Effects of PCSK-9 Inhibition by Alirocumab Treatments on Biliary Cirrhotic Rats

Hyperlipidemia and oxidative stress with elevated oxidized low-density lipoprotein (ox-LDL) exacerbate hepatic inflammation and fibrosis. The plasma level of low-density lipoprotein (LDL) is controlled by proprotein convertase subtilisin/kexin 9 (PCSK9). Alirocumab is a monoclonal antibody that decreases LDL via inhibiting PCSK9 function. Apart from lipid-lowering effects, alirocumab exerts anti-inflammation, anti-angiogenesis and anti-oxidant effects. This study aims to investigate the impact of alirocumab treatment on common bile duct ligation (BDL)-induced biliary cirrhotic rats. After a 4-week treatment of alirocumab, the hemodynamic data, blood biochemistry, ox-LDL level, oxidative stress markers, severity of hepatic encephalopathy and abnormal angiogenesis of BDL rats were measured and compared to the control group. BDL rats presented cirrhotic pictures and elevated ammonia, total cholesterol, LDL and ox-LDL levels compared to the control group. Alirocumab decreased plasma levels of total cholesterol, LDL, and oxidative stress markers; however, it did not affect the hemodynamics, liver and renal biochemistry, and the plasma levels of ammonia and ox-LDL. The motor activities, portal-systemic collaterals and mesenteric vascular density were not significantly different between alirocumab-treated and control groups. In addition, it did not affect hepatic inflammation, intrahepatic angiogenesis, liver fibrosis and free cholesterol accumulation in the liver of BDL rats. In conclusion, PCSK9 inhibition by alirocumab treatment ameliorates hyperlipidemia and systemic oxidative stress in biliary cirrhotic rats. However, it does not affect the plasma level of ox-LDL, intrahepatic inflammation and fibrosis. In addition, PCSK9 inhibition has a neutral effect on abnormal angiogenesis and hepatic encephalopathy in biliary cirrhotic rats.     

Docosahexaenoic Acid Attenuates Cardiovascular Risk Factors via a Decline in Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Plasma Levels

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulating protein that regulates cholesterol metabolism by promoting LDL receptor degradation in the liver and has recently been proposed as a therapeutic target in the management of hyperlipidaemia. We investigated the impact of dietary fat on the metabolism of sterols and on plasma PCSK9 concentrations to explore likely clinical usefulness. In a post hoc analysis of a double‐blind randomised crossover controlled feeding trial, the Canola Oil Multicenter Intervention Trial (COMIT), volunteers (n = 54) with at least one condition related to metabolic syndrome consumed diets with one of the following treatment oils in beverages: (1) conventional canola oil (Canola); (2) canola oil rich in docosahexanoic acid (DHA) (CanolaDHA); and (3) high‐oleic acid canola oil (CanolaOleic). The enrichment in oleic acid resulted in lower plasma cholesterol concentration compared with diets enriched in DHA. Contrarily, DHA‐enriched oil significantly decreased plasma PCSK9 and triacylglycerols levels, but increased circulating levels of sterols. The variations in lathosterol, sitosterol, and campesterol indicate that plasma PCSK9 levels are sensitive to changes in cholesterol synthesis and/or absorption. There was a significant correlation between plasma PCSK9 levels and plasma triacylglicerol and apolipoprotein B levels, which was not affected by dietary fat. Therefore, our results suggest that the impact of dietary fats should not be discarded as complementary treatment in the management of patients with hyperlipidaemia. These findings should be considered in the analysis of ongoing studies and may represent a cautionary note in the treatment of patients with cardiovascular risk.     

Platelet-Derived PCSK9 Is Associated with LDL Metabolism and Modulates Atherothrombotic Mechanisms in Coronary Artery Disease

Platelets play a significant role in atherothrombosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is critically involved in the regulation of LDL metabolism and interacts with platelet function. The effect of PCSK9 in platelet function is poorly understood. The authors of this article sought to characterize platelets as a major source of PCSK9 and PCSK9’s role in atherothrombosis. In a large cohort of patients with coronary artery disease (CAD), platelet count, platelet reactivity, and platelet-derived PCSK9 release were analyzed. The role of platelet PCSK9 on platelet and monocyte function was investigated in vitro. Platelet count and hyper-reactivity correlated with plasma LDL in CAD. The circulating platelets express on their surface and release substantial amounts of PCSK9. Release of PCSK9 augmented platelet-dependent thrombosis, monocyte migration, and differentiation into macrophages/foam cells. Platelets and PCSK9 accumulated in tissue derived from atherosclerotic carotid arteries in areas of macrophages. PCSK9 inhibition reduced platelet activation and platelet-dependent thrombo-inflammation. The authors identified platelets as a source of PCSK9 in CAD, which may have an impact on LDL metabolism. Furthermore, platelet-derived PCSK9 contributes to atherothrombosis, and inhibition of PCSK9 attenuates thrombo-inflammation, which may contribute to the reported beneficial clinical effects.     

