Protection against atherosclerosis by estrogen is independent of plasma cholesterol levels in LDL receptor-deficient mice

Low density lipoprotein (LDL) receptor-deficient (LDLR-/-) mice consuming a high fat diet were used to assess the effect of endogenous and exogenous estradiol (E2) on atherosclerosis. Sexually mature female mice were ovariectomized (OVX) and implanted with subcutaneous, slow-release pellets designed to release 6 microg/day of exogenous 17beta-estradiol (17beta-E2 ), 17alpha-estradiol (17alpha-E2 ), or placebo (E2- deficient). Sham-operated control female (endogenous E2 ) and male mice were studied as controls. Aortic atherosclerotic lesion area was reduced by physiologic amounts of both endogenous and exogenous E2 compared to E2-deficient female mice. Although plasma cholesterol levels were reduced by exogenous E2 despite the absence of the LDL receptor, endogenous E2 was not associated with any cholesterol changes. In contrast, only 17alpha-E2 was associated with decreased fasting triglyceride. In subgroup analyses matched for time-averaged plasma total cholesterol, aortic lesion area was reduced by the presence of estradiol (E2 ). E2 protected LDLR-/- female mice from atherosclerosis and this protection was independent of changes in plasma cholesterol levels.     

Dietary cholesterol exacerbates hepatic steatosis and inflammation in obese LDL receptor-deficient mice

Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, can progress to steatohepatitis (NASH) and advanced liver disease. Mechanisms that underlie this progression remain poorly understood, partly due to lack of good animal models that resemble human NASH. We previously showed that several metabolic syndrome features that develop in LDL receptor-deficient (LDLR-/-) mice fed a diabetogenic diet are worsened by dietary cholesterol. To test whether dietary cholesterol can alter the hepatic phenotype in the metabolic syndrome, we fed LDLR-/- mice a high-fat, high-carbohydrate diabetogenic diet (DD) without or with added cholesterol (DDC). Both groups of mice developed obesity and insulin resistance. Hyperinsulinemia, dyslipidemia, hepatic triglyceride, and alanine aminotransferase (ALT) elevations were greater with DDC. Livers of DD-fed mice showed histological changes resembling NAFLD, including steatosis and modest fibrotic changes; however, DDC-fed animals developed micro- and macrovesicular steatosis, inflammatory cell foci, and fibrosis resembling human NASH. Dietary cholesterol also exacerbated hepatic macrophage infiltration, apoptosis, and oxidative stress. Thus, LDLR-/- mice fed diabetogenic diets may be useful models for studying human NASH. Dietary cholesterol appears to confer a second "hit" that results in a distinct hepatic phenotype characterized by increased inflammation and oxidative stress.     

Effects of coexpression of the LDL receptor and apoE on cholesterol metabolism and atherosclerosis in LDL receptor-deficient mice

The low density lipoprotein receptor (LDLR) plays a major role in regulation of plasma cholesterol levels as a ligand for apolipoprotein B-100 and apolipoprotein E (apoE). Consequently, LDLR-deficient mice fed a Western-type diet develop significant hypercholesterolemia and atherosclerosis. ApoE not only mediates uptake of atherogenic lipoproteins via the LDLR and other cell-surface receptors, but also directly inhibits atherosclerosis. In this study, we examined the hypothesis that coexpression of the LDLR and apoE would have greater effects than either one alone on plasma cholesterol levels and the development of atherosclerosis in LDLR-deficient mice. LDLR-deficient mice fed a Western-type diet for 10 weeks were injected with recombinant adenoviral vectors encoding the genes for human LDLR, human apoE3, both LDLR and apoE3, or lacZ (control). Plasma lipids were analyzed at several time points after vector injection. Six weeks after injection, mice were analyzed for extent of atherosclerosis by two independent methods. As expected, LDLR expression alone induced a significant reduction in plasma cholesterol due to reduced VLDL and LDL cholesterol levels, whereas overexpression of apoE alone did not reduce plasma cholesterol levels. When the LDLR and apoE were coexpressed in this model, the effects on plasma cholesterol levels were no greater than with expression of the LDLR alone. However, coexpression did result in a substantial increase in large apoE-rich HDL particles. In addition, although the combination of cholesterol reduction and apoE expression significantly reduced atherosclerosis, its effects were no greater than with expression of the LDLR or apoE alone. In summary, in this LDLR-deficient mouse model fed a Western-type diet, there was no evidence of an additive effect of expression of the LDLR and apoE on cholesterol reduction or atherosclerosis.     

