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 anacetra- pib 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 concen- trations used in this