Study of the macrophage mitochondrial function during atherosclerosis

L'athérosclérose est l'une des principales causes de mortalité cardiovasculaire. Elle fait suite à la pénétration et l'oxydation des lipoprotéines de faible densité (LDL) au niveau de la paroi artérielle. La captation non régulée de ces LDL oxydées par les macrophages aboutit à une accumulation de cholestérol intracellulaire et à leur transformation en cellules spumeuses. Ces évènements sont à l'origine de la constitution des plaques d'athérome.Plusieurs arguments tendent à montrer que la progression de l'athérosclérose est liée à une dysfonction mitochondriale. L'étude de plaques d'athérome humaines a permis d'observer in vivo, une réduction de la respiration mitochondriale, associée à des altérations de l'ADNmt et une diminution de l'expression des facteurs transcriptionnels régulant la biogenèse mitochondriale (PGC-1α, NRF1, TFAM). De plus, le métabolisme mitochondrial joue un rôle essentiel dans la régulation de l'homéostasie lipidique au sein des macrophages. En effet, il a été montré que l'efflux du cholestérol hors des cellules spumeuses est dépendant de la production d'ATP par la chaine respiratoire mitochondriale. D'autre part, la mitochondrie est une source essentielle d'oxystérols qui sont d'importants modulateurs de l'efflux du cholestérol.L'objectif de ce travail a donc été de caractériser la fonction mitochondriale dans les macrophages spumeux. En effet, la surcharge en cholestérol, ainsi que l'import de produits d'oxydation lipidiques suite à l'internalisation des LDL oxydées, sont susceptibles d'altérer le fonctionnement mitochondrial. Afin d'étudier ces mécanismes, nous avons utilisé des macrophages de la lignée cellulaire THP-1 qui ont été exposés, soit à des LDL acétylées afin d'obtenir une surcharge intracellulaire en cholestérol, soit à des LDL oxydées transportant à la fois du cholestérol et des produits d'oxydation lipidiques. La fonction mitochondriale a été évaluée dans ces différentes conditions par la mesure de marqueurs du métabolisme énergétique, de la phosphorylation oxydative, du stress oxydant, de la biogenèse et de la dynamique mitochondriale.Nous avons observé que l'exposition des macrophages aux LDL oxydées entraine une réduction significative de la respiration mitochondriale, des activités des complexes I et III, et de la quantité d'ADNmt, associée à un état de stress oxydant, induisant la mise en place d'un cercle vicieux conduisant à la mort cellulaire. A l'inverse, la surcharge en cholestérol induite par l'exposition aux LDL acétylées se caractérise par le maintien d'une phosphorylation oxydative efficace en dépit de la diminution des activités de certaines enzymes du cycle de Krebs : l'aconitase, l'isocitrate déshydrogénase et l'α-cétoglutarate déshydrogénase. Afin de mieux comprendre les mécanismes sous-jacents à ces altérations mitochondriales, nous avons également exposé les macrophages à différents oxystérols et analysé leurs effets sur la consommation de glucose et la production de lactate. Toutefois, nous n'avons pas observé de modifications significatives du métabolisme énergétique dans ces conditions.Ce travail met en évidence les différences phénotypiques exprimées par les macrophages afin de s'adapter à l'environnement lipidique auxquels ils sont exposés. Nous rapportons ainsi la capacité des macrophages à maintenir leur fonction énergétique malgré l'internalisation massive de cholestérol provoquée par la captation de LDL acétylées et la réduction de l'activité de plusieurs enzymes du cycle de Krebs. De ce fait, cette étude apporte de nouveaux éléments nécessaires à la compréhension des mécanismes à l'origine de la dysfonction mitochondriale observée au cours des processus d'athérosclérose. De plus, l'identification et la modulation des paramètres affectant la fonction mitochondriale dans les macrophages pourraient constituer une nouvelle cible thérapeutique de stimulation de l'efflux du cholestérol.Atherosclerosis is a major cause of cardiovascular mortality. It occurs following the penetration and oxidation of low density lipoproteins (LDL) in the arterial wall. The unregulated uptake of these oxidized LDL by macrophages results in an accumulation of intracellular cholesterol and their transformation into foam cells. These events are at the origin of the atherosclerotic plaques.Several arguments suggest that the progression of atherosclerosis is linked to a mitochondrial dysfunction. In human atherosclerotic plaques, a reduction of mitochondrial respiration, associated with mtDNA alterations and the decreased expression of the transcriptional factors regulating mitochondrial biogenesis (PGC-1α, NRF1, TFAM) have been reported in vivo. Moreover, mitochondrial metabolism plays an essential role in the regulation of lipid homeostasis within macrophages. Indeed, it has been demonstrated that the cholesterol efflux out of the foam cells is dependent of the ATP production by the mitochondrial respiratory chain. Besides, mitochondria are an essential source of oxysterols, which are major cholesterol efflux modulators.The aim of the study was to characterize the mitochondrial function in foam cells. Indeed, the cholesterol overload, as well as the lipid oxidation products carried by the oxidized LDL, are susceptible to alter the mitochondrial function of macrophages. In order to study these mechanisms, we used macrophages from the THP-1 cell line which were exposed either to acetylated LDL, in order to obtain an intracellular cholesterol overload, or to oxidized LDL carrying both cholesterol and lipid oxidation products. Mitochondrial function was assessed in these different conditions by measuring markers of the energetic metabolism, the oxidative phosphorylation, the oxidative stress, the mitochondrial biogenesis and dynamics.We observed that the exposure of macrophages to oxidized LDL leads to a significant reduction of mitochondrial respiration, of complexes I and III activities, and of mtDNA copy number, in association with oxidative stress state, inducing the setting up of a vicious circle, leading to cell death. On the other hand, the cholesterol overload induced by exposure to acetylated LDL is characterized by the maintenance of oxidative phosphorylation efficiency despite the decreased activities of several tricarboxylic acids (TCA) cycle enzymes : aconitase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. In order to better understand the mechanisms underlying these mitochondrial alterations, we also exposed macrophages to different oxysterols and we analyzed their effects on glucose consumption and lactate production. However, we did not observe significant changes in energy metabolism under these conditions.This work reveals the phenotypic differences expressed by macrophages according to the lipid environment to which they are exposed. Thus, we report the ability of macrophages to maintain their energetic function despite the massive internalization of cholesterol caused by exposure to acetylated LDL and the reduction of the activity of several enzymes of the TCA cycle. Therefore, this study provides new findings to understand the mechanisms causing the mitochondrial dysfunction observed during the atherosclerotic processes. Moreover, the identification and modulation of the parameters affecting the mitochondrial function in macrophages could constitute a new therapeutic target for the stimulation of cholesterol efflux

