Erasmo Laura
email: laura.derasmo@uniroma1.it

Di Costanzo Alessia

Antonio Gallo

Eric Bruckert

Arca Marcello

Laura D ' Erasmo

Alessia Di Costanzo
email: alessia.dicostanzo@uniroma1.it

APOCIII: A MULTIFACETED PROTEIN IN CARDIOMETABOLIC DISEASE

Keywords: APOC3 gene, ApoCIII protein, triglycerides, triglycerides (TG)-rich lipoproteins (TRL), APOC3 gene expression, cardiovascular disease risk, therapeutic target ApoB, apolipoprotein B, ApoCIII, Apolipoprotein CIII, APOE, apolipoprotein E, ASCVD, atherosclerotic cardiovascular disease, ASO, antisense oligonucleotide, CHD, coronary heart disease, ChREBP, Carbohydrate-responsive element-binding protein, CM, chylomicrons, CVD, cardiovascular disease, DHA, docosahexaenoic acid, EPA, eicosapentaenoic acid, ERRa, estrogen-related receptor a, FCS, Familial Hyperchylomicronemia, FOXO1,

ApoCIII has a well-recognized role in triglyceride-rich lipoproteins metabolism. A considerable amount of data has clearly highlighted that high levels of ApoCIII lead to hypertriglyceridemia and, thereby, may influence the risk of cardiovascular disease. However, recent findings indicate that ApoCIII might also act beyond lipid metabolism. Indeed, ApoCIII has been implicated in other physiological processes such as glucose homeostasis, monocyte adhesion, activation of inflammatory pathways, and modulation of the coagulation cascade. As the inhibition of ApoCIII is emerging as a new promising therapeutic strategy, the complete understanding of multifaceted pathophysiological role of this apoprotein may be relevant. Therefore, the purpose of this work is to review available evidences not only related to genetics and biochemistry of ApoCIII, but also highlighting the role of this apoprotein in triglyceride and glucose metabolism, in the inflammatory process and coagulation cascade as well as in cardiovascular disease.

Introduction

Apolipoprotein CIII (ApoCIII) is one of the most abundant apolipoproteins in the blood, where it is present as an exchangeable component between high-density lipoprotein (HDL) and apolipoprotein B (ApoB)-containing lipoprotein particles (very-low and low density lipoproteins, VLDL and LDL). While in normolipidemic subjects, the majority of plasma ApoCIII is bound to HDL, in hypertriglyceridemic subjects it is bound to triglyceride (TG) rich lipoprotein (TRL), VLDL and chylomicrons (CM) [START_REF] Schonfeld | Apolipoprotein C-II and C-III levels in hyperlipoproteinemia[END_REF][START_REF] Fredenrich | Plasma lipoprotein distribution of apoC-III in normolipidemic and hypertriglyceridemic subjects: comparison of the apoC-III to apoE ratio in different lipoprotein fractions[END_REF].

ApoCIII was first isolated and characterized nearly 50 years ago by Brown et al. [START_REF] Brown | Inhibition of lipoprotein lipase by an apoprotein of human very low density lipoprotein[END_REF]. Studies on protein structure and dynamics revealed that it is an 8.8 kDa glycoprotein synthesized mainly by the liver, and to a lesser extent, by the intestine [START_REF] Cohn | Rate of production of plasma and very-low-density lipoprotein (VLDL) apolipoprotein C-III is strongly related to the concentration and level of production of VLDL triglyceride in male subjects with different body weights and levels of insulin sensitivity[END_REF][START_REF] Brewer | The complete amino acid sequence of alanine apolipoprotein (apoC-3), and apolipoprotein from human plasma very low density lipoproteins[END_REF][START_REF] Yassine | The Association of Human Apolipoprotein C-III Sialylation Proteoforms with Plasma Triglycerides[END_REF][START_REF] Vaith | Characterization of the oligosaccharide side chain of apolipoprotein C-III from human plasma very low density lipoproteins[END_REF]. For several years, ApoCIII has been investigated mainly for its strong correlation with TG concentrations. Firstly, ApoCIII was recognized as an inhibitor of lipoprotein lipase (LPL) and hepatic lipase (HL) [START_REF] Chapman | Triglyceride-rich lipoproteins and high-density lipoprotein cholesterol in patients at high risk of cardiovascular disease: evidence and guidance for management[END_REF][START_REF] Havel | Cofactor activity of protein components of human very low density lipoproteins in the hydrolysis of triglycerides by lipoproteins lipase from different sources[END_REF][START_REF] Landis | Influence of apolipoprotein E on soluble and heparin-immobilized hepatic lipase[END_REF], key enzymes in the hydrolysis of TG contained in TRL [START_REF] Chatterjee | Hepatic lipase, high density lipoproteins, and hypertriglyceridemia[END_REF]. Secondly, in addition to its extracellular functions, ApoCIII was found to play an intracellular role by promoting VLDL-TG secretion by hepatocytes [START_REF] Chan | Apolipoprotein B-100 kinetics in visceral obesity: associations with plasma apolipoprotein C-III concentration[END_REF]. Genome-wide association studies (GWAS) and Mendelian randomization studies have demonstrated a direct relationship between loss-of-function (LOF) mutations in ApoCIII-coding gene (APOC3) and reduced risk of atherosclerotic cardiovascular disease (ASCVD) [START_REF] Wulff | APOC3 Loss-of-Function Mutations, Remnant Cholesterol, Low-Density Lipoprotein Cholesterol, and Cardiovascular Risk: Mediation-and Meta-Analyses of 137 895 Individuals[END_REF]. Further evidences have highlighted how ApoCIII levels directly associate with endothelial cells (EC) dysfunction, monocyte adhesion, thrombosis, type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD). All these data allowed the identification of ApoCIII as a therapeutic target to treat hypertriglyceridemia (HTG), T2DM and to reduce ASCVD risk.

