Junichiro Tohyama

Expression of Cholesteryl Ester Transfer Protein in Mice Promotes Macrophage Reverse Cholesterol Transport

Background— Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from high-density lipoproteins to apolipoprotein (apo) B–containing lipoproteins and in humans plays an important role in lipoprotein metabolism. However, the role that CETP plays in mediation of reverse cholesterol transport (RCT) remains unclear. We used a validated in vivo assay of macrophage RCT to test the effect of CETP expression in mice (which naturally lack CETP) on macrophage RCT, including in mice that lack the low-density lipoprotein receptor or the scavenger receptor class B, type I. Method and Results— A vector based on adeno-associated virus serotype 8 (AAV8) with a liver-specific thyroglobulin promoter was used to stably express human CETP in livers of mice and was compared with an AAV8-lacZ control vector. The RCT assay was performed 4 weeks after vector injection and involved the intraperitoneal injection of acetylated low-density lipoprotein cholesterol–loaded and 3H-cholesterol–labeled J774 macrophages in mice with plasma sampling at several time points, liver and bile sampling at 48 hours, and continuous fecal collection to measure 3H-sterol as an integrated readout of macrophage RCT. In apobec-1–null mice, CETP expression reduced plasma high-density lipoprotein cholesterol levels but significantly increased fecal 3H-sterol excretion. In low-density lipoprotein receptor/apobec-1 double-null mice, CETP expression reduced high-density lipoprotein cholesterol levels and had no effect on fecal 3H-sterol excretion. Finally, in scavenger receptor class B, type I–null mice, CETP expression reduced high-density lipoprotein cholesterol levels and significantly increased fecal 3H-sterol excretion. Conclusion— The present results demonstrate that CETP expression promotes macrophage RCT in mice, that this effect is dependent on the low-density lipoprotein receptor, and that CETP expression restores to normal the impaired RCT in mice deficient in scavenger receptor class B, type I.

Lecithin: cholesterol acyltransferase expression has minimal effects on macrophage reverse cholesterol transport in vivo.

Background— Lecithin:cholesterol acyltransferase (LCAT) catalyzes the formation of plasma cholesteryl ester, plays a key role in high-density lipoprotein metabolism, and has been believed to be critical in the process of reverse cholesterol transport (RCT). Methods and Results— The role of LCAT in RCT from macrophages was quantified with a validated assay involving intraperitoneal injection in mice of 3H-cholesterol–labeled J774 macrophages and monitoring the appearance of tracer in plasma, liver, bile, and feces. Human LCAT overexpression in human apolipoprotein A-I transgenic mice substantially increased plasma high-density lipoprotein cholesterol levels but surprisingly did not increase macrophage RCT. Even in the setting of coexpression of scavenger receptor BI or cholesteryl ester transfer protein, both of which promoted the transfer of LCAT-derived high-density lipoprotein cholesterol ester to the liver, LCAT overexpression still had no effect on RCT. Serum from LCAT-overexpressing mice had reduced ability to promote cholesterol efflux from macrophages ex vivo via ABCA1. To determine the effect of LCAT deficiency on macrophage RCT, LCAT−/− and LCAT+/− mice were compared with wild-type mice. Despite extremely low plasma levels of high-density lipoprotein cholesterol, LCAT-deficient mice had only a 50% reduction in RCT. LCAT+/− mice had normal RCT despite a significant reduction in high-density lipoprotein cholesterol. Serum from LCAT-deficient mice had increased ability to promote ABCA1-mediated cholesterol efflux from macrophages ex vivo. Conclusions— These results demonstrate that LCAT overexpression does not promote an increased rate of macrophage RCT. Although LCAT activity does become rate limiting in the context of complete LCAT deficiency, RCT is reduced by only 50% even in the absence of LCAT. These data suggest that macrophage RCT may not be as dependent on LCAT activity as has previously been believed.

Anacetrapib lowers LDL by increasing ApoB clearance in mildly hypercholesterolemic subjects

