Local effects of human PCSK9 on the atherosclerotic lesion.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes atherosclerosis by increasing low-density lipoprotein (LDL) cholesterol levels through degradation of hepatic LDL receptor (LDLR). Studies have described the systemic effects of PCSK9 on atherosclerosis, but whether PCSK9 has local and direct effects on the plaque is unknown. To study the local effect of human PCSK9 (hPCSK9) on atherosclerotic lesion composition, independently of changes in serum cholesterol levels, we generated chimeric mice expressing hPCSK9 exclusively from macrophages, using marrow from hPCSK9 transgenic (hPCSK9tg) mice transplanted into apoE(-/-) and LDLR(-/-) mice, which were then placed on a high-fat diet (HFD) for 8 weeks. We further characterized the effect of hPCSK9 expression on the inflammatory responses in the spleen and by mouse peritoneal macrophages (MPM) in vitro. We found that MPMs from transgenic mice express both murine (m) Pcsk9 and hPCSK9 and that the latter reduces macrophage LDLR and LRP1 surface levels. We detected hPCSK9 in the serum of mice transplanted with hPCSK9tg marrow, but did not influence lipid levels or atherosclerotic lesion size. However, marrow-derived PCSK9 progressively accumulated in lesions of apoE(-/-) recipient mice, while increasing the infiltration of Ly6C(hi) inflammatory monocytes by 32% compared with controls. Expression of hPCSK9 also increased CD11b- and Ly6C(hi) -positive cell numbers in spleens of apoE(-/-) mice. In vitro, expression of hPCSK9 in LPS-stimulated macrophages increased mRNA levels of the pro-inflammatory markers Tnf and Il1b (40% and 45%, respectively) and suppressed those of the anti-inflammatory markers Il10 and Arg1 (30% and 44%, respectively). All PCSK9 effects were LDLR-dependent, as PCSK9 protein was not detected in lesions of LDLR(-/-) recipient mice and did not affect macrophage or splenocyte inflammation. In conclusion, PCSK9 directly increases atherosclerotic lesion inflammation in an LDLR-dependent but cholesterol-independent mechanism, suggesting that therapeutic PCSK9 inhibition may have vascular benefits secondary to LDL reduction.

Proprotein convertase subtilisin kexin type 9 promotes intestinal overproduction of triglyceride-rich apolipoprotein B lipoproteins through both low-density lipoprotein receptor-dependent and -independent mechanisms.

BACKGROUND: Proprotein convertase subtilisin kexin type 9 (PCSK9) promotes the degradation of the low-density lipoprotein (LDL) receptor (LDLR), and its deficiency in humans results in low plasma LDL cholesterol and protection against coronary heart disease. Recent evidence indicates that PCSK9 also modulates the metabolism of triglyceride-rich apolipoprotein B (apoB) lipoproteins, another important coronary heart disease risk factor. Here, we studied the effects of physiological levels of PCSK9 on intestinal triglyceride-rich apoB lipoprotein production and elucidated for the first time the cellular and molecular mechanisms involved. METHODS AND RESULTS: Treatment of human enterocytes (CaCo-2 cells) with recombinant human PCSK9 (10 µg/mL for 24 hours) increased cellular and secreted apoB48 and apoB100 by 40% to 55% each (P<0.01 versus untreated cells), whereas short-term deletion of PCSK9 expression reversed this effect. PCSK9 stimulation of apoB was due to a 1.5-fold increase in apoB mRNA (P<0.01) and to enhanced apoB protein stability through both LDLR-dependent and LDLR-independent mechanisms. PCSK9 decreased LDLR protein (P<0.01) and increased cellular apoB stability via activation of microsomal triglyceride transfer protein. PCSK9 also increased levels of the lipid-generating enzymes FAS, SCD, and DGAT2 (P<0.05). In mice, human PCSK9 at physiological levels increased intestinal microsomal triglyceride transfer protein levels and activity regardless of LDLR expression. CONCLUSIONS: PCSK9 markedly increases intestinal triglyceride-rich apoB production through mechanisms mediated in part by transcriptional effects on apoB, microsomal triglyceride transfer protein, and lipogenic genes and in part by posttranscriptional effects on the LDLR and microsomal triglyceride transfer protein. These findings indicate that targeted PCSK9-based therapies may also be effective in the management of postprandial hypertriglyceridemia.

