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

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

Combined PCSK9 and APOE Polymorphisms are Genetic Risk Factors Associated with Elevated Plasma Lipid Levels in a Thai Population

Proprotein convertase subtilisin/kexin type 9 (PCSK9) and apolipoprotein E (ApoE) play a key role in the regulation of lipid metabolism. We aimed to investigate the effects of PCSK9 (R46L, I474V, and E670G) and APOE polymorphisms on lipid levels in a Southern Thai population. A total of 495 participants (307 urban, 188 rural) were recruited for the study. Anthropometric and biochemical variables were evaluated. PCSK9 and APOE polymorphisms were analyzed using PCR–RFLP. The 46L urban male carriers had significantly higher diastolic blood pressure (DBP) and fasting blood sugar compared with non‐carriers. In contrast, the 46L urban female carriers had significantly lower total cholesterol (TC) and LDL‐C levels compared with non‐carriers. The 474V rural female carriers had significantly lower HDL‐C levels than non‐carriers. The 670G urban female carriers showed significantly higher TC and LDL‐C levels compared with non‐carriers. APOE4 carriers had increased TC and LDL‐C levels relative to APOE3 carriers in the urban males. APOE2 carriers had decreased TC and/or LDL‐C levels compared with APOE3 carriers in urban males and females. A significant trend of increased TC and LDL‐C levels was observed in non‐APOE4‐PCSK9 670EE carriers to APOE4‐PCSK9 670EG carriers in urban subjects. In summary, R46L, I474V, and E670G may be genetic risk factors for cardiovascular disease (CVD) in urban males, rural females, and urban females, respectively. In contrast, R46L had a favorable lipid profiles that may protect against CVD in urban females. The combination of PCSK9 E670G and APOE polymorphisms may represent an independent factor for the determination of lipid levels.     

A Genetic Polymorphism in RBP4 Is Associated with Coronary Artery Disease

Insulin resistance and obesity is influenced by the retinol binding protein 4 (RBP4) adipokine. This study aims to determine if genetic polymorphisms in RBP4 are associated with the risk of coronary artery disease (CAD) in Chinese patients. RBP4 polymorphisms were analyzed by high resolution melting (HRM) analysis in a case-control study of 392 unrelated CAD patients and 368 controls from China. The Gensini score was used to determine the severity of CAD. The genotypic and allelic frequencies of RBP4 single-nucleotide polymorphisms were evaluated for associations with CAD and severity of disease. The A allele frequency was significantly higher in CAD case groups compared to control groups (16.7% vs. 8.8%) at the RBP4 rs7094671 locus. Compared to the G allele, this allele was associated with a higher risk of CAD (OR = 2.07 (1.50–2.84)). Polymorphisms at rs7094671 were found to associate with CAD using either a dominant or recessive model (OR, 95% CI: 1.97, 1.38–2.81; 3.81, 1.53–9.51, respectively). Adjusting for sex, history of smoking, serum TC, TG, LDL-c, and HDL-c, the risk of CAD for carriers remained significantly higher in both dominant and recessive models (OR, 95% CI: 1.68, 1.12–2.51; 2.74, 1.00–7.52, respectively). However, this SNP was not significantly associated with severity of CAD using angiographic scores in multivariable linear regression models (p = 0.373). The RBP4 rs7094671 SNP is associated with CAD; however, our results do not indicate that this locus is associated with clinical severity of CAD or the extent of coronary lesions.     

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

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

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

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

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

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

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.     

