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

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.