Alternative C3 Complement System: Lipids and Atherosclerosis.

Familial hypercholesterolemia (FH) is increasingly associated with inflammation, a phenotype that persists despite treatment with lipid lowering therapies. The alternative C3 complement system (C3), as a key inflammatory mediator, seems to be involved in the atherosclerotic process; however, the relationship between C3 and lipids during plaque progression remains unknown. The aim of the study was to investigate by a systems biology approach the role of C3 in relation to lipoprotein levels during atherosclerosis (AT) progression and to gain a better understanding on the effects of C3 products on the phenotype and function of human lipid-loaded vascular smooth muscle cells (VSMCs). By mass spectrometry and differential proteomics, we found the extracellular matrix (ECM) of human aortas to be enriched in active components of the C3 complement system, with a significantly different proteomic signature in AT segments. Thus, C3 products were more abundant in AT-ECM than in macroscopically normal segments. Furthermore, circulating C3 levels were significantly elevated in FH patients with subclinical coronary AT, evidenced by computed tomographic angiography. However, no correlation was identified between circulating C3 levels and the increase in plaque burden, indicating a local regulation of the C3 in AT arteries. In cell culture studies of human VSMCs, we evidenced the expression of C3, C3aR (anaphylatoxin receptor) and the integrin αMß2 receptor for C3b/iC3b (RT-PCR and Western blot). C3mRNA was up-regulated in lipid-loaded human VSMCs, and C3 protein significantly increased in cell culture supernatants, indicating that the C3 products in the AT-ECM have a local vessel-wall niche. Interestingly, C3a and iC3b (C3 active fragments) have functional effects on VSMCs, significantly reversing the inhibition of VSMC migration induced by aggregated LDL and stimulating cell spreading, organization of F-actin stress fibers and attachment during the adhesion of lipid-loaded human VSMCs. This study, by using a systems biology approach, identified molecular processes involving the C3 complement system in vascular remodeling and in the progression of advanced human atherosclerotic lesions.

Profiling of immune-related gene expression in children with familial hypercholesterolaemia.

BACKGROUND: Innate and adaptive immune responses are pivotal in atherosclerosis, but their association with early-stage atherosclerosis in humans is incompletely understood. In this regard, untreated children with familial hypercholesterolaemia may serve as a human model to investigate the effect of elevated low-density lipoprotein (LDL)-cholesterol. OBJECTIVES: We aimed to study the immunological and inflammatory pathways involved in early atherosclerosis by examining mRNA molecules in peripheral blood mononuclear cells (PBMCs) from children with FH. METHODS: We analysed the level of 587 immune-related mRNA molecules using state-of-the-art Nanostring technology in PBMCs from children with (n = 30) and without (n = 21) FH, and from FH children before and after statin therapy (n = 10). RESULTS: 176 genes (30%) were differentially expressed between the FH and healthy children at P < 0.05. Compared to healthy children, the dysregulated pathways in FH children included the following: T cells (18/19); B cells (5/6); tumour necrosis factor super family (TNFSF) (6/8); cell growth, proliferation and differentiation (5/7); interleukins (5/9); toll-like receptors (2/5); apoptosis (3/7) and antigen presentation (1/7), where the ratio denotes higher expressed genes to total number of genes. Statin therapy reversed expression of thirteen of these mRNAs in FH children. CONCLUSION: FH children display higher PBMC expression of immune-related genes mapped to several pathways, including T and B cells, and TNFSF than healthy children. Our results suggest that LDL-C plays an important role in modulating expression of different immune-related genes, and novel data on the involvement of these pathways in the early atherosclerosis may represent future therapeutic targets for prevention of atherosclerotic progression.