Targeting mitochondrial function in macrophages: A novel treatment strategy for atherosclerotic cardiovascular disease?

International audienceAtherosclerotic cardiovascular disease is a major cause of morbidity and mortality due to chronic arterial injury caused by hyperlipidemia, hypertension, inflammation and oxidative stress. Recent studies have shown that the progression of this disease is associated with mitochondrial dysfunction and with the accumulation of mitochondrial alterations within macrophages of atherosclerotic plaques. These alterations contribute to processes of inflammation and oxidative stress. Among the many players involved, macrophages play a pivotal role in atherogenesis as they can exert both beneficial and deleterious effects due to their anti-and pro-inflammatory properties. Their atheroprotective functions, such as cholesterol efflux and efferocytosis, as well as the maintenance of their polarization towards an anti-inflammatory state, are particularly dependent on mitochondrial metabolism. Moreover, in vitro studies have demonstrated deleterious effects of oxidized LDL on macrophage mitochondrial function, resulting in a switch to a pro-inflammatory state and to a potential loss of atheroprotective capacity. Therefore, preservation of mitochondrial function is now considered a legitimate therapeutic strategy. This review focuses on the potential therapeutic strategies that could improve the mitochondrial function of macrophages, enabling them to maintain their atheroprotective capacity. These emerging therapies could play a valuable role in counteracting the progression of atherosclerotic lesions and possibly inducing their regression

