Welcome to the EAS 2022 Interactive Program

Background and Aims

PCSK9 is a plasma glycoprotein mainly produced by the liver which, interacting with lipids and lipoprotein receptors such as LDLR receptor(s) and CD36, can regulate their degradation. PCSK9 deficiency induces an increase in the uptake of lipoproteins, promoting the removal of circulating lipids and their accumulation in different tissues. Given the impact of PCSK9 inhibitors as a modulator of plasma lipoprotein, levels the present work was aimed to evaluate its role on cardiac functionality and metabolism.

Methods

WT, Pcsk9KO, liver selective KO and Pcsk9/Ldlr doubleKO (DKO) were fed with the SFD diet. Exercise intolerance, muscle strength, and cardiac structure were evaluated.

Results

Pcsk9 KO displays a reduced running endurance associated with echocardiographic alteration suggestive of heart failure with preserved ejection fraction (HFpEF). PCSK9 deficiency is affecting cardiac metabolism reducing mitochondrial oxygen consumption and ATP production. Concordantly, DKO show an increased LVPWT, thus excluding a contribution for LDLR on heart damage observed in Pcsk9KO mice. The cardiac function profile in the liver selective PCSK9KO model further ruled out the involvement of circulating PCSK9 in the development of HFpEF. In-vitro study on cardiomyocytes, both derived from human-iPSCs and neonatal mice confirm the crucial role of Triglycerides rich lipoprotein in cardiomyocytes lipid accumulation and mitochondrial mass depletion. Moreover, carriers of the R46L variant with loss of function for PCSK9 have an increased left ventricular mass, but a similar ejection fraction compared to control subjects.

Conclusions

PCSK9 deficiency, modulating other receptors than the LDLR, can influence cardiac lipid metabolism, thus favoring mitochondrial damage and the HFpEF development.

Background and Aims

PCSK9 may be involved in Alzheimer’s disease (AD), although the underlying mechanisms are not fully clarified. This study aims to investigate PCSK9' influence on brain cholesterol transport, essential to maintain neuronal functions, and neuroinflammation.

Methods

Human astrocytoma U373 cells exposed to exogenous PCSK9 and human neuroblastoma SH-SY5Y cells overexpressing PCSK9 have been used. The incubation with Amyloid β (Aβ) fibrils was used to reproduce AD-like conditions. Cholesterol synthesis, efflux and uptake were evaluated by radioisotopic assays, cholesterol content by a fluorometric assay, gene and protein expression through qRT-PCR and Western Blot (WB).

Results

In U373 PCSK9 increased cholesterol biosynthesis (p<0.05) and reduced LDLR and apoER2 expression (p<0.05). PCSK9 reduced intracellular cholesterol content (p<0.05), with a strengthened effect when co-incubated with Aβ (p<0.01). PCSK9 didn’t alter ABCA1- and ABCG1-mediated cholesterol efflux. Conversely, ABCA1-efflux and expression were reduced by Aβ (p<0.001). In PCSK9-overexpressing SH-SY5Y cells, apoE-HDL uptake was reduced (p<0.001) compared to control cells, independently of Aβ. PCSK9 overexpression reduced LDLR and ApoER2 expression (p<0.05). Cholesterol biosynthesis was reduced in PCSK9 overexpressing SH-SY5Y compared to controls (p<0.01), independently of Aβ. The overexpression of PCSK9 in SH-SY5Y furtherly increased the neurotoxicity induced by Aβ (p<0.05). In U373 PCSK9 enhanced the neuroinflammation induced by Aβ, increasing IL-6, IL-1β, TNF-α (p<0.05), and inflammasome Pyrin and NLRC4 gene expression although not statistically significant, with a minimal degrading effect on CD36 expression.

Conclusions

PCSK9, cooperating with Aβ, impairs brain cholesterol transport and worsened neuroinflammation, with negative consequences on neuronal survival. Hence, PCSK9 may be considered a pathogenic factor in AD.

Background and Aims

PCSK9 is the third gene involved in familial hypercholesterolemia, a major cause of atherosclerosis. Its role in regulating plasma cholesterol levels has been widely reported, and anti-PCSK9 drugs have been developed. Nevertheless, direct interactions between PCSK9 and atherosclerotic pathology are not fully elucidated.

We aim at demonstrating that PCSK9 promotes atherosclerosis through vascular smooth muscle cells (VSMC) involved in foam cells formation, atherosclerosis initiation, and progression.

