CETP (Forrest et al., 2008; Simic concentration of 1 mM whereas Forrest and colleagues used et al., 2012). The mechanisms underlying these actions torcatrapib and anacetrapib at 10 mg/kg administered in- are poorly understood, although some studies suggest that travenously over 30 minutes. hyperaldosteronism, down-regulation of endothelial nitric oxide Vascular contraction is a highly regulated process involving synthase, oxidative stress, and increased endothelin-1 produc- many signaling pathways, such as calcium-dependent regulation could be possible contributory factors (Forrest et al., 2008). tion of MLC, activation of redox-sensitive proteins due to Here we show that CETP inhibitors directly influence VSMC increased oxidative stress, and activation of Rho kinase (Reho function and vascular contractility. Torcetrapib, dalcetrapib, et al., 2014). Maintaining VSMCs in a mature differentiated and anacetrapib increased the sensitivity of vessels to phenyl- contractile phenotype is important for vascular contraction ephrine, indicating increased contractility. These findings differ and involves JAK-STAT3 (Kirchmer et al., 2014). We found from those of Forrest et al., (2008), who failed to demonstrate a that exaggerated contractile responses to CETP inhibitors direct action of torcetrapib on vascular tone in isolated arterial were attenuated in vessels treated with a ROS scavenger and vascular segments from mice infused with the drug. In that an inhibitor of STAT3, indicating the importance of redox- study, torcetrapib increased blood pressure but did not enhance sensitive processes and STAT3 signaling in CETP inhibitor contractile responses to vasoactive agents, did not influence vascular effects. Similar to others (Simic et al., 2012), we found endothelium-dependent vasorelaxation, and did not increase that endothelium-dependent vasorelaxation was impaired by pressure in an isolated perfused hindlimb preparation. Reasons torcetrapib, as evidenced by the shift of the acetylcholine- for the differences in results between our study and those of stimulated vasorelaxation curve. (Forrest et al. (2008), where it was concluded that blood To investigate in greater detail some of the molecular pressure effects are due to increased aldosterone production, mechanisms associated with vascular effects of CETP inhibitors, may relate to the variations in drug doses used and to we studied VSMCs stimulated with torcetrapib, dalcetrapib, differences in the experimental protocols. We studied direct and anacetrapib in the absence or presence of pharmacologic Vascular Effects of CETP Inhibitors 421

modulators. VSMC contraction is dependent on the increase of 21 intracellular concentration of calcium ([Ca ]i), which binds to calmodulin. This complex phosphorylates MLC kinase, en- abling the actin–myosin interaction and subsequent contraction. An additional mechanism underlying VSMC contraction is activation of Rho kinase, which phosphorylates and inacti- vates MLC phosphatase (MYPT1), thereby increasing the stability of the MLC kinase, resulting in contraction (Webb, 2003; Hilgers and Webb, 2005). Here we show that CETP inhibitors induced phosphorylation of both MLC and MYPT1. Despite all three compounds having a different chemical structure, they have similar molecular actions, possibly because they are all small molecules that target multiple common signaling pathways. Although we did not examine the effects on 21 [Ca ]i, others have shown that torcetrapib increases the 21 expression of L-type calcium channels and [Ca ]i (Hu et al., 2009; Clerc et al., 2010), which may be another mechanism

