Welcome to the EAS 2023 Interactive Program

Background and Aims

Hepatic lipid metabolism is a key determinant of plasma lipoprotein levels and its dysregulation is associated with the development of dyslipidemia and coronoary artery disease. Herein, the transcription factors Sterol-Regulatory Element Binding Proteins (SREBP) play a central role owing to their ability to govern cholesterol and fatty acid metabolism. In this study we aimed to identify novel physiological regulators of hepatic SREBP signaling and lipid metabolism.

Methods

To identify novel determinants of SREBP signaling we employed a genome-wide mammalian haploid genetic screen, which resulted in the identfication of SPRING1 (C12ORF49). We studied the consequence of gain- and loss-of SPRING function on SREBP signaling and lipid metabolism in cells. To study its physiolgical role we developed mice with liver-specific ablation of Spring.

Results

We found that abalation of SPRING in cells markedly attenuated SREBP signaling. This was a result of impaired retrograde transport of SCAP from the Golgi to the ER, as well as due to decreased maturation of a key protease, S1P, which is required for SREBP proteolytic activation. As a consequnce, cells lacking SPRING were unable to promote cholesterol biosynthesis or LDL uptake in response to sterol depletion. Mice lacking hepatic Spring had severely attenuated SREBP signaling associated with reduced cholesterol and fatty acid synthesis, reduced hepatic lipid levels, and a marked reduction in plasma cholesterol levels due, in part, to decreased VLDL secretion.

Conclusions

Our results position Spring as a cental physiological regulator of hepatic SREBP signaling and plasma lipoprotein levels.

Background and Aims

Autosomal Dominant Hypercholesterolemia (ADH) is a common monogenic disease (1/313)¹ characterized by isolated elevation of LDL-cholesterol levels. ADH is associated with a high risk of cardiovascular disease due to the early development of atherosclerosis. In 80% of cases, ADH is due to variants in 4 genes (LDLR, APOB, APOE, PCSK9) and for the 20% remaining (ADH/M-) the defect has to be identified. Inhibition of genes encoding U2-spliceosome (U2-S) has been shown to decreased LDL internalization in Huh-7 cell lines² and thus suggest these genes are candidates for ADH.

We aimed to investigate variants in the U2-S genes in a French ADH/M- cohort.

Methods

We performed next generation sequencing (NGS) of U2-S genes in a French cohort of 219 ADH/M- patients. Selected variants present a read depth >15, a <1% frequency in GnomAD³ and are predicted deleterious according to prediction tools (CADD⁴>20 or MutationTaster⁵).

Results

The sequencing highlighted 13 rare exonic variants predicted deleterious (Figure 1) and 150 in non-coding regions (131 intronic, 21 in UTRs). For the variants p.A67T in RBM25 and p.P675H in SF3A1, familial enlargement has been achieved and has shown a segregation with the ADH phenotype in each family.

Conclusions

These results suggest that variants in U2-S genes are involved in ADH/M- cases.

References :
1. Beheshti et al. J Am Coll Cardiol. 2020;75(20):2553-2566.
2. Zanoni et al. Circ Res. 2022;130(1):80-95.
3. https://gnomad.broadinstitute.org/
4. https://www.mutationtaster.org/
5. https://cadd.gs.washington.edu/snv

Background and Aims

PSCK9 and ANGPTL3 play a pivotal role in regulating circulating lipoprotein levels; however, their intracellular roles in modulating metabolic pathways are less established. Therapies aimed at targeting the gene expression of ANGPTL3 and PCSK9 are under development; therefore, the knowledge of the intracellular role of these proteins may be relevant. This study is part of a project aimed at understanding the role of the coordinated ANGPTL3 and PCSK9 modulation in cell metabolism.

Methods

We analyzed the intracellular kinases after overexpression of ANGPTL3 and PCSK9 or both in the HepG2 cell line. After 48h, we analyzed the phosphorylation levels of 49 different Receptor Tyrosine Kinase (RTKs), 37 intracellular kinases, and 2 total proteins using commercial proteome profilers.