Therefore, ApoCIII should be considered as a multifunctional protein with major physiological relevance beyond TRL metabolism and with a key role in cardiometabolic diseases.

The genetics of ApoCIII

APOC3 gene is a member of the APOA1/C3/A4/A5 gene cluster, an area in strong linkage with lipid metabolism. The gene encoding for ApoCIII is located on the long arm of chromosome 11 at position 23.3 (11q23.3).

Transcriptional control of gene expression

APOC3 gene expression is tightly regulated through several factors determining its tissue-specific expression. Scarce data exist regarding which factors regulate its expression in the intestine. By contrast, abundant research on mechanisms that control APOC3 expression in the liver has been conducted [START_REF] Qamar | Plasma apolipoprotein C-III levels, triglycerides, and coronary artery calcification in type 2 diabetics[END_REF][START_REF] Leff | A regulatory element in the ApoCIII promoter that directs hepatic specific transcription binds to proteins in expressing and nonexpressing cell types[END_REF][START_REF] Ogami | Promoter elements and factors required for hepatic and intestinal transcription of the human ApoCIII gene[END_REF][START_REF] Adamson | Hepatocyte nuclear factor-4alpha contributes to carbohydrate-induced transcriptional activation of hepatic fatty acid synthase[END_REF][START_REF] Chen | Transcriptional regulation of the apoC-III gene by insulin in diabetic mice: correlation with changes in plasma triglyceride levels[END_REF][START_REF] Schoonjans | Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression[END_REF][START_REF] Yc | Role of thyroid hormone in the expression of apolipoprotein A-IV and C-III genes in rat liver[END_REF][START_REF] Lacorte | Activation of CAAT enhancer-binding protein delta (C/EBPdelta) by interleukin-1 negatively influences apolipoprotein C-III expression[END_REF][START_REF] Raspé | Transcriptional regulation of apolipoprotein C-III gene expression by the orphan nuclear receptor RORalpha[END_REF][START_REF] Raspé | Modulation of rat liver apolipoprotein gene expression and serum lipid levels by tetradecylthioacetic acid (TTA) via PPARalpha activation[END_REF][START_REF] Caron | Transcriptional activation of apolipoprotein CIII expression by glucose may contribute to diabetic dyslipidemia[END_REF][START_REF] Altomonte | Foxo1 mediates insulin action on apoC-III and triglyceride metabolism[END_REF][START_REF] Poupeau | Cross-regulation of hepatic glucose metabolism via ChREBP and nuclear receptors[END_REF][START_REF] Biggs | Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1[END_REF][START_REF] Rena | Phosphorylation of the transcription factor forkhead family member FKHR by protein kinase B[END_REF][START_REF] Tocher | Omega-3 Long-Chain Polyunsaturated Fatty Acids, EPA and DHA: Bridging the Gap between Supply and Demand[END_REF][START_REF] Morton | The effect of omega-3 carboxylic acids on apolipoprotein CIII-containing lipoproteins in severe hypertriglyceridemia[END_REF][START_REF] Hertz | Mode of action of peroxisome proliferators as hypolipidemic drugs. Suppression of apolipoprotein C-III[END_REF][START_REF] Pavlic | Triglyceride-rich lipoprotein-associated apolipoprotein C-III production is stimulated by plasma free fatty acids in humans[END_REF][START_REF] Hernandez | Regulation of hepatic ApoC3 expression by PGC-1β mediates hypolipidemic effect of nicotinic acid[END_REF]. Table 1 summarises the main factors regulating APOC3 gene expression [START_REF] Chen | Transcriptional regulation of the apoC-III gene by insulin in diabetic mice: correlation with changes in plasma triglyceride levels[END_REF][START_REF] Caron | Transcriptional activation of apolipoprotein CIII expression by glucose may contribute to diabetic dyslipidemia[END_REF][START_REF] Altomonte | Foxo1 mediates insulin action on apoC-III and triglyceride metabolism[END_REF][START_REF] Poupeau | Cross-regulation of hepatic glucose metabolism via ChREBP and nuclear receptors[END_REF][START_REF] Morton | The effect of omega-3 carboxylic acids on apolipoprotein CIII-containing lipoproteins in severe hypertriglyceridemia[END_REF][START_REF] Hertz | Mode of action of peroxisome proliferators as hypolipidemic drugs. Suppression of apolipoprotein C-III[END_REF][START_REF] Pavlic | Triglyceride-rich lipoprotein-associated apolipoprotein C-III production is stimulated by plasma free fatty acids in humans[END_REF].

