Degradation Of Low-density Lipoprotein Receptor Research Articles

Engineering lentivirus envelope VSV-G for liver targeted delivery of IDOL-shRNA to ameliorate hypercholesterolemia and atherosclerosis

Lentiviral vectors (LVs) have been widely used as a tool for gene therapies. However, tissue-selective transduction after systemic delivery remains a challenge. Inducible degrader of low-density lipoprotein receptor is an attractive target for treating hypercholesterolemia. Here, a liver-targeted lentiviral vector CS8-LV-shIDOL is developed by incorporating a hepatocyte-targeted peptide derived from circumsporozoite protein (CSP) into the lentivirus envelope for liver-targeted delivery of IDOL-shRNA to alleviate hypercholesterolemia. Tail-vein injection of CS8-LV-shIDOL results in extremely high accumulation in liver, and nearly undetectable in other organs in mice. Besides, it shows superior therapeutic efficacy in lowering serum LDL-C and reducing atherosclerotic lesions over unmodified LV-shIDOL in hyperlipidemic mice. Mechanically, the envelope-engineered CS8-LV-shIDOL can enter liver cells via low density lipoprotein receptor-related protein (LRP). Thus, this study provides a novel approach for liver-targeted delivery of IDOL-shRNA to treat hypercholesterolemia by using an envelope-engineered lentiviral vector, and this delivery system has great potential for liver-targeted transgene therapy.

Is It Ever Wise to Edit Wild-Type Alleles? Engineered CRISPR Alleles Versus Millions of Years of Human Evolution.

The tremendous burden of lipid metabolism diseases, coupled with recent developments in human somatic gene editing, has motivated researchers to propose population-wide somatic gene editing of PCSK9 (proprotein convertase subtilisin/kexin type 9) within the livers of otherwise healthy humans. The best-characterized molecular function of PCSK9 is its ability to regulate plasma LDL (low-density lipoprotein) levels through promoting LDL receptor degradation. Individuals with loss-of-function PCSK9 variants have lower levels of plasma LDL and reduced cardiovascular disease. Gain-of-function variants of PCSK9 are strongly associated with familial hypercholesterolemia. A new therapeutic strategy delivers CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats; CRISPR-associated protein 9) specifically to liver cells to edit the wild-type alleles of PCSK9 with the goal of producing a loss-of-function allele. This direct somatic gene editing approach is being pursued despite the availability of US Food and Drug Administration-approved PCSK9 inhibitors that lower plasma LDL levels. Here, we discuss other characterized functions of PCSK9 including its role in infection and host immunity. We explore important factors that may have contributed to the evolutionary selection of PCSK9 in several vertebrates, including humans. Until such time that more fully understand the multiple biological roles of PCSK9, the ethics of permanently editing the gene locus in healthy, wild-type populations remains highly questionable.

Liver-Specific Ionizable Lipid Nanoparticles Mediated Efficient RNA Interference to Clear "Bad Cholesterol".

High-level low-density lipoprotein cholesterol (LDL-C) plays a vital role in the development of atherosclerotic cardiovascular disease. Low-density lipoprotein receptors (LDLRs) are scavengers that bind to LDL-C in the liver. LDLR proteins are regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9), which mediates the degradation of LDLR and adjusts the level of the plasma LDL-C. The low expression of PCSK9 leads to the up-regulation of liver LDLRs and the reduction of plasma LDL-C. Hepatocytes are attractive targets for small interfering RNA (siRNA) delivery to silence Pcsk9 gene, due to their significant role in LDL-C regulation. Here, a type of liver-specific ionizable lipid nanoparticles is developed for efficient siRNA delivery. This type of nanoparticles shows high stability, enabling efficient cargo delivery specifically to hepatocytes, and a membrane-active polymer that reversibly masks activity until an acidic environment is reached. Significantly, the siPcsk9 (siRNA targeting to Pcsk9)-loaded nanoparticles (GLP) could silence 90% of the Pcsk9 mRNA in vitro. In vivo study showed that the improved accumulation of GLP in the liver increased LDLR level by 3.35-fold and decreased plasma LDL-C by 35%. GLP has shown a powerful effect on reducing LDL-C, thus providing a potential therapy for atherosclerotic cardiovascular disease.

The Link between miRNAs and PCKS9 in Atherosclerosis.