Renal Ischemia/Reperfusion Early Induces Myostatin and PCSK9 Expression in Rat Kidneys and HK-2 Cells

During visceral interventions, the transient clampage of supraceliac aorta causes ischemia/reperfusion (I/R) in kidneys, sometime resulting in acute renal failure; preclinical studies identified redox imbalance as the main driver of I/R injury. However, in humans, the metabolic/inflammatory responses seem to prevail on oxidative stress. We investigated myostatin (Mstn) and proprotein convertase subtilisin/kexin type 9 (PCSK9), proatherogenic mediators, during renal I/R. Compared to sham-operated animals, the kidneys of rats who had experienced ischemia (30 min) had higher Mstn and PCSK9 expression after 4 h of reperfusion. After 24 h, they displayed tubular necrosis, increased nitrotyrosine positivity, and nuclear peroxisome proliferator-activated receptor gamma coactivator-1alpha relocation, markers of oxidative stress and mitochondria imbalance. Mstn immunopositivity was increased in tubuli, while PCSK9 immunosignal was depleted; systemically, PCSK9 was higher in plasma from I/R rats. In HK-2 cells, both ischemia and reperfusion enhanced reactive oxygen species production and mitochondrial dysfunction. H₂O₂ upregulated Mstn and PCSK9 mRNA after 1 and 3.5 h, respectively. Accordingly, ischemia early induced Mstn and PCSK9 mRNA; during reperfusion Mstn was augmented and PCSK9 decreased. Mstn treatment early increased PCSK9 expression (within 8 h), to diminish over time; finally, Mstn silencing restrained ischemia-induced PCSK9. Our study demonstrates that renal I/R enhances Mstn and PCSK9 expression and that Mstn induces PCSK9, suggesting them as therapeutic targets for vascular protection during visceral surgery.     

PCSK9 Affects Astrocyte Cholesterol Metabolism and Reduces Neuron Cholesterol Supplying In Vitro: Potential Implications in Alzheimer’s Disease

The Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) involvement in Alzheimer’s disease (AD) is poorly investigated. We evaluated the in vitro PCSK9 modulation of astrocyte cholesterol metabolism and neuronal cholesterol supplying, which is fundamental for neuronal functions. Moreover, we investigated PCSK9 neurotoxic effects. In human astrocytoma cells, PCSK9 reduced cholesterol content (−20%; p < 0.05), with a greater effect in presence of beta amyloid peptide (Aβ) (−37%; p < 0.01). PCSK9 increased cholesterol synthesis and reduced the uptake of apoE-HDL-derived cholesterol (−36%; p < 0.0001), as well as the LDL receptor (LDLR) and the apoE receptor 2 (ApoER2) expression (−66% and −31%, respectively; p < 0.01). PCSK9 did not modulate ABCA1- and ABCG1-cholesterol efflux, ABCA1 levels, or membrane cholesterol. Conversely, ABCA1 expression and activity, as well as membrane cholesterol, were reduced by Aβ (p < 0.05). In human neuronal cells, PCSK9 reduced apoE-HDL-derived cholesterol uptake (−41%; p < 0.001) and LDLR/apoER2 expression (p < 0.05). Reduced cholesterol internalization occurred also in PCSK9-overexpressing neurons exposed to an astrocyte-conditioned medium (−39%; p < 0.001). PCSK9 reduced neuronal cholesterol content overall (−29%; p < 0.05) and increased the Aβ-induced neurotoxicity (p < 0.0001). Our data revealed an interfering effect of PCSK9, in cooperation with Aβ, on brain cholesterol metabolism leading to neuronal cholesterol reduction, a potentially deleterious effect. PCSK9 also exerted a neurotoxic effect, and thus represents a potential pharmacological target in AD.     

