Degradation Of Low-density Lipoprotein Receptor Research Articles

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.

Potential dual inhibitors of PCSK-9 and HMG-R from natural sources in cardiovascular risk management.

Atherosclerotic cardiovascular disease (ASCVD) stands amongst the leading causes of mortality worldwide and has attracted the attention of world's leading pharmaceutical companies in order to tackle such mortalities. The low-density lipoprotein-cholesterol (LDL-C) is considered the most prominent biomarker for the assessment of ASCVD risk. Distinct inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-R), the chief hepatic cholesterogenic enzyme, are being used since last seven decades to manage hypercholesterolemia. On the other hand, discovery and the association of proprotein convertase subtilisin/kexin type-9 (PCSK-9) with increased ASCVD risk have established PCSK-9 as a novel therapeutic target in cardiovascular medicine. PCSK-9 is well reckoned to facilitate the LDL-receptor (LDL-R) degradation and compromised LDL-C clearance leading to the arterial atherosclerotic plaque formation. The currently available HMG-R inhibitors (statins) and PCSK-9 inhibitors (siRNA, anti-sense oligonucleotides, and monoclonal antibodies) have shown great promises in achieving LDL-C lowering goals, however, their life long prescriptions have raised significant concerns. These deficits associated with the synthetic HMG-R and PCSK-9 inhibitors called for the discovery of alternative therapeutic candidates with potential dual HMG-R and PCSK-9 inhibitory activities from natural origins. Therefore, this report firstly describes the mechanistic insights into the cholesterol homeostasis through HMG-R, PCSK-9, and LDL-R functionality and then compiles the pharmacological effects of natural secondary metabolites with special emphasis on their dual HMG-R and PCSK-9 inhibitory action. In conclusion, various natural products exhibit atheroprotective effects via targeting HMG-R and PCSK-9 activities and lipoprotein metabolism, however, further clinical assessments are still warranted prior their approval for ASCVD risk management in hypercholesterolemic patients.

PCSK9 mediates dyslipidemia induced by olanzapine treatment in schizophrenia patients.

It is controversial whether dyslipidemia induced by antipsychotics in schizophrenia patients is due to weight gain or direct effects of drug treatment. However, recent evidence showed that olanzapine can cause acute dyslipidemia independent of weight change, and the underlying mechanism remains unclear. To study the role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in olanzapine-induced dyslipidemia, we analyzed in schizophrenic patients and in experimental models involving mice and cells to understand the mechanism. Disturbances in lipid homeostasis caused by 8-week olanzapine treatment were prospectively evaluated in first-episode schizophrenic patients. Additionally, mice were administered olanzapine for 5 or 8 weeks to delineate liver actions for PCSK9 contributing to olanzapine-induced dyslipidemia. Olanzapine directly affected lipid metabolism, suggesting dyslipidemia is independent of weight gain in schizophrenia patients. Olanzapine administration significantly increased plasma PCSK9, which was positively correlated with the increment in low-density lipoprotein cholesterol (LDL-C) (r=0.77, p<0.001). Increased expression of PCSK9 in liver tissue of olanzapine-treated mice occurred prior to olanzapine-induced LDL-C abnormality. Hepatic sterol regulatory element binding protein-2 (SREBP-2) protein levels increased in mice treated with olanzapine but largely declined in olanzapine (10μM) treated HepG2 cells, which suggested high concentration of olanzapine-induced PCSK9 increase was not SREBP-2-dependent. However, expressions of sterol regulatory element binding protein-1c (SREBP-1c) significantly increased in the higher dose treated groups, which was consistent with PCSK9 increases. Activation of SREBP-1c after high-dose olanzapine treatment promotes PSCK9 expression, and consequently the degradation of low-density lipoprotein receptors results in LDL-C increase. Lipid disturbances caused by olanzapine are independent of weight gain. The study explored the relationship between SREBP-1c and PCSK9 in regulating lipoprotein metabolism after olanzapine treatment in vitro and in vivo. Further exploration of olanzapine-induced PCSK9 regulatory mechanisms may help identify control points for inhibition of olanzapine-mediated dyslipidemia.