Cardiovascular disease (CDV) represents the major cause of death globally. Atherosclerosis, as the primary cause of CVD, is a chronic immune-inflammatory disorder with complex multifactorial pathophysiology encompassing oxidative stress, enhanced immune-inflammatory cascade, endothelial dysfunction, and thrombosis. An initiating event in atherosclerosis is the subendothelial accumulation of low-density lipoprotein (LDL), followed by the localization of macrophages to fatty deposits on blood vessel walls, forming lipid-laden macrophages (foam cells) that secrete compounds involved in plaque formation. Given the fact that foam cells are one of the key culprits that underlie the pathophysiology of atherosclerosis, special attention has been paid to the investigation of the efficient therapeutic approach to overcome the dysregulation of metabolism of cholesterol in macrophages, decrease the foam cell formation and/or to force its degradation. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory serine proteinase that has emerged as a significant regulator of the lipid metabolism pathway. PCSK9 activation leads to the degradation of LDL receptors (LDLRs), increasing LDL cholesterol (LDL-C) levels in the circulation. PCSK9 pathway dysregulation has been identified as one of the mechanisms involved in atherosclerosis. In addition, microRNAs (miRNAs) are investigated as important epigenetic factors in the pathophysiology of atherosclerosis and dysregulation of lipid metabolism. This review article summarizes the recent findings connecting the role of PCSK9 in atherosclerosis and the involvement of various miRNAs in regulating the expression of PCSK9-related genes. We also discuss PCSK9 pathway-targeting therapeutic interventions based on PCSK9 inhibition, miRNA levels manipulation by therapeutic agents, and the most recent advances in PSCK9 gene editing using CRISPR/Cas9 platform, meganuclease, and base editors.

Efficacy and safety of alirocumab in children and adolescents with heterozygous familial hypercholesterolaemia inadequately controlled with statins

Abstract Introduction Alirocumab targets the low-density lipoprotein receptor (LDL-R) regulator proprotein convertase subtilisin/kexin type 9 (PCSK9), preventing LDL-R degradation and reducing LDL-cholesterol (LDL-C) level. Purpose This international Phase 3 trial assessed the efficacy and safety of alirocumab for paediatric patients with heterozygous familial hypercholesterolaemia (HeFH) inadequately controlled with statins. Methods Patients aged 8–17 years with HeFH and LDL-C ≥130 mg/dL who were receiving a stable dose of statin (or non-statin therapy if statin-intolerant) were randomised 2:1 in two consecutive cohorts (dosing every 2 weeks [Q2W] or every 4 weeks [Q4W]) to receive subcutaneous injections of alirocumab or placebo equivalent for 24 weeks (double-blind [DB] treatment period). Dosage was adjusted based on body weight (40 mg Q2W/150 mg Q4W if <50 kg; 75 mg Q2W/300 mg Q4W if ≥50 kg). Dosage was up-titrated from Week 12 if LDL-C was ≥110 mg/dL at Week 8. After completing Week 24, patients could enter an open-label (OL) period during which they received alirocumab treatment for up to 80 weeks. The primary endpoint was difference in LDL-C percentage change from baseline to Week 24 between alirocumab and placebo. Key secondary endpoints assessed the efficacy of alirocumab vs placebo for other lipid parameters. Results Of 153 patients enrolled, 74 were randomised to Q2W treatment (49 alirocumab, 25 placebo) and 79 to Q4W treatment (52 alirocumab, 27 placebo). Baseline characteristics were generally similar between treatment groups. For Q2W and Q4W cohorts, mean (standard deviation [SD]) age was 12.8 (2.6) vs 13.0 (3.0) years, 51% vs 62% were female, mean baseline LDL-C was 171.6 (47.7) vs 176.7 (52.0) mg/dL, and 3/74 vs 8/79 were statin intolerant. At Week 24, least squares (LS) mean difference was −43.3% (97.5% CI: −56.0, −30.7; p<0.0001) for alirocumab Q2W vs placebo Q2W and −33.8% (97.5% CI: −46.4, −21.2; p<0.0001) for alirocumab Q4W vs placebo Q4W (Figure 1). Alirocumab was associated with consistent reduction of LDL-C from baseline to Week 24 vs placebo across dosing cohorts. The first 12 key secondary endpoints in the testing hierarchy (Table 1) showed significant treatment differences in favour of alirocumab at Week 24 for both dosing regimens. Overall, 145 patients entered the OL treatment period; LS mean (SD) change in LDL-C from baseline to Week 104 was similar for alirocumab Q2W (−26.3% [28.0]) and alirocumab Q4W (−23.9% [33.5]). No clinically meaningful differences in AEs (any level) were observed between treatment groups in the DB or OL treatment periods, and no safety concerns were observed in the 4 patients (3 in DB and 1 in OL treatment period) who developed anti-alirocumab antibodies. Conclusions Both dosing regimens of alirocumab were associated with significantly reduced LDL-C and other atherogenic lipid parameters in paediatric patients with HeFH and were well tolerated, with efficacy sustained over time.