Methods

Healthy and atheromatous human aortas were collected, tissues were fixed, conditioned media were obtained after 24 hours incubation in serum-free culture medium, and primary VSMC were isolated from media of healthy aortas.

Results

We showed that PCSK9, although present in foam cells from fatty streaks, fibroatheromas and absent in healthy tissues, is not synthesized in the arterial wall. PCSK9 levels were significantly higher in conditioned media from atheromatous aortas than from healthy aortas. Thus, PCSK9 penetrates through the arterial wall by outward convection from the circulation. We showed that primary VSMC uptake PCSK9 when added to the media. Moreover, LRP1 gene was highly expressed in the arterial wall of healthy and affected aortas and blocking its function using the receptor-associated protein (RAP) significantly decreased the uptake of PCSK9 by VSMC. Also, PCSK9 and LOX-1 colocalized in VSMC and PCSK9 uptake was reduced by oxidized LDL.

Conclusions

We characterized the presence of PCSK9 in human atherosclerotic tissues and demonstrated that PCSK9 present in the aortic wall is not synthesized locally. Our results suggest that PCSK9 is being internalized and metabolized through scavenger receptors expressed by VSMC.

Background and Aims

Dyslipidemia contributes to the pronounced interconnection between chronic kidney disease (CKD) and cardiovascular disease (CVD). Discovery of the role of proprotein convertase subtilisin/kexin type 9 (PCSK9) led to the development of PCSK9 inhibitors that effectively lower LDL-cholesterol concentrations. Few studies with contradicting results have investigated the association between PCSK9 serum concentrations and cardiovascular outcomes in CKD patients.

Methods

The German Chronic Kidney Disease (GCKD) study is an ongoing study with a median follow-up of 6.5 years that includes 5,217 Caucasian patients with eGFR of 30-60 mL/min/1.73 m² or an eGFR>60 mL/min/1.73m² in presence of overt proteinuria. PCSK9 serum concentrations were determined in 5,138 participants and the association of PCSK9 with prevalent and incident CVD were assessed using logistic and Cox regression analyses.

Results

At baseline, 1,289 patients had already experienced a CVD event. Increasing quartiles of PCSK9 were significantly associated with increasing CVD risk in a model adjusted for major confounders (quartiles 3 and 4 with 36% and 51% higher odds). This association was consistent in non-statin users but not in statin users. During follow-up, 473 patients experienced 3-point-MACE. Patients in PCSK9 quartiles 2-4 had a 32%-47% higher risk compared to quartile 1 (p<0.05). Only in patients with baseline CVD, higher PCSK9 levels were significantly associated with 76%-79% increased risk in the upper three quartiles.

Conclusions

Higher baseline PCSK9 concentrations in patients with moderate CKD are associated with prevalent and incident CVD. This observation would argue for a pronounced lipid-lowering therapy especially in patients with prevalent CVD.

Background and Aims

Atherosclerosis is the central pathology in cardiovascular disease. This study evaluated the effect of lipid lowering, using a novel peptide inhibiting PCSK9 and a monoclonal antibody against angiopoietin-like 3 (evinacumab), either alone or in combination.

Methods

Transgenic female APOE*3Leiden.CETP mice fed a Western diet for 16 weeks were treated with either evinacumab (EVI) once weekly sc. plus daily injections of vehicle sc or with peptide (PEP) or vehicle (VEH) with daily sc. injections. One group received the combination treatment of EVI and PEP, n=17-18. Statistics: Mann Whitney U-test.

Results

The effects on body weight, plasma lipids and atherosclerotic lesion size and severity were assessed. Treatment with EVI led to a significant decrease in plasma cholesterol (CHOL) and triglycerides (TG) as compared to VEH (-44% and -55%, both p<0.001). PEP significantly decreased CHOL and TG (-69% and -68%, both p<0.001) and the combination of EVI and PEP led to a significant decrease in CHOL and TG (-74% and -81%, both p<0.001). Atherosclerotic lesion size was reduced in all treatment groups compared to VEH: EVI: -72%, PEP -97% and EVI+PEP -98%, all p<0.001. All treatments, led to a significant decrease in the number of lesions (EVI -28%, p=0.024; PEP -79%, p<0.001 and EVI+PEP -81%, p<0.001). Lesion severity was reduced by all treatments as compared to the VEH: EVI -46%, p=0.001, PEP -85%, p<0.001 and EVI+PEP: -85%, p<0.001.

Conclusions

The data show that treatment with EVI and PEP and the combination treatment EVI+PEP strongly reduced cholesterol and triglyceride levels and atherosclerosis development in APOE*3Leiden.CETP mice.