Case Series: Patients with Hypo-Responsiveness to PCSK9 Inhibitor Therapy

<h3>Lead Author's Financial Disclosures</h3> Nothing to disclose. <h3>Study Funding</h3> None. <h3>Background/Synopsis</h3> Proprotein convertase subtilisin/Kexin type 9 inhibitors (PCSK9i) [alirocumab, evolocumab] are fully human monoclonal antibodies that bind to plasma PCSK9 and prevent LDL receptor degradation. They reduce LDL-C by 50-70% and improve cardiovascular morbidity and mortality by 50% (Gouni-Berthold I.,2017). They are indicated as an adjunct to diet and maximally tolerated statin in individuals with familial hypercholesterolemia or clinical atherosclerotic cardiovascular disease who require additional LDL-C lowering. Evolocumab reduced LDL-C by 30% or greater in 99.8% patients, and alirocumab reduced LDL-C by atleast 15% in 98.9% patients (Qamar et al.,2019, Bays et al.,2018). About 87-98% achieve LDL-C goal with PCSK9i (Gouni-Berthold I.,2017). Hence, majority of patients on PCSK9i are expected to have a substantial and consistent LDL-C reduction. But, hypo-responsiveness to PCSK9i (less than 30% LDL-C reduction from baseline at all time points, i.e., beyond two standard deviations of mean for expected LDL-C reduction [Qamar et al.,2019]) are encountered more commonly in the clinical setting than expected. <h3>Objective/Purpose</h3> Assess factors contributing to hypo-responsiveness to PCSK9i. <h3>Methods</h3> A case series of five patients with hypo-responsiveness to PCSK9i reported in table 1. <h3>Results</h3> Unusual response to PCSK9i occur 3-fold higher in real-world than reported in clinical trials (Warden et al.,2020). Maximal PCSK9i LDL-C reduction occurs in 14-days (Chaudhary et al.,2017). In clinical trials, LDL-C was obtained at the end of dosing interval. Evolocumab reduces LDL-C by 85% at midpoint and by 60% at end of dosing interval (Qamar et al.,2019). Less than 15% LDL-C reduction in ODYSSEY trials was reported due to poor adherence to PCSK9i or concurrent lipid-lowering therapy (Bays et al.,2018). All our patients were adherent to treatment and had accurate injection techniques. Our patients have unique genetics and co-morbidities, which might have contributed to interindividual variation and hypo-responsiveness to PCSK9i. Elevated Lp(a) co-measured with LDL-C can contribute to suboptimal response as PCSK9i can lower Lp(a) by 15-30% only (Ruscica et al.,2020). PCSK9 and Lp(a) levels increase in nephrotic syndrome (Pavlakou et al.,2017, Hopewell et al.,2018). Disorders with very low LDL receptor activity can influence response to PCSK9i. There is a potential for immunogenicity, but anti-drug antibodies impact on clinical response to PCSK9i is unknown. There is no evidence of insufficient PCSK9 blockade, difference with switching PCSK9i, or changing injection site. <h3>Conclusions</h3> Careful medication reconciliation and injection technique assessment are the initial steps in evaluating hypo-responsiveness to PCSK9i. Understanding possible etiologies for hypo-responsiveness to PCSK9i might help advance lipid-lowering pharmacology.

Abstract 12089: Dual Actions of β 2 AR-Agonism Confer Protection Against Heart Failure and Renal Dysfunction via Inotropic and Lusitropic Effects and Normalized Cholesterol Homeostasis in a Mouse Model of Alport Syndrome

Introduction: Col4a3-/- Alport mice present a model of heart failure with preserved ejection fraction (HFpEF) secondary to chronic kidney disease(CKD) wherein etiological relationships have been established between hypertension, pulmonary edema, inflammation, cardiac hypertrophy and fibrosis, diastolic dysfunction and underlying abnormalities of elevated low-density lipoprotein receptor (LDLR) expression, excess LDL-cholesterol(LDL-C) accumulation, and mitochondrial dysfunction in renal tubules. Hypothesis: We tested the hypothesis that selective β2-Adrenoceptor (β2AR) modulation with salbutamol, a short-acting β2AR agonist, could alleviate symptoms of CKD and simultaneously augment cardiac function. Methods: Alport mice were injected i.p.with salbutamol or DMSO vehicle as a single bolus of 200μg/dose in short-term studies or daily with 100 μg/dose for 2 wks long-term. Cardiac and renal functions, cAMP levels, in vivo renal tubular LDL-C uptake and renal histology were evaluated post-injection. N=3-8 for all experiments. Results: Short-term, salbutamol improved renal function in parallel with decreased LDLR levels and reduced uptake of LDL-C into renal tubules. Long-term, cardiac diastolic function assessed by isovolumetric relaxation time, filling pressures, and myocardial performance index, and systolic function reflected by ejection fraction, stroke volume and cardiac output improved significantly in parallel with increased cardiac cAMP (p&lt;.05). Mechanistically, in the kidney, salbutamol activated IDOL and hence lysosomal ubiquitination and degradation of LDLR via a novel β2AR-mediated, cAMP-independent pathway involving the Rac1/Cdc42 β1PixGEF. β1Pix reversibly sequesters IDOL into a complex with LDLR, thereby blocking the degradation pathway. β2AR stimulation dissipates the complex reactivating IDOL-mediated LDLR degradation thereby re-establishing LDL-C homeostasis and renal function. Using flow cytometry in 293T cells, ectopic expression of bPix stabilized membrane LDLR, sensitive to IDOL- but not PCSK-mediated degradation. Conclusions: β2AR agonism represents a potential treatment strategy to alleviate progression of CKD and HF associated with HFpEF phenogroup 3.