Sirtuin-1 directly binds and deacetylates hepatic PCSK9 to inhibit low-density lipoprotein receptor degradation

Abstract Background Low-density lipoprotein cholesterol (LDL-C) is causally involved in atherosclerotic cardiovascular disease (ASCVD). We have previously shown that pharmacological activation of Sirtuin-1 (SIRT1), a NAD+-dependent deacetylase, reduces plasma LDL-C levels by decreasing PCSK9 and increasing hepatic LDLR expression, thus exerts atheroprotective effects in mice; however, the mechanism of SIRT1 action remains elusive. Purpose We found diminished levels of circulating SIRT1 in atherosclerotic mice compared to wild-type healthy controls. We hypothesized that this decrease in SIRT1 levels may promote dysregulated lipid metabolism and ASCVD in humans. Herein, we aimed to restore the reduced systemic Sirtuin-1 plasma levels in ASCVD by injecting recombinant murine Sirtuin-1 (rmSIRT1) in atherosclerotic mice. Methods Twelve-week-old apolipoprotein E-deficient (ApoE-/-) male mice fed a high-cholesterol diet (1.25% w/w) were randomized to receive rmSIRT1 (n=6; 0.3mg/kg BW i.p.) or vehicle treatment (n=6) every third day over 4-weeks. Mice were euthanized and organs were harvested to study lipid metabolism and ASCVD progression. Human hepatocytes (Huh7) cells were used to perform radioactive assays to study cellular uptake of radioiodinated LDL, surface plasmon resonance to study binding and site-directed mutagenesis to dissect the underlying molecular mechanism exerted by circulating SIRT1. Results Boosting circulating SIRT1 levels increased hepatic LDLR expression, reduced plasma LDL-C levels and progression of plaques in ApoE-/- mice (Figure 1). rmSIRT1 treatment did not change hepatic expression of total PCSK9 but increased its deacetylated status. Mechanistically, rmSIRT1 directly bound to hepatic PCSK9 and deacetylated PCSK9 at 3 sites: Lys243, Lys421 and Lys506 as shown by Surface Plasmon Resonance and mass spectrometry, respectively. In vitro mutagenesis to triple deacetylation mimetic (3KR) in hepatocytes (Huh7 cells) reduced SIRT1-induced PCSK9 activity, as evidenced by increased cellular binding and association of 125I-LDL through LDLR in Huh7cells. Finally, plasma levels of SIRT1 and PCSK9 were assessed at baseline in patients with acute coronary syndromes (ACS) where plasma SIRT1 showed an inverse correlation with PCSK9 and conferred a reduced risk of major adverse cardiovascular events. Conclusion SIRT1 directly binds PCSK9 and decreases its activity by deacetylation, thereby enhancing LDL-C clearance by hepatic LDLR upregulation. Increased circulating SIRT1 exerts atheroprotective effects and is associated with improved prognosis in patients with ACS.Figure 1

Role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in diabetic complications.

Cardiovascular disease (CVD) complications have remained a major cause of death among patients with diabetes. Hence, there is a need for effective therapeutics against diabetes-induced CVD complications. Since its discovery, proprotein convertase subtilisin/kexin type 9 (PCSK9) has been reported to be involved in the pathology of various CVDs, with studies showing a positive association between plasma levels of PCSK9, hyperglycemia, and dyslipidemia. PCSK9 regulates lipid homeostasis by interacting with low-density lipoprotein receptors (LDLRs) present in hepatocytes and subsequently induces LDLR degradation via receptor-mediated endocytosis, thereby reducing LDL uptake from circulation. In addition, PCSK9 also induces pro-inflammatory cytokine expression and apoptotic cell death in diabetic-CVD. Furthermore, therapies designed to inhibit PCSK9 effectively reduces diabetic dyslipidemia with clinical studies reporting reduced cardiovascular events in patients with diabetes and no significant adverse effect on glycemic controls. In this review, we discuss the role of PCSK9 in the pathogenesis of diabetes-induced CVD and the potential mechanisms by which PCSK9 inhibition reduces cardiovascular events in diabetic patients.

