Clinica Chimica Acta 431 (2014) 148–153

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Invited critical review

PCSK9 gene mutations and low-density lipoprotein cholesterol Na-Qiong Wu, Jian-Jun Li ⁎ Division of Dyslipidemia, State Key Laboratory of Cardiovascular Disease, Fu Wai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences, Peking Union Medical College, BeiLiShi Road 167, Beijing 100037, China

a r t i c l e

i n f o

Article history: Received 27 June 2013 Received in revised form 26 January 2014 Accepted 27 January 2014 Available online 8 February 2014 Keywords: Proprotein convertase subtilisin-like/ kexin type 9 Genetic variant Dyslipidemia Coronary heart disease

a b s t r a c t Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a newly-identified circulating protein in cholesterol metabolism in mammals, including humans, which has emerged as a new pharmacological target for hypocholesterolemia. It has been demonstrated that PCSK9 gene mutations are associated with hyperor hypocholesterolemia. In the latter case, the incidence of coronary heart disease (CHD) is markedly reduced, suggesting that low level of low-density lipoprotein cholesterol (LDL-C) at birth is highly beneficial. Loss-offunction PCSK9 mutations will result in lower LDL-C levels and protect against CHD. Conversely, patients harboring gain-of-function PCSK9 mutations will suffer from familial autosomal dominant hypercholesterolemia (ADH), a disease characterized by elevated LDL-C plasma concentration. Although compelling evidence has suggested that PCSK9 can impair the LDL receptor (LDLR) pathway, its biological role in cholesterol metabolism remains to be defined. According to data from previous studies, PCSK9 appears to be a promising therapeutic target due to its role as a major LDLR regulator. Specific pharmacological inhibitors of PCSK9 have demonstrated a significant impact on plasma LDL-C concentrations. Therefore, understanding the relationship between PCSK9 and its genetic variants, on one hand, and the level of plasma LDL-C, on the other hand, may be clinically useful due to the fact that this protein has become a key target of lipid-lowering therapy. In this manuscript we mainly review recent data with regard to the association between PCSK9 genetic variants and plasma LDL-C concentrations, and outline the clinical implications. © 2014 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Categories of PCSK9 genetic variants . . . . . . . . . . . . . . . . . . . 2.1. Loss-of-function mutations in the PCSK9 gene . . . . . . . . . . . . 2.2. PCSK9 gain-of-function mutations and polymorphisms . . . . . . . 2.3. Impact of PCSK9 genetic variants on CVD associated with LDL-C . . . 2.3.1. Protective effect of loss-of-function PCSK9 mutations on CVD 2.3.2. Causal effects of gain-of-function PCSK9 mutations on CVD . 3. Clinical prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⁎ Corresponding author. Tel.: +86 10 88396077; fax: +86 10 68331730. E-mail address: [email protected] (J.-J. Li). 0009-8981/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2014.01.043

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1. Introduction Cardiovascular diseases are the major cause of mortality in the Western world, and this situation is expected to persist for the foreseeable future [1]. Among cardiovascular diseases, coronary artery disease (CAD) is the most important underlying cause of cardiovascular mortality. Currently, treatments for patients with CAD mainly include risk factor management, drug therapy, and revascularization techniques [2]. It has been well recognized that the plasma concentration of lowdensity lipoprotein (LDL) cholesterol (LDL-C) is the main causal risk factor for atherosclerotic cardiovascular disease [1,2]. Lowering LDL-C levels is, therefore, one of the primary goals of strategies to either prevent or treat atherosclerotic cardiovascular diseases (ASCVD) [1]. Clinically, lipid-lowering therapy mainly relies, at this time, on treatment with 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors (statins). More recently, the development of new lipidlowering drugs is of great interest due to the target-achieved limitation of statins alone or even when combined with other currently available lipid-lowering drugs [2]. A promising new therapeutic target for further lowering LDL-C levels is proprotein convertase subtilisin-like/kexin type 9 (PCSK9), a protein strongly involved in LDL-C metabolism [3–5]. It has been established that PCSK9 plays a key role in regulating plasma LDL-C levels. PCSK9 seems to interfere with recycling the LDLreceptor (LDLR) by targeting the receptor to the lysosome for degradation, leading to reduced LDL-C clearance from the circulation [6–8]. In fact, the 2003 discovery of the first mutation in the PCSK9 gene, causing autosomal dominant hypercholesterolemia (ADH), shed light on an unknown factor in cholesterol metabolism that has since then been extensively investigated [9,10]. Until now, several different PCSK9 genetic variants have been identified in humans. Patients with gain-of-function mutations or PCSK9 variants will develop familiar hypercholesterolemia accompanied by increased LDL-C levels and cardiovascular risk [3]. In contrast, individuals with loss-of-function mutations or PCSK9 variants present reduced LDL-C levels and a decreased CAD risk [4]. In this review, we mainly discuss recent data with regard to the association of PCSK9 genetic variants with plasma LDL-C concentrations. Additionally, the spectrum of hyperor hypocholesterolemia phenotypes that are already known to be associated with PCSK9 mutations is also discussed in this review.

