Modulation of vascular endothelial inflammatory response by proprotein convertase subtilisin-kexin type 9.

BACKGROUND AND AIMS: Endotoxins carried within LDL are cleared from the circulation via hepatic LDL receptor (LDLR)-mediated endocytosis. Proprotein convertase subtilisin-kexin type 9 (PCSK9) reduces this clearance by down-regulating LDLR density on hepatocytes. In addition to hepatocytes, vascular endothelial cells also express receptor targets of PCSK9, including LDLR. Therefore, we hypothesized that PCSK9 may regulate vascular endothelial cell uptake of lipopolysaccharide (LPS) and alter the vascular endothelial cell inflammatory response. METHODS AND RESULTS: We found that LPS is internalized by human umbilical vein vascular endothelial cells (HUVECs) and LPS uptake dose-dependently increased with increasing LDL concentration. Intracellular LPS co-localized with LDL. PCSK9 and, separately, blocking antibodies against LDLR, dose-dependently decreased the vascular endothelial cell uptake of LPS and, furthermore, inhibition of endocytosis using Dynasore blocked LPS uptake. In contrast, blocking antibodies against TLR4 did not alter LPS uptake. PCSK9 decreased the LPS-induced proinflammatory response (IL-6 and IL-8 gene expression and protein secretion, and VCAM-1/ICAM-1 expression) in vascular endothelial cells. In addition, a decrease in PCSK9 and increase in LDLR, mediated by triciribine or siPCSK9, increased LPS uptake and the LPS-induced proinflammatory response. Similar results were also found in aortic vascular tissue from Pcsk9-/- mice after LPS injection. CONCLUSIONS: Our data suggest that, similar to PCSK9 treatment in hepatocytes, PCSK9 reduces vascular endothelial cell uptake of LPS via LDLR-mediated endocytosis. Consequently, PCSK9 decreases the LPS-induced proinflammatory response in vascular endothelial cells. These results raise the possibility that PCSK9 inhibition may have additional effects on vascular endothelial inflammation via this alternative pathway, beyond the known effects of PCSK9 inhibition on LDL lowering and hepatic endotoxin clearance.

Very Low Density Lipoprotein Receptor Sequesters Lipopolysaccharide Into Adipose Tissue During Sepsis.

OBJECTIVES: Obese patients have lower sepsis mortality termed the "obesity paradox." We hypothesized that lipopolysaccharide, known to be carried within lipoproteins such as very low density lipoprotein, could be sequestered in adipose tissue during sepsis; potentially contributing a survival benefit. DESIGN: Retrospective analysis. SETTING: University research laboratory. SUBJECTS AND PATIENTS: Vldlr knockout mice to decrease very low density lipoprotein receptors, Pcsk9 knockout mice to increase very low density lipoprotein receptor, and Ldlr knockout mice to decrease low density lipoprotein receptors. Differentiated 3T3-L1 adipocytes. Caucasian septic shock patients. INTERVENTIONS: We measured lipopolysaccharide uptake into adipose tissue 6 hours after injection of fluorescent lipopolysaccharide into mice. Lipopolysaccharide uptake and very low density lipoprotein receptor protein expression were measured in adipocytes. To determine relevance to humans, we genotyped the VLDLR rs7852409 G/C single-nucleotide polymorphism in 519 patients and examined the association of 28-day survival with genotype. MEASUREMENTS AND MAIN RESULTS: Lipopolysaccharide injected into mice was found in adipose tissue within 6 hours and was dependent on very low density lipoprotein receptor but not low density lipoprotein receptors. In an adipocyte cell line decreased very low density lipoprotein receptor expression resulted in decreased lipopolysaccharide uptake. In septic shock patients, the minor C allele of VLDLR rs7852409 was associated with increased survival (p = 0.010). Previously published data indicate that the C allele is a gain-of-function variant of VLDLR which may increase sequestration of very low density lipoprotein (and lipopolysaccharide within very low density lipoprotein) into adipose tissue. When body mass index less than 25 this survival effect was accentuated and when body mass index greater than or equal to 25 this effect was diminished suggesting that the effect of variation in very low density lipoprotein receptor function is overwhelmed when copious adipose tissue is present. CONCLUSIONS: Lipopolysaccharide may be sequestered in adipose tissue via the very low density lipoprotein receptor and this sequestration may contribute to improved sepsis survival.

