Original Research Low-Density Lipoprotein Receptor–Dependent and Low-Density Lipoprotein Receptor–Independent Mechanisms of Cyclosporin A–Induced Dyslipidemia Maaike Kockx, Elias Glaros, Betty Kan, Theodore W. Ng, Jimmy F.P. Berbée, Virginie Deswaerte, Diana Nawara, Carmel Quinn, Kerry-Anne Rye, Wendy Jessup, Patrick C.N. Rensen, Peter J. Meikle, Leonard Kritharides Objective—Cyclosporin A (CsA) is an immunosuppressant commonly used to prevent organ rejection but is associated with hyperlipidemia and an increased risk of cardiovascular disease. Although studies suggest that CsA-induced hyperlipidemia is mediated by inhibition of low-density lipoprotein receptor (LDLr)–mediated lipoprotein clearance, the data supporting this are inconclusive. We therefore sought to investigate the role of the LDLr in CsA-induced hyperlipidemia by using Ldlr-knockout mice (Ldlr−/−). Approach and Results—Ldlr−/− and wild-type (wt) C57Bl/6 mice were treated with 20 mg/kg per d CsA for 4 weeks. On a chow diet, CsA caused marked dyslipidemia in Ldlr−/− but not in wt mice. Hyperlipidemia was characterized by a prominent increase in plasma very low–density lipoprotein and intermediate-density lipoprotein/LDL with unchanged plasma high-density lipoprotein levels, thus mimicking the dyslipidemic profile observed in humans. Analysis of specific lipid species by liquid chromatography–tandem mass spectrometry suggested a predominant effect of CsA on increased very low–density lipoprotein–IDL/LDL lipoprotein number rather than composition. Mechanistic studies indicated that CsA did not alter hepatic lipoprotein production but did inhibit plasma clearance and hepatic uptake of [14C]cholesteryl oleate and glycerol tri[3H]oleate-double-labeled very low–density lipoprotein–like particles. Further studies showed that CsA inhibited plasma lipoprotein lipase activity and increased levels of apolipoprotein C-III and proprotein convertase subtilisin/kexin type 9. Conclusions—We demonstrate that CsA does not cause hyperlipidemia via direct effects on the LDLr. Rather, LDLr deficiency plays an important permissive role for CsA-induced hyperlipidemia, which is associated with abnormal lipoprotein clearance, decreased lipoprotein lipase activity, and increased levels of apolipoprotein C-III and proprotein convertase subtilisin/kexin type 9. Enhancing LDLr and lipoprotein lipase activity and decreasing apolipoprotein C-III and proprotein convertase subtilisin/kexin type 9 levels may therefore provide attractive treatment targets for patients with hyperlipidemia receiving CsA. (Arterioscler Thromb Vasc Biol. 2016;36:00-00. DOI: 10.1161/ATVBAHA.115.307030.) Key Words: apolipoprotein C-III ◼ hyperlipidemia ◼ immunosuppression ◼ lipolysis ◼ triglycerides yperlipidemia is observed in 40% to 60% of organ trans- hyperlipidemia in patients, such as post-transplantation obe- Hplant recipients and has been linked to the use of immu- sity, multiple drug therapy, and diabetes mellitus, CsA mono- nosuppressant agents such as corticosteroids, sirolimus, and therapy can independently lead to elevated plasma triglyceride cyclosporine A (CsA).1–3 Transplant hyperlipidemia is char- and cholesterol levels in humans which are reversible on ces- acterized by high plasma cholesterol and triglyceride levels.2 sation of immunosuppression therapy.4 Specifically, CsA increases very low–density lipoprotein The immunosuppressive effect of CsA is mediated by inhi- (VLDL) and low-density lipoprotein (LDL) concentrations bition of protein phosphatase 2B (calcineurin) and subsequent and shows variable effects on plasma high-density lipo- activation of the transcription factor nuclear factor of transcrip- protein. Although multiple factors potentially contribute to tion. The mechanism(s) by which CsA leads to hyperlipidemia Received on: December 11, 2015; final version accepted on: April 20, 2016. From the ANZAC Research Institute (M.K., D.N., W.J., L.K.) and Department of Cardiology (L.K.), Concord Hospital, University of Sydney, Sydney, Australia; Centre for Vascular Research (E.G., C.Q.) and Department of Pathology (B.K.), University of New South Wales, Sydney, Australia; Baker IDI Heart and Diabetes Institute, Melbourne, Australia (T.W.N., P.J.M.); Department of Medicine, Division Endocrinology, and Eindhoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Centre, Leiden, The Netherlands (J.F.P.B., P.C.N.R.); Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Australia (V.D.); Lipid Research Group, School of Medical Sciences, University of New South Wales Australia, Sydney, Australia (K.