The atherogenic role of immune cells in familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder of lipoprotein metabolism that mainly occurs due to mutations in the low-density lipoprotein receptor gene and is characterized by increased levels of low-density lipoprotein cholesterol, leading to accelerated atherogenesis and premature coronary heart disease. Both innate and adaptive immune responses, which mainly include monocytes, macrophages, neutrophils, T lymphocytes, and B lymphocytes, have been shown to play a key role for the initiation and progression of atherogenesis in the general population. In FH patients, these immune cells have been suggested to play specific pro-atherosclerotic activities, from the initial leukocyte recruitment to plaque rupture. In fact, the accumulation of cholesterol crystals and oxLDL in the vessels in FH patients is particularly high, with consequent abnormal mobilization of immune cells and secretion of various pro-inflammatory and chemokines. In addition, cholesterol accumulation in immune cells is exaggerated with chronic exposure to relevant pro-atherosclerotic triggers. The topics considered in this review may provide a more specific focus on the immune system alterations in FH and open new insights toward immune cells as potential therapeutic targets in FH.

PCSK9 monoclonal antibodies reverse the pro-inflammatory profile of monocytes in familial hypercholesterolaemia.

AIMS: Migration of monocytes into the arterial wall contributes to arterial inflammation and atherosclerosis progression. Since elevated low-density lipoprotein cholesterol (LDL-C) levels have been associated with activation of plasma monocytes, intensive LDL-C lowering may reverse these pro-inflammatory changes. Using proprotein convertase subtilisin/kexin type 9 (PCSK9) monoclonal antibodies (mAbs) which selectively reduce LDL-C, we studied the impact of LDL-C lowering on monocyte phenotype and function in patients with familial hypercholesterolaemia (FH) not using statins due to statin-associated muscle symptoms. METHODS AND RESULTS: We assessed monocyte phenotype and function using flow cytometry and a trans-endothelial migration assay in FH patients (n = 22: LDL 6.8 ± 1.9 mmol/L) and healthy controls (n = 18, LDL 2.9 ± 0.8 mmol/L). Monocyte chemokine receptor (CCR) 2 expression was approximaterly three-fold higher in FH patients compared with controls. C-C chemokine receptor type 2 (CCR2) expression correlated significantly with plasma LDL-C levels (r = 0.709) and was positively associated with intracellular lipid accumulation. Monocytes from FH patients also displayed enhanced migratory capacity ex vivo. After 24 weeks of PCSK9 mAb treatment (n = 17), plasma LDL-C was reduced by 49%, which coincided with reduced intracellular lipid accumulation and reduced CCR2 expression. Functional relevance was substantiated by the reversal of enhanced migratory capacity of monocytes following PCSK9 mAb therapy. CONCLUSIONS: Monocytes of FH patients have a pro-inflammatory phenotype, which is dampened by LDL-C lowering by PCSK9 mAb therapy. LDL-C lowering was paralleled by reduced intracellular lipid accumulation, suggesting that LDL-C lowering itself is associated with anti-inflammatory effects on circulating monocytes.

Current and emerging monoclonal antibodies for treating familial hypercholesterolemia in children.

INTRODUCTION: Heterozygous familial hypercholesterolemia (HeFH) is a common genetic disorder caused by pathogenic variants in the LDL-C metabolism. Lifelong exposure to elevated LDL-C levels leads to a high risk of premature cardiovascular disease. To reduce that risk, children with HeFH should be identified and treated with lipid-lowering therapy. The cornerstone consists of statins and ezetimibe, but not in all patients this lowers the LDL-C levels to treatment targets. For these patients, more intensive lipid-lowering therapy is needed. AREAS COVERED: In this review, we provide an overview of the monoclonal antibodies which are currently available or being tested for treating HeFH in childhood. EXPERT OPINION: Monoclonal antibodies that inhibit PCSK9 are first in line lipid-lowering treatment options if oral statin and ezetimibe therapy are insufficient, due to intolerance or very high baseline LDL-C levels. Both evolocumab and alirocumab have been shown to be safe and effective in children with HeFH. For children, evolocumab has been registered from the age of 10 years old and alirocumab from the age of 8 years old. The costs of these new agents are much higher than oral therapy, which makes it important to only use them in a selected patient population.

Effective treatment of familial hypercholesterolaemia in the mouse model using adenovirus-mediated transfer of the VLDL receptor gene.