Effects of emulsified policosanols with different chain lengths on cholesterol metabolism in heterozygous LDL receptor-deficient mice

Policosanol is a mixture of long-chain primary aliphatic saturated alcohols. Previous studies in humans and animals have shown that these compounds improved lipoprotein profiles. However, more-recent placebo-controlled studies could not confirm these promising effects. Octacosanol (C28), the main component of sugarcane-derived policosanol, is assumed to be the bioactive component. This has, however, never been tested in an in vivo study that compared individual policosanol components side by side. Here we present that neither the individual policosanol components (C24, C26, C28, or C30) nor the natural policosanol mixture (all 30 mg/100 g diet) lowered serum cholesterol concentrations in LDL receptor knock-out (LDLr(+/-)) mice. Moreover, there was no effect on gene expression profiles of LDLr, ABCA1, HMG-CoA synthase 1, and apolipoprotein A-I (apoA-I) in hepatic and small intestinal tissue of female LDLr(+/-) mice after the 7 week intervention period. Finally, none of the individual policosanols or their respective long-chain fatty acids or aldehydes affected de novo apoA-I protein production in vitro in HepG2 and CaCo-2 cells. Therefore, we conclude that the evaluated individual policosanols, as well as the natural policosanol mixture, have no potential for reducing coronary heart disease risk through effects on serum lipoprotein concentrations.     

Antisense oligonucleotide reduction of apoB-ameliorated atherosclerosis in LDL receptor-deficient mice

Chronic elevations of plasma apolipoprotein B (apoB) are strongly associated with cardiovascular disease. We have previously demonstrated that inhibition of hepatic apoB mRNA using antisense oligonucleotides (ASO) results in reductions of apoB, VLDL, and LDL in several preclinical animal models and humans. In this study, we evaluated the anti-atherogenic effects of a murine-specific apoB ASO (ISIS 147764) in hypercholesterolemic LDLr deficient (LDLr(-/-)) mice. ISIS 147764 was administered weekly at 25-100 mg/kg for 10-12 weeks and produced dose-dependent reductions of hepatic apoB mRNA and plasma LDL by 60-90%. No effects on these parameters were seen in mice receiving control ASOs. ApoB ASO treatment also produced dose-dependent reductions of aortic en face and sinus atherosclerosis from 50-90%, with high-dose treatment displaying less disease than the saline-treated, chow-fed LDLr(-/-) mice. No changes in intestinal cholesterol absorption were seen with apoB ASO treatment, suggesting that the cholesterol-lowering pharmacology of 147764 was primarily due to inhibition of hepatic apoB synthesis and secretion. In summary, ASO-mediated suppression of apoB mRNA expression profoundly reduced plasma lipids and atherogenesis in LDLr(-/-) mice, leading to the hypothesis that apoB inhibition in humans with impaired LDLr activity may produce similar effects.     

The bridging function of hepatic lipase clears plasma cholesterol in LDL receptor-deficient "apoB-48-only" and "apoB-100-only" mice

