Introduction

Cardiovascular disease (CVD) due to atherosclerosis is the foremost cause of premature mortality worldwide. It is generally accepted that elevated blood lipids play an important role in the development of atherosclerotic plaques and cardiovascular disease etiopathogenesis. Over the last two decades, the field of lipid study has paid attention to the study of proprotein convertase subtilisin/kexin type 9 (PCSK9). PCSK9 is one of the three most important genes involved in familial hypercholesterolemia, other than LDLR or apolipoprotein B (APOB). While PCSK9 gain-of-function (GOF) mutations associated with elevated blood lipids, carriers of loss of-function (LOF) mutations benefited from low-density lipoprotein cholesterol (LDL-C) reduction by up to 28%, accompanied by an 88% reduced risk of coronary artery disease [1]. Hepato-specific reduction LDL-C levels remains the first strategy in managing patients with familial hyperlipidemia and those with clinical atherosclerotic cardiovascular disease not reaching lipid-reducing goals [2]. To mimic PCSK9 natural inhibition, in 2015 PCSK9 monoclonal antibodies, the first strategy to inhibit PCSK9 therapeutically successfully approved in addition to statin therapy. More recently, silencing RNA (siRNA) reduced LDL-C by 45–60% [3]. Here, we systematically reviewed the available scientific evidence of genetic variation on PCSK9 and assessed the efficacy of PCSK9 inhibitors in cardiovascular outcomes, including cardiovascular death, myocardial infarction, and stroke.

PCSK9: Biological Function

PCSK9, the ninth member of the proprotein convertase family, is a serine protease that caught the attention of the scientific community in 2003 when the discovery of the first natural mutants of PCSK9 revealed the implication of an as-yet-unknown actor in cholesterol homeostasis [4, 5]. PCSK9 is mainly expressed on hepatocytes surface and has been shown to act both intracellularly playing a role as a chaperone in the degradation of the LDL receptor (LDLR), as well as a secreted factor promoting LDLR internalization from the hepatocellular surface [6]. PCSK9 regulates the degradation of the LDLR in response to cholesterol levels within the cell [7].

PCSK9 protein structure is characterized by a signal sequence, a prodomain, and a catalytic domain, followed by a C-terminal region. PCSK9 is synthesized as an inactive 75 kDa proenzyme that undergoes autocatalytic cleavage in the endoplasmic reticulum (ER) which produces an approximately 60-kDa catalytic fragment. Autocatalytic cleavage of the zymogen in the ER is essential for its transport from this compartment and for its secretion. PCSK9 favors LDLR degradation independently of its catalytic activity by involving mainly extracellular and possibly intracellular pathways [8]. PCSK9 catalytic domain (aa153–421) strongly interacts with the EGF-A domain (aa314–355) of the LDLR (extracellular domain, ECD). This prevents the LDLR from forming a closed conformation, making the receptor susceptible to enzymatic degradation, rather than being recycled to the cell surface (Fig. 1) [9].

Fig. 1
figure 1

a LDL-C uptake and recycling of LDLR. b PCSK9-mediated degradation of LDLR. The protein PCSK9 regulates the number of LDL receptors (LDL-Rs) on a cell’s surface. When PCSK9 binds to these receptors, the receptors do not get recycled but are broken down in cellular compartments called lysosomes instead. Adapted with permission from Publisher [10]

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PCSK9 Genetic Variants

The human PCSK9 gene is located on chromosome 1p32, and consist of 12 exons, which encode 692 amino acid protein (NP_777596.2). PCSK9 gene is highly polymorphic with the distribution of genetic variants in all its domains affecting PCSK9 synthesis, secretion, and activity [11,12,13] (Fig. 2). In fact, gain-of-function (GOF) mutations occurred less frequent than loss-of-function (LOF) mutations.