Liver directed gene transfer with adenoviral vectors is being considered for the treatment of several metabolic diseases, including familial hypercholesterolaemia (FH). Gene replacement therapy of human low density lipoprotein (LDL) receptor gene into the murine model of FH transiently corrected the dyslipidaemia; however, humoral and cellular immune responses to LDL receptor developed--possibly contributing to the associated hepatitis and extinguishing of transgene expression. We evaluated an alternative strategy of ectopic expression in the liver of the very low density lipoprotein (VLDL) receptor, which is homologous to the LDL receptor but has a different pattern of expression. Infusion of recombinant adenoviruses containing the VLDL receptor gene corrected the dsylipidaemia in the FH mouse and circumvented immune responses to the transgene leading to a more prolonged metabolic correction.

Lymphocyte count was significantly associated with hyper-LDL cholesterolemia independently of high-sensitivity C-reactive protein in apparently healthy Japanese.

The aim of this study was to investigate the association between leukocyte subtype counts and hyper-LDL cholesterolemia, hypertriglyceridemia, and hypo-HDL cholesterolemia. Logistic regressions using hyper-LDL cholesterolemia, hypertriglyceridemia, and hypo-HDL cholesterolemia as a dependent variable and total leukocyte, basophil, eosinophil, neutrophil, lymphocyte, and monocyte counts as an independent variable were calculated adjusting for age, body mass index (BMI), high-sensitivity C-reactive protein (hs-CRP), smoking, drinking, and physical activity in apparently healthy Japanese men (1,803) and women (1,150). The odds ratio (OR) of hyper-LDL cholesterolemia for total leukocyte, eosinophil, and lymphocyte counts, the OR of hypertriglyceridemia for total leukocyte, eosinophil, neutrophil, and lymphocyte counts, and the OR of hypo-HDL cholesterolemia for total leukocyte, neutrophil, and lymphocyte counts were significant in men, and the OR of hyper-LDL cholesterolemia, for lymphocyte count, and the OR of hypo-HDL cholesterolemia for eosinophil count were significant in women. Lymphocyte count was significantly associated with hyper-LDL cholesterolemia independently of hs-CRP in apparently healthy Japanese.

Beneficial effects of PCSK9 inhibition with alirocumab in familial hypercholesterolemia involve modulation of new immune players.

Familial hypercholesterolemia (FH) is associated with low-grade systemic inflammation, a key driver of premature atherosclerosis. We investigated the effects of inhibiting proprotein convertase subtilisin/kexin type 9 (PCSK9) function on inflammatory state, endothelial dysfunction and cardiovascular outcomes in patients with FH. Fourteen patients with FH were evaluated before and 8 weeks after administration of a PCSK9 blocking monoclonal antibody (alirocumab, 150 mg/subcutaneous/14 days). In vivo and ex vivo analysis revealed that alirocumab blunted the attachment of leukocytes to TNFα-stimulated human umbilical arterial endothelial cells (HUAEC) and suppressed the activation of platelets and most leukocyte subsets, which was accompanied by the diminished expression of CX3CR1, CXCR6 and CCR2 on several leukocyte subpopulations. By contrast, T-regulatory cell activation was enhanced by alirocumab treatment, which also elevated anti-inflammatory IL-10 plasma levels and lowered circulating pro-inflammatory cytokines. Plasma levels of IFNγ positively correlated with levels of total and LDL-cholesterol, whereas circulating IL-10 levels negatively correlated with these key lipid parameters. In vitro analysis revealed that TNFα stimulation of HUAEC increased the expression of PCSK9, whereas endothelial PCSK9 silencing reduced TNFα-induced mononuclear cell adhesion mediated by Nox5 up-regulation and p38-MAPK/NFκB activation, concomitant with reduced SREBP2 expression. PCSK9 silencing also decreased endothelial CX3CL1 and CXCL16 expression and chemokine generation. In conclusion, PCSK9 inhibition impairs systemic inflammation and endothelial dysfunction by constraining leukocyte-endothelium interactions. PCSK9 blockade may constitute a new therapeutic approach to control the inflammatory state associated with FH, preventing further cardiovascular events in this cardiometabolic disorder.