Human resistin stimulates hepatic overproduction of atherogenic ApoB-containing lipoprotein particles by enhancing ApoB stability and impairing intracellular insulin signaling.

RATIONALE: Obese individuals are at high risk for developing atherosclerosis primarily attributable to elevated plasma concentrations of apolipoprotein (apo)B-containing particles, including very-low-density lipoprotein (VLDL). Plasma levels of the adipose tissue adipokine resistin are increased in human obesity, and resistin expression is positively correlated with coronary atherosclerosis and VLDL levels. OBJECTIVE: We sought to determine for the first time whether resistin directly stimulates human hepatocyte production of apoB-containing particles and to elucidate the mechanisms responsible. METHODS AND RESULTS: Treatment of human hepatocytes with resistin at levels observed in human obesity stimulated apoB secretion up to 10-fold, because of increased microsomal triglyceride transfer protein (MTP) activity and decreased expression/phosphorylation of proteins in the insulin signaling pathways (insulin receptor substrate-2, Akt, and extracellular signal-regulated kinase). Resistin also increased hepatocyte lipid content by stimulating de novo lipogenesis via the SREBP1 and SREBP2 pathways. Furthermore, obese serum with elevated resistin levels induced greater hepatocyte stimulation of apoB secretion than lean human serum, an effect that was ameliorated by antibody immunoprecipitation removal of serum resistin. CONCLUSIONS: Resistin has a direct deleterious impact on human hepatic lipid and lipoprotein regulation. Resistin greatly increased hepatocyte VLDL apoB and lipid secretion because of MTP activation and induction of hepatocyte insulin resistance. Conversely, antibody removal of serum resistin ameliorated human serum stimulation of apoB secretion. Increased hepatic cellular lipids mediated by resistin reflects the fatty liver/steatosis observed with elevated resistin in humans. Thus, human resistin is a novel therapeutic target for mitigating common hepatic pathophysiological processes associated with human obesity, dyslipidemia and atherosclerosis.

Identification of a novel human cellular HDL biosynthesis defect.

AIMS: Severe high-density lipoprotein cholesterol (HDL-C) deficiency is attributed to mutations in several genes and may contribute to the genetic basis of coronary artery disease. To identify the cellular basis of a novel HDL-deficiency phenotype, we screened 54 subjects of French Canadian ancestry with severe HDL deficiency. METHODS AND RESULTS: We excluded individuals with mutations in genes currently associated with low HDL (ABCA1, LCAT, APOA-I, and SMPD1). We identified two patients in which cellular phospholipid efflux in the HDL biosynthesis process is impaired, whereas cholesterol efflux is normal. Two-dimensional gel electrophoresis analysis further showed that the two patients with impaired phospholipid efflux were defective primarily in the larger alpha-HDL subpopulations. In fibroblasts from affected subjects, oxysterol stimulation resulted in increased ABCA1 protein expression and normalized their defective phospholipid efflux defect. CONCLUSION: Our results indicate for the first time in humans that phospholipid and cholesterol efflux are two separate and distinct processes in cellular HDL biosynthesis. They further show for the first time that normal cellular phospholipid efflux is necessary for the formation of larger alpha-HDL particles. The defect in phospholipid efflux is due to defective ABCA1 protein regulation and can be corrected by treatment with physiological oxysterols, a current therapeutic target of interest, that may, with further studies, be used to raise HDL levels in patients with severe HDL deficiencies.

Should cholesterol-lowering medications be available in Canada without a prescription?

Cardiovascular disease (CVD) presents an enormous and growing burden on the Canadian health care system. Elevated serum low-density lipoprotein cholesterol levels are an established, major risk factor in the development of premature CVD. There is strong evidence that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, significantly lower both low-density lipoprotein cholesterol levels and CVD risk. However, there is currently a treatment gap, in that a large segment of the population who should be receiving statins due to elevated serum cholesterol levels are not. Individuals at moderate risk of developing CVD represent one large population segment that is currently being undertreated. This group may be a candidate for receiving over-the-counter (OTC) or behind-the-counter (BTC) statins, which may be a suitable primary prevention strategy. Nonetheless, it must be noted that hypercholesterolemia is a complex, chronic condition that must be carefully managed and requires close consultation with a health care practitioner. The advantages and disadvantages of OTC or BTC statin usage must therefore be carefully weighed before any potential introduction of OTC or BTC statins in Canada.