Cholesterol accumulation induced by acetylated LDL exposure modifies the enzymatic activities of the TCA cycle without impairing the respiratory chain functionality in macrophages

International audienceThe unregulated uptake of modified low-density lipoproteins (LDL) by macrophages leads to foam cell formation, promoting atherosclerotic plaque progression. The cholesterol efflux capacity of macrophages by the ATP-Binding Cassette transporters depends on the ATP mitochondrial production. Therefore, the mitochondrial function maintenance is crucial in limiting foam cell formation. Thus, we aimed to investigate the mechanisms involved in the mitochondrial dysfunction that may occur in cholesterol-laden macrophages. We incubated THP-1 macrophages with acetylated LDL (acLDL) to obtain cholesterol-laden cells or with mildly oxidized LDL (oxLDL) to generate cholesterol- and oxidized lipids-laden cells. Cellular cholesterol content was measured in each condition. Mitochondrial function was evaluated by measurement of several markers of energetic metabolism, oxidative phosphorylation, oxidative stress, mitochondrial biogenesis and dynamics. OxLDL-exposed macrophages exhibited a significantly reduced mitochondrial respiration and complexes I and III activities, associated to an oxidative stress state and a reduced mitochondrial DNA copy number. Meanwhile, acLDL-exposed macrophages featured an efficient oxidative phosphorylation despite the decreased activities of aconitase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. Our study revealed that mitochondrial function was differently impacted according to the nature of modified LDL. Exposure to cholesterol and oxidized lipids carried by oxLDL leads to a mitochondrial dysfunction in macrophages, affecting the mitochondrial respiratory chain functional capacity, whereas the cellular cholesterol enrichment induced by acLDL exposure results in a tricarboxylic acid cycle shunt while maintaining mitochondrial energetic production, reflecting a metabolic adaptation to cholesterol intake. These new mechanistic insights are of direct relevance to the understanding of the mitochondrial dysfunction in foam cells

Mitochondrial dysfunction caused by novel ATAD3A mutations

International audienceMitochondrial respiratory chain integrity depends on a number of proteins encoded by nuclear and mitochondrial genomes. Mutations of such factors can result in isolated or combined respiratory chain deficits, some of which can induce abnormal morphology of the mitochondrial network or accumulation of intermediary metabolites. Consequently, affected patients are clinically heterogeneous, presenting with central nervous system, muscular, or neurodegenerative disorders. ATAD3A is a nuclear-encoded ATPase protein of the AAA+ family and has been localized to the inner mitochondrial membrane. Recently reported mutations or large deletions in the ATDA3A gene in patients have been shown to induce altered mitochondrial structure and function and abnormal cholesterol metabolism in a recessive or dominant manner. Here, we report two siblings presenting axonal sensory-motor neuropathy associated with neonatal cataract. Genetic analyses identified two novel mutations in ATAD3A; a point mutation and an intronic 15 bp deletion affecting splicing and leading to exon skipping. Biochemical analysis in patient cells and tissues showed abnormal function of the mitochondrial respiratory chain in muscle and abnormal mitochondrial cristae structure. These new cases underline the large spectrum of biochemical and clinical presentations of ATAD3A deficiency and the different modes of inheritance, making it an atypical mitochondrial disorder

Homozygous MFN2 variants causing severe antenatal encephalopathy with clumped mitochondria

