Background and Aims

Non-Alcoholic Fatty Liver Disease (NAFLD) represents an increasing cause of liver disease worldwide. Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in these patients. Although NAFLD pathophysiology is not fully understood alterations in fat metabolism seem to play a role. Autoantibodies against apolipoprotein A-1 (anti-apoA-1 IgG) are a novel cardiovascular risk factor to which have been recently attributed a metabolic role and have a function as a disruptor of the cholesterol pathway. This study aims at evaluating a possible role of anti-apoA-1 IgG in NAFLD.

Methods

Serum from 137 NAFLD patients were tested for anti-apoA-1 IgG prevalence. In vitro, SREBP1, SREBP2 expressions were assessed in the human hepatic cell line HepaRG by western blot analysis and bodipy staining was used to evaluate the lipid droplet content. Oil Red O staining was used to detect lipid accumulation in liver sections from ApoE-/- mice.

Results

Elevated anti-apoA-1 IgG seropositivity was found in patients with NAFLD (46%). In vitro, anti-apoA-1 IgG and not control IgG induced lipid accumulation in hepatic cells (5.9 vs 2.5, p=0.0008) and this lipid overload was associated with a high SREBP1 but not SREBP2 expression. In vivo, anti-apoA-1 IgG and not control IgG also induced higher lipid accumulation in the livers of ApoE-/- mice (1.23 vs 0.53, p=0.03).

Conclusions

Anti-apoA-1 IgG are frequent in NAFLD and promote lipid accumulation through SREBP1 activation. We hypothesize that anti-apoA1 IgG may be a potential driver in the development of NAFLD.

Background and Aims

ApoA-I/HDL play a unique role in regulating cell cholesterol homeostasis and in modulating inflammatory response and immune cell activation. In the present study, we investigated the impact of genetic manipulation of apoA-I/HDL levels on lipid deposition in skin and heart vessels in relation to local and systemic immune-inflammatory activation.

Methods

ApoE deficient (EKO) mice, apoE/apoA-I double deficient (DKO) mice, DKO mice overexpressing human apoA-I (DKO/hA-I) and wild-type mice were fed chow diet until 30 weeks of age. Plasma lipids were quantified, atherosclerosis development at the aortic sinus and in coronary arteries was measured, skin ultrastructure was evaluated by electron microscopy. Blood and lymphoid organs were characterized through histological, immunocytofluorimetric and whole transcriptome analyses.

Results

DKO mice were characterized by almost complete HDL deficiency and by plasma total cholesterol levels comparable to those of control mice. Only DKO mice showed xanthoma formation and severe inflammation in the skin-draining lymph nodes, whose transcriptome analysis revealed a dramatic impairment in energy metabolism and fatty acid oxidation pathways. An increased presence of CD4⁺ T effector memory cells was detected in blood, spleen and in the skin-draining lymph nodes of DKO mice. A worsening of atherosclerosis at the aortic sinus and coronary arteries was also observed in DKO mice vs EKO mice. Human apoA-I overexpression in the DKO background was able to rescue the skin phenotype and to halt atherosclerosis development.

Conclusions

HDL deficiency, in the absence of hyperlipidemia, is associated with severe alterations of skin morphology, aortic and coronary atherosclerosis, local and systemic inflammation.

Background and Aims

Macrophage autophagy is a highly anti-atherogenic process that helps maintain cellular homeostasis. In foam cells, autophagy was demonstrated to contribute to the degradation of lipid droplets (LDs) via a selective form of autophagy called lipophagy. Selective autophagy relies on tags such as ubiquitin and selectivity factors to label specific cargo for degradation. Yet, how LDs are targeted for autophagy remains poorly defined. Our study was aimed at identifying LD factors responsible for lipophagy in macrophage foam cells.

Methods

To identify lipophagy factors in macrophage foam cells in an unbiassed manner, we employed mass spectrometry to qualify the LD proteome. Using siRNA array in combination with high-content microscopy and cholesterol efflux screens, we assessed the functional role of these candidate lipophagy factors.

Results

We confirmed the presence of known LD-associated structural and metabolic proteins in the LD proteome. Additionally, we found the association of several proteins related to the ubiquitination machinery and autophagy, along with other novel factors that could regulate lipophagy. We observed that knocking down several of these genes, including Maplc3b and Tfeb significantly reduced cholesterol efflux, suggesting a role for these proteins in lipophagy-mediated LD catabolism. Furthermore, we identified optineurin as a novel cargo receptor for lipophagy.