The history of APOC3 variants

Several APOC3 single nucleotide variants (SNVs) have been described in humans. Both LOF [START_REF] Maeda | Molecular cloning of a human apoC-III variant: Thr 74----Ala 74 mutation prevents O-glycosylation[END_REF][START_REF] Von Eckardstein | Apolipoprotein C-III(Lys58----Glu). Identification of an apolipoprotein C-III variant in a family with hyperalphalipoproteinemia[END_REF][START_REF] Pollin | A null mutation in human APOC3 confers a favorable plasma lipid profile and apparent cardioprotection[END_REF][START_REF] Crawford | Rare variant APOC3 R19X is associated with cardio-protective profiles in a diverse population-based survey as part of the Epidemiologic Architecture for Genes Linked to Environment Study[END_REF][START_REF] Timpson | A rare variant in APOC3 is associated with plasma triglyceride and VLDL levels in Europeans[END_REF][START_REF] Tg | Loss-of-function mutations in APOC3, triglycerides, and coronary disease[END_REF][START_REF] Jørgensen | Loss-of-function mutations in APOC3 and risk of ischemic vascular disease[END_REF][START_REF] Saleheen | Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity[END_REF][START_REF] Igartua | Rare non-coding variants are associated with plasma lipid traits in a founder population[END_REF] and gain-of function (GOF) [START_REF] Pullinger | A novel apolipoprotein C-III variant, apoC-III(Gln38-->Lys), associated with moderate hypertriglyceridemia in a large kindred of Mexican origin[END_REF][START_REF] Lin | Association between apolipoprotein C3 Sst I, T-455C, C-482T and C1100T polymorphisms and risk of coronary heart disease[END_REF][START_REF] Song | Associations of the APOC3 rs5128 polymorphism with plasma APOC3 and lipid levels: a meta-analysis[END_REF] variants have been discovered. Most of them were identified in isolated populations, but those within protein-coding sequences were subsequently tested to understand to what extent they contribute to TG variation in the general population [START_REF] Maeda | Molecular cloning of a human apoC-III variant: Thr 74----Ala 74 mutation prevents O-glycosylation[END_REF][START_REF] Von Eckardstein | Apolipoprotein C-III(Lys58----Glu). Identification of an apolipoprotein C-III variant in a family with hyperalphalipoproteinemia[END_REF][START_REF] Pollin | A null mutation in human APOC3 confers a favorable plasma lipid profile and apparent cardioprotection[END_REF][START_REF] Crawford | Rare variant APOC3 R19X is associated with cardio-protective profiles in a diverse population-based survey as part of the Epidemiologic Architecture for Genes Linked to Environment Study[END_REF][START_REF] Timpson | A rare variant in APOC3 is associated with plasma triglyceride and VLDL levels in Europeans[END_REF][START_REF] Tg | Loss-of-function mutations in APOC3, triglycerides, and coronary disease[END_REF][START_REF] Jørgensen | Loss-of-function mutations in APOC3 and risk of ischemic vascular disease[END_REF][START_REF] Saleheen | Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity[END_REF][START_REF] Igartua | Rare non-coding variants are associated with plasma lipid traits in a founder population[END_REF][START_REF] Pullinger | A novel apolipoprotein C-III variant, apoC-III(Gln38-->Lys), associated with moderate hypertriglyceridemia in a large kindred of Mexican origin[END_REF][START_REF] Lin | Association between apolipoprotein C3 Sst I, T-455C, C-482T and C1100T polymorphisms and risk of coronary heart disease[END_REF][START_REF] Song | Associations of the APOC3 rs5128 polymorphism with plasma APOC3 and lipid levels: a meta-analysis[END_REF]. Supplementary Figure 1 summarizes LOF and GOF variants in the APOC3 gene identified to date and their relative position in the protein sequence. Table 2 shows all significant studies evaluating the effect of LOF and GOF APOC3 variants on plasma ApoCIII concentrations and lipid profile in carriers vs. non-carriers. In vitro functional results, and kinetic studies were also reported [START_REF] Reyes-Soffer | Effects of APOC3 Heterozygous Deficiency on Plasma Lipid and Lipoprotein Metabolism[END_REF][START_REF] Liu | Characterization of the lipid-binding properties and lipoprotein lipase inhibition of a novel apolipoprotein C-III variant Ala23Thr[END_REF][START_REF] Sundaram | Functional analysis of the missense APOC3 mutation Ala23Thr associated with human hypotriglyceridemia[END_REF][START_REF] Qin | Missense mutation in APOC3 within the Cterminal lipid binding domain of human ApoC-III results in impaired assembly and secretion of triacylglycerolrich very low density lipoproteins: evidence that ApoC-III plays a major role in the formation of lipid precursors within the microsomal lumen[END_REF][START_REF] Sundaram | The apolipoprotein C-III (Gln38Lys) variant associated with human hypertriglyceridemia is a gain-of-function mutation[END_REF].