Reduced Proprotein convertase subtilisin/kexin 9 (PCSK9) function increases lipoteichoic acid clearance and improves outcomes in Gram positive septic shock patients.

Previous studies have shown lipopolysaccharide from Gram-negative bacteria is cleared from the circulation via LDL receptors on hepatocytes, which are downregulated by PCSK9. Whether clearance of Gram positive bacterial lipoteichoic acid (LTA) shows similar dependence on PCSK9, and whether this is clinically relevant in Gram positive human sepsis, is unknown. We examined survival data from three cohorts of patients who had Gram positive septic shock (n = 170, n = 130, and n = 59) and found that patients who carried a PCSK9 loss-of-function (LOF) allele had significantly higher 28-day survival (73.8%) than those with no LOF alleles (52.8%) (p = 0.000038). Plasma clearance of LTA was also found to be increased in PCSK9 knockout mice compared to wildtype control mice (p = 0.002). In addition, hepatocytes pre-treated with recombinant wildtype PCSK9 showed a dose-dependent decrease in uptake of fluorescently-labeled LTA (p < 0.01). In comparison to wildtype PCSK9, hepatocytes pre-treated with 3 different LOF variants of recombinant PCSK9 showed an increase in LTA uptake. This study shows the clearance of LTA follows a similar route as lipopolysaccharide, which is dependent on hepatic LDL receptors. This has important implications in health as strategies aimed at inhibiting PCSK9 function may be an effective treatment option for both Gram-positive and negative sepsis.

CETP genetic variant rs1800777 (allele A) is associated with abnormally low HDL-C levels and increased risk of AKI during sepsis.

High-density cholesterol (HDL-C) levels are influenced by genetic variation in several genes. Low levels of HDL-C have been associated with increased risk of acute kidney injury (AKI). We investigated whether genetic polymorphisms in ten genes known to regulate HDL-C levels are associated with both HDL-C levels and AKI development during sepsis. Two cohorts were retrospectively analyzed: Derivation Cohort (202 patients with sepsis enrolled at the Emergency Department from 2011 to 2014 in Vancouver, Canada); Validation Cohort (604 septic shock patients enrolled into the Vasopressin in Septic Shock Trial (VASST)). Associations between HDL-related genetic polymorphisms and both HDL-C levels, and risk for clinically significant sepsis-associated AKI (AKI KDIGO stages 2 and 3) were evaluated. In the Derivation Cohort, one genetic variant in the Cholesteryl Ester Transfer Protein (CETP) gene, rs1800777 (allele A), was strongly associated with lower HDL-C levels (17.4 mg/dL vs. 32.9 mg/dL, P = 0.002), greater CETP mass (3.43 µg/mL vs. 1.32 µg/mL, P = 0.034), and increased risk of clinically significant sepsis-associated AKI (OR: 8.28, p = 0.013). Moreover, the same allele was a predictor of sepsis-associated AKI in the Validation Cohort (OR: 2.38, p = 0.020). Our findings suggest that CETP modulates HDL-C levels in sepsis. CETP genotype may identify patients at high-risk of sepsis-associated AKI.

Survival benefit of a low ratio of visceral to subcutaneous adipose tissue depends on LDL clearance versus production in sepsis.

BACKGROUND: Patients with sepsis with a high ratio of visceral adipose tissue (VAT) to subcutaneous adipose tissue (SAT) have increased mortality. Our goal was to investigate the mechanism of this effect, noting that low LDL levels are also associated with increased sepsis mortality. Accordingly we tested for association between VAT/SAT, low-density lipoprotein (LDL) levels, and mortality. Then we examined the effect of statin treatment, which decreases LDL production, and the effect of PCSK9 genotype, which increases LDL clearance. METHODS: We performed retrospective analysis of a cohort of patients with sepsis from a tertiary care adult intensive care unit in Vancouver, Canada, who underwent abdominal computed tomography (CT) (n = 75) for clinical reasons. We compared LDL levels in patients with sepsis according to high versus low VAT/SAT and 90-day survival. We next examined the effects of statin therapy and PCSK9 loss-of-function genotype on survival. RESULTS: Patients with a low VAT/SAT had increased 90-day survival and were relatively protected against low LDL levels in sepsis compared to high VAT/SAT. Statin treatment abrogated the beneficial effects of low VAT/SAT; eliminating the difference in LDL levels and survival between patients with low and high VAT/SAT. PSCK9 loss-of-function genotype similarly eliminated the increased LDL levels in low VAT/SAT patients but, in contrast, increased the survival advantage of low VAT/SAT compared to high VAT/SAT. CONCLUSIONS: Low LDL levels per se are not simply associated with decreased sepsis survival because lowering LDL levels by inhibiting LDL production (statin treatment) is associated with adverse outcomes, while increased LDL clearance (PCSK9 loss-of-function genotype) is associated with improved outcomes in patients with low VAT/SAT.