Conclusions

Our study is the first to systematically identify several LD-associated proteins of the lipophagy machinery, a finding with important biological and therapeutic implications. Therapeutic targeting of these novel lipophagy factors may represent a means to enhance macrophage lipophagy to promote reverse cholesterol transport for the treatment of heart disease.

Background and Aims

TIR8/SIGIRR dampens the excessive activation mediated by ILRs and TLRs agonism and thus is a key regulator of inflammation. Aim of this study was to investigate the role of TIR8 in atherosclerosis.

Methods

8 weeks old-LDLR KO and TIR8/LDLR double KO (DKO) male mice were fed with standard diet (STD) or cholesterol-enriched diet (WTD) for 12 weeks. Plasma lipid profiling, extensive immunophenotyping and histological analysis of the atherosclerotic plaques were then performed.

Results

TIR8 deletion in STD-fed LDLR KO mice impacts circulating immune cell profile: decreased percentage of T lymphocytes (-29%, p<0.001) and increased percentage of B cells (+14%, p<0.05) were observed compared to LDLR KO mice, as well as increased mature Natural Killer cells (+13%, p<0.0001), as already described in the TIR8 KO mouse model. When fed a cholesterol rich diet for 12 weeks to induce atherosclerosis, in addition to changes observed on STD, also circulating levels of monocytes increased in DKO mice compared to LDL-R KO mice (mean 1464 vs 910 cells/ul, p<0.05). These changes in immune profile, however, did not affect atherosclerotic plaque area or stability. Similarly, no differences in plasma lipid profile were observed.

Conclusions

TIR8 deficiency in LDLR KO mice increases NKs and monocytes blood levels compared to LDL-R KO mice. Changes in these immune subsets, however, do not impact the development of atherosclerosis.

Background and Aims

Cardiovascular disease (CVD) is the leading cause of death worldwide and is often related to high plasma concentrations of low-density lipoprotein cholesterol (LDL-c). One of the most frequent dyslipidaemias is familial hypercholesterolemia (FH), which is mostly caused by mutations in the LDL receptor (LDLr). Although an early identification is essential to reduce premature mortality, only the 10 % of FH patients are properly diagnosed.

In vitro characterization of variants is time consuming and expensive, so computational predictors of pathogenicity of mutations are under constant development. The aim of our work was to create a machine learning-based model that can predict the pathogenicity of LDLr missense variants, the most common ones, on an easy and reliable way.

Methods

Using more than 700 LDLr missense variants characterized on ClinVar database and Excel solver Evolutionary algorithm, we created a Machine Learning model that predicts the pathogenicity of a variant based on seven characteristics of the mutated amino acid: Conservation of the residue, original and substituting amino acid, hydrophobicity, size, charge and affected domain.

We used a part of the dataset as training group for the obtention of pathogenicity frequency distribution and other essential parameters. Then, the model was tested with the other part of the dataset, the validation group.

Results

MLb-LDLr shows a sensitivity of 92.5% and a specificity of 91.5%, matching or even surpassing other predictor software such as PolyPhen-2 or SIFT.

Conclusions

With an accuracy higher than 90%, we conclude that in silico predictions are a reliable source of information about LDLr variant pathogenicity.

Background and Aims

The calcium score predicts cardiovascular mortality but the impact of calcification on plaque stability remains controversial. Tissue-nonspecific alkaline phosphatase (TNAP), the main enzyme involved in bone mineralization, is expressed in calcified mouse plaques, and is also elevated in the blood of individuals with metabolic syndrome, in whom it is associated with cardiovascular mortality. Therefore, we aimed to determine the involvement of TNAP in plaque calcification and progression.

Methods

TNAP activity was studied in aortic plaques, liver and blood of apoE-deficient mice fed a high fat diet from 10 weeks of age and sacrificed every two weeks from 17 to 31 weeks. Plaque calcification was imaged longitudinally with ¹⁸F-NaF PET and µCT and histologically with the calcium tracer osteosense. TNAP expression was also investigated in calcified and non-calcified human carotid plaques. TNAP was inhibited in mice using the inhibitor SBI-425 (30 mg/kg/day) from 10 weeks of age.

Results

Plaque calcification developed as cartilage metaplasia in association with TNAP activity in apoE-deficient mice. In human carotid plaques, calcification was also localized to TNAP-positive areas. In mice, short-term SBI-425 treatment prevented early plaque calcification, reduced inflammation, plaque growth and lipid accumulation, without exerting adverse effects on bone architecture. More unexpectedly, TNAP inhibition reduced serum cholesterol and triglycerides, suggesting that TNAP may slow down plaque development through direct and indirect effects.

Conclusions

In conclusion, this study demonstrates that TNAP activity strongly impacts plaque development, by effects probably not restricted to plaque calcification.