Background and Aims

Background: Inappropriate activation of platelet function is known to promote arterial thrombosis leading to myocardial infarction and ischaemic stroke. Dietary fat composition may represent an important modulator of platelet activation, but underlying mechanisms are unclear.

Aim: To determine the effects of replacement of dietary saturated fatty acids (SFA) with unsaturated fatty acids (UFA) on platelet function in healthy men.

Methods

Methods: In this secondary analysis of the RISSCI-1 study (ClinicalTrials.gov Identifier: NCT03270527), platelet function analysis was performed in a subgroup of men (n=51) at the University of Reading. Participants followed two sequential isoenergetic diets for 4 weeks each: high-SFA (18% of total energy (TE)) and low-SFA (≤10%TE). Light transmission platelet aggregometry and flow cytometric measurement of platelet P-selectin exposure, and fibrinogen binding were measured in response to a series of agonists at the end of each intervention diet. Platelet sensitivity to the agonists was established by calculating the EC₅₀.

Results

Results: Platelet sensitivity to a collagen receptor (GPVI) selective agonist was increased after the high compared with low SFA diet (P=0.007)(P-selectin binding, EC₅₀). A significant increase in the extent of platelet aggregation and the capacity to bind fibrinogen in response to ADP were also evident after the high compared with low SFA diet (P≤0.025). Platelet count increased, whereas platelet size decreased in whole blood after the high than low SFA diet (P≤0.01).

Conclusions

Conclusion: Our findings suggest that replacement of dietary SFA with UFA may have beneficial effects on platelet activation by decreasing the expression of P-selectin, fibrinogen binding and reducing platelet aggregation.

Background and Aims

PCSK6 is a protease strongly enriched in human liver however its function in liver has not been fully explored. Here, we aim to investigate the role of PCSK6 in lipid metabolism, and particularly in the context of atherosclerosis.

Methods

We used publically available datasets as well as biobanks to investigate the expression of PCSK6 in healthy and diseased tissues. In addition, we used Pcsk6^-/- to investigate the effect of PCSK6 ablation.

Results

Genetic analyses of the PCSK6 locus identified a variant rs7181043 that was significantly associated with PCSK6 mRNA expression in healthy human adipose tissue, liver and in atherosclerotic plaques. The same variant was associated specifically with plaque fat content and atherosclerotic patient’s plasma LDL levels. In addition, PCSK6 mRNA expression in plaques was positively correlated with total plasma cholesterol and LDL levels in atherosclerotic patients. Further analyses using public scRNAseq data of healthy human livers, revealed that PCSK6 is expressed in hepatocytes and stellate cells. Microarray comparison of the livers from Pcsk6^-/- mice and wild-type controls showed that VLDL particle assembly was one of the upregulated processes, in adipose tissue we found an increase in inflammatory infiltration and regulation of T cell mediated immunity. Preliminary in vivo studies showed that Pcsk6^-/- mice have higher plasma cholesterol and LPL levels at baseline compared to controls, and lower levels of LDLR in their liver.

Conclusions

Our data suggests that PCSK6 is involved in cholesterol and metabolic control. Further experiments are warranted in order to understand the role of PCSK6 in lipid metabolism.

Background and Aims

Background and Aims: Evolocumab and alirocumab (mAbs anti-PCSK9) block the function of PCSK9 but determine a significant increase of its plasma levels, an effect that can be determined by 1) an increased synthesis or 2) reduced clearance by the liver.

Methods

Methods: We have measured total PCSK9 plasma levels, by ELISA assay, in hypercholesterolemic patients at baseline and under treatment with mAbs anti-PCSK9. In vitro human hepatocarcinoma cell line (Huh7) was incubated with evolocumab or alirocumab and total PCSK9 and LDLR were determined.

Results

Results: Baseline plasma levels of PCSK9 (n=26 patients) was 458.8±297.4 ng/ml and increased to 1533.3±333.5 ng/ml in response to mAbs anti-PCSK9. In a second cohort of patients (n=30) we observed levels of PCSK9 equal to 1702.0±164.9 ng/ml which declined to 1360.0±344.8 ng/ml after 2 weeks post-injection.

