Acute Elevation of Plasma PLTP Activity Strongly Increases Pre-existing Atherosclerosis
Objective— A transgenic mouse model was generated that allows conditional expression of human PLTP, based on the tetracycline-responsive gene system, to study the effects of an acute increase in plasma PLTP activity as may occur in inflammation.
Methods and Results— The effects of an acute elevation of plasma PLTP activity on the metabolism of apolipoprotein B–containing lipoproteins and on diet-induced pre-existing atherosclerosis were determined in mice displaying a humanized lipoprotein profile (low-density lipoprotein receptor knockout background). Induced expression of PLTP strongly increases plasma VLDL levels in LDL receptor knockout mice, whereas VLDL secretion is not affected. The elevation in plasma triglyceride levels is explained by a PLTP-dependent inhibition of VLDL catabolism, which is caused, at least partly, by a decreased lipoprotein lipase activity. Together with the decreased plasma HDL levels, the acutely increased PLTP expression results in a highly atherogenic lipoprotein profile. Induction of PLTP expression leads to a further increase in size of pre-existing atherosclerotic lesions, even on a chow diet. In addition, the lesions contain more macrophages and less collagen relative to controls, suggesting a less stable lesion phenotype.
Conclusion— In conclusion, acute elevation of PLTP activity destabilizes atherosclerotic lesions and aggravates pre-existing atherosclerosis.
Studies in various atherosclerosis-prone mouse models have demonstrated that expression of human phospholipid transfer protein (PLTP) stimulates the development of atherosclerosis.1–3 This can be explained by effects of PLTP activity on lipoprotein composition and metabolism. PLTP increases hepatic VLDL secretion4–7 and decreases plasma HDL levels, resulting in a more atherogenic lipoprotein profile.7–9 Furthermore, PLTP unfavorably affects the antiinflammatory and antioxidative properties of HDL particles.6,10–12 Recently we developed a transgenic mouse model that allows conditional expression of human PLTP.13 We hypothesize that the physiological effect of an acute increase in plasma PLTP activity differs significantly from the effects seen in mice overexpressing human PLTP innately and life-long and which may have developed compensatory mechanisms. We tested this hypothesis in transgenic mice that conditionally overexpress human PLTP and that are deficient for the LDL receptor. The first objective was to test the effects of acute elevation of plasma PLTP activity on lipoprotein metabolism. The second objective was to study the effect of the acute expression of human PLTP on diet-induced pre-existing atherosclerosis.
Materials and Methods
The generation of inducible human PLTP transgenic mice is described in detail elsewhere.13 Briefly, 2 constructs were used to generate transgenic mice that allow inducible expression of PLTP: (1) hnRNP-rtTA-SV40, which consists of an improved version of the tetracycline-controlled transactivator rtTA2S-M2 under the control of an hnRNP A2 coding sequence, resulting in rtTAtg mice, and (2) pTet-PLTP-SV40, which consists of the human PLTP cDNA under the transcriptional control of a tetracycline operator and minimal cytomegalovirus promoter (TetO/Pcmv min), resulting in Tre-PLTPtg mice. Both Tre-PLTPtg mice and rtTAtg mice were crossed into an LDLR−/− background (Jackson Laboratory, Bar Harbor, ME, USA). Tre-PLTPtg/LDLR−/− mice and rtTAtg/LDLR−/− mice were crossbred, resulting in Tre-PLTPtg/rtTAtg/LDLR−/− mice. For convenience, these mice are referred to as “indPLTP” mice. Control mice are animals lacking either the Tre-PLTP transgene or the rtTA transgene. For determination of the genotype, genomic DNA was isolated from tail clips of 10-day-old mice and analyzed by polymerase chain reaction (PCR). Annealing temperatures and primer sequences are available on request.
After weaning, animals were fed a standard chow diet. Animals had free access to water and food. Blood samples were collected by orbital bleeding after removing food overnight. Male mice were used in all experiments. All procedures used in this study are in accordance with national and institutional guidelines.
Animals were subjected to 2 dietary regimes, referred to as treatment A and treatment B (supplemental Figure I, available online at http://atvb.ahajournals.org). Experiments were performed with mice of 10 to 15 weeks. Using treatment A, PLTP expression was induced immediately after switching from a high cholesterol to a normal chow diet, and its effect on lipoprotein metabolism was investigated. Two weeks after the switch from the high-fat high-cholesterol diet to the chow diet, PLTP expression was induced using treatment B and maintained for 5 weeks. This treatment was used to study the effect of PLTP expression on pre-existing atherosclerosis. In the online supplemental methods, the experimental setup is discussed in more detail.
