An Apolipoprotein A-I Mimetic Peptide Designed with a Reductionist Approach Stimulates Reverse Cholesterol Transport and Reduces Atherosclerosis in Mice

Apolipoprotein A-I (apoA-I) mimetic peptides are considered a promising novel therapeutic approach to prevent and/or treat atherosclerosis. An apoA-I mimetic peptide ELK-2A2K2E was designed with a reductionist approach and has shown exceptional activity in supporting cholesterol efflux but modest anti-inflammatory and anti-oxidant properties in vitro. In this study we compared these in vitro properties with the capacity of this peptide to modify rates of reverse cholesterol transport and development of atherosclerosis in mouse models. The peptide enhanced the rate of reverse cholesterol transport in C57BL/6 mice and reduced atherosclerosis in Apoe^−/− mice receiving a high fat diet. The peptide modestly reduced the size of the plaques in aortic arch, but was highly active in reducing vascular inflammation and oxidation. Administration of the peptide to Apoe^−/− mice on a high fat diet reduced the levels of total, high density lipoprotein and non-high density lipoprotein cholesterol and triglycerides. It increased the proportion of smaller HDL particles in plasma at the expense of larger HDL particles, and increased the capacity of the plasma to support cholesterol efflux. Thus, ELK-2A2K2E peptide reduced atherosclerosis in Apoe^−/− mice, however, the functional activity profile after chronic in vivo administration was different from that found in acute in vitro studies.     

RhoA/ROCK I signalling downstream of the P2Y13 ADP-receptor controls HDL endocytosis in human hepatocytes

Cell surface receptors for high-density lipoprotein (HDL) on hepatocytes are major partners in the regulation of cholesterol homeostasis. We have previously demonstrated on human hepatocytes that apolipoprotein A-I binding to an ectopic F(1)-ATPase stimulates the production of extracellular ADP that activates a P2Y(13)-mediated high-density lipoprotein (HDL) endocytosis pathway. However, P2Y(13)-dependent signalling pathway has never been described yet. The current study demonstrates a major role of cytoskeleton reorganization in F(1)-ATPase/P2Y(13)-dependent HDL endocytosis under the control of the small GTPase RhoA and its effector ROCK I. Indeed human hepatocytes (HepG(2) cells) stimulated by ADP or AR-C69931MX (both P2Y(13) agonists) showed a high specific activation of RhoA; in addition, inhibition of Rho proteins by C3 exoenzyme impairs HDL endocytosis whereas a constitutively active form of RhoA stimulates HDL endocytosis at the same level as under F(1)-ATPase/P2Y(13) activation. Pharmacological inhibition of ROCK activity decreased HDL endocytosis following stimulation by apoA-I (F(1)-ATPase ligand), ADP or AR-C69931MX and specific siRNA ROCK I extinction prevented the stimulation of HDL endocytosis without effect of ROCK II extinction. The functional involvement of ROCK I downstream F(1)-ATPase/P2Y(13) was confirmed by the strong enrichment of the membrane fraction in ROCK I and by the requirement of actin polymerization in hepatocyte HDL endocytosis. These results allow the identification of the molecular events downstream P2Y(13) receptor activation for a better understanding of hepatocyte HDL endocytosis, the latest step in reverse cholesterol transport.     

Angiopoietin-1 aggravates atherosclerosis by inhibiting cholesterol efflux and promoting inflammatory response

ObjectiveAngiopoietin-1 (Ang-1), a secreted protein, mainly regulates angiogenesis. Ang-1 has been shown to promote the development of atherosclerosis, whereas little is known about its effects on lipid metabolism and inflammation in this process.MethodAng-1 was transfected into ApoE^−/− mice via lentiviral vector or incubated with THP-1 derived macrophages. Oil red O and HE staining were performed to measure the size of atherosclerotic plaques in ApoE^−/− mice. Immunofluorescence was employed to show the expression of target proteins in aorta. [³H] labeled cholesterol was performed to examine the efficiency of cholesterol efflux and reverse cholesterol transport (RCT) both in vivo and vitro. Western blot and qPCR were used to quantify target proteins both in vivo and vitro. ELISA detected the levels of pro-inflammatory cytokines in mouse peritoneal macrophage.ResultsOur data showed that Ang-1 augmented atherosclerotic plaques formation and inhibited cholesterol efflux. The binding of Ang-1 to Tie2 resulted in downregulation of LXRα, ABCA1 and ABCG1 expression via inhibiting the translocation of TFE3 into nucleus. In addition, Ang-1 decreased serum HDL-C levels and reduced reverse cholesterol transport (RCT) in ApoE−/− mice. Furthermore, Ang-1 induced lipid accumulation followed by increasing TNF-α, IL-6, IL-1β,and MCP-1 produced by MPMs, as well as inducing M1 phenotype macrophage marker iNOS and CD86 expression in aorta of ApoE^−/− mice.ConclusionAng-1 has an adverse effect on cholesterol efflux by decreasing the expression of ABCA1 and ABCG1 via Tie2/TFE3/LXRα pathway, thereby promoting inflammation and accelerating atherosclerosis progression.     