Role of ApoCIII in TRL metabolism

All lipoproteins may contain ApoCIII, although this apoprotein is mainly represented in CM, VLDLs and their remnants and HDL. In its mature form, ApoCIII consists of 79 amino acids (from 99 amino acids of precursor protein), with 6 alpha amphipathic helices, capable of binding to lipids [START_REF] Brewer | The complete amino acid sequence of alanine apolipoprotein (apoC-3), and apolipoprotein from human plasma very low density lipoproteins[END_REF][START_REF] Vaith | Characterization of the oligosaccharide side chain of apolipoprotein C-III from human plasma very low density lipoproteins[END_REF][START_REF] Zannis | Genetic mutations affecting human lipoprotein metabolism[END_REF]. As well as the other apolipoproteins, ApoCIII is a key player in plasma TG metabolism.

Kinetic studies have clearly shown that increased levels of plasma ApoCIII are the main determinant of plasma TG levels in the population with impaired TRL catabolism [START_REF] Borén | Kinetic and Related Determinants of Plasma Triglyceride Concentration in Abdominal Obesity: Multicenter Tracer Kinetic Study[END_REF]. In fact, ApoCIII concentration in normolipidemic subjects are about 8-10 mg/dl while up to 3-times higher levels can be found in patients with HTG [START_REF] Brewer | The complete amino acid sequence of alanine apolipoprotein (apoC-3), and apolipoprotein from human plasma very low density lipoproteins[END_REF]. In normal conditions, most of the plasma ApoCIII is bound to HDL whereas, in HTG subjects, the majority is bound to TRL. Although ApoCIII is a relatively minor component of HDL protein, approximately 50% of total plasma ApoCIII may be contained in the HDL fraction [START_REF] Schonfeld | Apolipoprotein C-II and C-III levels in hyperlipoproteinemia[END_REF][START_REF] Fredenrich | Plasma lipoprotein distribution of apoC-III in normolipidemic and hypertriglyceridemic subjects: comparison of the apoC-III to apoE ratio in different lipoprotein fractions[END_REF][START_REF] Van Dijk | The role and mode of action of apolipoproteins CIII and AV: synergistic actors in triglyceride metabolism?[END_REF]. Therefore, while under normal conditions HDL particles work as the reservoir of ApoCIII, in HTG states there is an enrichment of ApoCIII in TRL and a concomitant shift within these lipoproteins from predominantly ApoE-containing particles (which are quickly removed from the circulation and have little tendency to form LDL) to ApoCIII-enriched particles [START_REF] Van Dijk | The role and mode of action of apolipoproteins CIII and AV: synergistic actors in triglyceride metabolism?[END_REF][START_REF] Jong | Role of ApoCs in lipoprotein metabolism: functional differences between ApoC1, ApoC2, and ApoC3[END_REF][START_REF] Chan | An ABC of apolipoprotein C-III: a clinically useful new cardiovascular risk factor?[END_REF][START_REF] Gordts | ApoC-III inhibits clearance of triglyceride-rich lipoproteins through LDL family receptors[END_REF].

As shown in Figure 1, ApoCIII influences TRL metabolism through different pathways: a) by promoting the assembly and production of VLDLs at the hepatic level b) by inhibiting the activity of LPL, c) by interfering with ApoE-mediated hepatic uptake of VLDL particles [START_REF] Ramms | Apolipoprotein C-III in triglyceride-rich lipoprotein metabolism[END_REF]. Studies in genetically modified HTG mice overexpressing ApoCIII, displayed increased hepatic secretion of VLDL particles [START_REF] Taskinen | Emerging Evidence that ApoC-III Inhibitors Provide Novel Options to Reduce the Residual CVD[END_REF]. By contrast, inhibition of ApoCIII synthesis in mice using antisense oligonucleotides (ASO) did not reduce VLDL secretion [START_REF] Graham | Antisense oligonucleotide inhibition of apolipoprotein C-III reduces plasma triglycerides in rodents, nonhuman primates, and humans[END_REF], so that the biological significance of ApoCIII as a regulator of hepatic VLDL assembly and release is still unclear, particularly in humans. Even though we can assume that ApoCIII raises plasma TG by inhibiting LPL [START_REF] Brown | Inhibition of lipoprotein lipase by an apoprotein of human very low density lipoprotein[END_REF][START_REF] Havel | Cofactor activity of protein components of human very low density lipoproteins in the hydrolysis of triglycerides by lipoproteins lipase from different sources[END_REF][START_REF] Krauss | Further observations on the activation and inhibition of lipoprotein lipase by apolipoproteins[END_REF][START_REF] Wang | Modulation of lipoprotein lipase activity by apolipoproteins. Effect of apolipoprotein C-III[END_REF], many in vitro and in vivo studies showed controversial results [START_REF] Gordts | ApoC-III inhibits clearance of triglyceride-rich lipoproteins through LDL family receptors[END_REF][START_REF] Aalto-Setälä | Mechanism of hypertriglyceridemia in human apolipoprotein (apo) CIII transgenic mice. Diminished very low density lipoprotein fractional catabolic rate associated with increased apo CIII and reduced apo E on the particles[END_REF][START_REF] Larsson | Apolipoproteins C-I and C-III inhibit lipoprotein lipase activity by displacement of the enzyme from lipid droplets[END_REF][START_REF] Larsson | Apolipoprotein C-III inhibits triglyceride hydrolysis by GPIHBP1-bound LPL[END_REF]. Some of them have reported only minor effects of ApoCIII on LPL activity [START_REF] Aalto-Setälä | Mechanism of hypertriglyceridemia in human apolipoprotein (apo) CIII transgenic mice. Diminished very low density lipoprotein fractional catabolic rate associated with increased apo CIII and reduced apo E on the particles[END_REF][START_REF] De Silva | Overexpression of human apolipoprotein C-III in transgenic mice results in an accumulation of apolipoprotein B48 remnants that is corrected by excess apolipoprotein E[END_REF]. A clinical study by Gaudet et al [START_REF] Gaudet | Targeting APOC3 in the familial chylomicronemia syndrome[END_REF] conducted in three patients with complete LPL deficiency showed that ASO-induced ApoCIII reduction was associated with a drastic reduction in plasma TG levels [START_REF] Gaudet | Targeting APOC3 in the familial chylomicronemia syndrome[END_REF]. This observation argues that ApoCIII may also act independently from LPL, suggesting the existence of other pathways of TG removal linked to ApoCIII. Indeed, recent human kinetic studies showed that ApoCIII-containing TRL are cleared more slowly than ApoCIIIlacking TRL. This effect does not involve any changes in LPL activity, but it represents the consequence of the increase in the hepatic uptake of remnants through their interaction with specific hepatic receptors, namely heparan sulphate proteoglycans receptor (HSPGs), syndecan-1 (SDC1), the LDL receptor (LDLR) and the LDLR-related protein 1 (LRP1) receptor [START_REF] Gordts | ApoC-III inhibits clearance of triglyceride-rich lipoproteins through LDL family receptors[END_REF][START_REF] Jong | Apolipoprotein C-III deficiency accelerates triglyceride hydrolysis by lipoprotein lipase in wild-type and apoE knockout mice[END_REF][START_REF] Williams | Recent insights into factors affecting remnant lipoprotein uptake[END_REF][START_REF] Foley | Hepatic heparan sulfate proteoglycans and endocytic clearance of triglyceride-rich lipoproteins[END_REF][START_REF] Zannis | Role of apoA-I, ABCA1, LCAT, and SR-BI in the biogenesis of HDL[END_REF].