Legacy perfluoro-alkyl substances impair LDL-cholesterol uptake independently from PCSK9-function

Perfluoro-alkyl substances (PFAS) are pollutants, whose exposure was associated with altered levels of low-density lipoproteins (LDL) in humans. Here we investigated this clinical outcome in two groups of young male adults residing in areas of respectively low and high environmental exposure to perfluoro-octanoic-acid (PFOA). From the Regional Authority data on pollution areas, 38 not-exposed and 59 exposed age-matched participants were evaluated for serum levels of total cholesterol (Total-Chol), LDL-Chol, high-density lipoprotein cholesterol (HDL-Chol), triglycerides (Tgl) and chromatography quantified PFOA. Human hepato-carcinoma cell line HepG2 was exposed to PFOA or perfluoro-octane-sulfonate (PFOS), as legacy PFAAs, and C6O4 as new generation compound. Fluorimetry was used to evaluate the cell-uptake of labelled-LDL. Proprotein Convertase Subtilisin/Kexin 9 (PCSK9)-mediated LDL-receptor (LDL-R) degradation and sub-cellular localization of LDL-R were evaluated by western blot analysis. Serum levels of PFOA, were positively and significantly correlated with Total-Chol (ρ = 0.312, P = 0.002), LDL-Chol (ρ = 0.333, P = 0.001) and Tgl (ρ = 0.375, P < 0.001). Participants with high serum LDL-Chol and Tgl levels, according to the cardiovascular risk, were more prevalent in exposed compared to not-exposed subjects (respectively: 23.7% vs 5.3%, P = 0.023 and 18,6% vs 0%, P = 0.006). Exposure of HepG2 cells to PFOA or C6O4 100 ng/mL was associated with a significantly lower LDL uptake than controls but no major impact of any PFAAs on PCSK9-mediated LDL-R degradation was observed. Compared to controls, exposure to PFAS showed an unbalanced LDL-R partition between membrane and cytoplasm. Endocytosis inducer sphingosine restored LDL-R partition only in samples exposed to C6O4. These data suggest a novel endocytosis-based mechanism of altered lipid trafficking associated with the exposure to legacy PFAS.

Correlation between Plasma PCSK9 and Blood Lipids in Patients with Newly Diagnosed Primary Nephrotic Syndrome

Abstract Background Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a major post-transcriptional regulator of low-density lipoprotein receptor degradation. Recently, PCSK9 was shown to be overexpressed by liver cells in rats with proteinuria. However, the levels of PCSK9 in newly diagnosed primary nephrotic syndrome (PNS) patients and correlations involving PCSK9 and blood lipids are not clearly understood. Aim of the Work The aim of this study is to assess the association between plasma pcsk 9 and blood lipids in patients with newly diagnosed primary nephrotic syndrome Patients and Methods This was a case control study in Ain Shams University hospital for 4 months on 50 patients, 35 control. Results in our study elevated plasma PCSK9 levels had a positive linear correlation with elevated serum TC (p value &amp;lt;0.001) and LDL-C (p value &amp;lt;0.001), triglycerides (p value 0.003), HDL (p value 0.012), VLDL(p value 0.001) A comparison of plasma PCSK9 levels of MCD, MN And primary focal segmental patients showed that they were not significantly different (p value 0.954). In our study, we found that the plasma levels of pcsk9 were significantly higher than in healthy controls 314.580 ± 73.835 ng/ml vs 253.429 ±36.666, and this difference was statistically significant P-value &amp;lt;0.001. In our study plasma PCSK9 levels in patients with PNS had no linear correlation with the amount of proteinuria per 24 h, p value 0.070 or with albumin p value 0.549 and there was no statistical difference between the the two groups of proteinuria (3.5-8 (g)/24 h vs &amp;gt; 8 (g)/24 h) p value 0.378. Conclusion Plasma PCSK9 levels in newly diagnosed primary nephrotic patients were markedly increased and positively correlated with total cholesterol, VLDL, LDL, HDL. suggesting that PCSK9 may emerge as a novel therapeutic target in nephrotic syndrome associated hypercholesterolemia

Inclisiran and cardiovascular events: a comprehensive review of efficacy, safety, and future perspectives.