Expression of SREBP-1c Requires SREBP-2-mediated Generation of a Sterol Ligand for LXR in Livers of Mice.

The synthesis of cholesterol and fatty acids (FA) in the liver is independently regulated by SREBP-2 and SREBP-1c, respectively. Here, we genetically deleted Srebf-2 from hepatocytes and confirmed that SREBP-2 regulates all genes involved in cholesterol biosynthesis, the LDL receptor, and PCSK9; a secreted protein that degrades LDL receptors in the liver. Surprisingly, we found that elimination of Srebf-2 in hepatocytes of mice also markedly reduced SREBP-1c and the expression of all genes involved in FA and triglyceride synthesis that are normally regulated by SREBP-1c. The nuclear receptor LXR is necessary for Srebf-1c transcription. The deletion of Srebf-2 and subsequent lower sterol synthesis in hepatocytes eliminated the production of an endogenous sterol ligand required for LXR activity and SREBP-1c expression. These studies demonstrate that cholesterol and FA synthesis in hepatocytes are coupled and that flux through the cholesterol biosynthetic pathway is required for the maximal SREBP-1c expression and high rates of FA synthesis.

Human PCSK9 promotes hepatic lipogenesis and atherosclerosis development via apoE- and LDLR-mediated mechanisms.

AIMS: Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of hepatic low-density lipoprotein (LDL) receptors (LDLR), thereby, decreasing hepatocyte LDL-cholesterol (LDL-C) uptake. However, it is unknown whether PCSK9 has effects on atherogenesis that are independent of lipid changes. The present study investigated the effect of human (h) PCSK9 on plasma lipids, hepatic lipogenesis, and atherosclerotic lesion size and composition in transgenic mice expressing hPCSK9 (hPCSK9tg) on wild-type (WT), LDLR⁻/⁻, or apoE⁻/⁻ background. METHODS AND RESULTS: hPCSK9 expression significantly increased plasma cholesterol (+91%), triglycerides (+18%), and apoB (+57%) levels only in WT mice. The increase in plasma lipids was a consequence of both decreased hepatic LDLR and increased hepatic lipid production, mediated transcriptionally and post-transcriptionally by PCSK9 and dependent on both LDLR and apoE. Despite the lack of changes in plasma lipids in mice expressing hPCSK9 and lacking LDLR (the main target for PCSK9) or apoE (a canonical ligand for the LDLR), hPCSK9 expression increased aortic lesion size in the absence of apoE (268 655 ± 97 972 µm² in hPCSK9tg/apoE⁻/⁻ vs. 189 423 ± 65 700 µm(2) in apoE⁻/⁻) but not in the absence of LDLR. Additionally, hPCSK9 accumulated in the atheroma and increased lesion Ly6C(hi) monocytes (by 21%) in apoE⁻/⁻ mice, but not in LDLR⁻/⁻ mice. CONCLUSIONS: PCSK9 increases hepatic lipid and lipoprotein production via apoE- and LDLR-dependent mechanisms. However, hPCSK9 also accumulate in the artery wall and directly affects atherosclerosis lesion size and composition independently of such plasma lipid and lipoprotein changes. These effects of hPCSK9 are dependent on LDLR but are independent of apoE.

Effect of atorvastatin on high-density lipoprotein apolipoprotein A-I production and clearance in the New Zealand white rabbit.