Elevated expression of phospholipid transfer protein in bone marrow derived cells causes atherosclerosis

Background

Phospholipid transfer protein (PLTP) is expressed by various cell types. In plasma, it is associated with high density lipoproteins (HDL). Elevated levels of PLTP in transgenic mice result in decreased HDL and increased atherosclerosis. PLTP is present in human atherosclerotic lesions, where it seems to be macrophage derived. The aim of the present study is to evaluate the atherogenic potential of macrophage derived PLTP.

Methods and Findings

Here we show that macrophages from human PLTP transgenic mice secrete active PLTP. Subsequently, we performed bone marrow transplantations using either wild type mice (PLTPwt/wt), hemizygous PLTP transgenic mice (huPLTPtg/wt) or homozygous PLTP transgenic mice (huPLTPtg/tg) as donors and low density lipoprotein receptor deficient mice (LDLR−/−) as acceptors, in order to establish the role of PLTP expressed by bone marrow derived cells in diet-induced atherogenesis. Atherosclerosis was increased in the huPLTPtg/wt→LDLR−/− mice (2.3-fold) and even further in the huPLTPtg/tg→LDLR−/− mice (4.5-fold) compared with the control PLTPwt/wt→LDLR−/− mice (both P<0.001). Plasma PLTP activity levels and non-HDL cholesterol were increased and HDL cholesterol decreased compared with controls (all P<0.01). PLTP was present in atherosclerotic plaques in the mice as demonstrated by immunohistochemistry and appears to co-localize with macrophages. Isolated macrophages from PLTP transgenic mice do not show differences in cholesterol efflux or in cytokine production. Lipopolysaccharide activation of macrophages results in increased production of PLTP. This effect was strongly amplified in PLTP transgenic macrophages.

Conclusions

We conclude that PLTP expression by bone marrow derived cells results in atherogenic effects on plasma lipids, increased PLTP activity, high local PLTP protein levels in the atherosclerotic lesions and increased atherosclerotic lesion size.     

In vivo tissue cholesterol efflux is reduced in carriers of a mutation in APOA1

Atheroprotection by high density lipoprotein (HDL) is considered to be mediated through reverse cholesterol transport (RCT) from peripheral tissues. We investigated in vivo cholesterol fluxes through the RCT pathway in patients with low plasma high density lipoprotein cholesterol (HDL-c) due to mutations in APOA1. Seven carriers of the L202P mutation in APOA1 (mean HDL-c: 20 ± 19 mg/dl) and seven unaffected controls (mean HDL-c: 54 ± 11 mg/dl, P < 0.0001) received a 20 h infusion of ¹³C₂-cholesterol (¹³C-C). Enrichment of plasma and erythrocyte free cholesterol and plasma cholesterol esters was measured. With a three-compartment SAAM-II model, tissue cholesterol efflux (TCE) was calculated. TCE was reduced by 19% in carriers (4.6 ± 0.8 mg/kg/h versus 5.7 ± 0.7 mg/kg/h in controls, P = 0.02). Fecal ¹³C recovery and sterol excretion 7 days postinfusion did not differ significantly between carriers and controls: 21.3 ± 20% versus 13.3 ± 6.3% (P = 0.33), and 2,015 ± 1,431 mg/day versus 1456 ± 404 mg/day (P = 0.43), respectively. TCE is reduced in carriers of mutations in APOA1, suggesting that HDL contributes to efflux of tissue cholesterol in humans. The residual TCE and unaffected fecal sterol excretion in our severely affected carriers suggest, however, that non-HDL pathways contribute to RCT significantly.     