2. Categories of PCSK9 genetic variants The gene encoding PCSK9 is highly polymorphic. The first example of ADH induced by a PCSK9 mutation was reported by Abifadel et al. in 2003 [10]. The first two PCSK9 mutations that were simultaneously detected in humans are p.S127R and p.F216R. The first one, p.S127R, occurs in exon 2, which is a highly conserved region across species, while the second one, p.F216L, is in exon 4 and was identified in a French family in which a man with 441 mg/dl total cholesterol and 356 mg/dl LDL-C died from myocardial infarction (MI) at age 49. From then, the role of PCSK9 mutations was subsequently established in vitro and in vivo. According to previously published data, PCSK9 gene mutations could be divided into two categories with respect to the functional outcome, loss-of-function and gain-of-function mutations. Studies from cell transfection and animal model experiments demonstrated that overexpression of wild-type or mutant PCSK9 was associated with hypercholesterolemia, resulting in a marked reduction of hepatic LDLR protein and hypercholesterolemia [11–13]. In another way, missense mutations in the PCSK9 gene, associated with hypercholesterolemia in humans, would increase the capacity of PCSK9 and limit the number of LDLR molecules, which may represent the gain-of-function mutations in the PCSK9 gene. In contrast, mice with targeted inactivation of the PCSK9 gene (pcsk9−/−) show an increased number of LDLR molecules in the liver, leading to increased removal of plasma LDL and reduced plasma LDL-C levels [14].