It has been also proposed that ApoCIII prevents the elimination of lipoprotein particles displacing ApoE from TRLs [START_REF] Jong | Apolipoprotein C-III deficiency accelerates triglyceride hydrolysis by lipoprotein lipase in wild-type and apoE knockout mice[END_REF][START_REF] Williams | Recent insights into factors affecting remnant lipoprotein uptake[END_REF]. However, this mechanism is questionable as anti-APOC3 ASO treatment did not affect ApoE levels on TRL [START_REF] Gordts | ApoC-III inhibits clearance of triglyceride-rich lipoproteins through LDL family receptors[END_REF].

However, the results by Ramms et al [START_REF] Ramms | ApoC-III ASO promotes tissue LPL activity in the absence of apoE-mediated TRL clearance[END_REF], suggested a new metabolic model in which ApoE determines the effects of ApoCIII in mediating either the inhibition of hepatic uptake of TRLs or the activity of LPL in adipose tissue.

Role of ApoCIII in HDL function and metabolism

In addition to LPL, it has been demonstrated that ApoCIII influences the activity of other enzymes, particularly LCAT and CETP, which are crucial in HDL metabolism and function [START_REF] Zhang | HDL-associated apoCIII plays an independent role in predicting postprandial hypertriglyceridemia[END_REF].

Experimental studies have shown that ApoCIII inhibits LCAT activity while stimulates CETP activity [START_REF] Jong | Role of ApoCs in lipoprotein metabolism: functional differences between ApoC1, ApoC2, and ApoC3[END_REF]. It has also been reported that ApoCIII in HDL can bind to scavenger-receptor BI (SR-BI) [START_REF] Xu | Apolipoproteins of HDL can directly mediate binding to the scavenger receptor SR-BI, an HDL receptor that mediates selective lipid uptake[END_REF], thus inhibiting the transfer of cholesterol from HDL and LDL to hepatocytes [START_REF] Xu | Apolipoproteins of HDL can directly mediate binding to the scavenger receptor SR-BI, an HDL receptor that mediates selective lipid uptake[END_REF]. Furthermore, biochemical studies revealed the presence of two types of HDL particles, those with ApoCIII and those without ApoCIII [START_REF] Jensen | Apolipoprotein C-III as a Potential Modulator of the Association Between HDL-Cholesterol and Incident Coronary Heart Disease[END_REF]. Nevertheless, the percentage of ApoCIII in HDL particles has been reported to be significantly correlated with the risk of coronary artery disease (CAD) [START_REF] Jensen | Apolipoprotein C-III as a Potential Modulator of the Association Between HDL-Cholesterol and Incident Coronary Heart Disease[END_REF]. In vitro experiments indicate that the deterioration of anti-apoptotic effects of HDL observed in CAD patients was associated with the remodeling of HDL proteome, and this was mainly characterized by the increase in ApoCIII content [START_REF] Riwanto | Altered activation of endothelial antiand proapoptotic pathways by high-density lipoprotein from patients with coronary artery disease: role of highdensity lipoprotein-proteome remodeling[END_REF].