Regulation of PCSK9 Expression and Function: Mechanisms and Therapeutic Implications.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes degradation of low-density lipoprotein receptor (LDLR) and plays a central role in regulating plasma levels of LDL cholesterol levels, lipoprotein(a) and triglyceride-rich lipoproteins, increasing the risk of cardiovascular disease. Additionally, PCSK9 promotes degradation of major histocompatibility protein class I and reduces intratumoral infiltration of cytotoxic T cells. Inhibition of PCSK9 increases expression of LDLR, thereby reducing plasma levels of lipoproteins and the risk of cardiovascular disease. PCSK9 inhibition also increases cell surface levels of major histocompatibility protein class I in cancer cells and suppresses tumor growth. Therefore, PCSK9 plays a vital role in the pathogenesis of cardiovascular disease and cancer, the top two causes of morbidity and mortality worldwide. Monoclonal anti-PCSK9 antibody-based therapy is currently the only available treatment that can effectively reduce plasma LDL-C levels and suppress tumor growth. However, high expenses limit their widespread use. PCSK9 promotes lysosomal degradation of its substrates, but the detailed molecular mechanism by which PCSK9 promotes degradation of its substrates is not completely understood, impeding the development of more cost-effective alternative strategies to inhibit PCSK9. Here, we review our current understanding of PCSK9 and focus on the regulation of its expression and functions.

PCSK9 Axis-Targeting Pseurotin A as a Novel Prostate Cancer Recurrence Suppressor Lead.

Prostate cancer (PC) is the most common malignancy and the second leading cause of cancer death in men. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an important role in the cholesterol metabolism by regulating the LDL receptor (LDLR) degradation. The PCSK9 axis is proved to be a potential novel therapeutic target in multiple cancer types. Pseurotin A (PS) is a small-molecule natural-product inhibitor of PCSK9 expression and PCSK9-LDLR protein-protein interaction (PPI). The in vitro results of this study show that PS treatments caused dose-dependent suppression of migration, colony formation, and PCSK9 expression in the PC cell lines PC-3 and 22Rv1. PS suppressed the in vivo progression of PC-3 cells orthotopically xenografted in nude mice and prevented locoregional and distant tumor recurrences after primary tumor surgical excision. Western blot analysis showed decreased PCSK9 expression in collected primary and recurred PC-3 tumors in PS-treated mice. PS treatments also reduced the hemoglobin content in collected treated tumors and the Matrigel-plug angiogenesis mouse model. PS treatments prevented metastasis to distant organs compared to vehicle-treated control mice. A reduction in mice plasma cholesterol levels was observed. Microarray analysis of collected treated primary PC-3 tumors showed a distinct gene signature that confirmed the targeting of PCSK9 and cholesterol metabolism. Thus, the PCSK9 axis is proposed as a novel PC pathogenesis molecular target, and PS is defined as a novel effective PCSK9-targeting lead potentially useful for the control of the castration-resistant PC recurrence and metastasis.