This review aims to offer an up-to-date evaluation of Inclisiran's (a small interfering RNA treatment) ability to decrease low-density lipoprotein cholesterol (LDL-C), as well as its safety and potential effects on decreasing cardiovascular risk. Inclisiran significantly lowers LDL-C levels, as shown by phase III studies, by inhibiting hepatic synthesis of proprotein convertase subtilisin kexin 9 (PCSK-9), a protein implicated in the degradation of LDL receptors. Inclisiran has the benefit of subcutaneous injection twice a year, which may reduce patient nonadherence when compared with other LDL-C reducing therapies such as statins and ezetimibe, which require daily dosing. When added on top of statins, a greater proportion of patients achieved recommended cholesterol goals. It has also demonstrated a good safety profile with few adverse effects. Inclisiran is a promising treatment for lowering LDL-C levels in people at high risk of atherosclerotic cardiovascular disease. It is a practical and well tolerated option for those who struggle to stick to medication regimes because of its twice-yearly dosage schedule and a good safety profile. Although it has been demonstrated to be effective in decreasing LDL-C, further research is needed to determine its impact on reducing cardiovascular events. Nonetheless, Inclisiran is a significant advancement in lipid-lowering medication and could improve patient outcomes.

The Effects of Statins, Ezetimibe, PCSK9-Inhibitors, Inclisiran, and Icosapent Ethyl on Platelet Function.

This review aims to examine the complex interaction between dyslipidemia, platelet function, and related drug treatments. In particular, the manuscript provides an overview of the effects of major hypolipidemic drugs on platelet function. Indeed, growing evidence supports the view that statins, ezetimibe, PCSK9 inhibitors, inclisiran, and icosapent ethyl also act as antithrombotics. It is known that platelets play a key role not only in the acute phase of coronary syndromes but also in the early phase of atherosclerotic plaque formation. The goal of cholesterol-lowering therapy is to reduce cardiovascular events. The direct effects of cholesterol-lowering drugs are widely described in the literature. Lowering LDL-c (low-density lipoprotein cholesterol) by 1 mmol/L results in a 22-23% reduction in cardiovascular risk. Numerous studies have examined the direct antithrombotic effects of these drugs on platelets, endothelium, monocytes, and smooth muscle cells, and thus, potentially independent of blood LDL-cholesterol reduction. We reviewed in vitro and in vivo studies evaluating the complex interaction between hypercholesterolemia, hypertriglyceridemia, platelet function, and related drug treatments. First, we discussed the role of statins in modulating platelet activation. Discontinuation of statin therapy was associated with increased cardiovascular events with increased ox-LDL, P-selectin, and platelet aggregation. The effect of PCSK9-I (inhibitors of proprotein convertase subtilisin/kexin type 9, PCSK9 involved in the degradation of LDL receptors in the liver) was associated with a statistically significant reduction in platelet reactivity, calculated in P2Y12 reaction units (PRU), in the first 14 days and no difference at 30 days compared to placebo. Finally, in patients with hypertriglyceridemia, the REDUCE-IT study showed that icosapent ethyl (an ethyl ester of eicosapentaenoic acid that reduces triglyceride synthesis and improves triglyceride clearance) resulted in a 25% reduction in ischemic events and cardiovascular death. However, to date, there is not yet clear clinical evidence that the direct antithrombotic effects of the drugs may have a beneficial impact on outcomes independently from the reduction in LDL-C or triglycerides.

Liver X Receptor-Inducible Host E3 Ligase IDOL Targets a Human Cytomegalovirus Reactivation Determinant.