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Accordingly, two categories of PCSK9 sequence variants produce mild to moderate (and opposing) phenotypes [15]. The loss-offunction PCSK9 sequence variants decrease LDLR degradation, and thereby reduce LDL-C concentration [16,17]. The gain-of-function PCSK9 sequence variants cause a reduction in the LDLR and lead to hypercholesterolemia [18] or ADH in the case of severe phenotypic variants [10,19]. 2.1. Loss-of-function mutations in the PCSK9 gene Loss-of-function mutations or polymorphisms in the PCSK9 gene are relatively common, and mainly present as decreased plasma LDL-C concentrations, lower circulating PCSK9 levels, and a higher density of liver LDLR. The evidence for loss-of-function PCSK9 sequence variants was gradually established both in vitro and in vivo from experimental observations and human studies. As early as 2005, Cohen et al. searched for mutations in subjects with low plasma levels of LDL-C (LDL-C b 58 mg/dl) [16]. The investigators found that two inactivating mutations in the PCSK9 gene (p.Y142X and p.C679X) were associated with a 30% to 40% reduction in plasma LDL-C levels in 2% to 2.6% of the black participants from the Dallas Heart Study population. Two nonsense mutations in the PCSK9 gene were reported in their study: p.Y142X, located in exon 3, deletes the last four-fifths of the protein, and p.C679X, located in exon 12, is predicted to truncate the protein by 14 amino acids. Subsequently, the p.Y142X mutation in the PCSK9 gene was found in 0.4% of the African Americans but not in any other ethnic groups. The p.C679X mutation in the PCSK9 gene was found in 1.4% of the African Americans, but very rarely in European Americans (b0.1%). Subjects with PCSK9 nonsense mutations had significantly lower plasma levels of total-cholesterol and LDL-C (about −28%), but not all of them were hypocholesterolemic. The authors proposed the concept that in humans, loss-of-function mutations in the PCSK9 gene would increase the number of LDLR molecules in the liver, and the receptor could mediate plasma LDL uptake and catabolism, as observed in pcsk9−/− mice. These loss-of-function mutations in the PCSK9 gene confer substantial protection against CAD [20]. The p.C679X mutation in the PCSK9 gene was also found in 3.7% of African women from Zimbabwe, and was reported to be associated with a 27% reduction in LDL-C [21]. Other mutations causing non-conservative amino acid substitutions in PCSK9 were found to be associated with a significant, although less pronounced, reduction of plasma LDL-C and with an increased response to statin therapy [17,20,22,23]. The p.R46L mutation belongs to the loss-of-function mutations in the PSCK9 gene, and has attracted much attention from researchers in the field. Published data have suggested that the cholesterol-lowering effect of the R46L PCSK9 mutation in familial hypercholesterolemia (FH) heterozygotes is less pronounced than in normocholesterolemic subjects. The R46L polymorphism of the PCSK9 gene has been associated with reductions in LDL-C of 0.28 to 0.54 mmol/L (9% to 15%) [20,24–27] and with a 47% reduction in the risk of IHD in carriers versus noncarriers [26]. Recently, a study screened for the PCSK9 gene R46L mutation in 1130 unrelated subjects with molecularly defined FH [28]. The authors also used cell culture experiments to evaluate the effect of high LDL concentrations on the PCSK9 binding to the LDLR [28]. They found that 2.7% of the subjects were carriers of the R46L mutation and had a nonsignificant 6% lower total serum cholesterol than non-carriers; this reduction was lower than the 8–9% reduction in total serum cholesterol that was previously observed in normo-cholesterolemic subjects. The results from cell culture experiments showed that the increase in LDL concentrations of medium decreased the amount of internalized PCSK9 and also decreased the PCSK9-mediated degradation of the LDLR. High LDL levels, as seen in untreated FH heterozygotes, completely prevented the binding of wild-type PCSK9 to the LDLR. Thus, in the presence of high LDL levels, wild-type PCSK9, which has twice the