PCSK9 Activity Is Potentiated Through HDL Binding.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) circulates in a free and lipoprotein-bound form, yet the functional consequence of the association between PCSK9 and high-density lipoprotein (HDL) remains unexplored. This study sought to interrogate the novel relationship between PCSK9 and HDL in humans. Comparing lipoprotein and apolipoprotein profiles by nuclear magnetic resonance and targeted mass spectrometry measurements with PCSK9 levels in the community-based Bruneck (n=656) study revealed a positive association of plasma PCSK9 with small HDL, alongside a highly significant positive correlation between plasma levels of PCSK9 and apolipoprotein-C3, an inhibitor of lipoprotein lipase. The latter association was replicated in an independent cohort, the SAPHIR study (n=270). Thus, PCSK9-HDL association was determined during the postprandial response in two dietary studies (n=20 participants each, 8 times points). Peak triglyceride levels coincided with an attenuation of the PCSK9-HDL association, a loss of apolipoprotein-C3 from HDL and lower levels of small HDL as measured by nuclear magnetic resonance. Crosslinking mass spectrometry (XLMS) upon isolated HDL identified PCSK9 as a potential HDL-binding partner. PCSK9 association with HDL was confirmed through size-exclusion chromatography and immuno-isolation. Quantitative proteomics upon HDL isolated from patients with coronary artery disease (n=172) returned PCSK9 as a core member of the HDL proteome. Combined interrogation of the HDL proteome and lipidome revealed a distinct cluster of PCSK9, phospholipid transfer protein, clusterin and apolipoprotein-E within the HDL proteome, that was altered by sex and positively correlated with sphingomyelin content. Mechanistically, HDL facilitated PCSK9-mediated low-density lipoprotein receptor degradation and reduced low-density lipoprotein uptake through the modulation of PCSK9 internalisation and multimerisation. This study reports HDL as a binder of PCSK9 and regulator of its function. The combination of -omic technologies revealed postprandial lipaemia as a driver of PCSK9 and apolipoprotein-C3 release from HDL.

Identification of a PCSK9-LDLR disruptor peptide with in vivo function

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by promoting hepatic LDL receptor (LDLR) degradation. Therapeutic antibodies that disrupt PCSK9-LDLR binding reduce LDL-C concentrations and cardiovascular disease risk. The epidermal growth factor precursor homology domain A (EGF-A) of the LDLR serves as a primary contact with PCSK9 via a flat interface, presenting a challenge for identifying small molecule PCSK9-LDLR disruptors. We employ an affinity-based screen of 1013invitro-translated macrocyclic peptides to identify high-affinity PCSK9 ligands that utilize a unique, induced-fit pocket and partially disrupt the PCSK9-LDLR interaction. Structure-based design led to molecules with enhanced function and pharmacokinetic properties (e.g., 13PCSK9i). In mice, 13PCSK9i reduces plasma cholesterol levels and increases hepatic LDLR density in a dose-dependent manner. 13PCSK9i functions by a unique, allosteric mechanism and is the smallest molecule identified to date with invivo PCSK9-LDLR disruptor function.

Asialoglycoprotein receptor 1 is a novel PCSK9-independent ligand of liver LDLR cleaved by furin

The hepatic carbohydrate-recognizing asialoglycoprotein receptor (ASGR1) mediates the endocytosis/lysosomal degradation of desialylated glycoproteins following binding to terminal galactose/N-acetylgalactosamine. Human heterozygote carriers of ASGR1 deletions exhibit ∼34% lower risk of coronary artery disease and ∼10% to 14% reduction of non-HDL cholesterol. Since the proprotein convertase PCSK9 is a major degrader of the low-density lipoprotein receptor (LDLR), we investigated the degradation and functionality of LDLR and/or PCSK9 by endogenous/overexpressed ASGR1 using Western blot and immunofluorescence in HepG2-naïve and HepG2-PCSK9-knockout cells. ASGR1, like PCSK9, targets LDLR, and both independently interact with/enhance the degradation of the receptor. This lack of cooperativity between PCSK9 and ASGR1 was confirmed in livers of wildtype (WT) and Pcsk9−/− mice. ASGR1 knockdown in HepG2-naïve cells significantly increased total (∼1.2-fold) and cell-surface (∼4-fold) LDLR protein. In HepG2-PCSK9-knockout cells, ASGR1 silencing led to ∼2-fold higher levels of LDLR protein and DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)-LDL uptake associated with ∼9-fold increased cell-surface LDLR. Overexpression of WT-ASGR1/2 primarily reduced levels of immature non-O-glycosylated LDLR (∼110 kDa), whereas the triple Ala-mutant of Gln240/Trp244/Glu253 (characterized by loss of carbohydrate binding) reduced expression of the mature form of LDLR (∼150 kDa), suggesting that ASGR1 binds the LDLR in both a sugar-dependent and -independent fashion. The protease furin cleaves ASGR1 at the RKMK103↓ motif into a secreted form, likely resulting in a loss of function on LDLR. Altogether, we demonstrate that LDLR is the first example of a liver-receptor ligand of ASGR1. We conclude that silencing of ASGR1 and PCSK9 may lead to higher LDL uptake by hepatocytes, thereby providing a novel approach to further reduce LDL cholesterol levels.