Less is known about a potential direct effect of ApoCIII on HDL-mediated cholesterol efflux. Recently, the work by Luo et al [START_REF] Luo | ApoCIII enrichment in HDL impairs HDL-mediated cholesterol efflux capacity[END_REF] highlighted that the presence of ApoCIII in HDL impairs the HDL-mediated cholesterol efflux capacity. Since both ApoCIIIHDL and ApoCIIIHDL ratio (apoCIIIHDL over plasma ApoCIII) inversely correlate with cholesterol efflux capacity, the authors hypothesized that elevated ApoCIII in HDL could competitively bind to SR-BI and interrupt, or impair, HDLmediated reverse cholesterol transport (RCT). In addition, when evaluating the interaction effect of ApoCIII and ApoE on HDL metabolism, Morton et al [START_REF] Morton | Apolipoproteins E and CIII interact to regulate HDL metabolism and coronary heart disease risk[END_REF] found that ApoE and ApoCIII cooperate in affecting HDL metabolism. Moreover, subspecies of HDL containing ApoE but not ApoCIII, uniquely participate in classical pathways of RCT. As ApoE promotes metabolic steps in RCT, when ApoCIII coexists with ApoE on HDL, abolishes these benefits [START_REF] Morton | Apolipoproteins E and CIII interact to regulate HDL metabolism and coronary heart disease risk[END_REF]. Overall, these observations strongly suggest that the increase of ApoCIII in HDL may deteriorate the atheroprotective functions of these particles, thus indicating another mechanism by which ApoCIII might influence ASCVD risk.

The role ApoCIII on vascular endothelial cells dysfunction, monocyte adhesion and inflammation

Basic science studies have highlighted that ApoCIII is also involved in several other crucial checkpoints promoting atherogenesis, such as monocyte adhesion to EC and smooth muscle cell (SMC) proliferation [START_REF] Luo | The emerging role of apolipoprotein C-III: beyond effects on triglyceride metabolism[END_REF][START_REF] Li | Aggravated restenosis and atherogenesis in ApoCIII transgenic mice but lack of protection in ApoCIII knockouts: the effect of authentic triglyceride-rich lipoproteins with and without ApoCIII[END_REF]. It is known that the activation of nuclear factor κB (NF-κB) is responsible for endothelial dysfunction and monocyte adhesion [START_REF] Kawakami | Apolipoprotein CIII induces expression of vascular cell adhesion molecule-1 in vascular endothelial cells and increases adhesion of monocytic cells[END_REF]. Kawakamy et al [START_REF] Kawakami | Apolipoprotein CIII induces expression of vascular cell adhesion molecule-1 in vascular endothelial cells and increases adhesion of monocytic cells[END_REF] have found that ApoCIII activates PKCβ and NF-κB thus increasing VCAM-1 and ICAM-1 expression in endothelial cells and promoting monocyte adhesion. Moreover, NF-κB also drives the enhancement of β1-integrin expression in monocytes, thus further favouring the adhesion of these cells to EC [START_REF] Kawakami | Apolipoprotein CIII-induced THP-1 cell adhesion to endothelial cells involves pertussis toxin-sensitive G protein-and protein kinase C alpha-mediated nuclear factor-kappaB activation[END_REF]. Consistently, it has been observed that ApoCIII-rich VLDL directly activates EC and monocytes recruitment [START_REF] Yingchun | Increased inflammation, endoplasmic reticulum stress and oxidative stress in endothelial and macrophage cells exacerbate atherosclerosis in ApoCIII transgenic mice[END_REF]. In an elegant study aimed at elucidating the atherogenic role of ApoCIII, Yingchun et al [START_REF] Yingchun | Increased inflammation, endoplasmic reticulum stress and oxidative stress in endothelial and macrophage cells exacerbate atherosclerosis in ApoCIII transgenic mice[END_REF] fed ApoCIIItg/LDLR-/-and LDLR-/-littermates with an atherogenic diet for 12 weeks.

Interestingly, authors found that ApoCIIItg mice had increased inflammation and endoplasmic reticulum (ER) stress in the aorta, with increased infiltration of macrophages and VCAM-1 expression, as compared to littermates. In fact, TRLs+ApoCIII can induce MCP1 expression in human umbilical vein EC, it promotes the expression of IL6 and MCP1 in peritoneal macrophages and, stimulated by LPS, can increase the expression of VCAM-1. These results suggested that TRL enriched in ApoCIII could induce a greater level of inflammation than TRL alone [START_REF] Yingchun | Increased inflammation, endoplasmic reticulum stress and oxidative stress in endothelial and macrophage cells exacerbate atherosclerosis in ApoCIII transgenic mice[END_REF]. In a subsequent study, the same authors examined whether statin treatment was able to suppress ApoCIII-induced EC activation in vitro and in vivo [START_REF] Zheng | Statins suppress apolipoprotein CIIIinduced vascular endothelial cell activation and monocyte adhesion[END_REF]. Despite a limited direct effect on ApoCIII levels, they found that statins were able to hinder ApoCIII's stimulation of vascular adhesiveness by downregulating VCAM-1. Additionally, Botteri et al [START_REF] Botteri | VLDL and apolipoprotein CIII induce ER stress and inflammation and attenuate insulin signalling via Toll-like receptor 2 in mouse skeletal muscle cells[END_REF] showed that the exposure of myotubes and isolated skeletal muscle to VLDL increases the levels of ER stress and inflammatory markers and attenuates the insulin signalling pathway [START_REF] Botteri | VLDL and apolipoprotein CIII induce ER stress and inflammation and attenuate insulin signalling via Toll-like receptor 2 in mouse skeletal muscle cells[END_REF].