PCSK9 Biology and Its Role in Atherothrombosis

It is now about 20 years since the first case of a gain-of-function mutation involving the as-yet-unknown actor in cholesterol homeostasis, proprotein convertase subtilisin/kexin type 9 (PCSK9), was described. It was soon clear that this protein would have been of huge scientific and clinical value as a therapeutic strategy for dyslipidemia and atherosclerosis-associated cardiovascular disease (CVD) management. Indeed, PCSK9 is a serine protease belonging to the proprotein convertase family, mainly produced by the liver, and essential for metabolism of LDL particles by inhibiting LDL receptor (LDLR) recirculation to the cell surface with the consequent upregulation of LDLR-dependent LDL-C levels. Beyond its effects on LDL metabolism, several studies revealed the existence of additional roles of PCSK9 in different stages of atherosclerosis, also for its ability to target other members of the LDLR family. PCSK9 from plasma and vascular cells can contribute to the development of atherosclerotic plaque and thrombosis by promoting platelet activation, leukocyte recruitment and clot formation, also through mechanisms not related to systemic lipid changes. These results further supported the value for the potential cardiovascular benefits of therapies based on PCSK9 inhibition. Actually, the passive immunization with anti-PCSK9 antibodies, evolocumab and alirocumab, is shown to be effective in dramatically reducing the LDL-C levels and attenuating CVD. While monoclonal antibodies sequester circulating PCSK9, inclisiran, a small interfering RNA, is a new drug that inhibits PCSK9 synthesis with the important advantage, compared with PCSK9 mAbs, to preserve its pharmacodynamic effects when administrated every 6 months. Here, we will focus on the major understandings related to PCSK9, from its discovery to its role in lipoprotein metabolism, involvement in atherothrombosis and a brief excursus on approved current therapies used to inhibit its action.     

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

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

PCSK9 Imperceptibly Affects Chemokine Receptor Expression In Vitro and In Vivo

Proprotein convertase subtilin/kexin type 9 (PCSK9) is a protease secreted mainly by hepatocytes and in lesser quantities by intestines, pancreas, and vascular cells. Over the years, this protease has gained importance in the field of cardiovascular biology due to its regulatory action on the low-density lipoprotein receptor (LDLR). However, recently, it has also been shown that PCSK9 acts independent of LDLR to cause vascular inflammation and increase the severity of several cardiovascular disorders. We hypothesized that PCSK9 affects the expression of chemokine receptors, major mediators of inflammation, to influence cardiovascular health. However, using overexpression of PCSK9 in murine models in vivo and PCSK9 stimulation of myeloid and vascular cells in vitro did not reveal influences of PCSK9 on the expression of certain chemokine receptors that are known to be involved in the development and progression of atherosclerosis and vascular inflammation. Hence, we conclude that the inflammatory effects of PCSK9 are not associated with the here investigated chemokine receptors and additional research is required to elucidate which mechanisms mediate PCSK9 effects independent of LDLR.     

Macadamia Nut Consumption Modulates Favourably Risk Factors for Coronary Artery Disease in Hypercholesterolemic Subjects

Macadamia nuts are rich source of monounsaturated fats (oleic and palmitoleic acids) and contain polyphenol compounds, therefore, their consumption can be expected to impart health benefits to humans. This study was conducted to examine the effects of consuming macadamia nuts in hypercholesterolemic male individuals on plasma biomarkers of oxidative stress, coagulation and inflammation. Seventeen hypercholesterolemic male subjects were given macadamia nuts (40–90 g/day), equivalent to 15% energy intake, for a period of 4 weeks. As expected, monounsaturated fatty acids (16:1n-7, 18:1n-9 and 20:1n-9) were elevated in the plasma lipids of all volunteers following intervention with macadamia nuts. Plasma markers of inflammation (leukotriene, LTB₄) and oxidative stress (8-isoprostane) were significantly lower (1,353 ± 225 vs. 1,030 ± 129 pg/mL and 876 ± 97 vs. 679 ± 116 pg/mL, respectively) within 4 weeks following macadamia nut intervention. There was a non-significant (23.6%) reduction in the plasma TXB₂/PGI₂ ratio following macadamia nut consumption. This study demonstrates, for the first time, that short-term macadamia nut consumption modifies favourably the biomarkers of oxidative stress, thrombosis and inflammation, the risk factors for coronary artery disease, despite an increase in dietary fat intake. These data, combined with our previous results on cholesterol-lowering effects of macadamia nuts, suggest that regular consumption of macadamia nuts may play a role in the prevention of coronary artery disease.     