Cytokines profile in serum of homozygous familial hypercholesterolemia is changed by LDL-apheresis.

OBJECTIVE: The effects of LDL-apheresis (LDLa) with dextran sulphate on plasma cytokines in 6 homozygous familial hypercholesterolemic (HozFH) patients, were evaluated. METHODS: Plasma IL-1α; IL-1ra; IL-4; IL-6; IL-10; IL-12(p40); IL-12(p70); TNF-α, sTNF-R, VEGF, VEGF-R1, E-Selectin (ESEL), and P-Selectin (PSEL) concentrations were measured before and after LDLa on three consecutive sessions for each patient. RESULTS: TNF-α was significantly reduced (-60%; P=0.01), while TNF-R was only slightly increased (+15%), although not significantly. Plasma VEGF was significantly reduced (-57%; P=1.87301E-05), while VEGF-R1 was significantly increased (+56%; P=0.05). ESEL and PSEL were reduced but not to a statistically significant extent (-19%, -15%, respectively). IL-1α level was dramatically reduced (-87%; P=0.0001). IL-1ra concentration was only slightly increased in plasma, but not significantly. IL-4 and IL-10 levels were significantly reduced in plasma after apheresis (-50%; P=0.03, and -55%; P=0.004, respectively). On the contrary, IL-6 concentration showed a slight decrease (-8%). Plasma IL-12p40 was significantly increased (+47%; P=0.0004). On the other hand, IL-12p70 was reduced, but the difference (-31%) was not statistically significant. CONCLUSIONS: Plasma cytokines imbalance is associated with inflammation and atherogenesis. In this study LDLa changed several circulating cytokines inducing anti-inflammatory and anti-atherogenic changes in cytokines plasma profile in HozFH patients with/without pre-existing angiographically demonstrated coronary heart disease (CHD) and aortic valvular disease (AVD).

Myeloperoxidase modulation by LDL apheresis in familial hypercholesterolemia.

BACKGROUND: Myeloperoxidase (MPO) is a marker of plaque vulnerability and a mechanistic bridge between inflammation and cardiovascular disease, and thus is a suitable target for therapeutic strategy against cardiovascular disease. METHODS: Since hypercholesterolemia is associated with atherosclerosis and inflammation, we tested whether MPO serum levels were up-regulated in Familial Hypercholesterolemia (FH) and whether acute reduction of total cholesterol (TC) would also reduce MPO concentration. FH subjects undergoing LDL-apheresis (LDL-A) treatment are a paradigmatic clinical model where TC rapidly plunges from extremely high to extremely low levels after selective LDL removal, and then spontaneously rebounds to baseline conditions. This clinical setting allows multiple intra-patient observations at different plasma TC concentrations. We measured MPO levels in serum by ELISA tests, and in peripheral leukocytes by immunofluorescence, to learn whether they were affected by the changes in TC levels. Serum MPO was measured before and serially up to the 14th day following LDL-A. RESULTS: In both serum and peripheral leukocytes, MPO concentrations were i) higher than in sex- and age-matched healthy controls (p < 0.01); ii) decreased with TC reduction; iii) parallel with TC time course; iv) correlated with plasma TC. At regression analysis, plasma TC was the only variable considered that influenced MPO serum levels (ß 0.022 ± 0.010, p < 0.0001). CONCLUSIONS: In FH the MPO serum levels were modulated through changes in the TC concentrations carried out by LDL-A. Further study is needed to determine whether reduced MPO levels obtained by LDL-A could have any therapeutic impact.

Vaccine targeting ANGPTL3 ameliorates dyslipidemia and associated diseases in mouse models of obese dyslipidemia and familial hypercholesterolemia.

