Showing papers by "Francesca Zimetti published in 2022"

Emerging role of HDL in brain cholesterol metabolism and neurodegenerative disorders.

Abstract: High-density lipoproteins (HDL) play a key role in cholesterol homeostasis maintenance in the central nervous system (CNS), by carrying newly synthesized cholesterol from astrocytes to neurons, to support their lipid-related physiological functions. As occurs for plasma HDL, brain lipoproteins are assembled through the activity of membrane cholesterol transporters, undergo remodeling mediated by specific enzymes and transport proteins, and finally deliver cholesterol to neurons by a receptor-mediated internalization process. A growing number of evidences indicates a strong association between alterations of CNS cholesterol homeostasis and neurodegenerative disorders, in particular Alzheimer's disease (AD), and a possible role in this relationship may be played by defects in brain HDL metabolism. In the present review, we summarize and critically examine the current state of knowledge on major modifications of HDL and HDL-mediated brain cholesterol transport in AD, by taking into consideration the individual steps of this process. We also describe potential and encouraging HDL-based therapies that could represent new therapeutic strategies for AD treatment. Finally, we revise the main plasma and brain HDL modifications in other neurodegenerative disorders including Parkinson's disease (PD), Huntington's disease (HD), and frontotemporal dementia (FTD).

PCSK9 Affects Astrocyte Cholesterol Metabolism and Reduces Neuron Cholesterol Supplying In Vitro: Potential Implications in Alzheimer’s Disease

Abstract: The Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) involvement in Alzheimer’s disease (AD) is poorly investigated. We evaluated the in vitro PCSK9 modulation of astrocyte cholesterol metabolism and neuronal cholesterol supplying, which is fundamental for neuronal functions. Moreover, we investigated PCSK9 neurotoxic effects. In human astrocytoma cells, PCSK9 reduced cholesterol content (−20%; p < 0.05), with a greater effect in presence of beta amyloid peptide (Aβ) (−37%; p < 0.01). PCSK9 increased cholesterol synthesis and reduced the uptake of apoE-HDL-derived cholesterol (−36%; p < 0.0001), as well as the LDL receptor (LDLR) and the apoE receptor 2 (ApoER2) expression (−66% and −31%, respectively; p < 0.01). PCSK9 did not modulate ABCA1- and ABCG1-cholesterol efflux, ABCA1 levels, or membrane cholesterol. Conversely, ABCA1 expression and activity, as well as membrane cholesterol, were reduced by Aβ (p < 0.05). In human neuronal cells, PCSK9 reduced apoE-HDL-derived cholesterol uptake (−41%; p < 0.001) and LDLR/apoER2 expression (p < 0.05). Reduced cholesterol internalization occurred also in PCSK9-overexpressing neurons exposed to an astrocyte-conditioned medium (−39%; p < 0.001). PCSK9 reduced neuronal cholesterol content overall (−29%; p < 0.05) and increased the Aβ-induced neurotoxicity (p < 0.0001). Our data revealed an interfering effect of PCSK9, in cooperation with Aβ, on brain cholesterol metabolism leading to neuronal cholesterol reduction, a potentially deleterious effect. PCSK9 also exerted a neurotoxic effect, and thus represents a potential pharmacological target in AD.

PCSK9 Confers Inflammatory Properties to Extracellular Vesicles Released by Vascular Smooth Muscle Cells