PCSK9 is Present in Human Cerebrospinal Fluid and is Maintained at Remarkably Constant Concentrations Throughout the Course of the Day

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a key regulator of serum low density lipoprotein cholesterol levels. PCSK9 is secreted by the liver and binds the hepatic low density lipoprotein receptor, causing its subsequent degradation. PCSK9 has also been shown to regulate the levels of additional membrane-bound proteins in vitro, including very low-density lipoprotein receptor, apolipoprotein E receptor 2, and beta-site amyloid precursor protein-cleaving enzyme 1, which are highly expressed in central nervous system (CNS) and have been implicated in Alzheimer’s disease. Previous studies have demonstrated that human circulating PCSK9 displays a diurnal rhythm. Currently, little is known about PCSK9 levels in human cerebrospinal fluid (CSF). In the present study, we measured PCSK9 concentrations in both serum and CSF collected from healthy human subjects at multiple time points throughout the day. While PCSK9 in serum manifested a distinct diurnal pattern, CSF PCSK9 levels were remarkably constant throughout the course of the day and were also consistently lower than corresponding serum PCSK9 concentrations. Our results indicate that regulation of PCSK9 in human CSF may be different than for plasma PCSK9, suggesting that further study of the role of PCSK9 in the CNS is warranted.     

Anti-Inflammatory Effect of Auranofin on Palmitic Acid and LPS-Induced Inflammatory Response by Modulating TLR4 and NOX4-Mediated NF-κB Signaling Pathway in RAW264.7 Macrophages

Chronic inflammation, which is promoted by the production and secretion of inflammatory mediators and cytokines in activated macrophages, is responsible for the development of many diseases. Auranofin is a Food and Drug Administration-approved gold-based compound for the treatment of rheumatoid arthritis, and evidence suggests that auranofin could be a potential therapeutic agent for inflammation. In this study, to demonstrate the inhibitory effect of auranofin on chronic inflammation, a saturated fatty acid, palmitic acid (PA), and a low concentration of lipopolysaccharide (LPS) were used to activate RAW264.7 macrophages. The results show that PA amplified LPS signals to produce nitric oxide (NO) and various cytokines. However, auranofin significantly inhibited the levels of NO, monocyte chemoattractant protein-1, and pro-inflammatory cytokines, such as interleukin (IL)-1β, tumor necrosis factor-α, and IL-6, which had been increased by co-treatment with PA and LPS. Moreover, the expression of inducible NO synthase, IL-1β, and IL-6 mRNA and protein levels increased by PA and LPS were reduced by auranofin. In particular, the upregulation of NADPH oxidase (NOX) 4 and the translocation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) induced by PA and LPS were suppressed by auranofin. The binding between the toll-like receptor (TLR) 4 and auranofin was also predicted, and the release of NO and cytokines was reduced more by simultaneous treatment with auranofin and TLR4 inhibitor than by auranofin alone. In conclusion, all these findings suggested that auranofin had anti-inflammatory effects in PA and LPS-induced macrophages by interacting with TLR4 and downregulating the NOX4-mediated NF-κB signaling pathway.     

Common and Rare PCSK9 Variants Associated with Low-Density Lipoprotein Cholesterol Levels and the Risk of Diabetes Mellitus: A Mendelian Randomization Study

PCSK9 is a candidate locus for low-density lipoprotein cholesterol (LDL-C) levels. The cause–effect relationship between LDL-C levels and diabetes mellitus (DM) has been suggested to be mechanism-specific. To identify the role of PCSK9 and genome-wide association study (GWAS)-significant variants in LDL-C levels and the risk of DM by using Mendelian randomization (MR) analysis, a total of 75,441 Taiwan Biobank (TWB) participants was enrolled for a GWAS to determine common and rare PCSK9 variants and their associations with LDL-C levels. MR studies were also conducted to determine the association of PCSK9 variants and LDL-C GWAS-associated variants with DM. A regional plot association study with conditional analysis of the PCSK9 locus revealed that PCSK9 rs10788994, rs557211, rs565436, and rs505151 exhibited genome-wide significant associations with serum LDL-C levels. Imputation data revealed that three rare nonsynonymous mutations—namely, rs151193009, rs768846693, and rs757143429—exhibited genome-wide significant association with LDL-C levels. A stepwise regression analysis indicated that seven variants exhibited independent associations with LDL-C levels. On the basis of two-stage least squares regression (2SLS), MR analyses conducted using weighted genetic risk scores (WGRSs) of seven PCSK9 variants or WGRSs of 41 LDL-C GWAS-significant variants revealed significant association with prevalent DM (p = 0.0098 and 5.02 × 10⁻⁷, respectively), which became nonsignificant after adjustment for LDL-C levels. A sensitivity analysis indicated no violation of the exclusion restriction assumption regarding the influence of LDL-C-level-determining genotypes on the risk of DM. Common and rare PCSK9 variants are independently associated with LDL-C levels in the Taiwanese population. The results of MR analyses executed using genetic instruments based on WGRSs derived from PCSK9 variants or LDL-C GWAS-associated variants demonstrate an inverse association between LDL-C levels and DM.     