Abstract MP257: Dual Actions Of β 2 Ar-agonism Confer Protection Against Heart Failure And Renal Dysfunction Via Inotropic And Lusitropic Effects And Normalized Cholesterol Homeostasis In A Mouse Model Of Alport Syndrome

Background: Col4a3 -/- Alport mice present a model of heart failure with preserved ejection fraction (HFpEF) secondary to chronic kidney disease (CKD) wherein etiological relationships have been established between hypertension, pulmonary edema, inflammation, cardiac hypertrophy and fibrosis, diastolic dysfunction and underlying abnormalities of elevated low-density lipoprotein receptor (LDLR) expression, excess LDL-cholesterol (LDL-C) accumulation, and mitochondrial dysfunction in renal tubules. HFpEF is characteristically unresponsive to pharmacological intervention. Here, we tested the hypothesis that selective β 2 -Adrenoceptor (β 2 AR) modulation with salbutamol, a short-acting β 2 AR agonist, could alleviate symptoms of CKD and simultaneously augment cardiac function. Secondarily, we investigated the mechanism of actions of such β 2 AR-mediated therapeutics on cardiac and renal functions. Methods: Alport mice were injected intraperitoneally with salbutamol or DMSO vehicle as a single bolus of 200μg/dose in short-term studies or daily with 100 μg/dose for 2 weeks long-term. Cardiac and renal functions, cAMP levels, in vivo renal tubular LDL-C uptake and renal histology were evaluated post-injection. In vitro mechanistic studies were performed in HK-2, Alport dog smooth muscle and tubular epithelial cells differentiated from Alport patient-derived iPSCs. Protein-protein interactions were studied using co-immunoprecipitation experiments and LDL-C uptake was measured by live-cell imaging. Results: Short-term, salbutamol improved renal function in parallel with decreased LDLR levels and reduced uptake of LDL-C into renal tubules. Long-term, cardiac diastolic function assessed by isovolumetric relaxation time (IVRT), filling pressures (E/E’), and myocardial performance index, and systolic function reflected by ejection fraction, stroke volume and cardiac output improved significantly in parallel with increased cardiac cAMP. Mechanistically, in the kidney, salbutamol activated IDOL and hence lysosomal ubiquitination and degradation of LDLR via a novel β 2 AR-mediated, cAMP-independent pathway involving the Rac1/Cdc42 β 1 PixGEF. β 1 Pix reversibly sequesters IDOL into a complex with LDLR, thereby blocking the degradation pathway. β 2 AR stimulation dissipates the complex reactivating IDOL-mediated LDLR degradation thereby re-establishing LDL-C homeostasis and renal function. Using flow cytometry in HEK293T cells, ectopic expression of bPix stabilized membrane LDLR, sensitive to IDOL- but not PCSK-mediated degradation. Conclusions: β 2 AR agonism represents a potential treatment strategy to alleviate progression of CKD and heart failure associated with HFpEF phenogroup 3.

Tollip controls atherogenesis through regulation of autophagy-mediated degradation of low-density lipoprotein receptor.

Background and Aims: Atherosclerosis is a chronic inflammatory disease, in which lipids accumulate in arterial walls and lead to plaque formation. This process is largely driven by the immune system and monocytes/macrophages are the main immune cells present in atherosclerotic lesions. In this context, high levels of circulating LDL induce its engulfment and retention by macrophages, forming foam cells. The modulation of LDL absorbance is thus wildly used to manage atherosclerosis. The complex process permitting the regulation of LDL is however not fully understood.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) association to LDL in patients treated with anti-PCSK9 monoclonal antibodies.

Background and Aims: Proprotein convertase subtilisin-kexin type 9 (PCSK9) increases low-density lipoprotein cholesterol (LDL-C) levels in plasma by enhancing the degradation of the hepatic LDL receptor (LDLR). Monoclonal antibodies (mAbs) targeting PCSK9 have shown efficacy in LDL-C reduction. PCSK9 associates to LDL in plasma. The aim of our study was to clarify whether mAbs administration could interfere with the PCSK9-LDL association.