Fig. 2
figure 2

Number of genetic variants in PCSK9 (transcript ID ENST00000302118.5) based on their functional effect: a distribution of mutations per domain; b types of mutation [11]

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Major findings confirmed the significant impact of PCSK9 genetic variants on plasma LDL-C concentrations by affecting the LDLR pathway [4, 14, 15]. Beyond LDLR, PCSK9 affects also other lipoproteins, lipoprotein(a) [Lp(a)], and HDL-cholesterol levels are associated with circulating PCSK9 levels [16,17,18,19]. PCSK9 may also play a role in the postprandial phase by modulating triglyceride-rich lipoprotein (TGRL) metabolism [20] (Supplementary file 1).

Gain-of-Function Mutations

Carriers of GOF PCSK9 mutations over-express PCSK9, and therefore have very high plasma levels of LDL-C due to increased LDLR degradation. Several GOF missense mutations have been associated with hypercholesterolemia and premature atherosclerosis with overt CVD, such as myocardial infarction (MI) and stroke (S127R; F216L, D374Y, N157K; E32K; E228K; F216L, R357H, R215H, R496W, N35Y) [4, 5, 14, 15, 21,22,23,24,25,26,27].

Variant p.Ser127Arg interferes with autocatalytic cleavage, which is crucial for secretion from the cell. The p.Arg218Ser, p.Phe216Leu, and p.Asp374Tyr mutations result in total (p.Arg218Ser) or partial loss of the furin/PC5/6A processing of PCSK9, which increases the stability of PCSK9 [28, 29]. PCSK9 S127R GOF mutation carriers demonstrated affected TG and LDL metabolism with a threefold increased production rate of apoB-100, compared with controls or LDLR-mutated patients, which led to direct overproduction of VLDL (threefold), intermediate-density lipoprotein (IDL, threefold), and LDL (fivefold) [30].

Elevated fasting plasma TG levels and increased cardiovascular risk were confirmed in carriers of another GOF PCSK9 mutation E670G (− 23968A > G, rs505151) located in exon 12 [12, 31]. 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, lower serum apoB levels, and higher apoAI/apoB ratio in females than the non-carriers of the G allele [32]. Interestingly, when PCSK9 mutations were investigated in a Canadian Caucasian population, carriers of the E670G variant showed a non-significant difference in serum PCSK9 but a significantly lower TG concentration (27.7%; p = 0.039), not observed even for LOF mutations [33]. In a Japanese population, PCSK9 GOF E32K was associated with over 30% increased plasma levels of PCSK9, whereas these results were confirmed also by media from transiently transfected HepG2 cells, compared to controls. In patients, homozygotes for the E32K mutation was a twofold higher LDL-C levels always present with a mutation in LDLR, and in the heterozygous carriers, almost half the effect was observed. This could suggest a PCSK9 E32K effect on LDL-C levels via increased mass and function of PCSK9 and could exacerbate the clinical phenotypes of patients carrying LDLR mutations [23]. Other PCSK9 GOF mutants (F216L, R357H, and R215H) were described in different FH French families with tendon xanthomas, CHD, premature MI, and stroke [4, 5, 22]. GOF mutation in catalytic domain R215 was described to segregate with hypercholesterolemia in the Norwich family [24]. D374H was detected in one Portuguese proband and R496W in an Italian proband, together with E228K in LDLR [25, 26]. Another study demonstrated that variant p.Leu108Arg exhibited a ∼twofold-enhanced degrading activity towards the LDLR, resulting in a GOF, while variant p.Asp35Tyr created a novel Tyr-sulfation site, which may enhance the intracellular activity of PCSK9 [14]. GOF mechanism could represent a higher activity of the LDLR-degrading function of PCSK9. However, a more recent hypothesis suggests that post-translational modifications within residues 31–60 may affect the inhibitory activity of this segment and represent a mechanism for fine-tuning the activity of PCSK9 towards the LDLR. GOF mutations in the catalytic domain, which involve charged residues, could affect the positioning of this segment [27].