Influence of Hyperglycemia and Diabetes on Cardioprotection by Humoral Factors Released after Remote Ischemic Preconditioning (RIPC)

Remote ischemic preconditioning (RIPC) protects hearts from ischemia–reperfusion (I/R) injury in experimental studies; however, clinical RIPC trials were unsatisfactory. This discrepancy could be caused by a loss of cardioprotection due to comorbidities in patients, including diabetes mellitus (DM) and hyperglycemia (HG). RIPC is discussed to confer protective properties by release of different humoral factors activating cardioprotective signaling cascades. Therefore, we investigated whether DM type 1 and/or HG (1) inhibit the release of humoral factors after RIPC and/or (2) block the cardioprotective effect directly at the myocardium. Experiments were performed on male Wistar rats. Animals in part 1 of the study were either healthy normoglycemic (NG), type 1 diabetic (DM1), or hyperglycemic (HG). RIPC was implemented by four cycles of 5 min bilateral hind-limb ischemia/reperfusion. Control (Con) animals were not treated. Blood plasma taken in vivo was further investigated in isolated rat hearts in vitro. Plasma from diseased animals (DM1 or HG) was administered onto healthy (NG) hearts for 10 min before 33 min of global ischemia and 60 min of reperfusion. Part 2 of the study was performed vice versa—plasma taken in vivo, with or without RIPC, from healthy rats was transferred to DM1 and HG hearts in vitro. Infarct size was determined by TTC staining. Part 1: RIPC plasma from NG (NG Con: 49 ± 8% vs. NG RIPC 29 ± 6%; p < 0.05) and DM1 animals (DM1 Con: 47 ± 7% vs. DM1 RIPC: 38 ± 7%; p < 0.05) reduced infarct size. Interestingly, transfer of HG plasma showed comparable infarct sizes independent of prior treatment (HG Con: 34 ± 9% vs. HG RIPC 35 ± 9%; ns). Part 2: No infarct size reduction was detectable when transferring RIPC plasma from healthy rats to DM1 (DM1 Con: 54 ± 13% vs. DM1 RIPC 53 ± 10%; ns) or HG hearts (HG Con: 60 ± 16% vs. HG RIPC 53 ± 14%; ns). These results suggest that: (1) RIPC under NG and DM1 induces the release of humoral factors with cardioprotective impact, (2) HG plasma might own cardioprotective properties, and (3) RIPC does not confer cardioprotection in DM1 and HG myocardium.     

Sphingosine 1-Phosphate and Apolipoprotein M Levels and Their Correlations with Inflammatory Biomarkers in Patients with Untreated Familial Hypercholesterolemia

High-density lipoprotein (HDL)-bound apolipoprotein M/sphingosine 1-phosphate (ApoM/S1P) complex in cardiovascular diseases serves as a bridge between HDL and endothelial cells, maintaining a healthy endothelial barrier. To date, S1P and ApoM in patients with untreated heterozygous familial hypercholesterolemia (HeFH) have not been extensively studied. Eighty-one untreated patients with HeFH and 32 healthy control subjects were included in this study. Serum S1P, ApoM, sCD40L, sICAM-1, sVCAM-1, oxLDL, and TNFα concentrations were determined by ELISA. PON1 activities were measured spectrophotometrically. Lipoprotein subfractions were detected by Lipoprint. We diagnosed FH using the Dutch Lipid Clinic Network criteria. Significantly higher serum S1P and ApoM levels were found in HeFH patients compared to controls. S1P negatively correlated with large HDL and positively with small HDL subfractions in HeFH patients and the whole study population. S1P showed significant positive correlations with sCD40L and MMP-9 levels and PON1 arylesterase activity, while we found significant negative correlation between sVCAM-1 and S1P in HeFH patients. A backward stepwise multiple regression analysis showed that the best predictors of serum S1P were large HDL subfraction and arylesterase activity. Higher S1P and ApoM levels and their correlations with HDL subfractions and inflammatory markers in HeFH patients implied their possible role in endothelial protection.     