Abstract: Vascular smooth muscle cells (VSMCs) are key participants in both early- and late-stage atherosclerosis and influence neighbouring cells possibly by means of bioactive molecules, some of which are packed into extracellular vesicles (EVs). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is expressed and secreted by VSMCs. This study aimed to unravel the role of PCSK9 on VSMCs-derived EVs in terms of content and functionality. EVs were isolated from human VSMCs overexpressing human PCSK9 (VSMCPCSK9-EVs) and tested on endothelial cells, monocytes, macrophages and in a model of zebrafish embryos. Compared to EVs released from wild-type VSMCs, VSMCPCSK9-EVs caused a rise in the expression of adhesion molecules in endothelial cells and of pro-inflammatory cytokines in monocytes. These acquired an increased migratory capacity, a reduced oxidative phosphorylation and secreted proteins involved in immune response and immune effector processes. Concerning macrophages, VSMCPCSK9-EVs enhanced inflammatory milieu and uptake of oxidized low-density lipoproteins, whereas the migratory capacity was reduced. When injected into zebrafish embryos, VSMCPCSK9-EVs favoured the recruitment of macrophages toward the site of injection. The results of the present study provide evidence that PCSK9 plays an inflammatory role by means of EVs, at least by those derived from smooth muscle cells of vascular origin.

Effects of PCSK9 inhibitors on HDL cholesterol efflux and serum cholesterol loading capacity in familial hypercholesterolemia subjects: a multi-lipid-center real-world evaluation

Abstract: Proprotein convertase subtilisin/kexin type 9 (PCSK9), beyond regulating LDL cholesterol (LDL-c) plasma levels, exerts several pleiotropic effects by modulating lipid metabolism in extrahepatic cells such as macrophages. Macrophage cholesterol homeostasis depends on serum lipoprotein functions, including the HDL capacity to promote cell cholesterol efflux (CEC) and the serum capacity to promote cell cholesterol loading (CLC). The aim of this observational study was to investigate the effect of PCSK9 inhibitors (PCSK9-i) treatment on HDL-CEC and serum CLC in patients with familial hypercholesterolemia (FH). 31 genetically confirmed FH patients were recruited. Blood was collected and serum isolated at baseline and after 6 months of PCSK9-i treatment. HDL-CEC was evaluated through the main pathways with a radioisotopic cell-based assay. Serum CLC was assessed fluorimetrically in human THP-1 monocyte-derived macrophages. After treatment with PCSK9-i, total cholesterol and LDL-c significantly decreased (−41.6%, p < 0.0001 and −56.7%, p < 0.0001, respectively). Total HDL-CEC was not different between patients before and after treatment. Conversely, despite no changes in HDL-c levels between the groups, ABCG1 HDL-CEC significantly increased after treatment (+22.2%, p < 0.0001) as well as HDL-CEC by aqueous diffusion (+7.8%, p = 0.0008). Only a trend towards reduction of ABCA1 HDL-CEC was observed after treatment. PCSK9-i significantly decreased serum CLC (−6.6%, p = 0.0272). This effect was only partly related to the reduction of LDL-c levels. In conclusion, PCSK9-i treatment significantly increased HDL-CEC through ABCG1 and aqueous diffusion pathways and reduced the serum CLC in FH patients. The favorable effect of PCSK9-i on functional lipid profile could contribute to the cardiovascular benefit of these drugs in FH patients.

Serum cholesterol loading capacity on macrophages is linked to coronary atherosclerosis and cardiovascular event risk in rheumatoid arthritis

Abstract: Objectives Cholesterol loading capacity (CLC) describes the ability of serum to deliver cholesterol to cells. It is linked to foam cell formation, a pivotal step in atherosclerotic plaque development. We evaluate the associations of CLC with coronary atherosclerosis presence, burden and cardiovascular risk in patients with rheumatoid arthritis (RA). Methods Coronary atherosclerosis (any, high-risk low-attenuation plaque and obstructive plaque) was evaluated with CT angiography in 141 patients. Participants were prospectively followed for 6.0±2.4 years and cardiovascular events including cardiac death, myocardial infarction, unstable angina, stroke, claudication, revascularisation and hospitalised heart failure were recorded. CLC was quantified as intracellular cholesterol in human macrophages after incubation with patient serum. Results CLC was not linked to overall plaque presence or burden after adjustments for atherosclerotic cardiovascular disease (ASCVD) score, statin use and low-density lipoprotein cholesterol. However, CLC associated with presence and numbers of any, low-attenuation and obstructive plaques exclusively in biologic disease-modifying antirheumatic drugs (bDMARD) non-users (p for interaction ≤0.018). CLC associated with cardiovascular event risk overall after adjustments for ASCVD and number of segments with plaque (HR=1.76 (95% CI 1.16 to 2.67) per 1 SD increase in CLC, p=0.008). Additionally, bDMARD use modified the impact of CLC on event risk; CLC associated with events in bDMARD non-users (HR=2.52 (95% CI 1.36 to 4.65) per 1SD increase in CLC, p=0.003) but not users. Conclusion CLC was linked to long-term cardiovascular event risk in RA and associated with high-risk low attenuation and obstructive coronary plaque presence and burden in bDMARD non-users. Its prospective validation as a predictive biomarker may be, therefore, warranted.