Another GOF variant G516V (c.1547G > T) was found in five index patients, and cascade screening identified 15 additional carriers. LDL-C levels were higher in those 15 carriers compared with the 27 non-carriers (236 ± 73 versus 124 ± 35 mg/dL; P < 0.001). In vitro studies demonstrated the pathogenicity of the G516V variant. Analysing LDL-C differences using generalized estimating equations revealed that the differences between carriers and non-carriers exceeded 39 mg/dL only for the G516V mutation [34].

Loss-of-Function Mutations

Contrariwise, genetic PCSK9 deficiency has been strongly associated with low plasma cholesterol levels and decreased cardiovascular risk, clearly demonstrated from several observational, randomization, and clinical studies [1, 12, 35,36,37,38,39,40]. Most of the LOF mutations result from either a deficiency in the synthesis or in the secretion of the PCSK9 protein, due to failure to exit the endoplasmic reticulum or failure to undergo the autocatalytic cleavage. Some LOF mutations could dramatically lower plasma PCSK9 up to 79%, and result in no immuno-detectable circulating PCSK9 levels (R104C/V114A, C679*; Y142X/290_292delGCC; Y142X/C679X; Q619X, Q679X, Y142X). However, these null carriers appeared healthy and fertile, with no observable signs of illness due to the absence of PCSK9. PCSK9 null compound heterozygotes did not differ from heterozygotes in any other cardio-metabolic trait, notwithstanding lower median LDL-C [13, 41,42,43,44].

Mendelian randomization studies have showed that PCSK9 genetic variants linked with low LDL-C levels were associated with reduced cardiovascular and all-cause mortality. For instance, the Copenhagen Population Study and Copenhagen City Heart Study have shown a 1 mmol/L (38.7 mg/dL) reduction in LDL-C levels due to the carriage of PCSK9 variants (R46L, R237W, I474V, and E670G) was associated with a 67% reduction in cardiovascular death (risk ratio 0.33, 95% confidence interval 0.19–0.58; p < 0.001) and a 28% reduction in all-cause death (risk ratio 0.72, 95% confidence interval 0.60–0.88; p = 0.001) [45].

LOF R46L represents one of the low-frequent PCSK9 variants (the MAF of the T allele ranged from 1 to 3.2%; 4.8% in a French–Canadian population). Despite its more moderate LDL-lowering effect (9 − 15%), the PCSK9 R46L allele was associated with a significant reduction in the incidence of CHD (47%) [1]. Homozygous R46L carriers showed a decreased trend of fasting TG levels (115 mg/dL) and higher HDL levels (70 mg/dl). In FH subjects carrying the 46L allele, significantly decreased apo-B and non-HDL concentrations with less severe clinical features were observed [46]. Interestingly, PCSK9 R46L carriers were observed an allele-dependent lower effect on lipoprotein(a) (Lp(a)) levels versus non-carriers (9 mg/dL in heterozygote, 8 mg/dL in homozygous vs. 10 mg/dL in non-carriers). The application of results regarding the link among PCSK9, LDLR, and Lp(a) metabolism is questionable, especially since Lp(a) plasma levels differ among different ethnic groups [47,48,49].

There is also evidence that PCSK9 LOF R46L according to apoE genotype would reveal some metabolic relationships. TG concentrations decreased significantly in the apoE3/E3 carriers with the R46L mutation with invariable plasma free-fatty acid (FFA) concentrations, compared with the apoE3/E2 and apoE3/E4 carriers. Subjects with both the R46L and apoE3/E2 genotypes showed a tendency toward insulin resistance, as indicated by a twofold increase in insulin, and leptin concentrations, compared with those without apoE3/E2 [39]. Emerging evidence suggests that PCSK9 LOF may also influence glucose homeostasis and insulin sensitivity. Beyond cardiovascular benefits, LOF R46L may increase the risk of diabetes in individuals with impaired fasting glucose levels [50]. Furthermore, carriers of PCSK9 and insertion of insLEU within positions 15 to 21 of the signal peptide of PCSK9 showed also increased occurrence of prediabetes and diabetes status [51]. Another study on animal models showed that glucose clearance is significantly impaired in PCSK9 KO mice fed a standard or a high-fat diet for 20 weeks compared with wild-type animals without affecting insulin sensitivity. PCSK9 KO mice presented larger islets with increased accumulation of cholesteryl esters, paralleled by increased intracellular insulin levels and decreased plasma insulin and C-peptide levels. This phenotype was completely reverted in PCSK9/LDLR DKO mice, implying the LDLR as PCSK9 target responsible for the phenotype observed [52]. Interestingly, results from the genetic and preclinical studies showing an increased risk of diabetes do not extrapolate to randomized trials results, but definitive long-term data are still lacking.