Emerging Insights on the Diverse Roles of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in Chronic Liver Diseases: Cholesterol Metabolism and Beyond

Chronic liver diseases are commonly associated with dysregulated cholesterol metabolism. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease of the proprotein convertase family that is mainly synthetized and secreted by the liver, and represents one of the key regulators of circulating low-density lipoprotein (LDL) cholesterol levels. Its ability to bind and induce LDL-receptor degradation, in particular in the liver, increases circulating LDL-cholesterol levels in the blood. Hence, inhibition of PCSK9 has become a very potent tool for the treatment of hypercholesterolemia. Besides PCSK9 limiting entry of LDL-derived cholesterol, affecting multiple cholesterol-related functions in cells, more recent studies have associated PCSK9 with various other cellular processes, including inflammation, fatty acid metabolism, cancerogenesis and visceral adiposity. It is increasingly becoming evident that additional roles for PCSK9 beyond cholesterol homeostasis are crucial for liver physiology in health and disease, often contributing to pathophysiology. This review will summarize studies analyzing circulating and hepatic PCSK9 levels in patients with chronic liver diseases. The factors affecting PCSK9 levels in the circulation and in hepatocytes, clinically relevant studies and the pathophysiological role of PCSK9 in chronic liver injury are discussed.     

Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) Deletion but Not Inhibition of Extracellular PCSK9 Reduces Infarct Sizes Ex Vivo but Not In Vivo

Hypoxia upregulates PCSK9 expression in the heart, and PCSK9 affects the function of myocytes. This study aimed to investigate the impact of PCSK9 on reperfusion injury in rats and mice fed normal or high-fat diets. Either the genetic knockout of PCSK9 (mice) or the antagonism of circulating PCSK9 via Pep2-8 (mice and rats) was used. Isolated perfused hearts were exposed to 45 min of ischemia followed by 120 min of reperfusion. In vivo, mice were fed normal or high-fat diets (2% cholesterol) for eight weeks prior to coronary artery occlusion (45 min of ischemia) and reperfusion (120 min). Ischemia/reperfusion upregulates PCSK9 expression (rats and mice) and releases it into the perfusate. The inhibition of extracellular PCSK9 does not affect infarct sizes or functional recovery. However, genetic deletion largely reduces infarct size and improves post-ischemic recovery in mice ex vivo but not in vivo. A high-fat diet reduced the survival rate during ischemia and reperfusion, but in a PCSK9-independent manner that was associated with increased plasma matrix metalloproteinase (MMP)9 activity. PCSK9 deletion, but not the inhibition of extracellular PCSK9, reduces infarct sizes in ex vivo hearts, but this effect is overridden in vivo by factors such as MMP9.     

Blocking of SGLT2 to Eliminate NADPH-Induced Oxidative Stress in Lenses of Animals with Fructose-Induced Diabetes Mellitus

Chronic hyperglycemia triggers an abnormal rise in reactive oxygen species (ROS) that leads to blindness in patients with diabetes mellitus (DM) and cataracts. In this study, the effects of dapagliflozin, metformin and resveratrol on ROS production were investigated in lens epithelial cells (LECs) of animals with fructose-induced DM. LECs were isolated from patients without DM, or with DM devoid of diabetic retinopathy. Animals were treated with 10% fructose for 8 weeks to induce DM, which was verified by monitoring blood pressure and serum parameters. For drug treatments, 1.2 mg/day of dapagliflozin was given for 2 weeks, 500 mg/kg/day of metformin was given, and 10 mg/kg/day of resveratrol was given. Dihydroethidium was used to stain endogenous O₂˙⁻ production in vivo of the LECs. Superoxide production was expressed in the cataract of DM, or patients without DM. Sodium–glucose cotransporter 2 (SGLT2), glucose transporter 1 (GLUT1), GLUT5, the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit p47/p67-phox, NOX4 and RAGE were significantly increased in LECs with DM. In addition, the dapagliflozin treatment reduced GLUT5, p47/p67-phox, NADPH oxidase 4 (NOX4) and receptor for advanced glycation end products (RAGE) expressions. On the contrary, metformin or resveratrol inhibited p47-phox, GLUT5, and SGLT2 expressions, but not nuclear factor erythroid 2–related factor 2 (NRF2). In summary, dapagliflozin, metformin or resveratrol down-regulated p47-phox expression through SGLT2 inactivation and ROS reduction. These important findings imply that SGLT2 can be blocked to ameliorate oxidative stress in the cataracts of DM patients.     