Op0136 serum cholesterol loading capacity on macrophages and interactions with treatments on coronary atherosclerosis burden and event risk in rheumatoid arthritis

Abstract: Cholesterol loading capacity (CLC) describes the ability of serum to deliver cholesterol to cells. It is linked to foam cell formation, a pivotal step in atherosclerotic plaque development. Rheumatoid arthritis (RA) serum promoted foam cell formation significantly more than control serum. Likewise, RA patients display greater plaque burden and higher-risk features than non-RA controls. bDMARDs and statins lower cardiovascular risk by reducing new coronary plaque formation, promoting regression, altering the composition and stabilizing prevalent atherosclerotic lesions.To evaluate the associations between CLC, coronary plaque burden and cardiovascular event risk in patients with RA. We further explored the conditioning effects of RA treatments on these relationships.140 patients underwent coronary CT angiography for atherosclerosis evaluation and were prospectively followed for cardiovascular events over 6.0±2.4 years. Coronary artery calcium score (CAC), number of segments with plaque (segment involvement score [SIS]) and plaque composition were assessed. CLC was the macrophage cholesterol content, measured by fluorometric assay, after a 24-hour incubation with whole serum. Robust linear regression examined the effects of CLC and the interaction between CLC and bDMARD use on SIS and CAC. Negative binomial regression evaluated CLC and CLC × bDMARD interaction effects on number of high-risk (low-attenuation) plaques. With data discretized into 1-month intervals, weighted pooled logistic regression models with robust variance estimation evaluated CLC and time-varying bDMARD use as predictors of event risk, and the effect of CLC × time-varying bDMARD use on risk. Stabilized inverse probability of treatment and censoring weights were estimated as a function of ASCVD risk, SIS, RA duration, and baseline and time-varying CRP and statin use.Mean (SD) CLC was 12.67 (2.83) μg/mg protein. In analyses adjusting for ASCVD score, HDL, prednisone and statin use, CLC (per 1-SD unit) was not related to SIS (β -0.05 [95%CI -1.19,0.09]), number of high-risk plaques (rate ratio [RR] 1.20 [95%CI 0.80-1.80]) or ln-transformed CAC (β 0.017 [95%CI -0.133,0.147]). However, in analyses stratified by baseline bDMARD use, CLC (per 1-SD unit) was positively related to number of high-risk plaques (RR 2.14 [95%CI 1.04-4.40]) and ln-transformed CAC (β 0.21 [95%CI 0.01-0.41]) among bDMARD-naïve individuals (Figure 1). In addition, CLC inversely associated with SIS (per SD increment; β -0.16 [95%CI -0.32, -0.01]) only in bDMARD-treated patients. Baseline statin use did not significantly modify the effect of CLC on coronary plaque (not shown). CLC associated with cardiovascular event risk (per SD increment; adjusted odds ratio 2.02 [95%CI 1.27-3.50], p=0.011) covarying for ASCVD score and time-varying bDMARD use. The CLC × time-varying bDMARD use interaction also predicted event risk (p =0.010); current bDMARD use associated with lower event risk at higher (1 SD above the mean) CLC levels (p=0.037) but not average or lower (1 SD below the mean) CLC levels (p=0.064 and 0.756, respectively).CLC associated with greater CAC score and high-risk plaque burden in bDMARD-naïve RA patients and lower total plaque burden in bDMARD-treated patients at baseline. CLC also predicted long-term cardiovascular risk and its effect was mitigated by bDMARD use.George Karpouzas Speakers bureau: Sanofi-Genzyme-Regeneron, Janssen, Bristol-Meyer-Squibb, Consultant of: Sanofi-Genzyme-Regeneron, Janssen, Bristol-Meyer-Squibb, Grant/research support from: Pfizer, Bianca Papotti: None declared, Sarah Ormseth: None declared, Marcella Palumbo: None declared, Elizabeth Hernandez: None declared, Cinzia Marchi: None declared, Francesca Zimetti: None declared, Matthew Budoff Consultant of: Pfizer, Nicoletta Ronda: None declared