Dyslipidemia is a risk factor for cardiovascular disease (CVD), a major cause of death worldwide. Angiopoietin-like protein 3 (ANGPTL3), recognized as a new therapeutic target for dyslipidemia, regulates the metabolism of low-density lipoprotein-cholesterol (LDL-C) and triglycerides. Here, we design 3 epitopes (E1-E3) for use in development of a peptide vaccine targeting ANGPTL3 and estimate effects of each on obesity-associated dyslipidemia in B6.Cg-Lepob /J (ob/ob) mice. Vaccination with the E3 (32EPKSRFAMLD41) peptide significantly reduces circulating levels of triglycerides, LDL-C, and small dense (sd)-LDL-C in ob/ob mice and decreases obese-induced fatty liver. Moreover, E3 vaccination does not induce cytotoxicity in ob/ob mice. Interestingly, the effect of E3 vaccination on dyslipidemia attenuates development of atherosclerosis in B6.KOR/StmSlc-Apoeshl mice fed a high-cholesterol diet, which represent a model of severe familial hypercholesterolemia (FH) caused by ApoE loss of function. Taken together, ANGPTL3 vaccination could be an effective therapeutic strategy against dyslipidemia and associated diseases.

Adoptive transfer of CX3CR1 transduced-T regulatory cells improves homing to the atherosclerotic plaques and dampens atherosclerosis progression.

AIM: Loss of immunosuppressive response supports inflammation during atherosclerosis. We tested whether adoptive cell therapy (ACT) with Tregulatory cells (Tregs), engineered to selectively migrate in the atherosclerotic plaque, would dampen the immune-inflammatory response in the arterial wall in animal models of familial hypercholesterolaemia (FH). METHODS AND RESULTS: FH patients presented a decreased Treg suppressive function associated to an increased inflammatory burden. A similar phenotype was observed in Ldlr -/- mice accompanied by a selective increased expression of the chemokine CX3CL1 in the aorta but not in other districts (lymph nodes, spleen, and liver). Treg overexpressing CX3CR1 were thus generated (CX3CR1+-Tregs) to drive Tregs selectively to the plaque. CX3CR1+-Tregs were injected (i.v.) in Ldlr -/- fed high-cholesterol diet (western type diet, WTD) for 8 weeks. CX3CR1+-Tregs were detected in the aorta, but not in other tissues, of Ldlr -/- mice 24 h after ACT, corroborating the efficacy of this approach. After 4 additional weeks of WTD, ACT with CX3CR1+-Tregs resulted in reduced plaque progression and lipid deposition, ameliorated plaque stability by increasing collagen and smooth muscle cells content, while decreasing the number of pro-inflammatory macrophages. Shotgun proteomics of the aorta showed a metabolic rewiring in CX3CR1+-Tregs treated Ldlr -/- mice compared to controls that was associated with the improvement of inflammation-resolving pathways and disease progression. CONCLUSION: ACT with vasculotropic Tregs appears as a promising strategy to selectively target immune activation in the atherosclerotic plaque.

Atopic Dermatitis-Like Rash During Evolocumab Treatment of Familial Hypercholesterolemia.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that targets the low-density lipoprotein (LDL) receptor for lysosomal degradation. PCSK9 impedes the receptor-mediated clearance of LDL-cholesterol, thereby increasing serum LDL-cholesterol levels. Evolocumab, a human monoclonal antibody against PCSK9, effectively reduces serum LDL-cholesterol levels. We report the first known case of a patient who developed an atopic dermatitis (AD)-like rash during evolocumab therapy. A 43-year-old Japanese man with heterozygous familial hypercholesterolemia was treated with subcutaneous injection of 140 mg evolocumab biweekly, for 16 months. The therapy was then changed to subcutaneous injection of 420 mg evolocumab monthly. A few days after the first dose, the patient experienced pruritus and rash on his extremities. The rash worsened, while the pruritus subsided, then relapsed after the second and third doses. He had erythema and excoriation on his legs, lichenification over his popliteal fossa, xerosis on his forearms, an increased serum IgE level, and a family history of AD in his siblings. We made a provisional diagnosis of AD characterized by enhanced type 2 helper T (Th2) activity and treated him with topical corticosteroids and oral anti-histamines. His rash improved and did not relapse after the fifth dose; however, his LDL-cholesterol level increased. PCSK9 or oxidized LDL activates macrophages or dendritic cells, respectively, and enhances their activity to induce Th1 cells antagonizing Th2 cells. We hypothesized that high-dose evolocumab may suppress Th1 activity to antagonize Th2, and unmask Th2 disposition based on the patient's atopic diathesis, triggering the rash mimicking AD. Clinicians should be aware of rash development during evolocumab therapy.