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LDLR binding affinity of R46L PCSK9, may not be significantly more potent in degrading the LDLR than R46L PCSK9. These data may suggest that targeting PCSK9 as monotherapy in FH heterozygotes may not prove to be very effective. In 2010, Benn M et al. reported the association between the R46L genotype on one hand and LDL-C, the risk of IHD, MI, and mortality on the other hand, in the prospective Copenhagen City Heart Study (CCHS, n = 10,032) and validated the results in: 1) the cross-sectional CGPS (Copenhagen General Population Study, n = 26,013); and 2) the case–control CIHDS (Copenhagen Ischemic Heart Disease Study, n = 9654). A meta-analysis of the present and previous studies (n = 66,698) was also performed [29]. To date, this is a largest metaanalysis examining the association between R46L-PCSK9 and the level of plasma LDL-C, risk of IHD, MI, and mortality. The authors found that in carriers (2.6%), as compared to non-carriers, the 46L allele was associated with reductions in LDL-C of 0.35 to 0.55 mmol/l (11% to 16%) from 20 to 80 + years in the general population (CCHS and CGPS). Moreover, in 2008, Cameron J et al. [30] identified five novel PCSK9 mutations by sequencing DNA from subjects from Norway with hypoor hypercholesterolemia. The effect of the mutations that were identified on the autocatalytic cleavage and secretion of PCSK9, as well as the effect on the PCSK9-mediated degradation of the LDLR, were determined in HepG2 or HEK293 cells transiently transfected with mutant PCSK9-containing plasmids. Mutations G236S and N354I were loss-of-function mutations due to the failure of the protein to exit the endoplasmic reticulum and undergo autocatalytic cleavage, respectively. The findings were correlated with the clinical characteristics of the subjects possessing these mutations, and the phenotypic effects were analyzed in terms of the available structural data for PCSK9. Interestingly, in 2011, Mayne J et al. studied another novel PCSK9 loss-of-function sequence variant in a white French-Canadian family [15]. A PCSK9 sequence variant that produced the PCSK9 Q152H substitution was identified. Family members carrying this variant had mean decreases in circulating PCSK9 and LDL-C concentrations of 79% and 48%, respectively, when compared with unrelated non-carriers (n = 210). In cell culture, the pro-PCSK9-Q152H variant did not undergo efficient autocatalytic cleavage and was not secreted. Cells transiently transfected with PCSK9-Q152H cDNA had LDLR concentrations that were significantly higher than those in cells overproducing wild-type PCSK9. Co-transfection of PCSK9-Q152H and PCSK9-WT cDNAs produced a 78% decrease in the secreted PCSK9-WT protein compared to control cells. Collectively, the results demonstrate that the PCSK9Q152H variant markedly lowers plasma PCSK9 and LDL-C concentrations in heterozygous carriers via decreased autocatalytic processing and secretion and, hence, LDLR inactivity. 2.2. PCSK9 gain-of-function mutations and polymorphisms The first two PCSK9 mutations discovered by Abifadel et al. were both gain-of-function mutations, and they were associated with hypercholesterolemia [10]. Among five novel PCSK9 mutations identified by Cameron J et al. in Norway in 2008 [30], there was a gain-of-function mutation, R215H, which was causing hypercholesterolemia. By comparing the number of patients with gain-of-function PCSK9 mutations with the number of FH heterozygotes among subjects with hypercholesterolemia, the prevalence of subjects with gain-of-function PCSK9 mutations in Norway can be estimated to be one in 15,000. In 2010, Noguchi T et al. [31] attempted to find gain-of-function PCSK9 mutations in Japanese subjects, and determined the frequency and impacts of these mutations, especially on circulating PCSK9 and LDL-C levels. The authors detected seven PCSK9 variants, including E32K. The frequency of PCSK9 E32K in clinical FH (6.42%) was significantly higher than in controls (1.71%). Three cases representing homozygous FH phenotypes were double heterozygotes for PCSK9 E32K and LDLR C183S, C292X, or K790X. Two cases were true homozygotes for