whereby CETP inhibitors influence VSMC function and vascu- Downloaded from lar contractility. We previously showed that torcetrapib, dalcetrapib, and anacetrapib stimulate ROS production in adipocytes (Rios et al., 2015). Here we advance those findings by demonstrating that these drugs also increase ROS generation in VSMCs. Although all three agents significantly increased ROS pro- jpet.aspetjournals.org duction, the time course was variable, with torcetrapib and dalcetrapib having acute effects within 10 minutes of cell stimulation whereas anacetrapib actions were slightly delayed. These diverse responses may relate, in part, to differential activity of Nox, the primary source of vascular ROS. ROS effects of CETP inhibitors have functional significance because procontractile signaling pathways are sensitive to changes in intracellular redox status (Montezano and at ASPET Journals on October 1, 2021 Touyz, 2014). This is further supported in our studies where we show that ROS scavenging by NAC ameliorates hyper- contractility induced by CETP inhibitors and that NAC and GKT137831 reduce CETP inhibitor effects on MLC phosphorylation. Together these findings indicate that in VSMC torcetrapib, dalcetrapib, and anacetrapib stimulate Nox- derived ROS production, which may induce phosphorylation of MLC leading to vascular contraction. Exactly how CETP inhibitors regulate ROS-generating enzymes remains unclear, but it may be possible that they have binding sites that target oxidases, as suggested by computational analysis (Chang et al., 2010). In addition to influencing ROS production, CETP inhibitors affected the activation of STAT3, a transcription factor involved in VSMC activation. We found that the MLC phosphorylation induced by CETP inhibitors was reduced by STAT3 inhibition. STAT3 inhibition was also associated with attenuated contractile responses to torcetrapib, dalcetrapib, and anacetrapib. Similar findings were shown by Johnson et al. (2013) in angiotensin II-induced hypertension. Whether the effects mediated by STAT3 involve gene transcription are Fig. 6. Effects of torcetrapib, dalcetrapib, and anacetrapib on phosphorylated MLC are affected by inhibitors of ROS and STAT3. Cells unclear, but the fact that responses were rapid suggests that were pretreated with NAC, a ROS scavenger (10 mM),GKT137831,a the effects are likely nongenomic. Activation of STAT3 by Nox1/4 inhibitor (10 mM), or S3I-201, a STAT3 inhibitor (50 mM), for CETP inhibitors appears to be independent of redox-sensitive 30 minutes, followed by stimulation with torcetrapib, dalcetrapib, or anacetrapib (1 mM, 5 minutes). Phosphorylated MLC and STAT3 were processes because NAC and GKT137831 did not influence evaluated by Western blot analysis and normalized to the total forms of STAT3 activation. Others have also shown that STAT3 may be MLC or STAT3. Results are presented as fold change of the vehicle independent of ROS (Johnson et al., 2013). The effects of CETP 6 control group. Graphs show mean S.E.M., and the autoradiographs inhibitors on VSMC signaling pathways are highly regulated show one representative experiment. (n =6).*P , 0.05 versus control vehicle group. #P , 0.05 versus NAC, GKT137831, or S3I-201 pretreated and are not generalized phenomena because activation of group. ERK1/2, p38MAPK, and c-Src were unaffected by these 422 Rios et al. agents. Moreover, VSMC growth was not influenced by CETP Forrest MJ, Bloomfield D, Briscoe RJ, Brown PN, Cumiskey AM, Ehrhart J, Hershey JC, Keller WJ, Ma X, and McPherson HE, et al. (2008) Torcetrapib-induced blood inhibitors, as evidenced by no effect on proliferating cell pressure elevation is independent of CETP inhibition and is accompanied by in- nuclear antigen expression. creased circulating levels of aldosterone. Br J Pharmacol 154:1465–1473. Gotto AM, Jr, Kher U, Chatterjee MS, Liu Y, Li XS, Vaidya S, Cannon CP, Brinton In conclusion, our findings indicate that torcetrapib, EA, Moon JE, and Shah S, et al. DEFINE Investigators (2014) Lipids, safety pa- dalcetrapib, and anacetrapib have CETP-independent effects rameters, and drug concentrations after an additional 2 years of treatment with anacetrapib in the DEFINE study. J Cardiovasc Pharmacol Ther 19:543–549. that directly influence vascular function. In particular, we Hilgers RH and Webb RC (2005) Molecular aspects of arterial smooth muscle con- show that these drugs promote vasoconstriction through traction: focus on Rho. Exp Biol Med (Maywood) 230:829–835. Hu X, Dietz JD, Xia C, Knight DR, Loging WT, Smith AH, Yuan H, Perry DA, redox-sensitive and STAT3-dependent pathways. Although and Keiser J (2009) Torcetrapib induces aldosterone and cortisol production by an most vascular effects were similar for the three different intracellular calcium-mediated mechanism independently of cholesteryl ester agents, only torcetrapib impaired endothelial function. These transfer protein inhibition. Endocrinology 150:2211–2219. Inazu A, Brown ML, Hesler CB, Agellon LB, Koizumi J, Takata K, Maruhama Y, differential effects may relate to chemical structural differ- Mabuchi H, and Tall AR (1990) Increased high-density lipoprotein levels caused by ences of the drugs. Overall, our findings suggest that off-target a common cholesteryl-ester transfer protein gene mutation. N Engl J Med 323: 1234–1238. vascular effects may explain, at least in part, some of the Johnson AW, Kinzenbaw DA, Modrick ML, and Faraci FM (2013) Small-molecule adverse cardiovascular events, such as hypertension, ob- inhibitors of signal transducer and activator of transcription 3 protect against angiotensin II-induced vascular dysfunction and hypertension. Hypertension 61: served in CETP inhibitor clinical trials. 437–442. Kastelein JJ, van Leuven SI, Burgess L, Evans GW, Kuivenhoven JA, Barter PJ, Revkin JH, Grobbee DE, Riley WA, and Shear CL, et al.; RADIANCE 1 Investi- Acknowledgments gators (2007) Effect of torcetrapib on carotid atherosclerosis in familial hypercho- lesterolemia. N Engl J Med 356:1620–1630.