Results

In ANGPTL3 overexpressing cells, the RTK phosphorylation levels are overall reduced, while those of IGF-1R are increased. The analysis of intracellular kinases indicated the increase of phosphorylation levels of PLC-y and WNK1, with the consequent hyper-activation of STAT3, a transcriptional factor with a role in energy homeostasis. The cells overexpressing PCSK9 activate the pathways controlled by the VEGFR3, FGFR3 and 4, Tie-1, ALK, RET, insulin receptor, and IGF-1R. The intercellular kinases analysis showed decreased levels of GSK3a and b inhibitory phosphorylation sites and the activation of STAT1. The cells overexpressing both proteins activated the FGF receptors family. On the contrary, the activation of the intracellular kinases was restored at the same level found in control cells.

Conclusions

These findings suggest that ANGPTL3 and PCSK9 are regulators of intracellular signaling related to glycogen, triglycerides, and cholesterol metabolism.

Background and Aims

To evaluate the long-term hepatic safety of lomitapide in patients with homozygous familial hypercholesterolaemia (HoFH).

Methods

Patient data were aggregated from the pivotal phase 3 and extension phase clinical trial with lomitapide (n=29) (median 5.1 years; serum total bilirubin, transaminases, cytokeratin-18 [CK-18] and enhanced liver fibrosis [ELF] score; 8-year data from the Lomitapide Observational Worldwide Evaluation Registry (LOWER) (n=214) and real-world evidence from a cohort of patients treated with lomitapide in Italy from the Italian Lomitapide Working Group in HoFH (n=34) (hepatic ultrasound, hepatic elastography, and FIB-4 score for hepatic fibrosis).

Results

In the phase 3 trial and LOWER registry, mild/moderate elevations in liver transaminase levels were observed without elevations in bilirubin (i.e., no Hy’s law cases). Evaluating hepatic biomarkers from the phase 3 dataset confirmed there were no clinically relevant increases in CK-18, CK-18 fragments, or ELF score.

Data from LOWER affirmed that no specific statin or other lipid-lowering combinations were more prone to cause liver function tests elevations when prescribed with lomitapide.

In the Italian patients treated with lomitapide for up to 9.5 years, 68% had no change in their hepatic fat versus baseline and 32% had an increase, with all elevations in the mild to moderate range. Hepatic elasticity remained normal for all patients (Figure 1) and the mean FIB-4 score remained below the fibrosis threshold value of 2.67.

Conclusions

Overall, the hepatic safety of lomitapide remains favourable with no clinically significant elevations in hepatic biomarkers and hepatic elasticity remained normal for up to 9.5 years in patients with HoFH.

Background and Aims

Beyond lifestyle intervention and statin treatment, residual atherosclerotic cardiovascular disease risk persists in many patients, and hypertriglyceridemia has emerged as a contributing factor in this risk. Therefore, fibrates that target the nuclear receptor peroxisome proliferator-activated receptor α (PPARα), represent interesting therapeutic candidates, given their beneficial effects on lipid metabolism and inflammation. Pemafibrate, a novel highly potent and selective PPARα agonist, has potential in managing cardiovascular risk, but the mechanisms of its atheroprotective effects are not well understood. The aim of our study was to better understand these mechanisms of action, and especially to evaluate the importance of the liver in these effects.

Methods

LDLr^-/- PPARα^+/+ and LDLr^-/- PPARα^-/-, as well as LDLr^-/- PPARα^-/- mice injected either with an AAV8-TBG (Thyroxin Binding Globulin)-GFP (control), an AAV8-TBG-PPARα^WT (in order to restore PPARα selectively in the hepatocytes), or an AAV8-TBG-PPARα^DISS (mutant of PPARα only owning anti-inflammatory properties), were submitted to a western diet supplemented or not with pemafibrate during 8 weeks.

Results

Pemafibrate induced a strong reduction of atherosclerotic plaque surface (-50%) in LDLr^-/- PPARα^+/+, but not in LDLr^-/- PPARα^-/- mice. Under pemafibrate treatment, AAV8-TBG-PPARα^WT exhibited a strong reduction of dyslipidemia and atherosclerosis development (-42%), demonstrating a major role of hepatocyte PPARα on pemafibrate-induced atheroprotection. Surprisingly, in AAV8-TBG-PPARα^DISS mice, pemafibrate was still able to induce a strong reduction of atherosclerosis lesion surface (-42%), despite no effect on atherogenic dyslipidemia.

Conclusions

Hepatocyte PPARα is the key driver of the beneficial effects of pemafibrate on dyslipidemia and atherosclerosis in LDLr^-/- mice. Interestingly, modulation of hepatic inflammation through hepatocyte PPARα is sufficient to mediate pemafibrate-induced atheroprotection.