A Western-Fed Diet Increases Plasma HDL and LDL-Cholesterol Levels in ApoD–/– Mice

Objective

Plasma apolipoprotein (apo)D, a ubiquitously expressed protein that binds small hydrophobic ligands, is found mainly on HDL particles. According to studies of human genetics and lipid disorders, plasma apoD levels positively correlate with HDL-cholesterol and apoAI levels. Thus, we tested the hypothesis that apoD was a regulator of HDL metabolism.

Methods & Results

We compared the plasma lipid and lipoprotein profiles of wild-type (WT) C57BL/6 mice with apoD−/− mice on a C57BL/6 background after receiving a high fat-high cholesterol diet for 12 weeks. ApoD−/− mice had higher HDL-cholesterol levels (61±13-apoD−/− vs. 52±10-WT-males; 37±11-apoD−/− vs. 22±2 WT-female) than WT mice with sex-specific changes in total plasma levels of cholesterol and other lipids. Compared to WT, the HDL of apoD−/− mice showed an increase in large, lipid-rich HDL particles and according to size various quantities and sizes of LDL particles. Plasma levels of lecithin:cholesterol acyltransferase in the control and apoD−/− mice were not different, however, plasma phospholipid transfer protein activity was modestly elevated (+10%) only in male apoD−/− mice. An in vivo HDL metabolism experiment with isolated Western-fed apoD−/− HDL particles showed that female apoD−/− mice had a 36% decrease in the fractional catabolic rate of HDL cholesteryl ester. Hepatic SR-BI and LDLR protein levels were significantly decreased; accordingly, LDL-cholesterol and apoB levels were increased in female mice.

Conclusion

In the context of a high fat-high cholesterol diet, apoD deficiency in female mice is associated with increases in both plasma HDL and LDL-cholesterol levels, reflecting changes in expression of SR-BI and LDL receptors, which may impact diet-induced atherosclerosis.     

Chronic pharmacological activation of P2Y13 receptor in mice decreases HDL-cholesterol level by increasing hepatic HDL uptake and bile acid secretion

High level of high-density lipoprotein cholesterol (HDL-cholesterol) is inversely correlated to the risk of atherosclerotic cardiovascular disease. The protective effect of HDL is mostly attributed to their metabolic functions in reverse cholesterol transport (RCT), a process whereby excess cell cholesterol is taken up from peripheral cells and processed in HDL particles, and is later delivered to the liver for further metabolism and bile excretion. We have previously demonstrated that P2Y₁₃ receptor is critical for RCT and that intravenous bolus injection of cangrelor (AR-C69931MX), a partial agonist of P2Y₁₃ receptor, can stimulate hepatic HDL uptake and subsequent lipid biliary secretion without any change in plasma lipid levels. In the present study, we investigated the effect of longer-term treatment with cangrelor on lipoprotein metabolism in mice. We observed that continuous delivery of cangrelor at a rate of 35 μg/day/kg body weight for 3 days markedly decreased plasma HDL-cholesterol level, by increasing the clearance of HDL particles by the liver. These effects were correlated to an increase in the rate of biliary bile acid secretion. An increased expression of SREBP-regulated genes of cholesterol metabolism was also observed without any change of hepatic lipid levels as compared to non-treated mice. Thus, 3-day cangrelor treatment markedly increases the flux of HDL-cholesterol from the plasma to the liver for bile acid secretion. Taken together our results suggest that P2Y₁₃ appears a promising target for therapeutic intervention aimed at preventing or reducing cardiovascular risk.     

Procollagen C-endopeptidase Enhancer Protein 2 (PCPE2) Reduces Atherosclerosis in Mice by Enhancing Scavenger Receptor Class B1 (SR-BI)-mediated High-density Lipoprotein (HDL)-Cholesteryl Ester Uptake