BACKGROUND Individuals treated with the cholesteryl ester transfer protein (CETP) inhibitor anacetrapib exhibit a reduction in both LDL cholesterol and apolipoprotein B (ApoB) in response to monotherapy or combination therapy with a statin. It is not clear how anacetrapib exerts these effects; therefore, the goal of this study was to determine the kinetic mechanism responsible for the reduction in LDL and ApoB in response to anacetrapib. METHODS We performed a trial of the effects of anacetrapib on ApoB kinetics. Mildly hypercholesterolemic subjects were randomized to background treatment of either placebo (n = 10) or 20 mg atorvastatin (ATV) (n = 29) for 4 weeks. All subjects then added 100 mg anacetrapib to background treatment for 8 weeks. Following each study period, subjects underwent a metabolic study to determine the LDL-ApoB-100 and proprotein convertase subtilisin/kexin type 9 (PCSK9) production rate (PR) and fractional catabolic rate (FCR). RESULTS Anacetrapib markedly reduced the LDL-ApoB-100 pool size (PS) in both the placebo and ATV groups. These changes in PS resulted from substantial increases in LDL-ApoB-100 FCRs in both groups. Anacetrapib had no effect on LDL-ApoB-100 PRs in either treatment group. Moreover, there were no changes in the PCSK9 PS, FCR, or PR in either group. Anacetrapib treatment was associated with considerable increases in the LDL triglyceride/cholesterol ratio and LDL size by NMR. CONCLUSION These data indicate that anacetrapib, given alone or in combination with a statin, reduces LDL-ApoB-100 levels by increasing the rate of ApoB-100 fractional clearance. TRIAL REGISTRATION ClinicalTrials.gov NCT00990808. FUNDING Merck & Co. Inc., Kenilworth, New Jersey, USA. Additional support for instrumentation was obtained from the National Center for Advancing Translational Sciences (UL1TR000003 and UL1TR000040).

Macrophage Sortilin Promotes LDL Uptake, Foam Cell Formation, and Atherosclerosis

RATIONALE Noncoding gene variants at the SORT1 locus are strongly associated with low-density lipoprotein cholesterol (LDL-C) levels, as well as with coronary artery disease. SORT1 encodes a protein called sortilin, and hepatic sortilin modulates LDL metabolism by targeting apolipoprotein B-containing lipoproteins to the lysosome. Sortilin is also expressed in macrophages, but its role in macrophage uptake of LDL and in atherosclerosis independent of plasma LDL-C levels is unknown. OBJECTIVE To determine the effect of macrophage sortilin expression on LDL uptake, foam cell formation, and atherosclerosis. METHODS AND RESULTS We crossed Sort1(-/-) mice onto a humanized Apobec1(-/-); hAPOB transgenic background and determined that Sort1 deficiency on this background had no effect on plasma LDL-C levels but dramatically reduced atherosclerosis in the aorta and aortic root. To test whether this effect was a result of macrophage sortilin deficiency, we transplanted Sort1(-/-);LDLR(-/-) or Sort1(+/+);LDLR(-/-) bone marrow into Ldlr(-/-) mice and observed a similar reduction in atherosclerosis in mice lacking hematopoetic sortilin without an effect on plasma LDL-C levels. In an effort to determine the mechanism by which hematopoetic sortilin deficiency reduced atherosclerosis, we found no effect of sortilin deficiency on macrophage recruitment or lipopolysaccharide-induced cytokine release in vivo. In contrast, sortilin-deficient macrophages had significantly reduced uptake of native LDL ex vivo and reduced foam cell formation in vivo, whereas sortilin overexpression in macrophages resulted in increased LDL uptake and foam cell formation. CONCLUSIONS Macrophage sortilin deficiency protects against atherosclerosis by reducing macrophage uptake of LDL. Sortilin-mediated uptake of native LDL into macrophages may be an important mechanism of foam cell formation and contributor to atherosclerosis development.

Increased Protein Nitration Burden in the Atherosclerotic Lesions and Plasma of Apolipoprotein A-I–Deficient Mice

Apolipoprotein A-I (apoA-I), the major protein constituent within high-density lipoprotein (HDL), has been associated with antiatherogenic protection by mechanisms that include reverse cholesterol transport and antiinflammatory functions. To evaluate the proposed protective function of apoA-I, proteins modified by nitrating oxidants were evaluated in the aortic tissue and plasma of mice lacking the low-density lipoprotein receptor and apobec (LA) and LA mice with genetic deletion of apoA-I (LA–apoA-I−/−). The levels of nitrated proteins in aortic tissue quantified by liquid chromatography with online electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) were 6-fold higher in the LA–apoA-I−/− as compared with the LA mice. The quantitative analyses were corroborated by immunohistochemical and high-resolution immunoelectron microscopic evaluation of the lesions, which revealed abundant staining for nitrated proteins in the aortic root lesions of LA–apoA-I−/− as compared with the LA mice. Proteomic approaches based on affinity enrichment and site-specific adduct mapping identified unique specific protein targets for nitration in the plasma of LA–apoA-I−/− that were not present in the plasma of LA mice. In particular the nitration of fibrinogen was shown to accelerate fibrin clot formation. Another consequence of the augmented levels of nitrated proteins was the induction of humoral responses documented by the increased circulating immunoglobulins that recognize nitrotyrosine in LA–apoA-I−/− as compared with the LA mice. These data collectively support a protective function of apoA-I diminishing the burden of nitrative oxidants in these mice models of atherosclerosis.