Interleukin-6 Receptor rs7529229 T/C Polymorphism Is Associated with Left Main Coronary Artery Disease Phenotype in a Chinese Population

Left main coronary artery disease (LMCAD) is a particular severe phenotype of coronary artery disease (CAD) and heritability. Interleukin (IL) may play important roles in the pathogenesis of CAD. Although several single nucleotide polymorphisms (SNPs) identified in IL related genes have been evaluated for their roles in inflammatory diseases and CAD predisposition, the investigations between genetic variants and CAD phenotype are limited. We hypothesized that some of these gene SNPs may contribute to LMCAD phenotype susceptibility compared with more peripheral coronary artery disease (MPCAD). In a hospital-based case-only study, we studied IL-1A rs1800587 C/T, IL-1B rs16944 G/A, IL-6 rs1800796 C/G, IL-6R rs7529229 T/C, IL-8 rs4073 T/A, IL-10 rs1800872 A/C, and IL-10 rs1800896 A/G SNPs in 402 LMCAD patients and 804 MPCAD patients in a Chinese population. Genotyping was done using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and ligation detection reaction (LDR) method. When the IL-6R rs7529229 TT homozygote genotype was used as the reference group, the CC or TC/CC genotypes were associated with the increased risk for LMCAD (CC vs. TT, adjusted odds ratio(OR) = 1.46, 95% confidence interval (CI) = 1.02–2.11, p = 0.042; CC + TC vs. TT, adjusted OR = 1.31, 95% CI = 1.02–1.69, p = 0.037). None of the other six SNPs achieved any significant differences between LMCAD and MPCAD. The present study suggests that IL-6R rs7529229 T/C functional SNP may contribute to the risk of LMCAD in a Chinese population. However, our results were limited. Validation by a larger study from a more diverse ethnic population is needed.     

Brain-Derived Neurotrophic Factor (BDNF) Is Associated with Platelet Activity and Bleeding Tendency in Patients with Gaucher Disease

Bleeding tendency, a prominent feature of patients with Gaucher disease (GD), is associated with abnormal platelet function. Brain-derived neurotrophic factor (BDNF) is a protein with neuroprotective potential stored in alpha granules of circulating platelets. Here we studied BDNF levels in 50 patients with type I GD (GD1) and their correlation with platelet activity and bleeding tendency. Flow cytometry was used to test unstimulated and stimulated measurement of platelet surface-activated expression of αIIbβ3 integrin, P-selectin and lysosomal-associated membrane protein (LAMP3/CD63). Serum and plasma BDNF levels were quantified using ELISA. The bleeding history was recorded by a bleeding questionnaire. Serum BDNF levels were positively correlated with platelet count and moderately correlated with unstimulated and stimulated platelet P-selectin expression. Patients with more than one bleeding manifestation were shown to have lower serum BDNF levels, albeit similar platelet count. Plasma BDNF levels were significantly elevated in splenectomized patients and showed a moderate positive correlation with stimulated platelet CD63 expression. These observations demonstrate the first association between BDNF levels in the peripheral blood with platelet dysfunction and increased bleeding manifestation. The role of measuring serum BDNF for assessing platelet alpha degranulation defects and bleeding risk in patients with GD and the general population needs further study.     

Peripheral Artery Disease Is Associated with a Deficiency of Erythrocyte Membrane n-3 Polyunsaturated Fatty Acids

Population-based data suggest that individuals who consume large dietary amounts of n-3 polyunsaturated fatty acids (PUFA) have lower odds of peripheral artery disease (PAD); however, clinical studies examining n-3 PUFA levels in patients with PAD are sparse. The objective of this study is to compare erythrocyte membrane fatty acid (FA) content between patients with PAD and controls. We conducted a cross-sectional study of 179 vascular surgery outpatients (controls, 34; PAD, 145). A blood sample was drawn and the erythrocyte FA content was assayed using capillary gas chromatography. We calculated the ratio of the n-3 PUFA eicosapentaenoic acid (EPA) to the n-6 PUFA arachidonic acid (ARA) as well as the omega-3 index (O3I), a measure of erythrocyte content of the n-3 PUFA, EPA, and docosahexaenoic acid (DHA), expressed as a percentage of total erythrocyte FA. Compared with controls, patients with PAD smoked more and were more likely to have hypertension and hyperlipidemia (p < 0.05). Patients with PAD had a lower mean O3I (5.0 ± 1.7% vs 6.0 ± 1.6%, p < 0.001) and EPA:ARA ratio (0.04 ± 0.02 vs 0.05 ± 0.05, p < 0.001), but greater mean total saturated fats (39.5 ± 2.5% vs 38.5 ± 2.6%, p = 0.01). After adjusting for several patient characteristics, comorbidities, and medications, an absolute decrease of 1% in the O3I was associated with 39% greater odds of PAD (odds ratio [OR] 1.39, 95% confidence interval [CI] 1.03–1.86, and p = 0.03). PAD was associated with a deficiency of erythrocyte n-3 PUFA, a lower EPA:ARA ratio, and greater mean total saturated fats. These alterations in FA content may be involved in the pathogenesis or development of poor outcomes in PAD.     