Studies in human populations have shown a significant correlation between procollagen C-endopeptidase enhancer protein 2 (PCPE2) single nucleotide polymorphisms and plasma HDL cholesterol concentrations. PCPE2, a 52-kDa glycoprotein located in the extracellular matrix, enhances the cleavage of C-terminal procollagen by bone morphogenetic protein 1 (BMP1). Our studies here focused on investigating the basis for the elevated concentration of enlarged plasma HDL in PCPE2-deficient mice to determine whether they protected against diet-induced atherosclerosis. PCPE2-deficient mice were crossed with LDL receptor-deficient mice to obtain LDLr^−/−, PCPE2^−/− mice, which had elevated HDL levels compared with LDLr^−/− mice with similar LDL concentrations. We found that LDLr^−/−, PCPE2^−/− mice had significantly more neutral lipid and CD68+ infiltration in the aortic root than LDLr^−/− mice. Surprisingly, in light of their elevated HDL levels, the extent of aortic lipid deposition in LDLr^−/−, PCPE2^−/− mice was similar to that reported for LDLr^−/−, apoA-I^−/− mice, which lack any apoA-I/HDL. Furthermore, LDLr^−/−, PCPE2^−/− mice had reduced HDL apoA-I fractional clearance and macrophage to fecal reverse cholesterol transport rates compared with LDLr^−/− mice, despite a 2-fold increase in liver SR-BI expression. PCPE2 was shown to enhance SR-BI function by increasing the rate of HDL-associated cholesteryl ester uptake, possibly by optimizing SR-BI localization and/or conformation. We conclude that PCPE2 is atheroprotective and an important component of the reverse cholesterol transport HDL system.     

Atheroprotective Effect of Oleoylethanolamide (OEA) Targeting Oxidized LDL

Dietary fat-derived lipid oleoylethanolamide (OEA) has shown to modulate lipid metabolism through a peroxisome proliferator-activated receptor-alpha (PPAR-α)-mediated mechanism. In our study, we further demonstrated that OEA, as an atheroprotective agent, modulated the atherosclerotic plaques development. In vitro studies showed that OEA antagonized oxidized LDL (ox-LDL)-induced vascular endothelial cell proliferation and vascular smooth muscle cell migration, and suppressed lipopolysaccharide (LPS)-induced LDL modification and inflammation. In vivo studies, atherosclerosis animals were established using balloon-aortic denudation (BAD) rats and ApoE^-/- mice fed with high-caloric diet (HCD) for 17 or 14 weeks respectively, and atherosclerotic plaques were evaluated by oil red staining. The administration of OEA (5 mg/kg/day, intraperitoneal injection, i.p.) prevented or attenuated the formation of atherosclerotic plaques in HCD-BAD rats or HCD-ApoE^−/− mice. Gene expression analysis of vessel tissues from these animals showed that OEA induced the mRNA expressions of PPAR-α and downregulated the expression of M-CFS, an atherosclerotic marker, and genes involved in oxidation and inflammation, including iNOS, COX-2, TNF-α and IL-6. Collectively, our results suggested that OEA exerted a pharmacological effect on modulating atherosclerotic plaque formation through the inhibition of LDL modification in vascular system and therefore be a potential candidate for anti-atherosclerosis drug.     

Enlargement of high density lipoprotein in mice via liver X receptor activation requires apolipoprotein E and is abolished by cholesteryl ester transfer protein expression

The factors involved in the generation of larger high density lipoprotein (HDL) particles, HDL1 and HDLc, are still not well understood. Administration of a specific synthetic liver X receptor (LXR) agonist, T0901317, in mice resulted in an increase of not only HDL cholesterol but also HDL particle size (Cao, G., Beyer, T. P., Yang, X. P., Schmidt, R. J., Zhang, Y., Bensch, W. R., Kauffman, R. F., Gao, H., Ryan, T. P., Liang, Y., Eacho, P. I., and Jiang, X. C. (2002) J. Biol. Chem. 277, 39561-39565). We have investigated the roles that apoE and CETP may play in this process. We treated apoE-deficient, cholesterol ester transport protein (CETP) transgenic, and wild type mice with various doses of the LXR agonist and monitored their HDL levels. Fast protein liquid chromatography and apolipoprotein analysis revealed that in apoE knockout mouse plasma, there was neither induction of larger HDL formation nor increase of HDL cholesterol, suggesting that apoE is essential for the LXR agonist effects on HDL metabolism. In CETP transgenic mice, CETP expression completely abolished LXR agonist-mediated HDL enlargement and greatly attenuated HDL cholesterol levels. Analysis of HDL particles by electron microscope and nondenaturing gel electrophoresis revealed similar findings. In apoE-deficient mice, LXR agonist also produced a significant increase in very low density lipoprotein/low density lipoprotein cholesterol and apolipoprotein B content. Our studies provide direct evidence that apoE and CETP are intimately involved in the accumulation of the enlarged HDL (HDL1 or HDLc) particles in mice.     