Gypenoside LVI improves hepatic LDL uptake by decreasing PCSK9 and upregulating LDLR expression

BackgroundsAtherosclerotic Cardiovascular Disease (ASCVD) is defined as ischemic or endothelial dysfunction-various inflammatory diseases, which is mainly caused by excessive low-density lipoprotein cholesterol (LDL-C) in circulating blood. Gynostemma pentaphyllum is a traditional Chinese medicine, and total Gypenosides are used for the treatment of hyperlipidemia and to reduce circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) level. However, which gypenoside involved in the modulation of PCSK9 expression is still unknown. PurposeThis study aimed to discover effective PCSK9 inhibitors from Gypenosides in accordance with the 2019 ESC/EAS guidelines. MethodsHPLC was employed to determine major six components of Gypenosides. The inhibitory activity on secreted PCSK9 in HepG2 of six major compounds from Gypenosides were screened by ELISA. The level of low-density lipoprotein (LDL) receptor (LDLR) was determined by Flow cytometry and Immunofluorescence. The expression levels of PCSK9, LDLR and Sterol-regulatory element binding proteins-2 (SREBP-2) were analyzed by qPCR and Western blot. DiI-LDL was added to evaluated LDL uptake into HepG2. ResultsThe results suggested that the mRNA and protein levels of PCSK9 were down-regulated by Gypenoside LVI and the LDLR degradation in lysosomes system was inhibited, thereby leading to an increasing in LDL uptake into HepG2 cells. Furthermore, Gypenoside LVI decreased PCSK9 expression induced by stains. Altogether, Gypenoside LVI enhances LDL uptake into HepG2 cells by increased LDLR level on cell-surface and suppressed PCSK9 expression. ConclusionThis indicates that Gypenoside LVI can be used as a useful supplement for statins in the treatment of hypercholesterolemia. This is firstly reported that monomeric compound of G. pentaphyllum planted in Hunan province has the effect of increasing LDL-C uptake in hepatocytes via inhibiting PCSK9 expression.

Proprotein convertase subtilisin/kexin type 9 regulates the production of acute-phase reactants from the liver.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) controls blood cholesterol levels by fostering the LDL receptor (LDLR) degradation in hepatocytes. Additionally, PCSK9 has been suggested to participate in immunoregulation by modulating cytokine production. We studied the immunological role of PCSK9 in Streptococcus pneumoniae bacteraemia in vivo and in a human hepatocyte cell line. CRISPR/Cas9 mutagenesis was utilized to create pcsk9 knock-out (KO) zebrafish, which were infected with S pneumoniae to assess the role of PCSK9 for the survival of the fish and in the transcriptomic response of the liver. The direct effects of PCSK9 on the expression of acute-phase reaction (APR) genes were studied in HepG2 cells. The pcsk9 KO zebrafish lines (pcsk9tpu-13 and pcsk9tpu-2,+15 ) did not show developmental defects or gross phenotypical differences. In the S pneumoniae infected zebrafish, the mortality of pcsk9 KOs was similar to the controls. A liver-specific gene expression analysis revealed that a pneumococcal challenge upregulated pcsk9, and that the pcsk9 deletion reduced the expression of APR genes, including hepcidin antimicrobial peptide (hamp) and complement component 7b (c7b). Accordingly, silencing PCSK9 in vitro in HepG2 cells using small interfering RNAs (siRNAs) decreased HAMP expression. We demonstrate that PCSK9 is not critical for zebrafish survival in a systemic pneumococcal infection. However, PCSK9 deficiency was associated with the lower expression of APR genes in zebrafish and altered the expression of innate immunity genes in a human hepatocyte cell line. Overall, our data suggest an evolutionarily conserved function for PCSK9 in APR in the liver.

Hypercholesterolemia in Progressive Renal Failure Is Associated with Changes in Hepatic Heparan Sulfate - PCSK9 Interaction.

Dyslipidemia is an important risk factor in CKD. The liver clears triglyceride-rich lipoproteins (TRL) via LDL receptor (LDLR), LDLR-related protein-1 (LRP-1), and heparan sulfate proteoglycans (HSPGs), mostly syndecan-1. HSPGs also facilitate LDLR degradation by proprotein convertase subtilisin/kexin type 9 (PCSK9). Progressive renal failure affects the structure and activity of hepatic lipoprotein receptors, PCSK9, and plasma cholesterol. Uninephrectomy- and aging-induced CKD in normotensive Wistar rats and hypertensive Munich-Wistar-Frömter (MWF) rats. Compared with 22-week-old sex- and strain-matched rats, 48-week-old uninephrectomized Wistar-CKD and MWF-CKD rats showed proteinuria, increased plasma creatinine, and hypercholesterolemia (all P<0.05), which were most apparent in hypertensive MWF-CKD rats. Hepatic PCSK9 expression increased in both CKD groups (P<0.05), with unusual sinusoidal localization, which was not seen in 22-week-old rats. Heparan sulfate (HS) disaccharide analysis, staining with anti-HS mAbs, and mRNA expression of HS polymerase exostosin-1 (Ext-1), revealed elongated HS chains in both CKD groups. Solid-phase competition assays showed that the PCSK9 interaction with heparin-albumin (HS-proteoglycan analogue) was critically dependent on polysaccharide chain length. VLDL binding to HS from CKD livers was reduced (P<0.05). Proteinuria and plasma creatinine strongly associated with plasma cholesterol, PCSK9, and HS changes. Progressive CKD induces hepatic HS elongation, leading to increased interaction with PCSK9. This might reduce hepatic lipoprotein uptake and thereby induce dyslipidemia in CKD. Therefore, PCSK9/HS may be a novel target to control dyslipidemia.