Function and Immunogenicity of Gene-corrected iPSC-derived Hepatocyte-Like Cells in Restoring Low Density Lipoprotein Uptake in Homozygous Familial Hypercholesterolemia.

Gene correction of induced pluripotent stem cells (iPSCs) has therapeutic potential for treating homozygous familial hypercholesterolemia (HoFH) associated with low-density lipoprotein (LDL) receptor (LDLR) dysfunction. However, few data exist regarding the functional recovery and immunogenicity of LDLR gene-corrected iPSC-derived hepatocyte-like cells (HLCs) obtained from an HoFH patient. Therefore, we generated iPSC-derived HLCs from an HoFH patient harbouring a point mutation (NM_000527.4:c.901 G > T) in exon 6 of LDLR, and examined their function and immunogenicity. From the patient's iPSCs, one homozygous gene-corrected HoFH-iPSC clone and two heterozygous clones were generated using the CRISPR/Cas9 method. Both types of iPSC-derived HLCs showed recovery of the function of LDL uptake in immunofluorescence staining analysis. Furthermore, these gene-corrected iPSC-derived HLCs showed little immunogenicity against the patient's peripheral blood mononuclear cells in a cell-mediated cytotoxicity assay. These results demonstrate that LDL uptake of iPSC-derived HLCs from HoFH can be restored by gene correction without the appearance of further immunogenicity, suggesting that gene-corrected iPSC-derived HLCs are applicable to the treatment of HoFH.

Multiple crm- mutations in familial hypercholesterolemia. Evidence for 13 alleles, including four deletions.

The low density lipoprotein (LDL) receptors in fibroblasts from 132 subjects with the clinical syndrome of homozygous familial hypercholesterolemia were analyzed by immunoprecipitation with an anti-LDL receptor monoclonal antibody. 16 of the 132 cell strains (12%) synthesized no immunodetectable LDL receptor protein, indicating the presence of two mutant genes that failed to produce cross-reacting material (crm- mutations). DNA and mRNA from 15 of the 16 crm- patients, representing 30 crm- genes, were available for further study. Haplotype analysis based on 10 restriction fragment length polymorphisms (RFLPs) suggested that the 30 crm- genes represent 13 mutant alleles. Four of the alleles produced no mRNA. Three of these four mRNA- alleles had large deletions ranging from 6 to 20 kb that eliminated the promoter region of the gene. The fourth mRNA- allele did not contain any deletion or alteration in the promoter sequence; the reason for the mRNA- phenotype was not apparent. Nine alleles were positive for mRNAs, of which three encoded mRNAs of abnormal size. One of the abnormal mRNAs was produced by a gene harboring a deletion, and another was produced by a gene with a complex rearrangement. The third abnormal-sized mRNA (3.1 kb larger than normal) was produced by an allele that had no detectable alterations as judged by Southern blotting. The other six mRNA+ alleles appeared normal by Southern blotting and produced normal-sized mRNA but no receptor protein. The current studies demonstrate that mRNA analysis coupled with haplotype determination by Southern blot analysis can be used to classify crm- mutations at a genetic locus where multiple alleles exist.

Chemokines in children with heterozygous familiar hypercholesterolemia: selective upregulation of RANTES.