Activation of the Liver X Receptor Stimulates Trans-intestinal Excretion of Plasma Cholesterol

Recent studies have indicated that direct intestinal secretion of plasma cholesterol significantly contributes to fecal neutral sterol loss in mice. The physiological relevance of this novel route, which represents a part of the reverse cholesterol transport pathway, has not been directly established in vivo as yet. We have developed a method to quantify the fractional and absolute contributions of several cholesterol fluxes to total fecal neutral sterol loss in vivo in mice, by assessing the kinetics of orally and intravenously administered stable isotopically labeled cholesterol combined with an isotopic approach to assess the fate of de novo synthesized cholesterol. Our results show that trans-intestinal cholesterol excretion significantly contributes to removal of blood-derived free cholesterol in C57Bl6/J mice (33% of 231 μmol/kg/day) and that pharmacological activation of LXR with T0901317 strongly stimulates this pathway (63% of 706 μmol/kg/day). Trans-intestinal cholesterol excretion is impaired in mice lacking Abcg5 (−4%), suggesting that the cholesterol transporting Abcg5/Abcg8 heterodimer is involved in this pathway. Our data demonstrate that intestinal excretion represents a quantitatively important route for fecal removal of neutral sterols independent of biliary secretion in mice. This pathway is sensitive to pharmacological activation of the LXR system. These data support the concept that the intestine substantially contributes to reverse cholesterol transport.Reverse cholesterol transport (RCT)³ is defined as the flux of excess cholesterol from peripheral tissues toward the liver followed by biliary secretion and subsequent disposal via the feces (1). Accumulation of cholesterol in macrophages in the vessel wall is considered a primary event in the development of atherosclerosis and, therefore, removal of excess cholesterol from these cells is of crucial importance for prevention and/or treatment of atherosclerotic cardiovascular diseases. It is generally accepted that HDL is the obligate transport vehicle in RCT and that plasma HDL levels reflect the capacity to accommodate this flux. In line herewith, HDL-raising therapies are currently considered as a promising strategy for prevention and treatment of atherosclerotic cardiovascular diseases (2). In the “classical” scenario, the liver has a central role in RCT (3). Biliary secretion of free cholesterol, facilitated by the heterodimeric ABC-transporter ABCG5/ABCG8 (4), and hepatic conversion of cholesterol into bile acids followed by fecal excretion are referred to as the main routes for quantitatively important elimination of cholesterol from the body. Fecal excretion of sterols is stimulated upon whole body activation of the liver X receptor (LXR, NR1H2/3), a member of the nuclear receptor family for which oxysterols have been identified as natural ligands (5). LXR regulates expression of several genes involved in RCT and activation of LXR by synthetic agonists leads to elevated plasma HDL-cholesterol levels, increased hepatobiliary cholesterol secretion, reduced fractional intestinal cholesterol absorption and increased fecal sterol loss (6). LXR is thus considered an attractive target for therapeutic strategies aimed at stimulation of RCT, which, however, will require approaches to circumvent potential detrimental consequences of LXR activation such as induction of lipogenesis.Recent studies indicate that the classical concept of RCT may require reconsideration. Studies in apoA-I-deficient mice revealed that the magnitude of the centripetal cholesterol flux from the periphery to the liver is not related to the concentration of HDL-cholesterol or apoA-I in plasma (7). Furthermore, Abca1^−/− mice that completely lack plasma HDL show unaffected rates of hepatobiliary cholesterol secretion and fecal sterol loss (8). Additionally, mice lacking both Abcg5 and Abcg8 do not show a reduction in fecal neutral sterol excretion to the extent expected on the basis of their strongly reduced hepatobiliary cholesterol secretion (9). Recent studies by Plösch et al. (6) have revealed that increased fecal neutral sterol loss upon general LXR activation cannot be attributed to the increased hepatobiliary cholesterol secretion only, suggesting a major contribution of the intestine in excretion of cholesterol. This potential role of the intestine in cholesterol removal from the body has been corroborated by Kruit et al. (10), who showed that fecal sterol loss is not affected in Mdr2^−/− (Abcb4^−/−) mice that have a dramatic reduction in biliary cholesterol secretion (11). Moreover, intravenously administered [³H]cholesterol could be recovered in the neutral sterol fraction of the feces in these mice and fecal excretion of neutral sterols was stimulated upon treatment with an LXR agonist (10). However, the exact quantitative contribution of the direct intestinal pathway under physiological conditions has not directly been determined so far. Very recently, intestinal perfusion studies in mice revealed that, in the presence of mixed micelles as cholesterol acceptors in the intestinal lumen, murine enterocytes indeed have a high capacity to secrete cholesterol via a specific process that is most active in the proximal part of the small intestine (12). In addition, it was shown that direct trans-intestinal cholesterol excretion (TICE) could be stimulated by a high fat diet. The existence of a non-biliary route for fecal neutral sterol excretion is further supported by very recent studies by Brown et al. (13) in mice with targeted deletion of hepatic ACAT2.The present study provides insight into the relative and absolute contributions of several cholesterol fluxes relevant to total fecal sterol loss in mice, making use of a panel of stable isotope tracers. Our results show that TICE is a major route for removal of blood-derived free cholesterol and that pharmacological LXR activation strongly stimulates this arm of the reverse cholesterol transport pathway.     