Moreover, their experiment highlighted that ApoCIII simulates the effects of VLDL confirming the crucial role of this apolipoprotein in mediating VLDL actions [START_REF] Botteri | VLDL and apolipoprotein CIII induce ER stress and inflammation and attenuate insulin signalling via Toll-like receptor 2 in mouse skeletal muscle cells[END_REF].

Role of ApoCIII in coagulation cascade and thrombosis

Several lines of evidence suggest a direct effect of ApoCIII on thrombosis [START_REF] Kawakami | Apolipoprotein CIII induces expression of vascular cell adhesion molecule-1 in vascular endothelial cells and increases adhesion of monocytic cells[END_REF]. In a prospective study including 663 subjects with angiographically proven ASCVD, it was observed an independent positive association between elevated ApoCIII and enhanced thrombin generation [START_REF] Olivieri | Apolipoprotein C-III predicts cardiovascular mortality in severe coronary artery disease and is associated with an enhanced plasma thrombin generation[END_REF]. The same authors [START_REF] Olivieri | Factor II activity is similarly increased in patients with elevated apolipoprotein CIII and in carriers of the factor II 20210A allele[END_REF] found that elevated circulating levels of ApoCIII (but not other lipids or apolipoproteins) were involved in a progressive increase in factor II (FII) coagulant activity in the plasma of patients with or without CAD. In addition, ApoCIII was found to be strongly associated with the activated FVII-anti-thrombin (FVIIa-AT) complex. FVIIa-AT is an indirect marker of intravascular exposure of tissue factor (TF), thus indirectly suggesting an activation of the extrinsic coagulation pathway. In a recent study, including 127 patients with venous thromboembolism and 299 controls, levels of apolipoproteins CI, CII, CIII and E were associated with the plasma concentration of several coagulation factors such as vitamin K-dependent factors, factor XI, antithrombin, clot lysis time as well as factor VIII and von Willebrand factor levels [START_REF] Orsi | Association of apolipoproteins C-I, C-II, C-III and E with coagulation markers and venous thromboembolism risk[END_REF]. These results may suggest that increased concentration of ApoCIII might be potentially used as a biomarker for the identification of subjects with pro-thrombotic diathesis and increased risk of thrombosis [START_REF] Martinelli | Apolipoprotein C-III Strongly Correlates with Activated Factor VII-Anti-Thrombin Complex: An Additional Link between Plasma Lipids and Coagulation[END_REF].

The demonstration that the administration of volanesorsen, an anti-ApoCIII ASO, is able to cause a significant drop in platelets count [START_REF] Witztum | Volanesorsen and Triglyceride Levels in Familial Chylomicronemia Syndrome[END_REF] raised the possibility that the ApoCIII inhibition might unfavourably interfere with platelets production or activity. By analysing a population of 42.503 participants in the UK Biobank, Khetarpal et al [START_REF] Khetarpal | Volanesorsen, Familial Chylomicronemia Syndrome, and Thrombocytopenia[END_REF] tested whether heterozygous carriers of LOF mutations in APOC3 were at increased risk of thrombocytopenia [START_REF] Khetarpal | Volanesorsen, Familial Chylomicronemia Syndrome, and Thrombocytopenia[END_REF][START_REF] Witztum | Familial Chylomicronemia Syndrome, and Thrombocytopenia[END_REF]. They did not find any differences between carriers and non-carriers thus concluding that this hematologic abnormality might be a drug or classspecific side effect and not directly related to the inhibition of ApoCIII. Further studies are needed to clarify the role of ApoCIII in influencing platelet function and count.

Other potential atherogenic effects of ApoCIII

ApoCIII has been implicated in other potential proatherogenic effects. It has been shown that this apolipoprotein increases the affinity of LDL particles to proteoglycans in the artery wall [START_REF] Krauss | ApoC-III content of apoBcontaining lipoproteins is associated with binding to the vascular proteoglycan biglycan[END_REF][START_REF] Gustafsson | Retention of atherogenic lipoproteins in atherogenesis[END_REF], thus influencing the accumulation of atherogenic lipoproteins in the vessel wall. Since ApoCIII does not directly bind proteoglycans, the enhanced binding may result from a conformational change associated with increased ApoCIII content by which ApoB and/or ApoE become more accessible to proteoglycans [START_REF] Krauss | ApoC-III content of apoBcontaining lipoproteins is associated with binding to the vascular proteoglycan biglycan[END_REF][START_REF] Gustafsson | Retention of atherogenic lipoproteins in atherogenesis[END_REF]. This hypothesis has been confirmed in the work by Hiukka et al [START_REF] Hiukka | ApoCIII-enriched LDL in type 2 diabetes displays altered lipid composition, increased susceptibility for sphingomyelinase, and increased binding to biglycan[END_REF] showing that LDL particles with a high ApoCIII/ApoB ratio have reduced content of unesterified cholesterol, sphingomyelin and ceramide.