Liver X receptor (LXR) signaling broadly restricts virus replication; however, the mechanisms of restriction are poorly defined. Here, we demonstrate that the cellular E3 ligase LXR-inducible degrader of low-density lipoprotein receptor (IDOL) targets the human cytomegalovirus (HMCV) UL136p33 protein for turnover. UL136 encodes multiple proteins that differentially impact latency and reactivation. UL136p33 is a determinant of reactivation. UL136p33 is targeted for rapid turnover by the proteasome, and its stabilization by mutation of lysine residues to arginine results in a failure to quiet replication for latency. We show that IDOL targets UL136p33 for turnover but not the stabilized variant. IDOL is highly expressed in undifferentiated hematopoietic cells where HCMV establishes latency but is sharply downregulated upon differentiation, a stimulus for reactivation. We hypothesize that IDOL maintains low levels of UL136p33 for the establishment of latency. Consistent with this hypothesis, knockdown of IDOL impacts viral gene expression in wild-type (WT) HCMV infection but not in infection where UL136p33 has been stabilized. Furthermore, the induction of LXR signaling restricts WT HCMV reactivation from latency but does not affect the replication of a recombinant virus expressing a stabilized variant of UL136p33. This work establishes the UL136p33-IDOL interaction as a key regulator of the bistable switch between latency and reactivation. It further suggests a model whereby a key viral determinant of HCMV reactivation is regulated by a host E3 ligase and acts as a sensor at the tipping point between the decision to maintain the latent state or exit latency for reactivation. IMPORTANCE Herpesviruses establish lifelong latent infections, which pose an important risk for disease particularly in the immunocompromised. Our work is focused on the betaherpesvirus human cytomegalovirus (HCMV) that latently infects the majority of the population worldwide. Defining the mechanisms by which HCMV establishes latency or reactivates from latency is important for controlling viral disease. Here, we demonstrate that the cellular inducible degrader of low-density lipoprotein receptor (IDOL) targets a HCMV determinant of reactivation for degradation. The instability of this determinant is important for the establishment of latency. This work defines a pivotal virus-host interaction that allows HCMV to sense changes in host biology to navigate decisions to establish latency or to replicate.

A Review of Progress on Targeting LDL Receptor-Dependent and -Independent Pathways for the Treatment of Hypercholesterolemia, a Major Risk Factor of ASCVD.

Since the discovery of the LDL receptor in 1973 by Brown and Goldstein as a causative protein in hypercholesterolemia, tremendous amounts of effort have gone into finding ways to manage high LDL cholesterol in familial hypercholesterolemic (HoFH and HeFH) individuals with loss-of-function mutations in the LDL receptor (LDLR) gene. Statins proved to be the first blockbuster drug, helping both HoFH and HeFH individuals by inhibiting the cholesterol synthesis pathway rate-limiting enzyme HMG-CoA reductase and inducing the LDL receptor. However, statins could not achieve the therapeutic goal of LDL. Other therapies targeting LDLR include PCSK9, which lowers LDLR by promoting LDLR degradation. Inducible degrader of LDLR (IDOL) also controls the LDLR protein, but an IDOL-based therapy is yet to be developed. Among the LDLR-independent pathways, such as angiopoietin-like 3 (ANGPTL3), apolipoprotein (apo) B, apoC-III and CETP, only ANGPTL3 offers the advantage of treating both HoFH and HeFH patients and showing relatively better preclinical and clinical efficacy in animal models and hypercholesterolemic individuals, respectively. While loss-of-LDLR-function mutations have been known for decades, gain-of-LDLR-function mutations have recently been identified in some individuals. The new information on gain of LDLR function, together with CRISPR-Cas9 genome/base editing technology to target LDLR and ANGPTL3, offers promise to HoFH and HeFH individuals who are at a higher risk of developing atherosclerotic cardiovascular disease (ASCVD).

PCSK9 inhibition interrupts the cross-talk between keratinocytes and macrophages and prevents UVB-induced skin damage

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme that promotes the degradation of low-density lipoprotein receptors. It is involved in hyperlipidemia as well as other diseases, such as cancer and skin inflammation. However, the detailed mechanism for PCSK9 on ultraviolet B (UVB)-induced skin lesions was not clear. Thus, the role and possible action mechanism of PCSK9 in UVB-induced skin damage in mice were studied here using siRNA and a small molecule inhibitor (SBC110736) against PCSK9. Immunohistochemical staining revealed a significant increase in PCSK9 expression after UVB exposure, indicating the possible role of PCSK9 in UVB damage. Skin damage, increase in epidermal thickness, and keratinocyte hyperproliferation were significantly alleviated after treatment with SBC110736 or siRNA duplexes, compared with that in the UVB model group. Notably, UVB exposure triggered DNA damage in keratinocytes, whereas substantial interferon regulatory factor 3 (IRF3) activation was observed in macrophages. Pharmacologic inhibition of STING or cGAS knockout significantly reduced UVB-induced damage. In the co-culture system, supernatant from UVB-treated keratinocyte induced IRF3 activation in macrophages. This activation was inhibited with SBC110736 and by PCSK9 knockdown. Collectively, our findings reveal that PCSK9 plays a critical role in the crosstalk between damaged keratinocytes and STING activation in macrophages. The interruption of this crosstalk by PCSK9 inhibition may be a potential therapeutic strategy for UVB-induced skin damage.