The authors thank C. Jenkins, L. Serrato, J. Thomson, and A. Downloaded from Carswell for technical assistance. Kirchmer MN, Franco A, Albasanz-Puig A, Murray J, Yagi M, Gao L, Dong ZM, and Wijelath ES (2014) Modulation of vascular smooth muscle cell phenotype by STAT-1 and STAT-3. Atherosclerosis 234:169–175. Authorship Contributions Krishna R, Garg A, Panebianco D, Cote J, Bergman AJ, Van Hoydonck P, Laethem T, Van Dyck K, Chen J, and Chavez-Eng C, et al. (2009) Single-dose pharmacokinetics Participated in research design: Rios, Montezano, Touyz. and pharmacodynamics of anacetrapib, a potent cholesteryl ester transfer protein Conducted experiments: Rios, Montezano, Lopes, Camargo, Neves. (CETP) inhibitor, in healthy subjects. Br J Clin Pharmacol 68:535–545. Contributed new reagents or analytic tools: Touyz. Montezano AC and Touyz RM (2014) Reactive oxygen species, vascular Noxs, and hypertension: focus on translational and clinical research. Antioxid Redox Signal Performed data analysis: Rios, Montezano, Lopes, Camargo, 20:164–182. jpet.aspetjournals.org Neves, Touyz. Nissen SE, Tardif JC, Nicholls SJ, Revkin JH, Shear CL, Duggan WT, Ruzyllo W, Wrote or contributed to the writing of the manuscript: Rios, Montezano, Bachinsky WB, Lasala GP, and Tuzcu EM; ILLUSTRATE Investigators (2007) Effect of torcetrapib on the progression of coronary atherosclerosis. N Engl J Med Touyz. 356:1304–1316. Okamoto H, Yonemori F, Wakitani K, Minowa T, Maeda K, and Shinkai H (2000) A References cholesteryl ester transfer protein inhibitor attenuates atherosclerosis in rabbits. Nature 406:203–207. Barter PJ, Brewer HB, Jr, Chapman MJ, Hennekens CH, Rader DJ, and Tall AR Reho JJ, Zheng X, and Fisher SA (2014) Smooth muscle contractile diversity in the (2003) Cholesteryl ester transfer protein: a novel target for raising HDL and control of regional circulations. Am J Physiol Heart Circ Physiol 306:H163–H172. inhibiting atherosclerosis. Arterioscler Thromb Vasc Biol 23:160–167. Rios FJ, Neves KB, Nguyen Dinh Cat A, Even S, Palacios R, Montezano AC, at ASPET Journals on October 1, 2021 Barter PJ, Caulfield M, Eriksson M, Grundy SM, Kastelein JJ, Komajda M, Lopez- and Touyz RM (2015) Cholesteryl ester-transfer protein inhibitors stimulate al- Sendon J, Mosca L, Tardif JC, and Waters DD, et al.; ILLUMINATE Investigators dosterone biosynthesis in adipocytes through Nox-dependent processes. J Phar- (2007) Effects of torcetrapib in patients at high risk for coronary events. N Engl J macol Exp Ther 353:27–34. Med 357:2109–2122. Schwartz GG, Olsson AG, Abt M, Ballantyne CM, Barter PJ, Brumm J, Chaitman Bots ML, Visseren FL, Evans GW, Riley WA, Revkin JH, Tegeler CH, Shear CL, BR, Holme IM, Kallend D, and Leiter LA, et al.; dal-OUTCOMES Investigators Duggan WT, Vicari RM, and Grobbee DE, et al.; RADIANCE 2 Investigators (2007) (2012) Effects of dalcetrapib in patients with a recent acute coronary syndrome. N Torcetrapib and carotid intima-media thickness in mixed dyslipidaemia (RADIANCE Engl J Med 367:2089–2099. 