Hepatic lipase clears plasma cholesterol by lipolytic and nonlipolytic processing of lipoproteins. We hypothesized that the nonlipolytic processing (known as the bridging function) clears cholesterol by removing apoB-48- and apoB-100-containing lipoproteins by whole particle uptake. To test our hypotheses, we expressed catalytically inactive human HL (ciHL) in LDL receptor deficient "apoB-48-only" and "apoB-100-only" mice. Expression of ciHL in "apoB-48-only" mice reduced cholesterol by reducing LDL-C (by 54%, 46 +/- 6 vs. 19 +/- 8 mg/dl, P < 0.001). ApoB-48 was similarly reduced (by 60%). The similar reductions in LDL-C and apoB-48 indicate cholesterol removal by whole particle uptake. Expression of ciHL in "apoB-100-only" mice reduced cholesterol by reducing IDL-C (by 37%, 61 +/- 19 vs. 38 +/- 12 mg/dl, P < 0.003). Apo-B100 was also reduced (by 27%). The contribution of nutritional influences was examined with a high-fat diet challenge in the "apoB-100-only" background. On the high fat diet, ciHL reduced IDL-C (by 30%, 355 +/- 72 vs. 257 +/- 64 mg/dl, P < 0.04) but did not reduce apoB-100. The reduction in IDL-C in excess of apoB-100 suggests removal either by selective cholesteryl ester uptake, or by selective removal of larger, cholesteryl ester-enriched particles. Our results demonstrate that the bridging function removes apoB-48- and apoB-100-containing lipoproteins by whole particle uptake and other mechanisms.     

Effects of probucol on atherosclerosis of apoE-deficient or LDL receptor-deficient mice.

The effect of probucol on atheroma formation was evaluated using mouse models for atherosclerosis with different diet protocols. Dietary administration of probucol (0.5 %, wt/wt) for 12 weeks reduced total plasma cholesterol levels in both apolipoprotein E (apoE)-deficient mice fed a western diet and in low-density lipoprotein receptor (LDLR)-deficient mice fed a Paigen diet by 60 % and 30 % to 60 %, respectively. Probucol treatment also significantly reduced high-density lipoprotein (HDL) levels in apoE-deficient mice, but not in LDLR-deficient mice. Atherosclerotic plaques in the aortic sinus of probucol-treated apoE-deficient mice were two-fold larger than those in untreated apoE-deficient mice, while the lesions in probucol-treated LDLR-deficient mice were similar to those in untreated LDLR-deficient mice. A strong negative correlation between HDL cholesterol levels and lesion sizes at the aortic sinus was observed in apoE-deficient mice, but not in LDLR-deficient mice. Thus, in contrast to LDLR-deficient mice, probucol had a strong proatherogenic effect in the aortic sinus of apoE-deficient mice associated with the reduction of HDL levels in spite of the reduction of total plasma cholesterol levels. The varying effects of probucol on atherogenesis depend upon the portion of aorta and which animal model is evaluated, implicating that complex cellular events are involved in the effect of probucol.     

Dual benefit of reduced Cx43 on atherosclerosis in LDL receptor-deficient mice

Paracrine cell-to-cell interactions are crucial events during atherogenesis, however, little is known on the role of gap junctional communication during this process. We recently demonstrated increased expression of Cx43 in intimal smooth muscle cells and in a subset of endothelial cells covering the shoulder of atherosclerotic plaques. The purpose of this study was to examine the role of Cx43 in the development of atherosclerosis in vivo. Atherosclerosis-susceptible LDL receptor-deficient (LDLR(-/-)) mice were intercrossed with mice heterozygous for Cx43 (Cx43(+/-) mice). Male mice with normal (Cx43(+/+)LDLR(-/-)) or reduced (Cx43(+/-)LDLR(-/-)) Cx43 level of 10 weeks old were fed a cholesterol-rich diet (1.25%) for 14 weeks. Both groups of mice showed similar increases in serum lipids and body weight. Interestingly, the progression of atherosclerosis was reduced by 50% (P < 0.01) in the thoraco-abdominal aorta and in the aortic roots of Cx43(+/-)LDLR(-/-) mice compared with Cx43(+/+)LDLR(-/-) littermate controls. In addition, atheroma in Cx43(+/-)LDLR(-/-) mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells, important features associated, in human, with stable atherosclerotic lesions. Thus, reducing Cx43 expression in mice provides beneficial effects on both the progression and composition of the atherosclerotic lesions.     