PCSK9 R46L variant is also associated with a twofold increased prevalence of hepatic steatosis and higher epicardial adiposity in human carriers of the PCSK9 R46L mutation. A similar observation was recapitulated in PCSK9 KO mice, showing increased visceral adipose tissue compared to the native genetic form [18, 20].

Postprandial studies have shown that in vivo PCSK9 deficiency was associated with a twofold decrease in postprandial TG levels [16]. In two heterozygous carriers of LOF PCSK9 mutation R104C-V114A, no alteration in TG compared with non-carriers in the postprandial state, and also no change in PCSK9 levels following an oral fat load in a small ten-patient sample was present [53]. Another study showed that carriers of LOF variants A53V, I474V, and/or R46L had no differences in PCSK9 levels, significantly lower LDL-C levels, and slightly lower TG levels in the fasting state versus non-carriers. Postprandial, PCSK9 and LDL-C levels are decreased in LOF carriers vs. non-carriers, but TG levels raised significantly to similar levels after the oral fat load. Interestingly, the same author observed that LOF PCSK9 leads to an inhibitory action on adipocyte differentiation in vitro, in both fasting and postprandial states. Furthermore, PBMC from PCSK9 LOF variant subjects showed significantly increase mRNA levels of some pro-inflammatory markers after meal [24]. Moderate postprandial lipemia was observed in LOF carriers compared to non-carrier controls after an oral fat load. It is of note that LOF was considered for carriers of the L10ins/A53V and/or I474V and/or R46L mutations, without significant effect on PCSK9 plasma levels. Moreover, as TG, apoB48, and VLDL-C were already lower compared to non-carrier controls (a phenotypic trait not observed in LOF hit variants), it remains to be clarified whether the observed effect should be consistently attributed to PCSK9 deficiency per se. In any case, there was no significant difference in fasting HDL-C (p = 0.46), apoCII (p = 0.13), and apoCIII (p = 0.66) between the groups, or the CII/CIII ratio between groups, suggesting that alterations in lipolysis are less likely a mechanism for PCSK9’s action on TGRL clearance in studied population [54, 55].

PCSK9 Inhibitors and Cardiovascular Outcomes

Various approaches have been tried to mimic natural PCSK9 inhibition, such as FDA-approved monoclonal antibodies (mAb) and non-antibody approaches including small interfering RNA (siRNA). Other approaches use therapeutic genome editing via CRISPR/Cas9 technologies (clustered regularly interspaced short palindromic repeats), a base editor which promises prolonged or even permanent reduction in circulating LDL-C levels, small molecule inhibitors (peptides/adnectins), gene silencing using antisense oligonucleotides (ASO), or peptide-based anti-PCSK9 vaccination [56,57,58]. Recently, the first oral PCSK9 inhibitor, macrocyclic peptide MK-0616, was designed to lower LDL-C via the same biological mechanism as currently approved injectable PCSK9, and it has already demonstrated statistically significant reductions in LDL-C up to 60.9% in phase 2b randomized trial [59]. Moreover, the current pre-clinical evidence regarding novel mechanisms for LDL-C lowering reveals annexin A2 as a natural extrahepatic inhibitor of the PCSK9, while depletion of protein denitrosylase SCoR2 (S-nitroso-coenzyme A reductase 2; AKR1A1) in mice lowers serum cholesterol by inhibiting liver secretion of PCSK9 [60, 61].