Genetic Ablation of Adamts13 Gene Dramatically Accelerates the Formation of Early Atherosclerosis in a Murine Model

Objective—ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) cleaves von Willebrand factor, thereby modulating thrombosis and inflammation. Low plasma ADAMTS13 activity is associated with cardiovascular events, including myocardial and cerebral infarction. Here, we investigated the role of ADAMTS13 in the development of early atherosclerosis in a murine model. Methods and Results—Apolipoprotein E–null (ApoE−/−) and Adamts13-null (Adamts13−/−) ApoE−/− mice were fed with a high-fat Western diet for 12 weeks. Atherosclerotic lesions in the aorta and aortic roots were quantified after staining. Leukocyte rolling and adhesion onto cremaster venules after oxidative injury were determined by intravital microscopy. Although plasma cholesterol levels were largely similar in both groups, the extent of atherosclerotic lesions in the aorta en face and in the aortic roots in the Adamts13−/−ApoE−/− mice increased ≈5.5-fold (P=0.0017) and ≈6.1-fold (P=0.0037), respectively. In addition, the ratio of plasma high- to low-molecular-weight von Willebrand factor multimers increased ≈3-fold. The leukocyte rolling velocities were significantly reduced (P<0.001), with an increased number of leukocyte rolling (P=0.0026) and macrophage infiltration into the atherosclerotic lesions in the Adamts13−/−ApoE−/− mice. Conclusion—Our results suggest that ADAMTS13 plays a critical role in modulating the development of early atherosclerosis, likely through the proteolytic cleavage of ultra-large von Willebrand factor multimers, thereby inhibiting platelet deposition and inflammation.

Knockout of Adamts7, a Novel Coronary Artery Disease Locus in Humans, Reduces Atherosclerosis in Mice

Background— Genome-wide association studies have established ADAMTS7 as a locus for coronary artery disease in humans. However, these studies fail to provide directionality for the association between ADAMTS7 and coronary artery disease. Previous reports have implicated ADAMTS7 in the regulation of vascular smooth muscle cell migration, but a role for and the direction of impact of this gene in atherogenesis have not been shown in relevant model systems. Methods and Results— We bred an Adamts7 whole-body knockout mouse onto both the Ldlr and Apoe knockout hyperlipidemic mouse models. Adamts7−/−/Ldlr−/− and Adamts7−/−/Apoe−/− mice displayed significant reductions in lesion formation in aortas and aortic roots compared with controls. Adamts7 knockout mice also showed reduced neointimal formation after femoral wire injury. Adamts7 expression was induced in response to injury and hyperlipidemia but was absent at later time points, and primary Adamts7 knockout vascular smooth muscle cells showed reduced migration in the setting of tumor necrosis factor-&agr; stimulation. ADAMTS7 localized to cells positive for smooth muscle cell markers in human coronary artery disease lesions, and subcellular localization studies in cultured vascular smooth muscle cells placed ADAMTS7 at the cytoplasm and cell membrane, where it colocalized with markers of podosomes. Conclusions— These data represent the first in vivo experimental validation of the association of Adamts7 with atherogenesis, likely through modulation of vascular cell migration and matrix in atherosclerotic lesions. These results demonstrate that Adamts7 is proatherogenic, lending directionality to the original genetic association and supporting the concept that pharmacological inhibition of ADAMTS7 should be atheroprotective in humans, making it an attractive target for novel therapeutic interventions.

Peroxisome Proliferator-Activated Receptor-α Activation Promotes Macrophage Reverse Cholesterol Transport Through a Liver X Receptor–Dependent Pathway

Objective—Peroxisome proliferator-activated receptor-&agr; (PPAR&agr;) activation has been shown in vitro to increase macrophage cholesterol efflux, the initial step in reverse cholesterol transport (RCT). However, it remains unclear whether PPAR&agr; activation promotes macrophage RCT in vivo. Methods and Results—We demonstrated that a specific potent PPAR&agr; agonist GW7647 inhibited atherosclerosis and promoted macrophage RCT in hypercholesterolemic mice expressing the human apolipoprotein A-I (apoA-I) gene. We compared the effect of GW7647 on RCT in human apoA-I transgenic (hA-ITg) mice with wild-type mice and showed that the PPAR&agr; agonist promoted RCT in hA-ITg mice to a much greater extent than in wild-type mice, indicating that human apoA-I expression is important for PPAR&agr;-induced RCT. We further investigated the dependence of the macrophage PPAR&agr;–liver X receptor (LXR) pathway on the promotion of RCT by GW7647. Primary murine macrophages lacking PPAR&agr; or LXR abolished the ability of GW7647 to promote RCT in hA-ITg mice. In concert, the PPAR&agr; agonist promoted cholesterol efflux and ATP binding cassette transporter A1/G1 expression in primary macrophages, and this was also by the PPAR&agr;-LXR pathway. Conclusion—Our observations demonstrate that a potent PPAR&agr; agonist promotes macrophage RCT in vivo in a manner that is enhanced by human apoA-I expression and dependent on both macrophage PPAR&agr; and LXR expression.