International audiencePathogenic variants in the MFN2 gene are commonly associated with autosomal dominant (CMT2A2A) or recessive (CMT2A2B) Charcot-Marie-Tooth disease, with possible involvement of the CNS. Here, we present a case of severe antenatal encephalopathy with lissencephaly, polymicrogyria and cerebellar atrophy. Whole genome analysis revealed a homozygous deletion c.1717-274_1734 del (NM_014874.4) in the MFN2 gene, leading to exon 16 skipping and in-frame loss of 50 amino acids (p.Gln574_Val624del), removing the proline-rich domain and the transmembrane domain 1 (TM1). MFN2 is a transmembrane GTPase located on the mitochondrial outer membrane that contributes to mitochondrial fusion, shaping large mitochondrial networks within cells. In silico modelling showed that the loss of the TM1 domain resulted in a drastically altered topological insertion of the protein in the mitochondrial outer membrane. Fetus fibroblasts, investigated by fluorescent cell imaging, electron microscopy and time-lapse recording, showed a sharp alteration of the mitochondrial network, with clumped mitochondria and clusters of tethered mitochondria unable to fuse. Multiple deficiencies of respiratory chain complexes with severe impairment of complex I were also evidenced in patient fibroblasts, without involvement of mitochondrial DNA instability. This is the first reported case of a severe developmental defect due to MFN2 deficiency with clumped mitochondria

Distinct phospholipid and sphingolipid species are linked to altered HDL function in apolipoprotein A-I deficiency

International audienceBACKGROUND:Familial apolipoprotein A-I (apoA-I) deficiency (FAID) involving low levels of both apoA-I and high-density lipoprotein (HDL) cholesterol is associated with accelerated atherosclerosis.OBJECTIVE:The objective of this study was to define distinctive patterns in the lipidome of HDL subpopulations in FAID in relationship to antiatherogenic activities.METHODS:Five HDL subfractions were isolated by ultracentrifugation from plasma of FAID Caucasian patients (n = 5) and age-matched healthy normolipidemic Caucasian controls (n = 8), and the HDL lipidome (160 molecular species of 9 classes of phospholipids and sphingolipids) was quantitatively evaluated.RESULTS:Increased concentrations of numerous molecular species of lysophosphatidylcholine (up to 12-fold), ceramides (up to 3-fold), phosphatidylserine (up to 34-fold), phosphatidic acid (up to 71-fold), and phosphatidylglycerol (up to 20-fold) were detected throughout all five HDL subpopulations as compared with their counterparts from controls, whereas concentrations of phosphatidylethanolamine species were decreased (up to 5-fold). Moderately to highly abundant, within their lipid class, species of phosphatidylcholine, sphingomyelin, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, and ceramide featuring multiple unsaturations were primarily affected by apoA-I deficiency; their HDL content, particularly that of phosphatidylcholine (34:2), was strongly correlated with HDL function, impaired in FAID. Metabolic pathway analysis revealed that sphingolipid, glycerophospholipid, and linoleic acid metabolism was significantly affected by FAID.CONCLUSION:These data reveal that altered content of specific phospholipid and sphingolipid species is linked to deficient antiatherogenic properties of HDL in FAID

Droplet Digital PCR combined with minisequencing, a new approach to analyze fetal DNA from maternal blood: application to the non-invasive prenatal diagnosis of achondroplasia.

International audienceBackground - Achondroplasia is generally detected by abnormal prenatal ultrasound findings in the third trimester of pregnancy and then confirmed by molecular genetic testing of fetal genomic DNA obtained by aspiration of amniotic fluid. This invasive procedure presents a small but significant risk for both the fetus and mother. Therefore, non-invasive procedures using cell-free fetal DNA in maternal plasma have been developed for the detection of the fetal achondroplasia mutations. Methods - To determine whether the fetus carries the de novo mis-sense genetic mutation at nucleotide 1138 in FGFR3 gene involved in >99% of achondroplasia cases, we developed two independent methods: digital-droplet PCR combined with minisequencing, which are very sensitive methods allowing detection of rare alleles. Results - We collected 26 plasmatic samples from women carrying fetus at risk of achondroplasia and diagnosed to date a total of five affected fetuses in maternal blood. The sensitivity and specificity of our test are respectively 100% [95% confidence interval, 56.6-100%] and 100% [95% confidence interval, 84.5-100%]. Conclusions - This novel, original strategy for non-invasive prenatal diagnosis of achondroplasia is suitable for implementation in routine clinical testing and allows considering extending the applications of these technologies in non-invasive prenatal diagnosis of many other monogenic diseases. © 2016 John Wiley & Sons, Ltd