Among the therapeutic interventions targeting PCSK9 that are currently in use, PCSK9-mAb evolocumab (Repatha®, Amgen) and alirocumab (Praluent®, Sanofi-Aventis/Regeneron) target circulating PCSK9. This therapy is undoubtedly effective in lowering LDL-C, and results from several large outcomes studies have shown clinical benefit in very high-risk patients, including those with acute coronary syndromes. Treatment with PCSK9 inhibitors decreases LDL-C up to 60%, and favorably affects other lipid parameters such as nonHDL-C, TGs, and Lp(a). More precisely, a 40–50% decrease in apoB, a decrease in non-HDL-C by approximately 50%, and a decrease in Lp (a) by 30% were observed in most studies by positively affecting HDL-C and the level of TAGs. The treatment is very well tolerated and have shown a favorable safety profile (including frequently reported local reaction at the application site) [62]. More recently, inclisiran (LEQVIO®, a siRNA) reported similar lipid-lowering effects by over 50% and maintain its effectduring each the 6-month dosing interval vs. placebo [63].

Extensive evidence of impressive reductions in LDL-C and other lipid parameters has raised questions about whether this reduction translates into a decrease in the cardiovascular events, which is of great clinical interest. Addressing this question, data from clinical trial FOURIER (Further Cardiovascular Outcomes Research with PCSK9 Inhibition in Subjects with Elevated Risk) have demonstrated a significant relative risk reduction of MI (HR 0.73; 95% Cl 0.65 − 0.82) and stroke (HR 0.79; 95% Cl 0.66 − 0.95) [64]. Data from ODYSSEY trial (ODYSSEY Outcomes: Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment with Alirocumab) showed a reduction and secondary endpoints [62]. Analysis of the inclisiran trial suggests potential benefits for MACE reduction [65]. Further insights on how efficacy of PCSK9 inhibitors may affect CVD outcomes will bring results from currently ongoing cardiovascular outcomes trials, one of evolocumab (VESALIUS-CV [NCT03872401]) and three of inclisiran (ORION-4 [NCT03705234], VICTORION-1 and -2 Prevent [NCT05739383 and NCT05030428, respectively]).

Meta-analyses have evaluated the effects of different PCSK9 modulators compared with control groups. In a meta-analysis, randomizing 3783 patients from ORION clinical trials (1895 to inclisiran and 1888 to placebo), there were no significant reductions in cardiovascular ischemic endpoints with inclisiran in patients with hypercholesterolemia on maximum tolerated statins doses compared with placebo [66].