Enhanced Hepatic apoA-I Secretion and Peripheral Efflux of Cholesterol and Phospholipid in CD36 Null Mice

CD36 facilitates oxidized low density lipoprotein uptake and is implicated in development of atherosclerotic lesions. CD36 also binds unmodified high and very low density lipoproteins (HDL, VLDL) but its role in the metabolism of these particles is unclear. Several polymorphisms in the CD36 gene were recently shown to associate with serum HDL cholesterol. To gain insight into potential mechanisms for these associations we examined HDL metabolism in CD36 null (CD36^−/−) mice. Feeding CD36^−/− mice a high cholesterol diet significantly increased serum HDL, cholesterol and phospholipids, as compared to wild type mice. HDL apolipoproteins apoA-I and apoA-IV were increased and shifted to higher density HDL fractions suggesting altered particle maturation. Clearance of dual-labeled HDL was unchanged in CD36^−/− mice and cholesterol uptake from HDL or LDL by isolated CD36^−/− hepatocytes was unaltered. However, CD36^−/− hepatocytes had higher cholesterol and phospholipid efflux rates. In addition, expression and secretion of apoA-I and apoA-IV were increased reflecting enhanced PXR. Similar to hepatocytes, cholesterol and phospholipid efflux were enhanced in CD36^−/− macrophages without changes in protein levels of ABCA1, ABCG1 or SR-B1. However, biotinylation assays showed increased surface ABCA1 localization in CD36^−/− cells. In conclusion, CD36 influences reverse cholesterol transport and hepatic ApoA-I production. Both pathways are enhanced in CD36 deficiency, increasing HDL concentrations, which suggests the potential benefit of CD36 inhibition.     

The anti-inflammatory effects of exercise training promote atherosclerotic plaque stabilization in apolipoprotein E knockout mice with diabetic atherosclerosis

Physical exercise is the cornerstone of cardiovascular disease treatment. The present study investigated whether exercise training affects atherosclerotic plaque composition through the modification of inflammatoryrelated pathways in apolipoprotein E knockout (apoE^−/−) mice with diabetic atherosclerosis. Forty-five male apoE^−/− mice were randomized into three equivalent (n=15) groups: control (CO), sedentary (SED), and exercise (EX). Diabetes was induced by streptozotocin administration. High-fat diet was administered to all groups for 12 weeks. Afterwards, CO mice were euthanatized, while the sedentary and exercise groups continued high-fat diet for 6 additional weeks. Exercising mice followed an exercise program on motorizedtreadmill (5 times/week, 60 min/session). Then, blood samples and atherosclerotic plaques in the aortic root were examined. A considerable (P<0.001) regression of the atherosclerotic lesions was observed in the exercise group (180.339±75.613×10³µm²) compared to the control (325.485±72.302×10³ µm²) and sedentary (340.188±159.108×10³µm²) groups. We found decreased macrophages, matrix metalloproteinase-2 (MMP-2), MMP-3, MMP-8 and interleukin-6 (IL-6) concentrations (P<0.05) in the atherosclerotic plaques of the exercise group. Compared to both control and sedentary groups, exercise training significantly increased collagen (P<0.05), elastin (P<0.001), and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) (P<0.001) content in the atherosclerotic plaques. Those effects paralleled with increased fibrous cap thickness and less internal elastic lamina ruptures after exercise training (P<0.05), while body-weight and lipid parameters did not significantly change. Plasma MMP-2 and MMP-3 concentrations in atherosclerotic tissues followed a similar trend. From our study we can conclude that exercise training reduces and stabilizes atherosclerotic lesions in apoE^−/− mice with diabetic atherosclerosis. A favorable modification of the inflammatory regulators seems to explain those beneficial effects.Key words: diabetes, atherosclerosis, exercise, matrix metalloproteinases, plaque stability.     

Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice

LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(−/−)] mice, which have a secondary defect in cholesterol esterification. Scarab(−/−)×LCAT-null [Lcat(−/−)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(−/−)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(−/−)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(−/−)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(−/−) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles.     

Up-regulation of Cholesterol Absorption Is a Mechanism for Cholecystokinin-induced Hypercholesterolemia

Excessive absorption of intestinal cholesterol is a risk factor for atherosclerosis. This report examines the effect of cholecystokinin (CCK) on plasma cholesterol level and intestinal cholesterol absorption using the in vivo models of C57BL/6 wild-type and low density lipoprotein receptor knock-out (LDLR^−/−) mice. These data were supported by in vitro studies involving mouse primary intestinal epithelial cells and human Caco-2 cells; both express CCK receptor 1 and 2 (CCK1R and CCK2R). We found that intravenous injection of [Thr²⁸,Nle³¹]CCK increased plasma cholesterol levels and intestinal cholesterol absorption in both wild-type and LDLR^−/− mice. Treatment of mouse primary intestinal epithelial cells with [Thr²⁸,Nle³¹]CCK increased cholesterol absorption, whereas selective inhibition of CCK1R and CCK2R with antagonists attenuated CCK-induced cholesterol absorption. In Caco-2 cells, CCK enhanced CCK1R/CCK2R heterodimerization. Knockdown of both CCK1R and CCK2 or either one of them diminished CCK-induced cholesterol absorption to the same extent. CCK also increased cell surface-associated NPC1L1 (Niemann-Pick C1-like 1) transporters but did not alter their total protein expression. Inhibition or knockdown of NPC1L1 attenuated CCK-induced cholesterol absorption. CCK enhanced phosphatidylinositide 3-kinase (PI3K) and Akt phosphorylation and augmented the interaction between NPC1L1 and Rab11a (Rab-GTPase-11a), whereas knockdown of CCK receptors or inhibition of G protein βγ dimer (Gβγ) diminished CCK-induced PI3K and Akt phosphorylation. Inhibition of PI3K and Akt or knockdown of PI3K diminished CCK-induced NPC1L1-Rab11a interaction and cholesterol absorption. Knockdown of Rab11a suppressed CCK-induced NPC1L1 translocation and cholesterol absorption. These data imply that CCK enhances cholesterol absorption by activation of a pathway involving CCK1R/CCK2R, Gβγ, PI3K, Akt, Rab11a, and NPC1L.     

Hematopoietic Stem/Progenitor Cell Proliferation and Differentiation Is Differentially Regulated by High-Density and Low-Density Lipoproteins in Mice

Rationale

Hematopoietic stem/progenitor cells (HSPC) are responsible for maintaining the blood system as a result of their self-renewal and multilineage differentiation capacity. Recently, studies have suggested that HDL cholesterol may inhibit and impaired cholesterol efflux may increase HSPC proliferation and differentiation.

Objectives

We hypothesized that LDL may enhance HSPC proliferation and differentiation while HDL might have the opposing effect which might influence the size of the pool of inflammatory cells.

Methods and Results

HSPC number and function were studied in hypercholesterolemic LDL receptor knockout (LDLr^−/−) mice on high fat diet. Hypercholesterolemia was associated with increased frequency of HSPC, monocytes and granulocytes in the peripheral blood (PB). In addition, an increased proportion of BM HSPC was in G₂M of the cell cycle, and the percentage of HSPC and granulocyte-macrophage progenitors (GMP) increased in BM of LDLr^−/− mice. When BM Lin-Sca-1+cKit+ (i.e. “LSK”) cells were cultured in the presence of LDL in vitro we also found enhanced differentiation towards monocytes and granulocytes. Furthermore, LDL promoted lineage negative (Lin−) cells motility. The modulation by LDL on HSPC differentiation into granulocytes and motility was inhibited by inhibiting ERK phosphorylation. By contrast, when mice were infused with human apoA-I (the major apolipoprotein of HDL) or reconstituted HDL (rHDL), the frequency and proliferation of HSPC was reduced in BM in vivo. HDL also reversed the LDL-induced monocyte and granulocyte differentiation in vitro.