OBJECTIVE: Increasing data support the involvement of chemokines in atherogenesis. However, although several studies have shown increased chemokine levels in adult patients, the literature is virtually devoid of data on chemokines in children with hypercholesterolemia. METHODS AND RESULTS: We examined the gene expression of chemokines in peripheral blood mononuclear cells (PBMCs) from clinically healthy children with and without heterozygous familial hypercholesterolemia (FH). Our main findings were: (1) compared with healthy controls, PBMCs from FH children showed significantly higher mRNA levels of RANTES, but not of the other examined chemokines; (2) an opposite pattern was seen in adult FH subjects, with markedly enhanced expression of macrophage inflammatory peptide-1alpha, but not of RANTES; (3) this increased gene expression of RANTES in PBMCs from FH children seemed to reflect enhanced RANTES expression in monocytes but not in T cells; (4) FH children also had raised serum levels of neopterin, additionally suggesting monocyte/macrophage activation in these children; and (5) PBMCs from both FH children and controls showed enhanced release of interleukin 8 on RANTES stimulation in vitro. CONCLUSIONS: Our findings support a role of inflammation also in the early stages of atherogenesis possibly involving monocyte-derived RANTES as an important mediator.

Efficacy and safety of the proprotein convertase subtilisin/kexin type 9 monoclonal antibody alirocumab vs placebo in patients with heterozygous familial hypercholesterolemia.

BACKGROUND: Patients with heterozygous familial hypercholesterolemia (HeFH) are characterized by elevated low-density lipoprotein cholesterol (LDL-C) levels. Long-term effects of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition have not been thoroughly investigated in these patients. OBJECTIVE: We evaluated efficacy and safety of alirocumab, a PCSK9 inhibitor, vs placebo in patients with HeFH. METHODS: In total, 1257 patients with HeFH on maximally tolerated statin ± other lipid-lowering therapies from four 78-week ODYSSEY trials were analyzed. In FH I and II, patients with baseline LDL-C levels ≥70/100 mg/dL (n = 735), depending on documented cardiovascular disease history, received placebo or alirocumab 75 mg every 2 weeks (Q2W; with dose increase to 150 mg Q2W at week 12 if week 8 LDL-C was ≥70 mg/dL). Separately, data were pooled from HIGH FH (baseline LDL-C ≥160 mg/dL) and patients with HeFH from LONG TERM (baseline LDL-C ≥70 mg/dL), where patients received placebo or alirocumab 150 mg Q2W (n = 522). RESULTS: At week 24, alirocumab reduced LDL-C levels by -48.8% (75/150 mg Q2W; placebo: +7.1%) and -55.0% (alirocumab 150 mg Q2W; placebo: +1.3%) (both P < .0001 vs placebo; intention-to-treat analysis). Least-squares mean LDL-C levels of 69.1 to 75.6 mg/dL (alirocumab 75/150 mg/dL Q2W; baseline: 141.3 mg/dL) and 72.2 to 82.3 mg/dL (alirocumab 150 mg Q2W; baseline: 168.4 mg/dL) were achieved at weeks 24 to 78 (on-treatment analysis). Additional beneficial effects were observed in other lipids. Treatment-emergent adverse event rates were similar in the alirocumab (80.5%) and placebo groups (83.0%). CONCLUSIONS: In this large cohort of patients with HeFH, alirocumab significantly reduced LDL-C levels. Alirocumab was generally well tolerated.

Altered leukocyte distribution under hypercholesterolemia: A cross-sectional study in children with familial hypercholesterolemia.

BACKGROUND AND AIMS: Children with familial hypercholesterolemia (FH) have elevated LDL cholesterol from the first year of life, and represent a model of early-stage atherosclerosis. Data suggest that adults with FH have alterations in circulating monocyte subpopulations towards a more pro-inflammatory phenotype, but it is not known whether FH children have similar perturbations. In addition, there are no data on the distribution of lymphocyte subpopulations in FH children. The objective of the present study was to characterize the distributions of circulating monocyte and lymphocyte subpopulations in children with FH and healthy, normocholesterolemic children. METHODS: Using flow cytometry analysis, we analyzed whole blood B- and T-cell subpopulations and monocyte subpopulations in FH (n = 23) and healthy (n = 20) children. Moreover, we measured serum markers of leukocyte and endothelial cell activation using EIA. RESULTS: We found that FH children had monocytosis as well as a shift in the monocyte subpopulations. This shift was characterized by higher circulating pro-inflammatory and non-classical monocytes, and lower levels of classical monocytes, and seemed to be present only in FH children with low HDL cholesterol (HDL-C, below 1.3 mmol/L). Additionally, monocytes expressing CD18 and serum E-selectin were higher in FH children, in particular FH children with low HDL-C. CONCLUSIONS: FH children with low HDL-C had monocytosis as well as a shift in monocyte subpopulations towards a more pro-inflammatory phenotype. Our results suggest activation of monocytes at a very early stage of atherosclerosis in humans.