Serum cholesterol loading capacity on macrophages is regulated by seropositivity and C-reactive protein in rheumatoid arthritis patients.

Abstract: OBJECTIVE Excessive cholesterol accumulation in macrophages is the pivotal step underlying atherosclerotic plaque formation. We here explore factors in the serum of patients with rheumatoid arthritis (RA) and mechanisms through which they interact and influence cholesterol loading capacity (CLC) on macrophages. METHODS In a cross-sectional observational cohort of 104 patients with RA, CLC was measured as intracellular cholesterol content in human THP-1-derived macrophages after incubation with patient serum. LDL oxidation was measured as oxidized phospholipids on apoB100-containing particles (oxPL-apoB100). Antibodies against oxidized LDL (anti-oxLDL), proprotein convertase subtilisin/Kexin type-9 (PCSK9) and high-sensitivity CRP were also quantified. All analyses adjusted for atherosclerotic cardiovascular disease (ASCVD) risk score, obesity, total LDL, statin use, age at diagnosis, and anti-oxLDL IgM. RESULTS OxPL-apoB100, anti-oxLDL IgG and PCSK9 positively associated with CLC (all p< 0.020). OxPL-apoB100 directly influenced CLC only in dual rheumatoid factor and anti-citrullinated protein antibody positive patients (unstandardized b [95% bootstrap confidence interval]=2.08 [0.38-3.79]). An indirect effect of oxPL-apoB100 on CLC through anti-oxLDL IgG increased along with level of CRP (index of moderated mediation = 0.55 [0.05-1.17]). CRP also moderated yet another indirect effect of oxPL-apoB100 on CLC through upregulation of PCSK9, but only among dual seropositive patients (conditional indirect effect = 0.64 [0.13-1.30]). CONCLUSION Oxidized LDL can directly influence CLC in dual seropositive RA patients. Two additional and independent pathways-via anti-oxLDL IgG and PCSK9-may mediate the effects of oxPL-apoB100 on CLC depending on CRP and seropositivity status. If externally validated, these findings may have clinical implications for cardiovascular risk prevention.

HDL Cholesterol Efflux and Serum Cholesterol Loading Capacity Alterations Associate to Macrophage Cholesterol Accumulation in FH Patients with Achilles Tendon Xanthoma

Abstract: Achilles tendon xanthoma (ATX) formation involves macrophage cholesterol accumulation within the tendon, similar to that occurring in atheroma. Macrophage cholesterol homeostasis depends on serum lipoprotein functions, namely the high-density lipoprotein (HDL) capacity to promote cell cholesterol efflux (cholesterol efflux capacity, CEC) and the serum cholesterol loading capacity (CLC). We explored the HDL-CEC and serum CLC, comparing 16 FH patients with ATX to 29 FH patients without ATX. HDL-CEC through the main efflux mechanisms mediated by the transporters ATP binding cassette G1 (ABCG1) and A1 (ABCA1) and the aqueous diffusion (AD) process was determined by a cell-based radioisotopic technique and serum CLC fluorimetrically. Between the two groups, no significant differences were found in terms of plasma lipid profile. A trend toward reduction of cholesterol efflux via AD and a significant increase in ABCA1-mediated HDL-CEC (+18.6%) was observed in ATX compared to no ATX patients. In ATX-presenting patients, ABCG1-mediated HDL-CEC was lower (−11%) and serum CLC was higher (+14%) compared to patients without ATX. Considering all the patients together, ABCG1 HDL-CEC and serum CLC correlated with ATX thickness inversely (p = 0.013) and directly (p < 0.0001), respectively. In conclusion, lipoprotein dysfunctions seem to be involved in ATX physiopathology and progression in FH patients.