Hepatic lipase overexpression lowers remnant and LDL levels by a noncatalytic mechanism in LDL receptor-deficient mice

To address the role of the noncatalytic ligand function of hepatic lipase (HL) in low density lipoprotein (LDL) receptor-mediated lipoprotein metabolism, we characterized transgenic mice lacking the LDL receptor (LDLR) that express either catalytically active (Ldlr(-/-)HL) or inactive (Ldlr(-/-)HL(S145G)) human HL on both chow and high fat diets and compared them with nontransgenic Ldlr(-/-) mice. In mice fed a chow diet, apolipoprotein (apo)B-containing lipoprotein levels were 40-60% lower in Ldlr(-/-)HL and Ldlr(-/-)HL(S145G) mice than in Ldlr(-/-) mice. This decrease was mainly reflected by decreased apoB-48 levels in the Ldlr(-/-)HL mice and by decreased apoB-100 levels in Ldlr(-/-) HL(S145G) mice. These findings indicate that HL can reduce apoB-100-containing lipoproteins through a noncatalytic ligand activity that is independent of the LDLR. Cholesterol enrichment of the apoB-containing lipoproteins induced by feeding Ldlr(-/-)HL and Ldlr(-/-)HL(S145G) mice a cholesterol-enriched high fat (Western) diet resulted in parallel decreases in both apoB-100 and apoB-48 levels, indicating that HL is particularly efficient at reducing cholesterol-enriched apoB-containing lipoproteins through both catalytic and noncatalytic mechanisms. These data suggest that the noncatalytic function of HL provides an alternate clearance pathway for apoB-100- and apoB-48-containing lipoproteins that is independent of the LDLR and that contributes to the clearance of high density lipoproteins.     

Hyperlipidemia is a major determinant of neointimal formation in LDL receptor-deficient mice

LDL receptor-deficient (LDLR(-/-)) mice exhibit mild hyperlipidemia on a chow diet but develop severe hyperlipidemia on a high fat diet. In this study, we investigated neointimal formation after removal of the endothelium when LDLR(-/-) mice were fed chow or a Western diet containing 42% fat, 0.15% cholesterol, and 19.5% casein. At 10 weeks of age, female mice underwent endothelial denudation of the left common carotid artery. Two weeks after injury, neointimal formation was barely detectable in the injured vessel when mice developed mild hyperlipidemia on the chow diet. In contrast, neointimal lesions were obvious when mice developed severe hyperlipidemia on the Western diet. Immunohistochemical and histological analyses demonstrated the presence of macrophage foam cells and smooth muscle cells in neointimal lesions. The injured artery also exhibited a significant increase in medial area on the Western diet. Plasma levels of MCP-1 and soluble VCAM-1 were significantly elevated by feeding of the Western diet. These data indicate that hyperlipidemia aggravates neointimal growth in LDLR(-/-) mice by promoting foam cell formation and inflammation.     

Low density lipoprotein (LDL)-mediated suppression of Lewis lung carcinoma in hypercholesterolemic LDL receptor-deficient mice

An inverse relationship has been reported between cancer risk and cholesterol level, prompting the hypothesis that hypercholesterolemia may be protective against cancer. We tested this hypothesis by evaluating the growth of Lewis lung carcinoma in three different murine models of hypercholesterolemia: Pluronic treated mice, apolipoprotein E (ApoE) deficient mice, and low density lipoprotein receptor (LDL-R) deficient mice. Only the accumulation of LDL-cholesterol in LDL-R deficient mice suppressed tumor growth. Accumulation of chylomicrons, very low density lipoproteins (VLDL), and cholesterol-enriched remnants in the Pluronic treated mice and ApoE deficient mice did not inhibit tumor growth, even though mice in all three models were equally hypercholesterolemic. Taken together, the experimental evidence from our studies indicate that high plasma cholesterol in the form of LDL-cholesterol could have a beneficial effect against cancer in vivo.     

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.     