Tissue Uptake Mechanisms Involved in the Clearance of Non-Protein Nanoparticles that Mimic LDL Composition: A Study with Knockout and Transgenic Mice

Lipid core nanoparticles (LDE) resembling LDL behave similarly to native LDL when injected in animals or subjects. In contact with plasma, LDE acquires apolipoproteins (apo) E, A‐I and C and bind to LDL receptors. LDE can be used to explore LDL metabolism or as a vehicle of drugs directed against tumoral or atherosclerotic sites. The aim was to investigate in knockout (KO) and transgenic mice the plasma clearance and tissue uptake of LDE labeled with ³H‐cholesteryl ether. LDE clearance was lower in LDLR KO and apoE KO mice than in wild type (WT) mice (p < 0.05). However, infusion of human apoE3 into the apoE KO mice increased LDE clearance. LDE clearance was higher in apoA‐I KO than in WT. In apoA‐I transgenic mice, LDE clearance was lower than in apoA‐I KO and than in apoA‐I KO infusion with human HDL. Infusion of human HDL into the apoA‐I KO mice resulted in higher LDE clearance than in the apoA‐I transgenic mice (p < 0.05). In apoA‐I KO and apoA‐I KO infused human HDL, the liver uptake was greater than in WT animals and apoA‐I transgenic animals (p < 0.05). LDE clearance was lower in apoE/A‐I KO than in WT. Infusion of human HDL increased LDE clearance in those double KO mice. No difference among the groups in LDE uptake by the tissues occurred. In conclusion, results support LDLR and apoE as the key players for LDE clearance, apoA‐I also influences those processes.     

Protease-Activated Receptor 1 in Human Carotid Atheroma Is Significantly Related to Iron Metabolism,Plaque Vulnerability,and the Patient’s Age

(1) Background: Protease-activated receptor 1 (PAR1) has regulatory functions in inflammation, atherogenesis, and atherothrombosis. Chronic iron administration accelerates arterial thrombosis. Intraplaque hemorrhage and hemoglobin catabolism by macrophages are associated with dysregulated iron metabolism and atherosclerotic lesion instability. However, it remains unknown whether expressions of PAR1 in human atherosclerotic lesions are related to plaque severity, accumulation of macrophages, and iron-related proteins. We investigated the expression of PAR1 and its relation to the expression of ferritin and transferrin receptors in human carotid atherosclerotic plaques and then explored potential connections between their expressions, plaque development, and classical risk factors. (2) Methods: Carotid samples from 39 patients (25 males and 14 females) were immunostained with PAR1, macrophages, ferritin, and transferrin receptor. Double immunocytochemistry of PAR1 and ferritin was performed on THP-1 macrophages exposed to iron. (3) Results: PAR1 expression significantly increases with the patient’s age and the progression of human atherosclerotic plaques. Expressions of PAR1 are significantly correlated with the accumulation of CD68-positive macrophages, ferritin, and transferrin receptor 1 (TfR1), and inversely correlated with levels of high-density lipoprotein. In vitro, PAR1 is significantly increased in macrophages exposed to iron, and the expression of PAR1 is colocalized with ferritin expression. (4) Conclusions: PAR1 is significantly related to the progression of human atherosclerotic lesions and the patient’s age. PAR1 is also associated with macrophage infiltration and accumulation of iron metabolic proteins in human atherosclerotic lesions. Cellular iron-mediated induction of PAR1 and its colocalization with ferritin in macrophages may further indicate an important role of cellular iron in atherothrombosis.     