PCSK9 E32K. The PCSK9 E32K mutant was associated with over 30% increased levels of PCSK9 in plasma from the subjects and in media from transiently transfected HepG2 cells as compared to controls. Furthermore, LDL-C levels in the PCSK9 E32K true homozygotes and heterozygotes were 2.10-fold and 1.47-fold higher than those in controls with comparable circulating PCSK9 levels, respectively, suggesting enhanced PCSK9 E32K function. Two true homozygotes for PCSK9 E32K and 3 double heterozygotes for PCSK9 E32K were found and LDL-R mutations were linked to autosomal dominant hypercholesterolemia. These findings have demonstrated that PCSK9 E32K significantly affects LDL-C levels via increased mass and function of PCSK9, and could exacerbate the clinical phenotypes of patients carrying LDLR mutations. In a Chinese study that enrolled a total of 649 Guangxi Bai Ku Yao subjects (White Trousers Yao, one of the minor Chinese ethnic groups) and 646 Han participants, Aung LH et al. [32] found a significant difference in the genotypic and allelic frequencies of PCSK9 E670G between the Bai Ku Yao and the Han; between normal LDL-C and high LDL-C subgroups in the Bai Ku Yao; and between normal HDL-C and low HDL-C, between normal ApoAI and low ApoAI, or between normal ApoAI/ ApoB ratio and low ApoAI/ApoB ratio subgroups in the Han individuals. The G allele carriers in Han had higher serum HDL-C levels and ApoAI to ApoB ratios than the G allele non-carriers. The G allele carriers in Han had higher serum HDL-C and ApoAI levels than the G allele noncarriers in males, whereas the G allele carriers had lower serum ApoB levels and higher ApoAI to ApoB ratio than female G allele noncarriers. Serum HDL-C and ApoAI levels in Han were correlated with genotypes in males, and serum ApoB levels and the ratio of ApoAI to ApoB were associated with genotypes in females. The PCSK9 E670G polymorphism is mainly associated with some serum lipid parameters in the Han population. The G allele carriers had higher serum HDL-C and ApoAI levels in males, and lower serum ApoB levels, and higher ApoAI/ApoB ratio in females than the G allele non-carriers. Both alcohol consumption and PCSK9 genetic polymorphisms modulate serum lipid levels and, therefore, in the same study population [32], Aung LH et al. [33] also detected the association between PCSK9 E670G and several environmental factors, such as alcohol consumption and serum lipid levels. The authors found that serum triglyceride, highdensity lipoprotein cholesterol, and apolipoprotein (Apo) A1 levels, and the ApoA1 to ApoB ratio were higher in drinkers than in non-drinkers, whereas the levels of total cholesterol (TC), LDL-C, and ApoB were lower in drinkers than in non-drinkers. The genotypic and allelic frequencies of PCSK9 E670G were not different between drinkers and non-drinkers. Non-drinkers with the AA genotype had higher LDL-C serum levels than subjects with the AG genotype, whereas drinkers with the AG genotype had higher TC serum levels than subjects with the AA genotype (p b 0.05 for each). The effects of alcohol consumption on TC and LDL-C levels depended upon the genotype, drinkers with the AA genotype having lower serum TC and LDL-C levels than nondrinkers. Based on these findings, the authors concluded that alcohol consumption can modify the effects of the PCSK9 E670G polymorphism on serum TC and LDL-C levels. Subjects with the PCSK9 E670G AA genotype benefited more from alcohol consumption than subjects with the AG genotype in decreasing serum TC and LDL-C levels. However, Hsu LA et al. [34] performed a case–control study that enrolled an ethnic Chinese population from Taiwan, including 202 patients with CHD and 614 unrelated controls. Among controls, the authors noted a significantly lower level of LDL-C in 670G carriers than in noncarriers, even after adjusting for age, gender, smoking, hypertension, diabetes mellitus, body mass index, and the use of lipid-lowering agents. The data also showed that the 670G carrier status was identified less frequently in patients with CHD than in controls, but the difference was not significant in a multivariable logistic regression analysis. The G allele also occurred at similar frequencies in the two groups in this study. These results indicate that the PCSK9 E670G genetic polymorphism modulates plasma LDL-C levels, but that it is not a risk variant for CAD in ethnic Chinese from Taiwan.

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Table 1 Loss-of-function and gain-of-function PCSK9 mutations in different geographical patients and clinical features associated with the mutations. Mutations of PCSK9 Loss-offunction

Geographical origin

Change of LDL-C concentration

In vitro testing

References

Cell culture

Cohen JC et al. [16,20] Cohen JC et al. [20] Cohen JC et al. [20] Cameron J et al. [30] Mayne J et al. [15] Cameron J et al. [30] Abifadel et al. [10] Noguchi T et al. [31] Aung LH et al. [32] Hsu LA et al. [34]

C679X

French, American Reduce 88% risk of CAD

Decreased by 27%

R46L

Danish, American French

Reduce 47% risk of CAD

Decreased by 8–9%; 9–15%; 11–16% Cell culture

Hypocholesterolemia

Decreased by 28%

G236S Norway N354I Q152H French-Canadian family R215H Norway

Hypocholesterolemia Hypocholesterolemia

Not available Not available Decrease 48%

Hypercholesterolemia

Not available

S127R

French

Hypercholesterolemia

Not available

E32K

Japanese

Hypercholesterolemia

Transiently transfected HepG2 cells

E670G

Chinese

Increasing HDL-C and ApoA1/ ApoB in male; Reducing LDL-C

2.1-Fold, 1.4-fold higher than control, separately Not available Not available

Not available

Y142X

Gain-offunction

Clinical features

Taiwan

2.3. Impact of PCSK9 genetic variants on CVD associated with LDL-C As shown in Table 1 and Fig. 1, the association of the genetic variants of PCSK9 and the level of plasma LDL-C is very strong. In a brief summary, loss-of-function variants of PCSK9 can reduce plasma LDL-C levels by reducing LDLR degradation, while gain-of-function PCSK9 variants can increase plasma LDL-C levels by accelerating LDLR degradation. Hence, the loss-of-function variants can protect from CVD because of low levels of plasma LDL-C. On the contrary, the gain-of-function variants can cause atherosclerotic CVD due to hypercholesterolemia.