PCSK9-mAb significantly reduced the risk of stroke (RR 0.75; 95% CI 0.66–0.86, p < 0.0001) and MI (RR 0.81; 95% CI 0.76–0.87, p < 0.00001), but not the risk of cardiovascular death (RR 0.96; 95% CI 0.86–1.07, p = 0.45) compared with placebo [67]. Similarly, no significant reduction of all-cause mortality was found (RR 0.88; 95% CI 0.72–1.07; p = 0.182) in meta-analysis of 25 randomized controlled trials (RCTs) comparing PCSK9 mAbs alirocumab and evolocumab with placebos or active drugs in patients at high cardiovascular risk. Both alirocumab (RR 0.89; 95% CI 0.83–0.95; p < 0.001) and evolocumab (RR 0.86; 95% CI 0.80–0.92; p < 0.001) were associated with a lower risk of major cardiovascular events (MACEs), especially in secondary prevention (alirocumab group: RR 0.88; 95% CI 0.82–0.95; p < 0.001; evolocumab group: RR 0.86; 95% CI 0.80–0.92; p < 0.001). The reduction in MACEs was observed in Caucasians but not in Asian subjects [68]. Interestingly, the meta-analysis of clinical outcomes of PCSK9 modulators (evolocumab, alirocumab, inclisiran) in patients with established atherosclerotic cardiovascular disease (ASCVD) indicated a reduction in the composite outcomes of MI, stroke, and cardiovascular death (relative risk RR 0.80, 95% CI 0.73–0.87) and MI, stroke, unstable angina (requiring revascularization), and cardiovascular death (RR 0.85, 95% CI 0.74–0.97). However, individual effects on mortality, cardiovascular death, MI, and stroke remained nonsignificant [69]. Another finding from a metanalytic study of nine trials, including a total of 54,301 participants was that both alirocumab and evolocumab, were associated with reductions in MI (RR 0.86; 95% CI 0.77–0.95 and RR 0.73; 95% CI 0.65–0.82 respectively) and stroke (RR 0.76; 95% CI 0.60–0.96 and RR 0.79; 95% CI 0.66–0.94 respectively). Moreover, the use of alirocumab was associated with reductions in all-cause mortality compared with control (RR 0.83; 95% CI 0.72–0.95), while evolocumab was associated with increased all-cause mortality compared with alirocumab (RR 1.26, 95% CI 1.04–1.52) [70]. An earlier meta-analytic study of 67 RCTs including 259,429 participants with PCSK9 inhibitors plus statin confirmed the significantly reduced risk of non-fatal MI (RR 0.82; 95% CI 0.72–0.93, p = 0.003) or stroke (RR 0.74; 95% CI 0.65–0.85, p < 0.001) [71]. The results of the most recent study of Imran et al. align with overall previous findings in MACE. Moreover, alirocumab reduced all-cause mortality, which is likely due to the inclusion of new randomized trials that have been published since the previous studies were conducted, and longer follow-up periods [72]. In conclusion, the investigation into the efficacy of pharmacologic agents targeting PCSK9 suggests an effect rather for lowering the risk of MI and stroke than for cardiovascular mortality (Fig. 3).

Fig. 3
figure 3

Meta-analysis of randomized clinical trials assessing the effect of different modulators targeting PCSK9 on cardiovascular events (PCSK9-mAb, inclisiran vs. placebo). a Forest plot for cardiovascular death. b Forest plot treatment for myocardial infarction. c Forest plot for stroke. Stroke was significantly reduced following the treatment with a PCSK9 inhibitors (RR 0.78 [95% CI 0.63–0.98] p = 0.0352), a similar trend was observed for myocardial infarction (RR 0.79 [95% CI 0.58–1.01] p = 0.0951). No significant difference was observed in cardiovascular mortality (RR 0.98 [95% CI 0.86–1.11] p = 0.753). Egger’s test for a regression intercept gave a p-value of 0.43306 for CVD death, a p-value of 0.4905 for MI and p-value of 0.2436 for stroke, indicating no evidence of publication bias (Comprehensive Meta-Analysis software V4, Meta-Mar v 3.5.1). The meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. The process of study selection is detailed in Supplementary file 2. Overall estimates of effect were calculated with a fixed or random-effects model and expressed as RR with 95% CI. Statistical significance was set at p < 0.05. The assumption of homogeneity between the treatment effects in different trials was tested by Q statistic and I2 statistic. A significant heterogeneity was defined by a p < 0.10 at Q statistic; I2 ranging from 0 to 40% might indicate not important heterogeneity, from 30 to 60% might represent moderate heterogeneity, from 50 to 90% might indicate substantial heterogeneity, and from 75 to 100% might represent considerable heterogeneity. Abbreviations: CI, confidence interval. The black square represents the weight of each study, the canter of the square represents the risk ratio (RR), and the length of each line around the point represents its 95% confidence interval (95% CI). The overall summary estimate (a result of the meta-analysis) is represented by a lozenge (diamond shape) at the end of the graph

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Post hoc and secondary analysis of long-term studies indicate greater clinical benefits in subgroups of patients with a heavier burden of atherosclerosis or complex plaque morphology, with more recent ischemic events, or in those who reach very low LDL levels.