-A.R.). The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.115.307030/-/DC1. Correspondence to Leonard Kritharides, Department of Cardiology, Concord Repatriation General Hospital, Concord, New South Wales 2139, Australia. E-mail [email protected] © 2016 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.115.307030 Downloaded from http://atvb.ahajournals.org/1 at University of Sydney on May 11, 2016 2 Arterioscler Thromb Vasc Biol July 2016 Nonstandard Abbreviations and Acronyms Materials and Methods Materials and Methods are available in the online-only Data apo apolipoprotein Supplement. CE cholesteryl ester CO cholesteryl oleate Results CsA cyclosporin A CsA Treatment Hprt Hypoxanthine guanine phosphoribosyl transferase Mice were administered 20 mg/kg per d CsA or vehicle (con- IDL intermediate-density lipoprotein trol) via osmotic minipumps. This CsA dose has previously LDL low-density lipoprotein been shown to reduce transplant rejection in mice while not LDLr LDL receptor inducing liver and kidney toxicity.12,13 Whole-blood CsA lev- LPL lipoprotein lipase els were 1087±124 ng/mL and 711±91 ng/mL after 1 week PCSK9 proprotein convertase subtilisin/kexin type 9 and 4 weeks, respectively, consistent with levels reported by SM sphingomyelin others using a similar delivery method.14 Mouse weights were TO trioleate/triolein not affected (23.3±0.3 versus 23.2±0.3 g, for control and CsA, VLDL very low–density lipoprotein respectively). Histological examination of liver and kidney wt wild-type sections revealed no signs of toxicity after CsA treatment (Figure I in the online-only Data Supplement). −/− are most likely not related to nuclear factor of transcription It is known that Ldlr mice have increased hepatic lipid inhibition1,4 as tacrolimus (FK506), another nuclear factor of content compared with C57Bl/6 wild-type (wt) mice and transcription–inhibiting immunosuppressant, does not induce are more vulnerable to develop nonalcoholic steatohepatitis hyperlipidemia.3 Animal and cell studies have indicated that after high-fat feeding, although they do not develop fatty 15 CsA may affect many aspects of lipid metabolism.1 In 1996, livers on a chow diet. This was supported by our histologi- Rayyes5 and Winegard6 suggested that CsA increased plasma cal studies (Figure I in the online-only Data Supplement). LDL and VLDL levels via decreased low-density lipopro- Hepatic cholesterol, cholesteryl ester (CE), and triglyc- −/− tein receptor (LDLr) activity. The precise role of the LDLr eride contents were higher in Ldlr mice compared with in mediating CsA-mediated hypercholesterolemia, however, wt mice, most marked in triglyceride content (115 versus is unclear. Cell culture studies identified that CsA can inhibit 30 nmol/mg; P<0.05; Table 1). In the wt mice, CsA treat- ment increased hepatic CE content from 11 to 20 nmol/mg β-VLDL uptake via the LDLr in the human hepatoma HepG2 cell line.6 CsA was also found to reduce LDLr-mediated bind- (P<0.01) without significantly increasing other lipids. In Ldlr−/− mice, CsA caused no significant changes in hepatic ing and uptake of LDL in HepG2 cells, and this was associated lipid content. with a reduction in hepatic LDLr mRNA levels.5 However, in vivo studies did not corroborate these findings. Hepatic LDLr protein levels decreased in mice treated with CsA7 but were CsA Treatment Increases Plasma Lipid −/− unaffected in rats8 even though both models demonstrated Levels in Ldlr Mice but not in wt Mice CsA-induced hyperlipidemia. These studies suggest that CsA- CsA treatment did not affect plasma total cholesterol or tri- mediated hyperlipidemia may be related to effects on targets glyceride levels in wt mice (Figure 1A and 1B). However, −/− other than the LDLr, such as activation of hepatic cholesterol in Ldlr mice fed a chow diet, CsA markedly elevated total synthesis or inhibition of lipoprotein lipolysis. In vivo studies plasma cholesterol (263±12 versus 527±31 mg/dL, control in rats demonstrated that CsA reduced LDL production and versus CsA, respectively; P<0.0001) and triglyceride levels catabolism,9 while in renal transplant recipients reduced catab- olism of chylomicron-like emulsion particles, and a reduced Table 1. Hepatic Tissue Lipid Levels 10,11 fractional catabolic rate of VLDL has been observed. Control CsA Given the current and increasing clinical importance −/− of contemporary therapies such as statins and proprotein Ldlr convertase subtilisin/kexin type 9 (PCSK9) inhibitors in TG 115±30 86±28 promoting LDLr-mediated lipoprotein uptake, we sought Cholesterol 25±2 28±2 to investigate the role of the LDLr in CsA-induced hyper- CE 30±4 46±16 lipidemia by studying Ldlr-knockout mice. We report that CsA induces hyperlipidemia in chow-fed