Substantial PCSK9 inactivation in β-cells does not modify glucose homeostasis or insulin secretion in mice

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an important role in cholesterol homeostasis by promoting the degradation of the LDL receptor (LDLR). PCSK9 loss-of-function mutations are associated with increased fasting plasma glucose levels and slightly elevated risk of type 2-diabetes. Considering the known detrimental effects of cholesterol accumulation in β-cell, and the widespread use of PCSK9 inhibitors to treat hypercholesterolemia, it is important to gain insight into the role of pancreatic PCSK9 in glucose homeostasis and β-cell function. We generated the first β-cell-specific KO of PCSK9 (βKO). PCSK9 mRNA and protein expression were reduced by 48% and 78% in βKO islets, respectively, indicating that β-cells constitute a major site of PCSK9 expression. In islets, loss of β-cell PCSK9 resulted in unchanged LDLR protein levels, but reduced LDLR mRNA, indicating that cholesterol internalization is enhanced and that β-cell PCSK9 promotes LDLR degradation. In contrast, whole body PCSK9 KO mice exhibited 2-fold higher LDLR protein levels in islets and a stable expression of cholesterogenic genes. Whole body KO and βKO mice presented normal glucose tolerance, insulin release in response to glucose load and insulin sensitivity. Ex vivo glucose-stimulated insulin secretion in presence or absence of fatty acids was similar in WT and KO islets. Like KO mice, individuals carrying loss-of-function PCSK9 variants may be protected from cholesterol-induced toxicity due to reduced circulating cholesterol levels. Using both whole body KO or βKO models, our data demonstrate that PCSK9 deletion in mouse does not have any toxic effect on β-cell function and glucose homeostasis.

Circulating levels of proprotein convertase subtilisin/kexin type 9 (PCSK9) are associated with monocyte subsets in patients with stable coronary artery disease

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): FWF Background and aims Proprotein convertase subtilisin/kexin type-9 (PCSK9) is an enzyme promoting the degradation of low-density lipoprotein receptors (LDL-R) in hepatocytes. Inhibition of PCSK9 has emerged as a novel target for lipid-lowering therapy. Monocytes are crucially involved in the pathogenesis of atherosclerosis and can be divided into three subsets. The aim of this study was to examine whether circulating levels of PCSK9 are associated with monocyte subsets. Methods We included 69 patients with stable coronary artery disease. PCSK9 levels were measured and monocyte subsets were assessed by flow cytometry and divided into classical monocytes (CD14++CD16-; CM), intermediate monocytes (CD14++CD16+; IM) and non-classical monocytes (CD14 + CD16++; NCM). Results Mean age was 64 years and 80% of patients were male. Patients on statin treatment (n = 55) showed higher PCSK9-levels (245.4 (206.0-305.5) ng/mL) as opposed to those without statin treatment (186.1 (162.3-275.4) ng/mL; p = 0.05). In patients on statin treatment, CM correlated with circulating PCSK9 levels (R = 0.29; p = 0.04), while NCM showed an inverse correlation with PCSK9 levels (R=-0.33; p = 0.02). Patients with PCSK9 levels above the median showed a significantly higher proportion of CM as compared to patients with PCSK-9 below the median (83.5 IQR 79.2-86.7 vs. 80.4, IQR 76.5-85.2%; p = 0.05). Conversely, PCSK9 levels &amp;gt;median were associated with a significantly lower proportion of NCM as compared to those with PCSK9 &amp;lt;median (10.2, IQR 7.3-14.6 vs. 14.3, IQR 10.9-18.7%; p = 0.02). In contrast, IM showed no association with PCSK-9 levels. Conclusions We hereby provide a novel link between PCSK9 regulation, innate immunity and atherosclerotic disease in statin-treated patients.

Inclisiran: A Novel Agent for Lowering Apolipoprotein B-containing Lipoproteins.