Production of limit size nanoliposomal systems with potential utility as ultra-small drug delivery agents.

Previous studies from this group have shown that limit size lipid-based systems--defined as the smallest achievable aggregates compatible with the packing properties of their molecular constituents--can be efficiently produced using rapid microfluidic mixing technique. In this work, it is shown that similar procedures can be employed for the production of homogeneously sized unilamellar vesicular systems of 30-40 nm size range. These vesicles can be remotely loaded with the protonable drug doxorubicin and exhibit adequate drug retention properties in vitro and in vivo. In particular, it is demonstrated that whereas sub-40 nm lipid nanoparticle (LNP) systems consisting entirely of long-chain saturated phosphatidylcholines cannot be produced, the presence of such lipids may have a beneficial effect on the retention properties of limit size systems consisting of mixed lipid components. Specifically, a 33-nm diameter doxorubicin-loaded LNP system composed of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1,2-dipalmitoyl phosphatidylcholine (DPPC), cholesterol, and PEGylated lipid (DSPE-PEG2000) demonstrated adequate, stable drug retention in the circulation, with a half-life for drug release of ∼ 12 h. These results indicate that microfluidic mixing is the technique of choice for the production of bilayer LNP systems with sizes less than 50 nm that could lead to development of a novel class of ultra-small drug delivery vehicles.

Microfluidic Mixing: A General Method for Encapsulating Macromolecules in Lipid Nanoparticle Systems.

Previous work has shown that lipid nanoparticles (LNP) composed of an ionizable cationic lipid, a poly(ethylene glycol) (PEG) lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and small interfering RNA (siRNA) can be efficiently manufactured employing microfluidic mixing techniques. Cryo-transmission electron microscopy (cryo-TEM) and molecular simulation studies indicate that these LNP systems exhibit a nanostructured core with periodic aqueous compartments containing siRNA. Here we examine first how the lipid composition influences the structural properties of LNP-siRNA systems produced by microfluidic mixing and, second, whether the microfluidic mixing technique can be extended to macromolecules larger than siRNA. It is shown that LNP-siRNA systems can exhibit progressively more bilayer structure as the proportion of bilayer DSPC lipid is increased, suggesting that the core of LNP-siRNA systems can exhibit a continuum of nanostructures depending on the proportions and structural preferences of component lipids. Second, it is shown that the microfluidic mixing technique can also be extended to encapsulation of much larger negatively charged polymers such mRNA (1.7 kb) or plasmid DNA (6 kb). Finally, as a demonstration of the generality of the microfluidic mixing encapsulation process, it is also demonstrated that negatively charged gold nanoparticles (5 nm diameter) can also be efficiently encapsulated in LNP containing cationic lipids. Interestingly, the nanostructure of these gold-containing LNP reveals a "currant bun" morphology as visualized by cryo-TEM. This structure is fully consistent with LNP-siRNA structure predicted by molecular modeling.

Lipid nanoparticles for short interfering RNA delivery.

The discovery of RNA interference (RNAi) in mammalian cells has created a new class of therapeutics based on the reversible silencing of specific disease-causing genes. This therapeutic potential depends on the ability to deliver inducers of RNAi, such as short-interfering RNA (siRNA) and micro-RNA (miRNA), to cells of target tissues. This chapter reviews various challenges and delivery strategies for siRNA, with a particular focus on the development of lipid nanoparticle (LNP) delivery technologies. Currently, LNP delivery systems are the most advanced technology for systemic delivery of siRNA, with numerous formulations under various stages of clinical trials. We also discuss methods to improve gene silencing potency of LNP-siRNA, as well as application of LNP technologies beyond siRNA to the encapsulation of other nucleic acids such as mRNA and clustered regularly interspaced short palindromic repeats (CRISPR).