2.3.1. Protective effect of loss-of-function PCSK9 mutations on CVD The impact of PCSK9 variants in the clinic was ascertained by the protection against CHD found to be associated with three cholesterollowering PCSK9 variants (p.C679X, p.Y142X, and p.R46L) in the Atherosclerosis Risk in Communities (ARIC) study. The comparison of the incidence of CHD (MI, fatal CHD, and coronary revascularization) over a 15-year interval, according to the presence or absence of these PCSK9 variants, was established in 3363 Black and 9523 White participants aged 45 to 64 years old from the ARIC study, comprising four American communities [20]. In this study, the authors found that the nonsense mutations were associated with an 88% reduction in the CHD risk, which was mainly attributed to a 28% reduction in mean LDL-C. Similarly, p.R46L was associated with a 47% reduction in the CHD risk, which was also mainly related with a 15% reduction in LDL-C. These PCSK9 alleles were also associated with a reduced risk of carotid atherosclerosis, and with a mean intima-media thickness slightly but significantly lower among carriers than among non-carriers among both the Black and the White groups of participants [20]. In the meta-analysis performed by Benn M et al., the authors observed that the IHD risk reduction in 46L allele carriers was 6% in the CCHS study, 46% in the CGPS study, 18% in the CIHDS study, and 30% in the studies combined. In the CCHS study, the HR for mortality was 1.18. In this meta-analysis, 46L allele carriers had a 12% (0.43 mmol/l) reduction in LDL-C and a 28% reduction in the risk of IHD, similar to results from the CCHS, CGPS, and CIHDS studies combined. However, the observed 12% (0.43 mmol/l) reduction in LDL-C theoretically predicted only a 5% reduction in IHD risk. The reduction in the IHD risk was larger than predicted by the observed reduction of LDL-C alone. This could be because the genotype is a better predictor of lifelong exposure to LDL-C than the LDL-C measured during adult life [29].

Determined in HepG2 or HEK293 cells transiently transfected Cells transiently transfected with PCSK9Q152H cDNA Determined in HepG2 or HEK293 cells transiently transfected

Not available

2.3.2. Causal effects of gain-of-function PCSK9 mutations on CVD Clinical findings reported in PCSK9 heterozygous carriers are those related to hypercholesterolemia: texon xanthomas, CHD, premature MI, and stroke. In vivo kinetics of apo B100-containing lipoproteins conducted in two French subjects carrying the p.S127R PCSK9 mutation showed that this mutant dramatically increased the production rate of apoB-100 (three-fold) compared with controls or LDLR-mutated patients, and led to a higher direct overproduction of VLDL (threefold), intermediate-density lipoprotein (IDL, three-fold), and LDL (five-fold) [35]. As mentioned above, in the Chinese study that enrolled the Bai Ku Yao and Han populations [32], the authors also evaluated the impact of the p.E670G mutation on the level of lipid profiles including LDL-C, ApoA1, ApoB, and ApoA1/ApoB, and concluded that the influence on the lipid profiles was different in males as compared to females. However, in the Chinese study that enrolled the population from Taiwan, the results were opposite to those obtained for the population from mainland China, and the authors concluded that p.E670G was not a risk variant for CAD in Chinese populations from Taiwan due to decreased LDL-C levels. In summary, the loss-of-function and gain-of-function PCSK9 mutations were found in patients from different geographical origins with hypo- or hypercholesterolemia and examined in vitro in recent studies. Both categories of PCSK9 mutations were found to be associated with the development of CVD due to the impact on plasma LDL-C.