2 study): a randomised, double-blind trial. Lancet 370:153–160. Simic B, Hermann M, Shaw SG, Bigler L, Stalder U, Dörries C, Besler C, Lüscher TF, Brinton EA, Kher U, Shah S, Cannon CP, Davidson M, Gotto AM, Ashraf TB, and Ruschitzka F (2012) Torcetrapib impairs endothelial function in hypertension. McCrary Sisk C, Dansky H, and Mitchel Y, et al.; DEFINE Investigators (2015) Eur Heart J 33:1615–1624. Effects of anacetrapib on plasma lipids in specific patient subgroups in the DEFINE Tall AR (1986) Plasma lipid transfer proteins. J Lipid Res 27:361–367. (Determining the Efficacy and Tolerability of CETP INhibition with AnacEtrapib) Teramoto T, Takeuchi M, Morisaki Y, Ruotolo G, and Krueger KA (2014) Efficacy, trial. J Clin Lipidol 9:65–71. safety, tolerability, and pharmacokinetic profile of evacetrapib administered as Chang RL, Xie L, Xie L, Bourne PE, and Palsson BO (2010) Drug off-target effects monotherapy or in combination with atorvastatin in Japanese patients with dys- predicted using structural analysis in the context of a metabolic network model. lipidemia. Am J Cardiol 113:2021–2029. PLOS Comput Biol 6:e1000938. Touyz RM, Tolloczko B, and Schiffrin EL (1994) Mesenteric vascular smooth muscle Clerc RG, Stauffer A, Weibel F, Hainaut E, Perez A, Hoflack JC, Bénardeau A, cells from spontaneously hypertensive rats display increased calcium responses to Pflieger P, Garriz JM, and Funder JW, et al. (2010) Mechanisms underlying off- angiotensin II but not to endothelin-1. J Hypertens 12:663–673. target effects of the cholesteryl ester transfer protein inhibitor torcetrapib involve Webb RC (2003) Smooth muscle contraction and relaxation. Adv Physiol Educ 27: L-type calcium channels. J Hypertens 28:1676–1686. 201–206. Dalvie D, Chen W, Zhang C, Vaz AD, Smolarek TA, Cox LM, Lin J, and Obach RS (2008) Pharmacokinetics, metabolism, and excretion of torcetrapib, a cholesteryl ester transfer protein inhibitor, in humans. Drug Metab Dispos 36:2185–2198. Address correspondence to: Dr. Rhian M Touyz, Institute of Cardiovascular Derks M, Abt M, Phelan M, Turnbull L, Meneses-Lorente G, Bech N, White AM, & Medical Sciences, BHF Glasgow Cardiovascular Research Centre, Univer- and Parr G (2010) Coadministration of dalcetrapib with pravastatin, rosuvastatin, sity of Glasgow, 126 University Place, Glasgow, G12 8TA. E-mail: Rhian. or simvastatin: no clinically relevant drug-drug interactions. J Clin Pharmacol 50: [email protected] 1188–1201. JPET #230748 1