Identification of mildly oxidized low-density lipoprotein (electronegative LDL) and its auto-antibodies IgG in children and adolescents hypercholesterolemic offsprings.

BACKGROUND: Oxidative modification of low-density lipoproteins (LDL) is an essential step in atherogenesis, generating minimally oxidized LDL, also called electronegative LDL [LDL(-)], which has chemotactic, cytotoxic and immunogenic properties. METHODS AND RESULTS: Serum LDL(-) and anti-LDL(-) auto-antibodies (IgG) were evaluated in 28 children and adolescents with familial hypercholesterolemia (FH) antecedents, with or without early coronary artery disease in first-degree relatives (eCAD), hypercholesterolemic (hc) or normocholesterolemic (nc) versus a control group of normocholesterolemic children without pathologic antecedents (C). ELISA method was used for detection of LDL(-) and anti-LDL(-) IgG. LDL(-) serum levels did not differ among the four groups (FH-eCAD-hc 41.4 +/- 24.9 microg/dl; FH-hc 38.3 +/- 11.2 microg/dl; FH-nc 47.3 +/- 17.0 microg/dl and C 44.2 +/- 28.8 microg/dl, p = 0.659). However, IgG anti-LDL(-) auto-antibodies were significantly higher in the control group in comparison to the FH groups with or without eCAD, independent of hypercholesterolemia or normocholesterolemia (FH-eCAD-hc 0.825 +/- 0.289 microg/dl; FH-hc 0.667 +/- 0.307 microg/dl; FH-nc 0.763 +/- 0.204 microg/dl and C 1.105 +/- 0.233 microg/dl, p = 0.006). When the auto-antibodies of groups with FH, with or without eCAD and with or without hypercholesterolemia were compared, no differences were found (p = 0.509). CONCLUSION: These results showed that FH and/or eCAD children and adolescents have lower titers of auto-antibodies anti-LDL(-) than children from normal families, independent of serum LDL-cholesterol or serum LDL(-).

Anti-sense oligonucleotide therapies for the treatment of hyperlipidaemia.

INTRODUCTION: Anti-sense oligonucleotide (ASO) therapies are a new development in clinical pharmacology offering greater specificity compared to small molecule inhibitors and the ability to target intracellular process' not susceptible to antibody-based therapies. AREAS COVERED: This article reviews the chemical biology of ASOs and related RNA therapeutics. It then reviews the data on their use to treat hyperlipidaemia. Data on mipomersen - an ASO to apolipoprotein B-100(apoB) licensed for treatment of homozygous familial hypercholesterolaemia (FH) is presented. Few effective therapies are available to reduce atehrogenic lipoprotein (a) levels. An ASO therapy to apolipoprotein(a) (ISIS Apo(a)Rx) specifically reduced lipoprotein (a) levels by up to 78%. Treatment options for patients with familial chylomicronaemia syndrome (lipoprotein lipase deficiency; LPLD) or lipodystrophies are highly limited and often inadequate. Volanesorsen, an ASO to apolipoprotein C-3, shows promise in the treatment of LPLD and severe hypertriglyceridaemia as it increases clearance of triglyceride-rich lipoproteins and can normalise triglycerides in these patients. EXPERT OPINION: The uptake of the novel ASO therapies is likely to be limited to selected niche groups or orphan diseases. These will include homozygous FH, severe heterozygous FH for mipomersen; LPLD deficiency and lipodystrophy syndromes for volanesorsen and treatment of patients with high elevated Lp(a) levels.