Effects of Antirheumatic Treatment on Cell Cholesterol Efflux and Loading Capacity of Serum Lipoproteins in Spondylarthropathies

Abstract: Spondyloarthropathies (SpA) are associated with increased cardiovascular risk. Among possible mechanisms is the dysfunction of serum lipoproteins in regulating cell cholesterol homeostasis. Cholesterol efflux capacity (CEC)—the atheroprotective ability of HDL (high density lipoproteins) to accept cholesterol from macrophages—might predict cardiovascular disease independently of HDL-cholesterol levels. We aimed at evaluating modifications of CEC and of the atherogenic cholesterol loading capacity (CLC) of serum lipoproteins in psoriatic arthritis (PsA) and ankylosing spondylitis (AS) following anti-rheumatic treatment. A total of 62 SpA patients (37 PsA and 25 AS) were evaluated before and after treatment with tumor necrosis factor inhibitor and/or methotrexate. CEC and CLC were measured by radioisotopic and fluorometric techniques, respectively. Endothelial function was assessed by finger plethysmography (Endopat). In the whole SpA group, total and HDL-cholesterol increased after treatment, while lipoprotein(a) decreased and CLC was unchanged. Treatment was associated with increased Scavenger Receptor class B type I (SR-BI)-mediated CEC in the AS group. SR-BI- and ABCG1-mediated CEC were negatively associated with inflammatory parameters and positively related to coffee consumption. SR-BI CEC and CLC were positively and negatively associated with endothelial function, respectively. Our pilot study suggests that anti-rheumatic treatment is associated with favorable modulation of lipoprotein quality and function in SpA, particularly in AS, in spite of the induced increase in total cholesterol levels. If confirmed in a larger population, this might represent an atheroprotective benefit beyond what is reflected by conventional serum lipid profile.

HDL metabolism and functions impacting on cell cholesterol homeostasis are specifically altered in patients with abdominal aortic aneurysm

Abstract: Background The etiopathogenesis of abdominal aortic aneurysm (AAA) is still unclarified, but vascular inflammation and matrix metalloproteases activation have a recognized role in AAA development and progression. Circulating lipoproteins are involved in tissue inflammation and repair, particularly through the regulation of intracellular cholesterol, whose excess is associated to cell damage and proinflammatory activation. We analyzed lipoprotein metabolism and function in AAA and in control vasculopathic patients, to highlight possible non-atherosclerosis-related, specific abnormalities. Methods We measured fluorometrically serum esterified/total cholesterol ratio, as an index of lecithin-cholesterol acyltransferase (LCAT) activity, and cholesteryl ester transfer protein (CETP) activity in patients referred to vascular surgery either for AAA (n=30) or stenotic aortic/peripheral atherosclerosis (n=21) having similar burden of cardiovascular risk factors and disease. We measured high-density lipoprotein (HDL)-cholesterol efflux capacity (CEC), through the ATP-binding cassette G1 (ABCG1) and A1 (ABCA1) pathways and serum cell cholesterol loading capacity (CLC), by radioisotopic and fluorimetric methods, respectively. Results We found higher LCAT (+23%; p < 0.0001) and CETP (+49%; p < 0.0001) activity in AAA sera. HDL ABCG1-CEC was lower (−16%; p < 0.001) and ABCA1-CEC was higher (+31.7%; p < 0.0001) in AAA. Stratification suggests that smoking may partly contribute to these modifications. CEC and CETP activity correlated with CLC only in AAA. Conclusions We demonstrated that compared to patients with stenotic atherosclerosis, patients with AAA had altered HDL metabolism and functions involved in their anti-inflammatory and tissue repair activity, particularly through the ABCG1-related intracellular signaling. Clarifying the relevance of this mechanism for AAA evolution might help in developing new diagnostic parameters and therapeutic targets for the early management of this condition.