The PINK1 p.Asn521Thr Variant Is Associated with Earlier Disease Onset in GRN/C9orf72 Frontotemporal Lobar Degeneration

Genetic frontotemporal lobar degeneration (FTLD) is characterized by heterogeneous phenotypic expression, with a disease onset highly variable even in patients carrying the same mutation. Herein we investigated if variants in lysosomal genes modulate the age of onset both in FTLD due to GRN null mutations and C9orf72 expansion. In a total of 127 subjects (n = 74 GRN mutations and n = 53 C9orf72 expansion carriers), we performed targeted sequencing of the top 98 genes belonging to the lysosomal pathway, selected based on their high expression in multiple brain regions. We described an earlier disease onset in GRN/C9orf72 pedigrees in subjects carrying the p.Asn521Thr variant (rs1043424) in PTEN-induced kinase 1 (PINK1), a gene that is already known to be involved in neurodegenerative diseases. We found that: (i) the PINK1 rs1043424 C allele is significantly associated with the age of onset; (ii) every risk C allele increases hazard by 2.11%; (iii) the estimated median age of onset in homozygous risk allele carriers is 10–12 years earlier than heterozygous/wild type homozygous subjects. A replication study in GRN/C9orf72 negative FTLD patients confirmed that the rs1043424 C allele was associated with earlier disease onset (−5.5 years in CC versus A carriers). Understanding the potential mechanisms behind the observed modulating effect of the PINK1 gene in FTLD might prove critical for identifying biomarkers and/or designing drugs to modify the age of onset, especially in GRN/C9orf72-driven disease.     

HOXB9 Overexpression Promotes Colorectal Cancer Progression and Is Associated with Worse Survival in Liver Resection Patients for Colorectal Liver Metastases

As is known, HOXB9 is an important factor affecting disease progression and overall survival (OS) in cancer. However, its role in colorectal cancer (CRC) remains unclear. We aimed to explore the role of HOXB9 in CRC progression and its association with OS in colorectal liver metastases (CRLM). We analysed differential HOXB9 expression in CRC using the Tissue Cancer Genome Atlas database (TCGA). We modulated HOXB9 expression in vitro to assess its impact on cell proliferation and epithelial-mesenchymal transition (EMT). Lastly, we explored the association of HOXB9 protein expression with OS, using an institutional patient cohort (n = 110) who underwent liver resection for CRLM. Furthermore, HOXB9 was upregulated in TCGA-CRC (n = 644) vs. normal tissue (n = 51) and its expression levels were elevated in KRAS mutations (p < 0.0001). In vitro, HOXB9 overexpression increased cell proliferation (p < 0.001) and upregulated the mRNA expression of EMT markers (VIM, CDH2, ZEB1, ZEB2, SNAI1 and SNAI2) while downregulated CDH1, (p < 0.05 for all comparisons). Conversely, HOXB9 silencing disrupted cell growth (p < 0.0001). High HOXB9 expression (HR = 3.82, 95% CI: 1.59–9.2, p = 0.003) was independently associated with worse OS in CRLM-HOXB9-expressing patients after liver resection. In conclusion, HOXB9 may be associated with worse OS in CRLM and may promote CRC progression, whereas HOXB9 silencing may inhibit CRC growth.     

Deposition of Free Cholesterol in the Blood Vessels of Patients with Coronary Artery Disease: a Possible Novel Mechanism for Atherogenesis

A cholesterol-rich nanoemulsion (LDE) that mimics the composition of low-density lipoprotein (LDL) acquires apoE in the plasma and is taken-up by the cells by LDL receptors. In this study, to verify whether free cholesterol (FC) and the cholesteryl ester (CE) components of LDL are taken-up differently by the vessels. LDE labeled with ³H-cholesterol and ¹⁴C-cholesteryl oleate was injected into 20 coronary artery disease patients 24 h before a scheduled myocardial coronary artery bypass grafting. The plasma kinetics of both radiolabels was determined from plasma samples collected over 24 h, and fragments of vessels discarded during surgery were collected and analyzed for radioactivity. LDE FC was removed faster than CE. The radioactive counting of LDE CE was greater than that of LDE FC in the blood, but the uptake of FC was markedly greater than that of CE in all fragments: fivefold greater in the aorta (p = 0.04), fourfold greater in the internal thoracic artery (p = 0.03), tenfold greater in the saphenous vein (p = 0.01) and threefold in the radial artery (p = 0.05). In conclusion, the greater removal from plasma of FC compared with CE and the remarkably greater vessel tissue uptake of FC compared with CE suggests that, in the plasma, FC dissociates from the nanoemulsion particles and precipitates in the vessels. Considering LDE as an artificial nanoemulsion model for LDL, our results suggest that dissociation of FC from lipoprotein particles and deposition in the vessel wall may play a role as an independent mechanism in atherogenesis.