Analysis of Plasma Activity of PLTP, Hepatic Lipase, and Lipoprotein Lipase
Plasma samples were collected by orbital puncture. Activities of PLTP, pre- and postheparin hepatic lipase (HL), and lipoprotein lipase (LPL) were analyzed as described in the supplemental methods.
Analysis of Plasma Lipids and Lipoproteins
Measurements of plasma concentration of lipids, and isolation and analysis of plasma lipoproteins were performed as described in the supplemental methods.
Determination of VLDL Secretion and VLDL Decay
VLDL secretion experiments were performed as described in the supplemental methods. VLDL decay experiments were performed with [3H]cholesteryl oleyl ether labeled murine VLDL, injected intravenously in mice as a tracer. See supplemental methods for details.
Histology and Measurement of Atherosclerotic Lesions
Histological analysis of atherosclerotic lesions were performed as described in the supplemental methods.
Data are expressed as means±SE. Differences were analyzed by 2-sample Wilcoxon rank sum tests using Intercooled Stata 8.2/SE software (Stata Corporation).
PLTP-Effects on Lipoprotein Metabolism
For the first set of experiments, designed to determine the effect of acutely increased PLTP activity on lipoprotein metabolism, treatment A was followed (supplemental Figure I). At 0 weeks, no differences were observed in plasma PLTP activity, triglycerides, cholesterol, or phospholipid levels between indPLTP and control mice (Figure 1, Table). After 9 weeks of Western diet, PLTP activity, triglycerides, cholesterol, and phospholipids were increased in both groups to a similar extent (Figure 1, Table). Subsequently, Western diet was stopped and doxycycline was administered for 2 weeks. This resulted in a further 4.5-fold increase in PLTP activity in the indPLTP group, whereas PLTP activity remained unchanged in the control group (Figure 1A). Induction of PLTP in animals fed a chow diet resulted in a 3-fold increase in plasma triglycerides in the indPLTP mice, whereas in the control mice triglyceride levels returned to basal levels (Figure 1B). Similar effects were observed for cholesterol and phospholipids levels in the control group (Table). In contrast, plasma cholesterol and phospholipid levels remained elevated in the indPLTP group: the plasma cholesterol was 20.0±8.3 mmol/L and the phospholipid level 7.1±1.1 mmol/L in the indPLTP mice, versus 8.5±1.5 mmol/L and 4.4±0.6 mmol/L in the controls.
The effects of induced expression of human PLTP on plasma lipids were studied in more detail by separation of HDL and non-HDL using density ultracentrifugation. Feeding the Western diet did not influence HDL-cholesterol or HDL-phospholipid levels in either group, whereas it strongly increased non–HDL-cholesterol and non–HDL-phospholipid levels (Table, 9 weeks). The subsequent 2 weeks administration of doxycycline on a chow diet did not induce any change in HDL levels in the control mice (Table). Non–HDL-phospholipid levels returned to basal values, non–HDL-cholesterol level strongly decreased but remained slightly elevated compared to basal level (6.4±0.8 mmol/L versus 4.3±0.7 mmol/L). In contrast, HDL levels dramatically decreased in the indPLTP mice on PLTP induction by doxycycline treatment (Table, treatment A: HDL-cholesterol from 2.1±0.5 to 0.3±0.1 mmol/L, HDL-phospholipid from 1.9±0.4 to 0.4±0.0 mmol/L). Non–HDL-lipids remained increased when human PLTP expression was induced (Table).
To study the effects of expression of human PLTP on lipoprotein distribution further, pooled plasma samples obtained from 8 to 10 mice were subjected to gel filtration by fast protein liquid (FPLC) at 0, 9, and 11 weeks. At 0 weeks, cholesterol profiles of the control group and the indPLTP group did not differ (Figure 2). At 9 weeks cholesterol levels in the non-HDL size range (fractions 1 to 12) strongly increased in both groups. At 11 weeks (treatment A) the non-HDL peak strongly declined in the control mice, although the level of LDL-sized particles remained elevated (Figure 2A). In the indPLTP mice the non-HDL peak overall also declined (Figure 2B). However, in these animals a substantial lipoprotein fraction with VLDL size was clearly present after induction of PLTP expression (fractions 1 to 4).