Cerebrospinal fluid exposure to bictegravir/emtricitabine/tenofovir in HIV-1-infected patients with CNS impairment

International audienceObjectives: The penetration of antiretroviral drugs into deep compartments, such as the CNS, is a crucial component of strategies towards an HIV cure. This study aimed to determine CSF concentrations of bictegravir, emtricitabine and tenofovir in patients with HIV-related CNS impairment (HCI) enrolled in a real-life observational study. Methods: Patients with HCI treated by optimized ART, including bictegravir/emtricitabine/tenofovir alafenamide (BIC/FTC/TAF) for at least 1 month were enrolled. Plasma and CSF concentrations were measured by quality control-validated assays (LC-MS/MS). The inhibitory quotient (IQARV) was calculated as the ratio of unbound (bictegravir) or total (emtricitabine and tenofovir) concentration to half (or 90%) maximal inhibitory concentration for bictegravir (or emtricitabine and tenofovir). All numerical variables are expressed as median (range). Results: Twenty-four patients (nine women) were enrolled. The age was 45 (26-68) years. Unbound bictegravir and total emtricitabine and tenofovir CSF concentrations were 4.4 (1.6-9.6), 84.4 (28.6-337.4) and 1.6 (0.7-4.3) ng/mL, respectively. The unbound bictegravir CSF fraction was 34% (15%-82%) versus 0.33% (0.11%-0.92%) in plasma. Three patients had an IQARV above unity for the three antiretrovirals. Factors positively associated with the CSF concentration (unbound for bictegravir) were age and total plasma concentration for the three antiretrovirals. Patients aged over 51 years had higher CSF concentrations (unbound for bictegravir). Conclusions: We observed low CSF exposure to bictegravir, emtricitabine and tenofovir. These results suggest that BIC/FTC/TAF should be used with caution as first-line treatment for people living with HIV with HCI under 51 years of age

Resistance to Insulin in Patients with Gitelman Syndrome and a Subtle Intermediate Phenotype in Heterozygous Carriers: A Cross-Sectional Study

BACKGROUND: Gitelman syndrome is a salt-losing tubulopathy caused by mutations in the SLC12A3 gene, which encodes the thiazide-sensitive sodium-chloride cotransporter. Previous studies suggested an intermediate phenotype for heterozygous carriers. METHODS: To evaluate the phenotype of heterozygous carriers of pathogenic SLC12A3 mutations, we performed a cross-sectional study of patients with Gitelman syndrome, heterozygous carriers, and healthy noncarriers. Participants measured their BP at home for three consecutive days before hospital admission for blood and urine sampling and an oral glucose tolerance test. RESULTS: We enrolled 242 participants, aged 18-75 years, including 81 heterozygous carriers, 82 healthy noncarriers, and 79 patients with Gitelman syndrome. The three groups had similar age, sex ratio, and body mass index. Compared with healthy noncarriers, heterozygous carriers showed significantly higher serum calcium concentration (P=0.01) and a trend for higher plasma aldosterone (P=0.06), but measures of home BP, plasma and urine electrolytes, renin, parathyroid hormone, vitamin D, and response to oral glucose tolerance testing were similar. Patients with Gitelman syndrome had lower systolic BP and higher heart rate than noncarriers and heterozygote carriers; they also had significantly higher fasting serum glucose concentration, higher levels of markers of insulin resistance, and a three-fold higher sensitivity to overweight. According to oral glucose tolerance testing, approximately 14% of patients with Gitelman syndrome were prediabetic, compared with 5% of heterozygous carriers and 4% of healthy noncarriers. CONCLUSIONS: Heterozygous carriers had a weak intermediate phenotype, between that of healthy noncarriers and patients with Gitelman syndrome. Moreover, the latter are at risk for development of type 2 diabetes, indicating the heightened importance of body weight control in these patients