3. Clinical prospects As known, genetic mutations resulting in altered cholesterol homeostasis provide valuable information regarding novel approaches for treating hypercholesterolemia. PCSK9 mutations were linked to altered LDL-C levels, illustrating this protein's role in lipid metabolism. Several ongoing clinical trials recently proposed to evaluate the clinical outcomes of PCSK9 inhibition as monotherapy or in combination with statins, and the effects on plasma LDL-C levels [36–40]. The results of these clinical trials are expected to demonstrate the effect of PCSK9 inhibition on lowering LDL-C levels and improving the outcomes of CVD. AMG145 is a fully humanized monoclonal immunoglobulin G2 antibody that binds specifically to human PCSK9 and inhibits its interaction with the LDLR. The PCSK9 Monoclonal Antibody Inhibition Combined With Statin Therapy-Thrombolysis In Myocardial Infarction 57

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Fig. 1. Correlation of PCSK9 genetic variants with plasma LDL-C levels and the impact on CVD risk.

(LAPLACE-TIMI 57; NCT01380730) is a 12-week randomized, doubleblind, dose-ranging, placebo-controlled study designed to assess the safety and efficacy of AMG145 when added to statin therapy in patients with hypercholesterolemia [36]. The results suggest that PCSK9 inhibition could become a new model in lipid management. Inhibition of PCSK9 warrants assessments in phase 3 clinical trials [37]. In the Heterozygous Familial Hypercholesterolemia Disorder (RUTHERFORD) randomized trial, the authors found that AMG145 administered every 4 weeks yielded rapid and substantial reductions in LDL-C in heterozygous familial hypercholesterolemia patients despite intensive statin use, with or without ezetimibe, with minimal adverse events and good tolerability [38]. In the LAPLACE-TIMI57 trial, AMG145 also significantly reduced Lp(a), by up to 32%, among subjects with hypercholesterolemia receiving statin therapy, offering an additional, complementary benefit beyond robust LDL-C reduction with regard to a patient's atherogenic lipid profile [39]. It has been demonstrated that a monoclonal antibody to PCSK9 was capable of lowering LDL-C by up to 70% over the levels achieved by statin therapy [40]. There are at least 3 monoclonal antibodies that inhibit PCSK9 activity as well as an antisense approach in human trials. The monoclonal antibody approach is expected to meet an important clinical need for LDL-C lowering in patients with statin intolerance, those who cannot achieve an adequate LDL-C level with existing therapies, refractory hypercholesterolemia, and those who may otherwise require LDL apheresis. Clinical outcome trials of PCSK9 monoclonal antibodies are highly anticipated. It is now established that statins induce an up-regulation of PCSK9 that might attenuate their cholesterol lowering effect by reducing LDL

receptor abundance at the cell surface [41]. Some studies demonstrated that statins could increase plasma levels of PCSK9 [42,43]. Thus, it was suggested that a combined statin and anti-PCSK9 therapeutic regimen could overcome this effect and enhance cholesterol reduction. Initial proof-of-concept was provided by statin administration to pcsk9 (−/−) mice that produced an exaggerated increase in LDLR in the liver and enhanced LDL clearance from the plasma [14]. Since the loss of a functional copy of PCSK9 is not associated with apparent deleterious effects in humans, this protease is an attractive target for developing plasma LDL-C-lowering agents, either alone or in combination with statins. Inhibition of PCSK9 is emerging as a novel strategy for treating hypercholesterolemia, and data obtained from pre-clinical studies show that use of monoclonal antibodies, antisense oligonucleotides, and short interfering RNA is effective in reducing LDL-C. Clinical studies, accompanied by a better understanding of PCSK9 biology, are now necessary to address whether these new compounds will have a future in clinical practice [4–8]. In conclusion, the discovery of PCSK9 in 2003 and its identification as the third protagonist responsible for ADH opened many new avenues in cardiovascular research. The discovery of the first natural mutants in PCSK9 has revealed the implication of an as-yet-unknown actor in cholesterol homeostasis. The abundance of results obtained by research teams worldwide has provided insight into its physiological and pathogenic role in cholesterol metabolism. However, the existence of additional roles in other physiologic pathways has yet to be investigated. The discovery of the role that PCSK9 plays in cholesterol homeostasis opened the way towards a better understanding of the physiopathology of ADH and CHD to improve the prevention and

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