BACKGROUND: HMG-CoA reductase inhibitors reduce the incidence of cardiovascular disease predominantly by their LDL-lowering effect. Recently, there has been great interest in the pleiotropic effects of statins, which appear to differ among the various agents in this class. Unlike other statins, atorvastatin exhibits a decline in its HDL-raising effect at higher doses in humans. Whether atorvastatin-mediated alterations in HDL turnover in vivo contribute to this effect has not previously been investigated. We therefore studied the effect of atorvastatin on HDL apolipoprotein (apo) A-I production and clearance in normolipidemic male New Zealand White rabbits. METHODS AND RESULTS: Kinetic studies of HDL-apoA-I radiolabeled with 131I were performed in chow-fed rabbits after 3 weeks of atorvastatin treatment of 5 mg x kg(-1) x d(-1) (n=7) versus placebo-treated rabbits (n=7). Our results showed a significantly (P<0.001) more rapid clearance ( approximately 2-fold) of HDL apoA-I in atorvastatin-treated animals compared with the control group (0.121+/-0.012 versus 0.061+/-0.004 pools/h, respectively), accompanied by a lesser 48% increase in the apoA-I production rate (3.84+/-0.38 versus 2.59+/-0.41 mg x kg(-1) x h(-1), P=0.06). Accordingly, plasma apoA-I levels in atorvastatin-treated animals declined significantly (P<0.05, n=8 animals) after 3 weeks of treatment (173.5+/-1.8 mg/dL) from baseline values. CONCLUSIONS: These data suggest that the effect on apoA-I levels observed with atorvastatin at higher drug doses in humans may be caused at least in part by enhanced HDL apoA-I catabolism, which is not entirely offset by a concomitant increase in apoA-I production. Whether this finding results from an effect of atorvastatin on HDL particle composition or on receptors involved in circulating HDL holoparticle clearance will require further study.

Expression of human hepatic lipase in the rabbit model preferentially enhances the clearance of triglyceride-enriched versus native high-density lipoprotein apolipoprotein A-I.

BACKGROUND: We have shown previously that triglyceride (TG) enrichment of HDL, as occurs in hypertriglyceridemic states, contributes to HDL lowering in humans by enhancing the clearance of HDL apolipoprotein (apo) A-I from the circulation. In the New Zealand White rabbit, an animal naturally deficient in hepatic lipase (HL), we demonstrated that TG enrichment of HDL per se is not sufficient to enhance HDL clearance in the absence of ex vivo lipolysis by HL. Here, we examined in the rabbit the interaction between in vivo HL lipolytic action and HDL TG enrichment on the subsequent metabolic clearance of HDL apoA-I. METHODS AND RESULTS: The clearance of HDL, TG-enriched with human VLDL (12% mass TG), was compared with a simultaneously injected native rabbit HDL tracer (8% TG) 5 to 7 days after injection of recombinant (r) adenovirus expressing either the human HL or lacZ transgene (n=6 animals each). In rHL-Adv rabbits, HL activity levels were 2- to 7-fold higher (versus rlacZ-Adv controls; P<0.01), and there were significant (P<0.05) reductions in HDL TG (-18%), cholesterol (-21%), cholesteryl ester (-24%), and phospholipid (-14%). Moreover, the clearance of TG-enriched versus native HDL was significantly greater (by 50%; 0.122+/-0.022 versus 0.081+/-0.015 pools/h; P<0.01) in rHL-Adv rabbits but not in controls. CONCLUSIONS: These studies have shown that TG enrichment of HDL in the presence but not in the absence of in vivo expression of moderate levels of lipolytically active HL results in enhanced HDL clearance, demonstrating the important interaction between TG enrichment and HL action in the pathogenesis of HDL lowering in hypertriglyceridemic states.

The mechanisms of differential glucocorticoid and mineralocorticoid action in the brain and peripheral tissues.

Mineralocorticoids and glucocorticoids are key categories of adrenocorticosteroid hormones that mediate distinct physiological responses. While the primary role of aldosterone, the major mineralocorticoid, is in regulating sodium homeostasis, the major role of the glucocorticoids is mediating the catabolic response to stress. Over the past two decades, these adrenocorticosteroid hormones have been the subject of considerable attention due to the paradox that despite exerting greatly different physiological effects, they act through very closely related receptors and a common DNA response element. This review will examine the research focused on the mechanisms of selective adrenocorticosteroid action. In general, it has been demonstrated that differential adrenocorticosteroid action is mediated at pre-receptor, receptor, and post-receptor levels, depending on the target tissue and physiological environment. The marked neuroendocrine pathophysiologies resulting from perturbations in this complex system make it imperative that further research into mechanisms of coordination of the three levels of adrenocorticosteroid control be conducted.