Effects of nevirapine and efavirenz on HDL cholesterol levels and reverse cholesterol transport in mice.

OBJECTIVE The mechanism by which non-nucleoside reverse transcriptase inhibitors (NNRTIs) increase HDL cholesterol (HDL-C) in HIV+ patients and the benefits of this with respect to cardiovascular risk are not known. Studies were conducted to test the hypothesis that NNRTIs have a beneficial effect on HDL-C and reverse cholesterol transport (RCT). METHODS LDLr-/- and hA-I transgenic mice were fed a Western diet containing either nevirapine (20mg/kg per day), efavirenz (10mg/kg per day), or diet alone. hA-I transgenic mice underwent a study to measure RCT (measured by excretion of macrophage [(3)H]-cholesterol into HDL and feces) at 8 weeks. RESULTS LDLr-/- and hA-I transgenic mice treated with nevirapine and efavirenz had a significant increase in HDL-C level (up to 23% in hA-I transgenic) at 4 weeks. However, there was no difference in HDL levels beyond 4 weeks of treatment. At 4 weeks, the FPLC profile of hA-I transgenic mice showed an increase in large HDL. hApoA-I transgenic mice treated with efavirenz for 4 weeks had increased expression of human apoA-I in liver and an increased human apoA-I production rate. Incubation of plasma from hA-I transgenic mice treated for 4 weeks with [(3)H]-cholesterol-labeled macrophages revealed increased cholesterol efflux to plasma from mice treated with efavirenz and nevirapine. Following injection of hA-I transgenic mice treated for 8 weeks with [(3)H]-cholesterol-labeled macrophages, RCT was increased in the efavirenz (p=0.01) group and trended towards an increase in the nevirapine (p=0.15) group. CONCLUSION Nevirapine and efavirenz transiently increased HDL-C in LDLr-/- and hA-I transgenic mice fed a Western diet that was associated with increased apoA-I production. An increase in RCT in hA-I transgenic mice at 8 weeks despite no difference in HDL levels indicates that these drugs affect additional factors in the RCT pathway that enhance cholesterol efflux from the macrophage and peripheral tissues to plasma and delivery to liver for excretion. These results suggest that treatment with NNRTIs has a beneficial effect on cholesterol efflux and RCT.

Knockout of Adamts7, A Novel CAD Locus in Humans, Reduces Atherosclerosis in Mice

Background— Genome-wide association studies have established ADAMTS7 as a locus for coronary artery disease in humans. However, these studies fail to provide directionality for the association between ADAMTS7 and coronary artery disease. Previous reports have implicated ADAMTS7 in the regulation of vascular smooth muscle cell migration, but a role for and the direction of impact of this gene in atherogenesis have not been shown in relevant model systems. Methods and Results— We bred an Adamts7 whole-body knockout mouse onto both the Ldlr and Apoe knockout hyperlipidemic mouse models. Adamts7−/−/Ldlr−/− and Adamts7−/−/Apoe−/− mice displayed significant reductions in lesion formation in aortas and aortic roots compared with controls. Adamts7 knockout mice also showed reduced neointimal formation after femoral wire injury. Adamts7 expression was induced in response to injury and hyperlipidemia but was absent at later time points, and primary Adamts7 knockout vascular smooth muscle cells showed reduced migration in the setting of tumor necrosis factor-&agr; stimulation. ADAMTS7 localized to cells positive for smooth muscle cell markers in human coronary artery disease lesions, and subcellular localization studies in cultured vascular smooth muscle cells placed ADAMTS7 at the cytoplasm and cell membrane, where it colocalized with markers of podosomes. Conclusions— These data represent the first in vivo experimental validation of the association of Adamts7 with atherogenesis, likely through modulation of vascular cell migration and matrix in atherosclerotic lesions. These results demonstrate that Adamts7 is proatherogenic, lending directionality to the original genetic association and supporting the concept that pharmacological inhibition of ADAMTS7 should be atheroprotective in humans, making it an attractive target for novel therapeutic interventions.