Hypercholesterolemia is a leading cause of cardiovascular morbidity and mortality. Accordingly, efforts to lower apolipoprotein B-containing lipoproteins in plasma are the centerpiece of strategies for cardiovascular prevention and treatment in primary and secondary management. Despite the importance of this endeavor, many patients do not achieve appropriate low-density lipoprotein cholesterol (LDL-C) and non-high-density lipoprotein cholesterol (non-HDL-C) goals, even among those who have experienced atherosclerotic cardiovascular disease. The development of new LDL-C-lowering medications with alternative mechanisms of action will facilitate improved goal achievement in high-risk patients. Inclisiran is a novel small interfering RNA-based drug that is experimental in the United States and approved for clinical use in the European Union. It lowers LDL-C and other apolipoprotein B-containing lipoproteins by reducing production of proprotein convertase subtilisin/kexin Type 9 (PCSK9), a protein that normally contributes to LDL-receptor degradation, thereby increasing LDL-receptor density and recycling in hepatocytes. Although the lipid-lowering efficacy of inclisiran is comparable with results achieved with PCSK9-blocking monoclonal antibodies (alirocumab and evolocumab), there are several important differences between the 2 drug classes. First, inclisiran reduces levels of PCSK9 both intracellularly and extracellularly by blocking translation of and degrading PCSK9 messenger RNA. Second, the long biological half-life of inclisiran produces sustained LDL-C lowering with twice yearly dosing. Third, although PCSK9-blocking monoclonal antibodies drugs are proven to reduce atherosclerotic cardiovascular disease events, clinical outcomes trials with inclisiran are still in progress. In this article, we review the clinical development of inclisiran, its mechanism of action, lipid-lowering efficacy, safety and tolerability, and potential clinical role of this promising new agent.

Surf4 Knockdown Protects Against Cardiovascular Disease

Plasma low-density lipoprotein cholesterol (LDL-C) levels are positively correlated with risk of cardiovascular disease. LDL is produced from catabolism of very low-density lipoprotein (VLDL) that is exclusively secreted from hepatocytes. Inhibition of VLDL secretion reduces plasma LDL-C levels. LDL is cleared from circulation via hepatic LDL receptor (LDLR). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is mainly secreted from the liver and promotes LDLR degradation, increasing plasma LDL-C levels. Understanding mechanisms of VLDL and PCSK9 secretion are crucial for identifying novel therapeutic targets. Surfeit 4 (Surf4) is a cargo receptor that resides in the endoplasmic reticulum (ER). Here, we investigated its role in VLDL and PCSK9 secretion. We generated Surf4 liver specific knockout (Surf4LKO) mice and found that knockout of Surf4 did not affect PCSK9 secretion but significantly reduced plasma levels of cholesterol, triglyceride, and apolipoprotein B (apoB). In cultured human hepatocytes, Surf4 co-immunoprecipitated and co-localized with apoB100, and Surf4 silencing reduced secretion of apoB100. VLDL secretion was impaired in Surf4LKO mice, causing the retention of small lipid particles in the hepatic ER lumen. However, liver lipid levels and plasma alanine aminotransferase activity were comparable in Surf4LKO and Surf4FLOXmice. Furthermore, knockdown of Surf4 in LDLR knockout (Ldlr-/-) mice significantly reduced triglyceride secretion and plasma levels of apoB and non-HDL cholesterol but had no effect on plasma HDL cholesterol or apoA-I levels. Surf4 silencing markedly reduced the development of atherosclerosis without causing notable liver damage. Expression of stearoyl-CoA-1 (SCD1) was reduced in the liver of Surf4LKO mice and Surf4 knockdown Ldlr-/- mice. In summary, Surf4 is not required for PCSK9 secretion, instead it interacts with apoB100 and facilitates VLDL secretion. Hepatic deficiency of Surf4 reduces VLDL secretion and the development of atherosclerosis but does not cause hepatic lipid accumulation or liver damage. Thus, hepatic Surf4 can be a novel therapeutic target to reduce LDL production and the risk of cardiovascular disease. Translational perspective. Patients with homozygous familial hypercholesterolemia harboring LDLR null mutations or with autosomal recessive hypercholesterolemia cannot be effectively treated by currently lipid-lowering drugs, statins and PCSK9 inhibitors. Inhibition of VLDL secretion can markedly reduce plasma levels of cholesterol and the development of atherosclerosis. Our findings that Surf4 facilitated VLDL secretion, and inhibition of Surf4 drastically reduced plasma cholesterol levels and the development of atherosclerosis without causing notable liver damage provide critical information for the development of novel LDL production-based therapies through inhibiting hepatic Surf4.