Lipolytically modified triglyceride-enriched HDLs are rapidly cleared from the circulation.

The precise biochemical mechanisms underlying the reduction of HDL levels in hypertriglyceridemic states are currently not known. In humans, we showed that triglyceride (TG) enrichment of HDL, as occurs in hypertriglyceridemic states, enhances the clearance of HDL-associated apolipoprotein A-I (apoA-I) from the circulation. In the New Zealand White rabbit (an animal model naturally deficient in hepatic lipase [HL]), however, TG enrichment of HDL is not sufficient to alter the clearance of either the protein or lipid moieties of HDL. In the present study, therefore, we determined in the New Zealand White rabbit the combined effects of ex vivo TG enrichment and lipolytic transformation of HDL by HL on the subsequent metabolic clearance of HDL apoA-I. Results of the in vivo kinetic studies (n=18 animals) showed that apoA-I associated with TG-enriched rabbit HDL modified ex vivo by catalytically active HL was cleared 22% more rapidly versus TG-enriched HDL incubated with heat-inactivated HL, and 26% more rapidly than fasting (TG-poor) HDL incubated with active HL (P<0.05 for both). Furthermore, a strong correlation was observed between the HDL TG content and apoA-I fractional catabolic rate (0.59, P<0.05) in the combined active HL groups. These data establish that TG enrichment of HDL with subsequent lipolysis by HL enhances HDL apoA-I clearance, but neither TG enrichment of HDL without HL lipolysis nor HL lipolysis in the absence of previous TG enrichment of HDL is sufficient to enhance HDL clearance. These data further support the important interaction between HDL TG enrichment and HL action in the pathogenesis of HDL lowering in hypertriglyceridemic states.

On the function and homeostasis of PCSK9: reciprocal interaction with LDLR and additional lipid effects.

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a circulatory ligand that terminates the lifecycle of the low-density lipoprotein (LDL) receptor (LDLR) thus affecting plasma LDL-cholesterol (LDL-C) levels. Recent evidence shows that in addition to the straightforward mechanism of action, there are more complex interactions between PCSK9, LDLR and plasma lipoprotein levels, including: (a) the presence of both parallel and reciprocal regulation of surface LDLR and plasma PCSK9; (b) a correlation between PCSK9 and LDL-C levels dependent not only on the fact that PCSK9 removes hepatic LDLR, but also due to the fact that up to 40% of plasma PCSK9 is physically associated with LDL; and (c) an association between plasma PCSK9 production and the assembly and secretion of triglyceride-rich lipoproteins. The effect of PCSK9 on LDLR is being successfully utilized toward the development of anti-PCSK9 therapies to reduce plasma LDL-C levels. Current biochemical research has uncovered additional mechanisms of action and interacting partners for PCSK9, and this opens the way for a more thorough understanding of the regulation, metabolism, and effects of this interesting protein.

Elevated cholesteryl ester transfer protein (CETP) activity, a major determinant of the atherogenic dyslipidemia, and atherosclerotic cardiovascular disease in South Asians.

AIMS: Why South Asians are at increased risk of premature atherosclerotic cardiovascular diseases compared with other ethnic groups is not fully understood. Atherogenic dyslipoproteinemia - hypertriglyceridemia, elevated numbers of low-density lipoprotein (LDL) particles and low high-density lipoprotein cholesterol (HDL-C) - is more common in South Asians but the mechanisms responsible have not been explicated. Here we examined whether the circulating lipid transfer protein, cholesteryl ester transfer protein (CETP), plays a role in the pathogenesis of the atherogenic dyslipoproteinemia among South Asians. METHODS AND RESULTS: CETP activity was determined by exogenous substrate assay in the serum of healthy, metabolically well-characterized individuals of South Asian and European descent (N = 244 and 238, respectively). Serum and lipoprotein lipids and apolipoproteins were measured and lipoprotein particle number and size were quantified via nuclear magnetic resonance spectroscopy. All the elements of the atherogenic dyslipoproteinemia were more severe in South Asians and CETP activity was significantly greater by 30% in South Asians compared with Europeans, adjusted for age, sex, body mass index and waist circumference (p < 0.0001). CETP activity was directly associated with serum triglycerides and inversely with HDL-C in the whole population. CETP activity was also directly related to apoB and LDL particle number. Finally, increased CETP activity was associated with pro-atherogenic reductions in HDL and LDL particle size. CONCLUSIONS: We identified novel associations between elevated CETP activity and the triad of quantitative and qualitative lipoprotein abnormalities in the atherogenic dyslipidemia in South Asians, a major contributor of increased atherosclerotic cardiovascular diseases in South Asians.