Development of lipid nanoparticle formulations of siRNA for hepatocyte gene silencing following subcutaneous administration.

Recently developed lipid nanoparticle (LNP) formulations of siRNA have proven to be effective agents for hepatocyte gene silencing following intravenous administration with at least three LNP-siRNA formulations in clinical trials. The aim of this work was to develop LNP-siRNA systems for hepatocyte gene silencing that can be administered subcutaneously (s.c.). Three parameters were investigated, namely LNP size, residence time of the polyethylene glycol (PEG)-lipid coating and the influence of hepatocyte-specific targeting ligands. LNP sizes were varied over the range of 30 to 115 nm in diameter and PEG-lipid that dissociates rapidly (PEG-DMG) and slowly (PEG-DSG) were employed. In mice, results show that large (~80 nm) LNP exhibited limited accumulation in the liver and poor Factor VII (FVII) gene silencing at 1mg siRNA/kg body weight. Conversely, small (~30 nm) LNP systems showed maximal liver accumulation yet still had minimal activity. Interestingly, intermediate size (~45 nm) LNP containing PEG-DSG exhibited nearly equivalent liver accumulation as the smaller systems following s.c. administration but reduced FVII levels by 80% at 1mg siRNA/kg body weight. Smaller systems (~35 nm diameter) containing either PEG-DMG or PEG-DSG were less active; however addition of 0.5 mol.% of a GalNAc-PEG lipid to these smaller systems improved activity to levels similar to that observed for the ~45 nm diameter systems. In summary, this work shows that appropriately designed LNP-siRNA systems can result in effective hepatocyte gene silencing following s.c administration.

Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core.

Lipid nanoparticles (LNP) containing ionizable cationic lipids are the leading systems for enabling therapeutic applications of siRNA; however, the structure of these systems has not been defined. Here we examine the structure of LNP siRNA systems containing DLinKC2-DMA(an ionizable cationic lipid), phospholipid, cholesterol and a polyethylene glycol (PEG) lipid formed using a rapid microfluidic mixing process. Techniques employed include cryo-transmission electron microscopy, (31)P NMR, membrane fusion assays, density measurements, and molecular modeling. The experimental results indicate that these LNP siRNA systems have an interior lipid core containing siRNA duplexes complexed to cationic lipid and that the interior core also contains phospholipid and cholesterol. Consistent with experimental observations, molecular modeling calculations indicate that the interior of LNP siRNA systems exhibits a periodic structure of aqueous compartments, where some compartments contain siRNA. It is concluded that LNP siRNA systems formulated by rapid mixing of an ethanol solution of lipid with an aqueous medium containing siRNA exhibit a nanostructured core. The results give insight into the mechanism whereby LNP siRNA systems are formed, providing an understanding of the high encapsulation efficiencies that can be achieved and information on methods of constructing more sophisticated LNP systems.

Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing.

Limit size systems are defined as the smallest achievable aggregates compatible with the packing of the molecular constituents in a defined and energetically stable structure. Here we report the use of rapid microfluidic mixing for the controlled synthesis of two types of limit size lipid nanoparticle (LNP) systems, having either polar or nonpolar cores. Specifically, limit size LNP consisting of 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC), cholesterol and the triglyceride triolein were synthesized by mixing a stream of ethanol containing dissolved lipid with an aqueous stream, employing a staggered herringbone micromixer. Millisecond mixing of aqueous and ethanol streams at high flow rate ratios (FRR) was used to rapidly increase the polarity of the medium, driving bottom-up synthesis of limit size LNP systems by spontaneous assembly. For POPC/triolein systems the limit size structures consisted of a hydrophobic core of triolein surrounded by a monolayer of POPC where the diameter could be rationally engineered over the range 20-80 nm by varying the POPC/triolein ratio. In the case of POPC and POPC/cholesterol (55/45; mol/mol) the limit size systems achieved were bilayer vesicles of approximately 20 and 40 nm diameter, respectively. We further show that doxorubicin, a representative weak base drug, can be efficiently loaded and retained in limit size POPC LNP, establishing potential utility as drug delivery systems. To our knowledge this is the first report of stable triglyceride emulsions in the 20-50 nm size range, and the first time vesicular systems in the 20-50 nm size range have been generated by a scalable manufacturing method. These results establish microfluidic mixing as a powerful and general approach to access novel LNP systems, with both polar or nonpolar core structures, in the sub-100 nm size range.