Huh7 were incubated with simvastatin (5 µM), evolocumab (10 µg/ml) and alirocumab (10 µg/ml) for 4, 24 and 48 h. Simvastatin induced both the LDLR and PCSK9 in a time-dependent manner with maximal effect at 48 h (2.15 and 10.8-fold for LDLR and PCSK9, respectively). mAbs anti-PCSK9 induced the LDLR already after 4 h incubation (+46% and +43% for evolocumab and alirocumab, respectively). On the contrary, evolocumab and alirocumab strongly reduced both intracellular and extracellular PCSK9 at 48 h, as determined by western blot and ELISA assays (0.29 and 0.16 vs basal).

Conclusions

Conclusions: Our in vitro results suggest that alirocumab and evolocumab increase PCSK9 plasma levels by reducing its hepatic clearance.

Background and Aims

The Proprotein convertase subtilisin/kexin type 9 (PCSK9) seems to be involved in Alzheimer’s disease (AD) pathogenesis, although the mechanisms are still unknown. We investigated PCSK9 influence on cerebral lipid metabolism and neuroinflammation in human cell models of astrocytes and neurons.

Methods

The following models have been utilized: 1) human astrocytoma cells (U-373) exposed to human recombinant PCSK9; 2) human neuroblastoma cells (SH-SY5Y) overexpressing human PCSK9 and differentiated to neurons with retinoic acid.

Results

In U-373, exogenous PCSK9 reduced the expression of the LDL receptor (LDLr) and of the apoE receptor 2 (ApoER2); (-91 and -37%, respectively; p<0.05) and increased cholesterol synthesis (+44%; p<0.01), but the total cholesterol content was reduced (- 20%; p<0.05). In U373, PCSK9 did not affect plasma membrane cholesterol distribution nor efflux to isolated apoE or apoE-containing HDL. PCSK9 also worsened the inflammatory response induced by Aβ fibrils, further increasing the gene expression of IL-1β and TNF-α (p<0.05). In PCSK9-overexpressing SH-SY5Y, the uptake apoE-HDL-derived [³H]-cholesterol was significantly reduced compared to control cells (-30%; p<0.001). PCSK9 overexpression also reduced the interaction between fluorescein labelled-apoE and cells. Moreover, the expression of the ApoER2 and of the LDLr was significantly reduced (-33% and -57%, respectively; p<0.05). PCSK9 expression was associated to a reduced cell viability in Aβ fibrils-treated SH-SY5Y (-20%; p<0.05).

Conclusions

These results suggest a possible deleterious influence of PCSK9 on cerebral cholesterol metabolism and neuroinflammation. Ongoing studies will reveal whether the highlighted in vitro effects may lead to neurodegeneration in vivo, evaluating PCSK9 influence on cognitive function in AD animal models.

Background and Aims

Current methods for determining “LDL-C” in clinical practice measure the cholesterol content of both LDL and lipoprotein(a) [Lp(a)-C]. Prior studies suggested Lp(a) mass contains ~30% cholesterol. A high-throughput, sensitive and rapid method to quantitate Lp(a)-C and improve the accuracy of “LDL-C” was developed.

Methods

Lp(a) was isolated on magnetic beads linked to monoclonal antibody LPA4 recognizing apolipoprotein(a) and the cholesterol content measured with standard techniques. This method does not detect cholesterol in plasma samples lacking Lp(a) and is linear up to 747 nM Lp(a). LDL-C_corr was determined as LDL-C - Lp(a)-C.

Results

Lp(a)-C and LDL-C_corr were determined in 29 participants from a completed clinical trial of Lp(a) lowering with an antisense oligonucleotide. Baseline Lp(a) ranged from 9.0-822.8 nM, Lp(a)-C ranged from 0.6-35.0 mg/dL and correlated significantly with Lp(a) molar concentration (r=0.76, p<0.001). The percent Lp(a)-C relative to Lp(a) mass varied from 5.8–57.3% and was not affected by Lp(a) lowering. Baseline LDL-C_corr was significantly lower than “LDL-C” (mean [SD]) 102.2(31.8) versus 119.2(32.4) mg/dL, p<0.001) and did not correlate with Lp(a)-C. Three commercially-available “direct LDL-C” assays could not differentiate LDL-C from Lp(a)-C.

Conclusions

In conclusion, we developed a novel method to quantitate Lp(a)-C that suggests a broader range of Lp(a)-C relative to mass than previously reported. The use of this method to report a more accurate LDL-C will allow a re-assessment of LDL-C_corr in clinical medicine.