Mechanisms of HDL lowering in insulin resistant, hypertriglyceridemic states: the combined effect of HDL triglyceride enrichment and elevated hepatic lipase activity.

Hypertriglyceridemia, low plasma concentrations of high density lipoproteins (HDL) and qualitative changes in low density lipoproteins (LDL) comprise the typical dyslipidemia of insulin resistant states and type 2 diabetes. Although isolated low plasma HDL-cholesterol (HDL-c) and apolipoprotein A-I (apo A-I, the major apolipoprotein component of HDL) can occur in the absence of hypertriglyceridemia or any other features of insulin resistance, the majority of cases in which HDL-c is low are closely linked with other clinical features of insulin resistance and hypertriglyceridemia. We and others have postulated that triglyceride enrichment of HDL particles secondary to enhanced CETP-mediated exchange of triglycerides and cholesteryl ester between HDL and triglyceride-rich lipoproteins, combined with the lipolytic action of hepatic lipase (HL), are driving forces in the reduction of plasma HDL-c and apoA-I plasma concentrations. The present review focuses on these metabolic alterations in insulin resistant states and their important contributions to the reduction of HDL-c and HDL-apoA-I plasma concentrations.

The mechanism of HDL lowering in hypertriglyceridemic, insulin-resistant states.

Hypertriglyceridemia and reduced plasma levels of high-density lipoprotein cholesterol (HDL-c) are the most frequent forms of dyslipidemia observed in insulin-resistant states, such as obesity, impaired fasting glucose, and Type 2 diabetes, and are highly atherogenic in these settings. The hypertriglyceridemia of insulin resistance is primarily due to an overproduction of very low-density lipoproteins (VLDL), and in some instances, is also due to reduced VLDL clearance and postprandial accumulation of VLDL, chylomicrons, and their remnants [i.e., triglyceride (TG)-rich lipoproteins]. TG-rich lipoproteins actively exchange their core lipids with HDL in vivo, a process that is facilitated by cholesteryl ester (CE) transfer protein (CETP), and in hypertriglyceridemic states, this process is enhanced. This results in TG enrichment of HDL in hypertriglyceridemic states. There is accumulating evidence that TG enrichment of HDL plays an important role in determining the rate at which HDL particles are cleared from the circulation. Here, we review the evidence that TG-enriched HDL, when modulated by lipolytic enzymes in the circulation, are catabolized more rapidly than native HDL, and may ultimately explain the lowering of HDL-c in insulin-resistant, hypertriglyceridemic states. Since we have recently reviewed in detail the evidence by Lamarche et al. [Clin. Chim. Acta 286 (1999) 145; J. Clin. Invest. 103 (8) (1999) 1191.] to support this hypothesis, in the present brief review, we will focus predominantly on our own recent research in this area.

Alirocumab: PCSK9 inhibitor for LDL cholesterol reduction.

The proof of concept that proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition affects cholesterol levels was first established after the demonstration that PCSK9 loss-of-function mutations result in a significant drop in circulating LDL cholesterol levels. Subsequent studies revealed that PCSK9 binds the epidermal growth factor precursor homology domain-A on the surface LDL Receptor (LDLR) and directs LDLR and PCSK9 for lysosomal degradation. Alirocumab (also known as SAR236553/REGN727) is a monoclonal antibody that binds circulating PCSK9 and blocks its interactions with surface LDLR. Alirocumab clinical trials with different doses on different administration schedules were shown to significantly reduce LDL cholesterol both as a mono-therapy and in combination with statins or ezetimibe. Although there is great potential for anti-PCSK9 therapies in the management of cholesterol metabolism, there is no clear evidence yet that blocking PCSK9 reduces cardiovascular disease outcome. This is being investigated in ongoing Phase III clinical trials with alirocumab.