Supplemental Data

Off-target vascular effects of cholesteryl ester transfer protein (CETP) inhibitors involve redox-sensitive and STAT3-dependent pathways.

Francisco J. Rios, Rheure A. Lopes, Karla B. Neves, Livia L. Camargo, Augusto C. Montezano, Rhian M. Touyz

Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, United Kingdom (F.J.R., R.A.L., K.B.N., L.L.C., A.C.M., R.M.T.) Ribeirao Preto Medical School (R.A.L.) and Faculty of Pharmaceutical Sciences of Ribeirao Preto (K.B.N.), University of Sao Paulo, Ribeirao Preto, Brazil

Corresponding author: Rhian M Touyz MBBCh, PhD, FRCP, FRSE. Institute of Cardiovascular & Medical Sciences BHF Glasgow Cardiovascular Research Centre University of Glasgow 126 University Place Glasgow, G12 8TA Tel: + 44 (0)141 330 7775/7774 Fax: + 44 (0)141 330-3360 Email: [email protected]

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Supplemental Figure S1

* 5 2.0 * 4 1.5 3 * 1.0 2 fold increase fold increasefold - - 2 2

O 0.5 O 1

0 _ 0.0 _ 0.1 1 10 0.1 1 10 Torcetrapib (M) 5 min Dalcetrapib (M) 5 min

Supplemental figure S1. Torcetrapib and dalcetrapib induce ROS generation in VSMC from WKY rats. Cells were treated with 0.1-10 µM of - torcetrapib and dalcetrapib for 5 min and the ROS production (O2 ) was evaluated in the cell lysate by lucigenin chemiluminescence assay. Values were normalized by total protein concentration of the cell lysate. Data are presented as fold change of the control vehicle values and expressed as mean ± SEM (n=6); *p<0.05 vs control vehicle group.

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Supplemental figure S2. PCNA expression is not affected by torcetrapib, dalcetrapib, and anacetrapib. Rat VSMC were treated with torcetrapib, dalcetrapib or anacetrapib (1 µM). PCNA was evaluated by western blot after 24 h and normalized to β-actin. Values are presented as fold change of the vehicle control group. Graph show mean ± SEM and autoradiograph show one representative experiment (n=6). Significance was determined using a one-way ANOVA with Newman-Keuls multiple comparisons test. *p<0.05 vs control vehicle group.

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Supplemental figure S3. Phosphorylation of MYPT1 and STAT3 induced by torcetrapib, dalcetrapib or anacetrapib are independent on ROS production. Rat VSMC were pre-treated with N-acetylcysteine (NAC – ROS scavenger) (10 µM) and GKT137831 (Nox1/4 inhibitor) (10 µM) for 30 min, followed by stimulation with torcetrapib, dalcetrapib or anacetrapib (1 µM). p- MYPT1 (A) and p-STAT3 (B) were evaluated by western blot and normalized to β-actin or total-STAT3. Values are presented as fold change of the vehicle control group. Graph show mean ± SEM and autoradiograph show one representative experiment (n=7). Significance was determined using a one-way ANOVA with Newman-Keuls multiple comparisons test. *p<0.05 vs control vehicle group. JPET #230748 5

Supplemental figure S4. Phosphorylation of MYPT1 induced by torcetrapib, dalcetrapib or anacetrapib is independent on STAT3 activation. Cells were pre-treated with S3I-201 (STAT3 inhibitor) (50 µM) for 30 min, followed by stimulation with torcetrapib, dalcetrapib or anacetrapib (1 µM). p-MYPT1 was evaluated by western blot and normalized to β-actin. Values are presented as fold change of the vehicle control group. Graph show mean ± SEM and autoradiograph show one representative experiment (n=8). Significance was determined using a one-way ANOVA with Newman-Keuls multiple comparisons test. *p<0.05 vs control vehicle group.