Exploring Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Autoproteolysis Process by Molecular Simulations: Hints for Drug Design

Proprotein convertase subtilisin/kexin 9 (PCSK9) is a notable target for the treatment of hypercholesterolemia because it regulates the population of the low-density lipoprotein receptor (LDLR) on liver cells. The PCSK9 zymogen is a serine protease that spontaneously undergoes a double self-cleavage step. Available X-ray structures depict the PCSK9 mature state, but the atomic details of the zymogen state of the enzyme are still unknown. Additionally, why the protease activity of PCSK9 is blocked after the second autoprocessing step remains unclear, as this deviates from other members of the PCSK family. By performing constant-pH molecular dynamics (MD) simulations, we investigated the protonation state of the catalytic triad of PCSK9 and found that it strongly influences the catalytic properties of the enzyme. Moreover, we determined the final step of the maturation process by classical and steered MD simulations. This study could facilitate the identification of ligands capable of interfering with the PCSK9 maturation process.     

The Course of AαVal541 as a Proteinase 3 Specific Neo-Epitope after Alpha-1-Antitrypsin Augmentation in Severe Deficient Patients

In alpha-1-antitrypsin deficiency (AATD), neutrophil serine proteases such as elastase and proteinase 3 (PR3) are insufficiently inhibited. A previous study in AATD patients showed a higher plasma level of the specific PR3-generated fibrinogen-derived peptide AαVal541, compared with healthy controls. Here, we analyzed the course of AαVal541 plasma levels during 4 weeks after a single iv dose of 240 mg/kg AAT in ten patients with genotype Z/Rare or Rare/Rare. To this end, we developed an immunoassay to measure AαVal541 in plasma and applied population pharmacokinetic modeling for AAT. The median AαVal541 plasma level before treatment was 140.2 nM (IQR 51.5–234.8 nM)). In five patients who received AAT for the first time, AαVal541 levels decreased to 20.6 nM (IQR 5.8–88.9 nM), and in five patients who already had received multiple infusions before, it decreased to 26.2 nM (IQR 22.31–35.0 nM). In 9 of 10 patients, AαVal541 levels were reduced to the median level of healthy controls (21.4 nM; IQR 16.7–30.1 nM). At 7–14 days after treatment, AαVal541 levels started to increase again in all patients. Our results show that fibrinopeptide AαVal541 may serve as a biochemical footprint to assess the efficacy of in vivo inhibition of PR3 activity in patients receiving intravenous AAT augmentation therapy.     

A Systematic Approach to Assess the Activity and Classification of PCSK9 Variants

Background: Gain of function (GOF) mutations of PCSK9 cause autosomal dominant familial hypercholesterolemia as they reduce the abundance of LDL receptor (LDLR) more efficiently than wild-type PCSK9. In contrast, PCSK9 loss of function (LOF) variants are associated with a hypocholesterolemic phenotype. Dozens of PCSK9 variants have been reported, but most remain of unknown significance since their characterization has not been conducted. Objective: Our aim was to make the most comprehensive assessment of PCSK9 variants and to determine the simplest approach for the classification of these variants. Methods: The expression, maturation, secretion, and activity of nine well-established PCSK9 variants were assessed in transiently transfected HEK293 cells by Western blot and flow cytometry. Their extracellular activities were determined in HepG2 cells incubated with the purified recombinant PCSK9 variants. Their binding affinities toward the LDLR were determined by solid-phase immunoassay. Results: LDLR expression increased when cells were transfected with LOF variants and reduced when cells were transfected with GOF variants compared with wild-type PCSK9. Extracellular activities measurements yielded exactly similar results. GOF and LOF variants had increased, respectively reduced, affinities for the LDLR compared with wild-type PCSK9 with the exception of one GOF variant (R218S) that showed complete resistance to inactivation by furin. All variants were expressed at similar levels and underwent normal maturation and secretion patterns except for two LOF and two GOF mutants. Conclusions: We propose that transient transfections of HEK293 cells with a plasmid encoding a PCSK9 variant followed by LDLR expression assessment by flow cytometry is sufficient to reliably determine its GOF or LOF status. More refined experiments should only be used to determine the underlying mechanism(s) at hand.