Safety of living with very low levels of LDL-C concentrations achieved (30 mg/dL with the evolocumab; < 15 mg/dL with the alirocumab) was reported as well as benefits in reducing the risk of angina, MI, or cerebrovascular disorder and total mortality [64, 73]. Patients closer to the most recent MI, a history of multiple MIs, or multivessel coronary artery disease gained greater absolute risk reductions (3.5 to 1%) from evolocumab [74]. Furthermore, evolocumab reduced the risk of MI related to plaque rupture [75]. The risk of ischemic stroke was significantly reduced by evolocumab (1.2 vs. 1.6%), in subgroup analyses of patients with prior ischemic stroke vs. patients without [76]. In the ODYSSEY OUTCOMES subanalysis, the estimated absolute risk reductions with alirocumab were numerically greater in patients with previous MI (MACE, 1.91% vs. 1.42%; death, 1.35% vs. 0.41%) [77]. A further post hoc analysis evaluating the efficacy of alirocumab according to metabolic risk factors concluded consistently reduced MACE across categories defined by the number of metabolic risk factors, but absolute risk reduction (aRR) increased with the number of metabolic risk factors findings revealed larger aRR in cardiovascular events compared to individuals without diabetes (7.7–14.6%) and metabolic syndrome (0.91 to 3.82%) [78]. Results are in concordance with post hoc analyses from the FOURIER trial (patients with atherosclerotic cardiovascular disease and metabolic syndrome, MetS). Evolocumab reduced the risk of CVD events in patients with MetS (evolocumab vs. placebo [HR 0.83, 95% CI 0.76–0.91]), patients with vs. without MetS [HR 0.89, 95% CI 0.79–1.01] pinteraction = 0.39) [79]. PCSK9 mAb appears to be effective in reducing the risk of MACE by 18% (OR 0.82, 95% CI 0.74–0.90) in subjects with diabetes and dyslipidemia [80].

Moreover, in patients with MetS, evolocumab and alirocumab did not result in an increased risk of developing new-onset diabetes (NOD), or worsening glycemia [78, 81]. The use of PCSK9 inhibitors raised concerns about the risk of NOD, especially taking into consideration results from genetic studies. Safety reports from RCTs showed that evolocumab and alirocumab were primarily related to mild hyperglycemia rather than diabetes, with most of the hyperglycaemic events occurring during the first 6 months of treatment [82]. This effect disappears after drug withdrawal and treatment with PCSK9 inhibitors should be of minimal concern [83]. A meta-analysis by Khan et al. did not show any significant association between PCSK9 inhibitors and NOD (HR 1.00, 95% CI 0.93–1.07; p = 0.96) [84]. A recent meta-analysis of 39 randomized clinical trials including 35,896 participants treated with alirocumab or evolocumab did not find any association between the use of these drugs and NOD (p = 0.97) [85]. Finally, besides effects on lipids and traditional cardiovascular outcomes, a recent post hoc analysis highlights the positive effects of both PCSK9-mAb on venous thromboembolic events (31% relative risk reduction) and aortic stenosis progression [86, 87] (Table 1).

Table 1 Overview of post hoc analyses and meta-analyses involving PCSK9 inhibitors
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Conclusions

Genetic studies have clearly demonstrated that PCSK9 is a major determinant of cholesterol homeostasis, and several GOF and LOF PCSK9 mutations have been described, highlighting a key role for PCSK9 in regulating lipid metabolism. In this regard, PSCK9-targeting agents have become the most promising therapeutic approach to manage hypercholesterolemia and related diseases and reduce the risk of some cardiovascular outcomes. However, many challenging issues related to PCSK9 still exist and a better understanding of the genetic variations in PCSK9 may clarify its biologic role, especially as a promising tool for novel lipid-lowering therapies.