Comprehensive computational study in the identification of novel potential cholesterol lowering agents targeting proprotein convertase subtilisin/kexin type 9

The enzymatic target proprotein convertase subtilisin/kexin type 9 (PCSK9) is critically involved in the regulation of the lipoprotein metabolism leading to the degradation of low-density lipoprotein receptors (LDLRs) upon binding. Drugs that lower LDL cholesterol (LDL-C) through the inhibition of PCSK9 are useful in the management of hypercholesterolemia which greatly reduces the associated risk of atherosclerotic cardiovascular disease (CVD). In 2015, anti-PCSK9 monoclonal antibodies (mAbs), alirocumab and evolocumab were approved but owing to their high costs their prior authorization practices were impeded, reducing their long-term adherence. This has drawn considerable attention for the development of small-molecule PCSK9 inhibitors. In this research work, novel and diverse molecules with affinity towards PCSK9 thereby having ability to lower cholesterol. A hierarchical multistep docking was implemented to identify small molecules from chemical libraries with a score cutoff −8.00 kcal/mol, thereby weeding all the non-potential molecules. A set of seven representative molecules Z1139749023, Z1142698190, Z2242867634, Z2242893449, Z2242894417, Z2242909019, and Z2242914794 have been identified from a comprehensive computational study which included assessment of pharmacokinetics and toxicity profiles and binding interactions along with in-depth analysis of structural dynamics and integrity using prolong molecular dynamics (MD) simulation (in-duplicate). Furthermore the binding affinity of these PCSK9 inhibitory candidates molecules was ascertained over 1000 trajectory frames using MM-GBSA calculations. The molecules reported herein are propitious candidates for further development through necessary experimental considerations. Communicated by Ramaswamy H. Sarma

Delivery of low-density lipoprotein from endocytic carriers to mitochondria supports steroidogenesis.

The low-density lipoprotein (LDL) is a major cholesterol carrier in circulation and is internalized into cells through LDL receptor (LDLR)-mediated endocytosis. The LDLR protein is highly expressed in the steroidogenic organs and LDL cholesterol is an important source for steroidogenesis. Cholesterol must be transported into the mitochondria, where steroid hormone biosynthesis initiates. However, how LDL cholesterol is conveyed to the mitochondria is poorly defined. Here, through genome-wide small hairpin RNA screening, we find that the outer mitochondrial membrane protein phospholipase D6 (PLD6), which hydrolyses cardiolipin to phosphatidic acid, accelerates LDLR degradation. PLD6 promotes the entrance of LDL and LDLR into the mitochondria, where LDLR is degraded by mitochondrial proteases and LDL-carried cholesterol is used for steroid hormone biosynthesis. Mechanistically, the outer mitochondrial membrane protein CISD2 binds to the cytosolic tail of LDLR and tethers LDLR+ vesicles to the mitochondria. The fusogenic lipid phosphatidic acid generated by PLD6 facilitates the membrane fusion of LDLR+ vesicles with the mitochondria. This intracellular transport pathway of LDL-LDLR bypasses the lysosomes and delivers cholesterol to the mitochondria for steroidogenesis.

PCSK9 inhibitors for acute coronary syndrome: the era of early implementation.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, a new cholesterol-lowering strategy, can decrease low-density lipoprotein cholesterol (LDL-C) levels by inhibiting PCSK9 and reducing the degradation of LDL receptors; thus, they are impacting the management of dyslipidemia to the prevention of cardiovascular events. Recent guidelines recommend PCSK9 inhibitors for patients who fail to achieve target lipids after ezetimibe/statin therapy. As PCSK9 inhibitors have been demonstrated to significantly and safely reduce LDL-C, discussions have begun to explore its optimal timing in coronary artery disease, especially in subjects with acute coronary syndrome (ACS). Also, their additional benefits, such as anti-inflammatory effects, plaque regression effects, and cardiovascular event prevention, have become the focus of recent research. Several studies, including EPIC-STEMI, suggest the lipid-lowering effects of early PCSK9 inhibitors in ACS patients, while some studies such as PACMAN-AMI suggest that early PCSK9 inhibitors can decelerate plaque progression and reduce short-term risks of cardiovascular events. Thus, PCSK9 inhibitors are entering the era of early implementation. In this review, we are committed to summarizing the multidimensional benefits of early implementation of PCSK9 inhibitors in ACS.