To evaluate possible mechanisms explaining this observation, we investigated VLDL secretion in indPLTP and control mice at 11 weeks. All mice had been given drinking water containing both doxycycline and 5% sucrose during the last 2 weeks. Plasma triglycerides (at t=0 minutes) strongly differed between controls and indPLTP (Figure 3A), as observed before (Figure 1B). However, both groups had equal rates of VLDL triglyceride secretion. Therefore, the increase in VLDL after induction of PLTP in indPLTP mice cannot be explained by an increase in formation and secretion of these particles. Next, we evaluated possible differences in VLDL degradation. Murine VLDL was labeled with [3H]-cholesteryl oleoyl ether, and tracer amounts were injected intravenously in doxycycline-treated control mice and indPLTP mice at 11 weeks of treatment A. Subsequently, the disappearance of radioactivity from the blood was monitored (Figure 3B). There is a big difference between the 2 groups in VLDL clearance during the first 15 minutes after labeled VLDL injection. After 15 minutes, already 39% of labeled VLDL had been cleared from the plasma of control mice, whereas in the indPLTP mice there had not been any clearance yet. At later time points [3H]-VLDL disappeared slowly and linearly in both control and indPLTP mice. Four hours after injection, 51±5% and 84±10% of the label still remained in the plasma of the control mice and the indPLTP mice, respectively. Separation of lipoprotein particles by FPLC demonstrated that both in the indPLTP and the control mice a substantial part of the injected VLDL particles had been converted into IDL and LDL (data not shown). The tissue distribution of injected label was studied after sacrificing the animals at t=4 hours (Figure 3C). In both groups, almost all injected label that had disappeared from plasma was detected in the liver. To get more insight in the mechanism behind the initially delayed decay of VLDL-particles in the human PLTP expressing mice, we measured plasma lipase activities in doxycycline-treated indPLTP mice and control mice. Hepatic lipase activity in preheparin plasma did not differ between the indPLTP group and the control group. However, LPL activity in postheparin plasma was significantly decreased in the indPLTP group compared to the control group (Figure 4).
The Effect of PLTP Expression on Preexisting Atherosclerosis
The second main objective was to determine the effect of acute elevation of PLTP activity on pre-existing atherosclerosis. For this purpose, treatment B was followed (supplemental Figure I). Plasma PLTP activity levels or triglyceride levels did not differ between the control mice and the indPLTP mice at 0, 9, and 11 weeks (ie, before PLTP induction; Figure 1). From 11 weeks on, doxycycline was administered for an additional 6 weeks. This resulted in a 5.5-fold increase in PLTP activity in the indPLTP group, whereas PLTP activity did not change in the control group (Figure 1A). The increased PLTP activity in the indPLTP group resulted in strongly increased plasma levels of triglycerides, cholesterol, and phospholipids (Figure 1B, Table). Separation of lipoprotein classes using density ultracentrifugation showed that on induction of PLTP activity, levels of HDL-cholesterol and HDL-phospholipids were substantially decreased whereas levels of non-HDL-cholesterol and non-HDL-phospholipids were substantially increased (Table). Atherosclerotic lesion development was determined at 9 and 17 weeks. In the control mice, discontinuing the Western diet for 8 weeks did not influence atherosclerotic lesion area significantly (Figure 5A and supplemental Figure II; 2.8±1.5×104 μm2 at 9 weeks and 2.4±0.7×104 μm2 at 17 weeks), but lesion composition changed. The macrophage content of the lesion decreased by 40% on switching the Western diet to a chow diet (Figure 5B and supplemental Figure II), whereas the collagen content increased 4-fold (Figure 5C and supplemental Figure II), suggesting a significant stabilization of the lesion. In the indPLTP mice however, induction of human PLTP expression resulted in a further increase in mean lesion area (Figure 5A and supplemental Figure II; from 3.1±1.2×104 μm2 at 9 weeks to 5.2±1.3×104 μm2 at 17 weeks), even though the mice were on a chow diet. In these mice, the relative macrophage content of the lesion remained unchanged (Figure 5B and supplemental Figure II). The collagen content increased but was significantly lower when compared to that seen in the control mice (Figure 5C and supplemental Figure II; 38.9±14.8% versus 27.2±9.5%).