Transgenic expression of CYP7A1 in LDL receptor-deficient mice blocks diet-induced hypercholesterolemia

Constitutive expression of a cholesterol-7alpha-hydroxylase (CYP7A1) transgene in LDL receptor-deficient mice blocked the ability of a cholesterol-enriched diet to increase plasma levels of apolipoprotein B-containing lipoproteins. LDL receptor-deficient mice expressing the CYP7A1 transgene exhibited complete resistance to diet-induced hypercholesterolemia and to the accumulation of cholesterol in the liver. Hepatic mRNA expression of liver X receptor-inducible ABCG5 and ABCG8 was decreased in CYP7A1 transgenic, LDL receptor-deficient mice fed a cholesterol-enriched diet. Thus, increased biliary cholesterol excretion could not account for the maintenance of cholesterol homeostasis. CYP7A1 transgenic, LDL receptor-deficient mice fed the cholesterol-enriched diet exhibited decreased jejunal Niemann-Pick C1-Like 1 protein (NPC1L1) mRNA expression, an important mediator of intestinal cholesterol absorption. A taurocholate-enriched diet also decreased NPC1L1 mRNA expression in a farnesoid X receptor-independent manner. Reduced expression of NPC1L1 mRNA was associated with decreased cholesterol absorption ( approximately 20%; P < 0.05) exhibited by CYP7A1 transgenic LDL receptor-deficient mice fed the cholesterol-enriched diet. The combined data show that enhanced expression of CYP7A1 is an effective means to prevent the accumulation of cholesterol in the liver and of atherogenic apolipoprotein B-containing lipoproteins in plasma.     

PANDORA-Seq unveils the hidden small noncoding RNA landscape in atherosclerosis of LDL receptor-deficient mice

Small noncoding RNAs (sncRNAs) play diverse roles in numerous biological processes. While the widely used RNA sequencing (RNA-Seq) method has advanced sncRNA discovery, RNA modifications can interfere with the complementary DNA library construction process, preventing the discovery of highly modified sncRNAs including transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs) that may have important functions in disease development. To address this technical obstacle, we recently developed a novel PANDORA-Seq (Panoramic RNA Display by Overcoming RNA Modification Aborted Sequencing) method to overcome RNA modification-elicited sequence interferences. To identify novel sncRNAs associated with atherosclerosis development, LDL receptor-deficient (LDLR^−/−) mice were fed a low-cholesterol diet or high-cholesterol diet (HCD) for 9 weeks. Total RNAs isolated from the intima were subjected to PANDORA-Seq and traditional RNA-Seq. By overcoming RNA modification-elicited limitations, PANDORA-Seq unveiled an rsRNA/tsRNA-enriched sncRNA landscape in the atherosclerotic intima of LDLR^−/− mice, which was strikingly different from that detected by traditional RNA-Seq. While microRNAs were the dominant sncRNAs detected by traditional RNA-Seq, PANDORA-Seq substantially increased the reads of rsRNAs and tsRNAs. PANDORA-Seq also detected 1,383 differentially expressed sncRNAs induced by HCD feeding, including 1,160 rsRNAs and 195 tsRNAs. One of HCD-induced intimal tsRNAs, tsRNA-Arg-CCG, may contribute to atherosclerosis development by regulating the proatherogenic gene expression in endothelial cells. Overall, PANDORA-Seq revealed a hidden rsRNA and tsRNA population associated with atherosclerosis development. These understudied tsRNAs and rsRNAs, which are much more abundant than microRNAs in the atherosclerotic intima of LDLR^−/− mice, warrant further investigations.     