Effect of type 2 diabetes on the inducible degrader of LDL receptor

The inducible degrader of LDL receptor (IDOL) acts as a post-transcriptional degrader of the LDL receptor (LDLR). IDOL is functionally active in the liver and in peripheral tissues. We have evaluated IDOL expression in circulating monocytes in subjects with and without type 2 diabetes and determined whether changes in IDOL expression could affect macrophage function like cytokine production in vitro. One hundred forty individuals with type 2 diabetes and 110 healthy control subjects were recruited. Cellular expression of IDOL and LDLR in peripheral blood CD14+ monocytes was measured by flow cytometry. The expression of intracellular IDOL was lower in individuals with diabetes than control (21.3 ± 4.6 mean fluorescence intensity × 1,000 vs. 23.8 ± 6.2, P < 0.01), and this was accompanied by an increase in cell surface LDLR (5.2 ± 3.0 mean fluorescence intensity × 1,000 vs. 4.3 ± 1.5, P < 0.01), LDL binding, and intracellular lipid (P < 0.01). IDOL expression correlated with HbA1c (r = −0.38, P < 0.01) and serum fibroblast growth factor-21 (FGF21) (r = −0.34, P < 0.01). Multivariable regression analysis, including age, sex, BMI, smoking, HbA1c, and log(FGF21), showed that HbA1c and FGF21 were significant independent determinants of IDOL expression. IDOL knockdown human monocyte-derived macrophages produced higher concentrations of interleukin 1 beta, interleukin 6, and TNFα than control macrophages upon stimulation with lipopolysaccharide (all P < 0.01). In conclusion, the expression of IDOL in CD14+ monocytes was decreased in type 2 diabetes and was associated with glycemia and serum FGF21 concentration.

Inclisiran-A Revolutionary Addition to a Cholesterol-Lowering Therapy.

Hypercholesterolemia plays a crucial role in the development of atherosclerosis, but it remains an undertreated and underdiagnosed disease. Taking into consideration the high prevalence of lipid disorders, long duration of the asymptomatic course of the disease, life-threatening complications resulting from inaccurate therapy, and stringent treatment goals concerning LDL cholesterol level in the prevention of cardiovascular events, novel lipid-lowering therapies have been introduced in the last few years. In this article, a drug belonging to the group of small interfering RNA (siRNA) called inclisiran is described. It is a novel molecule that increases the number of LDL receptors (LDLRs) on the surface of hepatic cells by preventing the formation of proprotein convertase subtilisin/kexin type 9 (PCSK9) responsible for the degradation of LDLRs. With great potential for lowering plasma LDL cholesterol level, high liver specificity, comfortable dosing regimen, and good tolerance without significant adverse effects, it could play an important part in future hypolipemic therapies.

Research on Hepatocyte Regulation of PCSK9-LDLR and Its Related Drug Targets.

The prevalence of hyperlipidemia has increased significantly due to genetic, dietary, nutritional and pharmacological factors, and has become one of the most common pathological conditions in humans. Hyperlipidemia can lead to a range of diseases such as atherosclerosis, stroke, coronary heart disease, myocardial infarction, diabetes, and kidney failure, etc. High circulating low-density lipoprotein cholesterol (LDL-C) is one of the causes of hyperlipidemia. LDL-C in the blood binds to LDL receptor (LDLR) and regulates cholesterol homeostasis through endocytosis. In contrast, proprotein convertase subtilisin/kexin type 9 (PCSK9) mediates LDLR degradation via the intracellular and extracellular pathways, leading to hyperlipidemia. Targeting PCSK9-synthesizing transcription factors and downstream molecules are important for development of new lipid-lowering drugs. Clinical trials regarding PCSK9 inhibitors have demonstrated a reduction in atherosclerotic cardiovascular disease events. The purpose of this review was to explore the target and mechanism of intracellular and extracellular pathways in degradation of LDLR and related drugs by PCSK9 in order to open up a new pathway for the development of new lipid-lowering drugs.