Pos0596 serum cholesterol loading capacity on macrophages is linked to oxidized low-density lipoprotein and regulated by seropositivity and c-reactive protein in patients with rheumatoid arthritis

Abstract: Excessive cholesterol accumulation in macrophages underlies foam cell formation, initiation and progression of atherosclerosis. LDL oxidation and unregulated uptake of oxidized LDL by macrophages are critical in foam cell development. Cholesterol loading capacity (CLC) is the ability of serum to deliver cholesterol to cells and is related to foam cell formation. Rheumatoid arthritis (RA) serum increased cholesterol content in macrophages and promoted foam cell formation significantly more than control serum1. Although inflammation, LDL oxidation and antibodies to oxidized LDL (anti-oxLDL) may be higher in RA, their relationships and their individual and synergistic contributions to CLC in RA are unknown.To explore determinants and moderators of serum CLC in patients with RA. We also investigated whether oxidized LDL influences CLC directly or indirectly through anti-oxLDL IgG and proprotein convertase subtilisin/Kexin type-9 (PCSK9), independently or conditionally on RA-related autoantibodies such as rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) or level of inflammation.In an observational study of 104 patients, CLC was measured fluorimetrically as intracellular cholesterol content in human THP-1-derived macrophages after incubation with patient serum. Oxidized LDL was measured as oxidized phospholipids on apoB100 particles (oxPL-apoB100). Anti-oxLDL, PCSK9 and C-reactive protein (CRP) were also quantified. Associations of oxPL-apoB100, anti-oxLDL IgG and PCSK9 with CLC were examined with multivariable linear regression. A two-stage dual moderated mediation model explored whether an indirect association of oxPL-apoB100 with CLC through parallel mediators anti-oxLDL IgG and PCSK9 varied as a function of moderators CRP and RF/ACPA positivity.OxPL-apoB100, anti-oxLDL IgG and PCSK9 positively associated with CLC (all adjusted p<0.020). In the final dual moderated mediation model oxPL-apoB100 was directly linked to CLC only in dual seropositive patients (unstandardized b [95% bootstrap confidence interval]=2.08 [0.38-3.79], Figure 1). An indirect effect of oxPL-apoB100 on CLC through anti-oxLDL IgG was present and increased along with level of CRP (index of moderated mediation=0.55 [0.05-1.17]). CRP also moderated the other indirect effect of oxPL-apoB100 on CLC through PCSK9, but only in dual seropositive patients (conditional indirect effect=0.64 [0.13-1.30]).Oxidized LDL can directly influence CLC in dual seropositive RA patients, regardless of CRP. This suggests that targeting LDL oxidation in addition to inflammation may enable a more comprehensive reduction of atherosclerotic risk in these patients. Depending on CRP level, oxidized LDL also affected CLC indirectly via anti-oxLDL IgG and via PCSK9 in dual seropositive patients. If externally validated, our findings may have clinical implications for cardiovascular risk stratification and prevention.[1]Voloshyna I et al. Plasma from rheumatoid arthritis patients promotes pro-atherogenic cholesterol transport gene expression in THP-1 human macrophages. Exp Biol Med (Maywood) 2013;238:1192–7.George Karpouzas Speakers bureau: Sanofi-Genzyme-Regeneron, Janssen, Bristol-Meyer-Squibb, Consultant of: Sanofi-Genzyme-Regeneron, Janssen, Bristol-Meyer-Squibb, Grant/research support from: Pfizer, Bianca Papotti: None declared, Sarah Ormseth: None declared, Marcella Palumbo: None declared, Elizabeth Hernandez: None declared, Cinzia Marchi: None declared, Francesca Zimetti: None declared, Matthew Budoff Consultant of: Pfizer, Nicoletta Ronda: None declared