Lipolytic and ligand-binding functions of hepatic lipase protect against atherosclerosis in LDL receptor-deficient mice

To elucidate the separate contributions of the lipolytic versus ligand-binding functions of hepatic lipase (HL) to lipoprotein metabolism and atherosclerosis, and to investigate the role of the low density lipoprotein receptor (LDLr) in these processes, we compared mice expressing catalytically active HL (HL-WT) with mice expressing inactive HL (HL-S145G) in a background lacking endogenous HL and the LDLr (LDLr-KOxHL-KO). HL-WT and HL-S145G reduced (P < 0.05 for all) cholesterol (55% vs. 20%), non-HDL-cholesterol (63% vs. 22%), and apolipoprotein B (apoB; 34% vs. 16%) by enhancing the catabolism of autologous (125)I-apoB-intermediate density lipoprotein (IDL)/LDL (fractional catabolic rate in day(-1): 6.07 +/- 0.25, LDLr-KOxHL-WT; 4.76 +/- 0.30, LDLr-KOxHL-S145G; 3.70 +/- 0.13, LDLr-KOxHL-KO); HL-WT had a greater impact on the concentration, composition, particle size, and catabolism of apoB-containing lipoproteins (apoB-Lps) and HDL. Importantly, consistent with the changes in apoB-Lps, atherosclerosis in LDLr-KOxHL-KO mice fed a regular chow diet (RCD) was reduced by both HL-WT and HL-S145G (by 71% and 51% in cross-sectional analysis, and by 85% and 67% in en face analysis; P < 0.05 for all). These data identify physiologically relevant but distinct roles for the lipolytic versus ligand-binding functions of HL in apoB-Lp metabolism and atherosclerosis and demonstrate that their differential effects on these processes are mediated by changes in catabolism via non-LDLr pathways. These changes, evident even in the presence of apoE, establish an antiatherogenic role of the ligand-binding function of HL in LDLr-deficient mice.     

Anacetrapib reduces (V)LDL cholesterol by inhibition of CETP activity and reduction of plasma PCSK9

Recently, we showed in APOE*3-Leiden cholesteryl ester transfer protein (E3L.CETP) mice that anacetrapib attenuated atherosclerosis development by reducing (V)LDL cholesterol [(V)LDL-C] rather than by raising HDL cholesterol. Here, we investigated the mechanism by which anacetrapib reduces (V)LDL-C and whether this effect was dependent on the inhibition of CETP. E3L.CETP mice were fed a Western-type diet alone or supplemented with anacetrapib (30 mg/kg body weight per day). Microarray analyses of livers revealed downregulation of the cholesterol biosynthesis pathway (P < 0.001) and predicted downregulation of pathways controlled by sterol regulatory element-binding proteins 1 and 2 (z-scores −2.56 and −2.90, respectively; both P < 0.001). These data suggest increased supply of cholesterol to the liver. We found that hepatic proprotein convertase subtilisin/kexin type 9 (Pcsk9) expression was decreased (−28%, P < 0.01), accompanied by decreased plasma PCSK9 levels (−47%, P < 0.001) and increased hepatic LDL receptor (LDLr) content (+64%, P < 0.01). Consistent with this, anacetrapib increased the clearance and hepatic uptake (+25%, P < 0.001) of [¹⁴C]cholesteryl oleate-labeled VLDL-mimicking particles. In E3L mice that do not express CETP, anacetrapib still decreased (V)LDL-C and plasma PCSK9 levels, indicating that these effects were independent of CETP inhibition. We conclude that anacetrapib reduces (V)LDL-C by two mechanisms: 1) inhibition of CETP activity, resulting in remodeled VLDL particles that are more susceptible to hepatic uptake; and 2) a CETP-independent reduction of plasma PCSK9 levels that has the potential to increase LDLr-mediated hepatic remnant clearance.     