Conclusion

Our data suggest that LDL and HDL have opposing effects on HSPC proliferation and differentiation. It will be of interest to determine if breakdown of HSPC homeostasis by hypercholesterolemia contributes to inflammation and atherosclerosis progression.     

Stabilizing Role of Platelet P2Y12 Receptors in Shear-Dependent Thrombus Formation on Ruptured Plaques

Background

In most models of experimental thrombosis, healthy blood vessels are damaged. This results in the formation of a platelet thrombus that is stabilized by ADP signaling via P2Y₁₂ receptors. However, such models do not predict involvement of P2Y₁₂ in the clinically relevant situation of thrombosis upon rupture of atherosclerotic plaques. We investigated the role of P2Y₁₂ in thrombus formation on (collagen-containing) atherosclerotic plaques in vitro and in vivo, by using a novel mouse model of atherothrombosis.

Methodology

Plaques in the carotid arteries from Apoe ^−/− mice were acutely ruptured by ultrasound treatment, and the thrombotic process was monitored via intravital fluorescence microscopy. Thrombus formation in vitro was assessed in mouse and human blood perfused over collagen or plaque material under variable conditions of shear rate and coagulation. Effects of two reversible P2Y₁₂ blockers, ticagrelor (AZD6140) and cangrelor (AR-C69931MX), were investigated.

Principal Findings

Acute plaque rupture by ultrasound treatment provoked rapid formation of non-occlusive thrombi, which were smaller in size and unstable in the presence of P2Y₁₂ blockers. In vitro, when mouse or human blood was perfused over collagen or atherosclerotic plaque material, blockage or deficiency of P2Y₁₂ reduced the thrombi and increased embolization events. These P2Y₁₂ effects were present at shear rates >500 s⁻¹, and they persisted in the presence of coagulation. P2Y₁₂-dependent thrombus stabilization was accompanied by increased fibrin(ogen) binding.

Conclusions/Significance

Platelet P2Y₁₂ receptors play a crucial role in the stabilization of thrombi formed on atherosclerotic plaques. This P2Y₁₂ function is restricted to high shear flow conditions, and is preserved in the presence of coagulation.     

Control of hypercholesterolemia and atherosclerosis using the cholesterol recognition/interaction amino acid sequence of the translocator protein TSPO

The translocator protein (18-kDa) TSPO is an ubiquitous high affinity cholesterol-binding protein reported to be present in the endothelial and smooth muscle cells of the blood vessels; its expression dramatically increased in macrophages found in atherosclerotic plaques. A domain in the carboxy-terminus of TSPO was identified and characterized as the cholesterol recognition/interaction amino acid consensus (CRAC). The ability of the CRAC domain to bind to cholesterol led us to hypothesize that this peptide could be used as an hypocholesterolemic, with potential anti-atherogenic properties, agent. We report herein the therapeutic benefit that resulted for the administration of the VLNYYVWR human CRAC sequence to guinea pigs fed with a high cholesterol diet and ApoE knock-out B6.129P2-Apoetm1Unc/J mice. CRAC treatment (3 and 30 mg/kg once daily for 6 weeks) resulted in reduced circulating cholesterol levels in guinea pigs fed with 2% high cholesterol diet and ApoE knock-out B6.129P2-Apoetm1Unc/J mice. In high cholesterol fed guinea pigs, CRAC treatment administered once daily induced an increase in circulating HDL, decreased total, free and LDL cholesterol, and removed atheroma deposits in the aorta in a dose-dependent manner. The treatment also prevented the high cholesterol diet-induced increase in serum creatine kinase, total and isoforms, markers of neurological, cardiac and muscular damage. No toxicity was observed. Taken together these results support a role of TSPO in lipid homeostasis and atherosclerosis and indicate that CRAC may constitute a novel and safe treatment of hypercholesterolemia and atherosclerosis.