Our first objective was to investigate the effect of conditional PLTP expression on lipoprotein metabolism after changing from a hyperlipidaemic state, induced by a high-fat high-cholesterol diet, to normolipidaemia induced by chow diet. HDL levels strongly decreased on induction of expression of human PLTP. In addition, we observed an important novel effect of elevated PLTP expression on VLDL catabolism, probably caused by induction of decreased LPL activity. Induction of elevated PLTP activity strongly increased plasma triglyceride, cholesterol, and phospholipid levels in the non-HDL fractions. Separation of lipoproteins by FPLC revealed that the majority of the lipids was present in VLDL-sized particles. To our knowledge this is the first demonstration that the plasma levels of apoB-containing lipoproteins or plasma triglyceride levels are actually higher after induction of PLTP expression. Apparently, the metabolic effects of an acute increase in PLTP activity differ from the effect of life-long increase in plasma PLTP activity levels in human PLTP transgenic mice. Indeed, in the acute model, the triglyceride secretion rate by the liver was not affected by overexpression of PLTP, whereas we previously showed that life-long enhanced PLTP expression results in increased hepatic triglyceride secretion.5,7 The high lipid levels in the non-HDL fractions therefore cannot be explained by an increase in VLDL production. To examine whether these observations could be explained by a decreased catabolism of VLDL, we performed VLDL decay studies using [3H]-cholesteryl oleyl ether labeled VLDL as a tracer. The absolute amount of injected VLDL-cholesterol was only 0.5% to 1% of total circulating cholesterol, an amount that was not expected to affect endogenous lipoprotein metabolism. We found that plasma clearance of [3H]-labeled VLDL was delayed, and that the hepatic uptake of [3H]-labeled cholesterol was strongly decreased in indPLTP mice when compared with control mice.
It is unlikely that hepatic clearance of VLDL is inhibited due to a decreased apoE content in indPLTP mice, as protein analysis of FPLC fractions showed that VLDL in doxycycline-treated indPLTP mice contained normal amounts of apoE (data not shown). Already a very low level of apoE is sufficient to normalize plasma cholesterol levels in apoE-deficient mice.14 Next, we examined whether PLTP may affect VLDL lipolysis. Lipoprotein lipase (LPL) plays an important role in this process by hydrolyzing the triglycerides in VLDL particles.15–17 In addition, HL and LPL may act as a ligand between low density lipoproteins and hepatic lipoprotein binding sites. Indeed, plasma LPL activity measured in postheparin plasma under optimal conditions was significantly decreased in indPLTP mice. Thus the delayed VLDL clearance could, at least partly, be explained by a decrease in the plasma level of LPL, causing a limited turnover of VLDL triglycerides in peripheral tissues.
Our second main objective was to determine the effect of increased PLTP activity on pre-existing atherosclerotic lesions, which had been induced by feeding a high-fat high-cholesterol diet for 9 weeks. Induction of high plasma PLTP activity levels in the indPLTP mice not only decreased HDL levels but also strongly increased plasma triglyceride, cholesterol, and phospholipid levels in the non-HDL fraction. In the control mice, the lesion size at 9 and 17 weeks was similar. Lesion macrophage content was strongly decreased, whereas collagen content was significantly increased. This is in accordance with previous experiments in which the regression of pre-existing atherosclerotic lesions was studied.18–21 A decrease in lesion area on withdrawal of an atherosclerosis-inducing diet is only observed in foam cell–containing fatty streaks. For lesions containing more advanced characteristics, such as fibrous caps and necrotic cores, a decrease in lesion area may not be observed, but generally macrophage content of the lesions strongly decreases on withdrawal of the high-fat high-cholesterol diet. In addition, collagen content of the lesions increases,22 resulting in lesion stabilization. In contrast to the situation observed in the control mice, the unfavorable lipoprotein profile observed in the indPLTP resulted in a further increase in lesion area. Although the Western diet had been stopped, the lesion area had almost doubled compared to the situation seen at 9 weeks, indicating a strong atherogenic effect of PLTP. No regression of the macrophage percentage was observed. Also the collagen percentage was remarkably lower than that seen in control mice, indicating that high plasma PLTP activity levels stimulate an increase in lesion size and also inhibit lesion stabilization, which may be caused by the elevated plasma VLDL levels. Besides, a PLTP-induced inflammatory response might very well contribute to the formation of atherosclerotic lesion. Plasma PLTP activity is increased during acute systemic inflammation,23 a situation that is comparable with the acute increase in plasma PLTP activity that is induced in our mouse model.
In conclusion, using a novel mouse model with inducible expression of PLTP we found that increased plasma PLTP activity strongly enhances plasma VLDL levels by a PLTP-dependent inhibition of VLDL catabolism. In combination with the PLTP-dependent decrease in plasma HDL levels, this results in a strongly atherogenic lipoprotein profile and may account for the PLTP-dependent size increase of pre-existing atherosclerotic lesions and the decreased lesion stability, even after replacement of the Western diet by chow diet. Our study supplies new evidence that high systemic PLTP activity unfavorably affects the cardiovascular system.
A.J. Zonneveld, Department of Clinical Chemistry, Erasmus MC is gratefully thanked for the determination of lipase activities.
Original received December 10, 2007; final version accepted April 9, 2008.
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