Plasma PCSK9 preferentially reduces liver LDL receptors in mice

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that regulates the expression of LDL receptor (LDLR) protein. Gain-of-function mutations in PCSK9 cause hypercholesterolemia, and loss-of-function mutations result in lower plasma LDL-cholesterol. Here, we investigate the kinetics and metabolism of circulating PCSK9 relative to tissue levels of LDLRs. The administration of recombinant human PCSK9 (32 microg) to mice by a single injection reduced hepatic LDLRs by approximately 90% within 60 min, and the receptor levels returned to normal within 6 h. The half-life of the PCSK9 was estimated to be approximately 5 min. Continuous infusion of PCSK9 (32 microg/h) into wild-type mice caused a approximately 90% reduction in hepatic LDLRs within 2 h and no associated change in the level of LDLR in the adrenals. Parallel studies were performed using a catalytically inactive form of PCSK9, PCSK9(S386A), and similar results were obtained. Infusion of PCSK9(D374Y), a gain-of-function mutation, resulted in accelerated clearance of the mutant PCSK9 and a greater reduction in hepatic LDLRs. Combined, these data suggest that exogenously administrated PCSK9 in plasma preferentially reduces LDLR protein levels in liver at concentrations found in human plasma and that PCSK9's action on the LDLR is not dependent on catalytic activity in vivo.     

Common and rare gene variants affecting plasma LDL cholesterol

The plasma level of LDL cholesterol is clinically important and genetically complex. LDL cholesterol levels are in large part determined by the activity of LDL receptors (LDLR) in the liver. Autosomal dominant familial hypercholesterolaemia (FH) - with its high LDL cholesterol levels, xanthomas, and premature atherosclerosis - is caused by mutations in either the LDLR or in APOB - the protein in LDL recognised by the LDLR. A third, rare form - autosomal recessive hypercholesterolaemia - arises from mutations in the gene encoding an adaptor protein involved in the internalisation of the LDLR. A fourth variant of inherited hypercholesterolaemia was recently found to be associated with missense mutations in PCSK9, which encodes a serine protease that degrades LDLR. Whereas the gain-of-function mutations in PCSK9 are rare, a spectrum of more frequent loss-of-function mutations in PCSK9 associated with low LDL cholesterol levels has been identified in selected populations and could protect against coronary heart disease. Heterozygous familial hypobetalipoproteinaemia (FHBL) - with its low LDL cholesterol levels and resistance to atherosclerosis - is caused by mutations in APOB. In contrast to other inherited forms of severe hypocholesterolaemia such as abetalipoproteinaemia - caused by mutations in MTP - and homozygous FHBL, a deficiency of PCSK9 appears to be benign. Rare variants of NPC1L1, the gene encoding the putative intestinal cholesterol receptor, have shown more modest effects on plasma LDL cholesterol than PCSK9 variants, similar in magnitude to the effect of common APOE variants. Taken together, these findings indicate that heritable variation in plasma LDL cholesterol is conferred by sequence variation in various loci, with a small number of common and multiple rare gene variants contributing to the phenotype.     

Cathepsin G activity lowers plasma LDL and reduces atherosclerosis

Cathepsin G (CatG), a serine protease present in mast cells and neutrophils, can produce angiotensin-II (Ang-II) and degrade elastin. Here we demonstrate increased CatG expression in smooth muscle cells (SMCs), endothelial cells (ECs), macrophages, and T cells from human atherosclerotic lesions. In low-density lipoprotein (LDL) receptor-deficient (Ldlr^–/–) mice, the absence of CatG reduces arterial wall elastin degradation and attenuates early atherosclerosis when mice consume a Western diet for 3 months. When mice consume this diet for 6 months, however, CatG deficiency exacerbates atherosclerosis in aortic arch without affecting lesion inflammatory cell content or extracellular matrix accumulation, but raises plasma total cholesterol and LDL levels without affecting high-density lipoprotein (HDL) or triglyceride levels. Patients with atherosclerosis also have significantly reduced plasma CatG levels that correlate inversely with total cholesterol (r = –0.535, P < 0.0001) and LDL cholesterol (r = –0.559, P < 0.0001), but not with HDL cholesterol (P = 0.901) or triglycerides (P = 0.186). Such inverse correlations with total cholesterol (r = –0.504, P < 0.0001) and LDL cholesterol (r = –0.502, P < 0.0001) remain significant after adjusting for lipid lowering treatments among this patient population. Human CatG degrades purified human LDL, but not HDL. This study suggests that CatG promotes early atherogenesis through its elastinolytic activity, but suppresses late progression of atherosclerosis by degrading LDL without affecting HDL or triglycerides.