These changes in lipid composition lead to higher membrane fluidity, thus allowing ApoB to acquire a conformation that is more favorable for proteoglycan binding [START_REF] Segrest | Apolipoprotein B-100: conservation of lipid-associating amphipathic secondary structural motifs in nine species of vertebrates[END_REF]. In the artery wall, LDL is exposed to several enzymes, including sphingomyelinases (SMase) that promote aggregation and fusion of retained lipoproteins [START_REF] Schissel | Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral pH. Implications for atherosclerotic lesion development[END_REF]. ApoCIII is an activator of SMase and ApoCIII-enriched LDL displays increased susceptibility to hydrolysis and aggregation by SMase [START_REF] Hiukka | ApoCIII-enriched LDL in type 2 diabetes displays altered lipid composition, increased susceptibility for sphingomyelinase, and increased binding to biglycan[END_REF].

The role of ApoCIII in the risk of clinical complications of ASCVD

In addition to the above-mentioned experimental evidence linking ApoCIII to atherogenesis, a bulk of studies have convincingly demonstrated that ApoCIII contributes to clinical manifestations of ASCVD in humans (13, 36, 39 ,40 ,74, 98-104). To this regard, of special interest are the genetic studies testing the impact of LOF APOC3 variants with large TGlowering effect on CVD risk. It has been reported that the R19X LOF mutation ( 36) is associated with 60% decrease in the risk of coronary artery calcification. In the study by Crosby et al [START_REF] Tg | Loss-of-function mutations in APOC3, triglycerides, and coronary disease[END_REF], the risk of coronary heart disease (CHD) among 498 carriers of any rare LOF APOC3 mutation was 40% lower than the risk among 110,472 non-carriers [START_REF] Tg | Loss-of-function mutations in APOC3, triglycerides, and coronary disease[END_REF]. Moreover, LOF mutations were also associated with a 46% reduction in levels of ApoCIII and 41% and 36% risk reduction of ASCVD and ischemic heart disease (IHD), respectively [START_REF] Jørgensen | Loss-of-function mutations in APOC3 and risk of ischemic vascular disease[END_REF]. A still open question is whether the reduced ASCVD risk found in APOC3

LOF carriers is driven by lowering plasma TG levels or by other lipid-related effect, such reduction of LDL-C, cholesterol remnants or increased HDL-C [START_REF] Cohen | APOC3, coronary disease, and complexities of Mendelian randomization[END_REF]. Recently, Wulff AB et al [START_REF] Wulff | APOC3 Loss-of-Function Mutations, Remnant Cholesterol, Low-Density Lipoprotein Cholesterol, and Cardiovascular Risk: Mediation-and Meta-Analyses of 137 895 Individuals[END_REF] performed a mediation and meta-analysis study, showing that the low risk of ASCVD observed in APOC3 LOF mutation carriers was accompanied by a reduction of remnant cholesterol (mean 43%) without any changes in LDL-C (mean 4%). Although not significant, the percentage of non-carriers taking lipid lowering therapies (9%) was higher than that of APOC3 LOF mutation carriers (6%). Moreover, ApoB, a marker of the total number of atherogenic lipoprotein particles, was 13% lower in LOF heterozygotes compared to non-carriers [START_REF] Wulff | APOC3 Loss-of-Function Mutations, Remnant Cholesterol, Low-Density Lipoprotein Cholesterol, and Cardiovascular Risk: Mediation-and Meta-Analyses of 137 895 Individuals[END_REF][START_REF] Taskinen | Why Is Apolipoprotein CIII Emerging as a Novel Therapeutic Target to Reduce the Burden of Cardiovascular Disease?[END_REF] showing that an increase in HDL enriched in ApoCIII was associated with a higher CVD risk [START_REF] Jensen | Apolipoprotein C-III as a Potential Modulator of the Association Between HDL-Cholesterol and Incident Coronary Heart Disease[END_REF]. Similarly, the presence of ApoCIII on LDL correlated with the development of CVD (103). These findings suggest that the presence or absence of ApoCIII on lipoprotein particles may turn HDL into an atherogenic lipoprotein, and the dose of ApoCIII on LDL and VLDL may determine the degree of their atherogenicity (103).

Furthermore, the impact of ApoCIII levels on ASCVD risk has been recently confirmed in a meta-analysis including a total of 3163 cases of cardiovascular events from 5 retrospectives and 7 prospective studies (104). Authors have shown that while 5-mg/dL increase in total plasma ApoCIII translates into a relative risk estimate of 1.33 (95% CI 1.07-1.66) for cardiovascular events, a comparable increase in the non-HDL ApoCIII fraction translates into a2.48-fold increased risk (95% CI 1.48-4.32) (104).

These results suggest that the robust association between ApoCIII and ASCVD might be explained by the ApoCIII content in atherogenic lipoproteins so that ApoCIII might be considered as a potent marker of the atherogenicity of lipoprotein particles.

ApoCIII, glucose metabolism and T2DM

Emerging evidence suggests that ApoCIII may also be involved in the pathophysiology of T2DM through pancreatic beta cell apoptosis and glucose homeostasis [START_REF] Qamar | Plasma apolipoprotein C-III levels, triglycerides, and coronary artery calcification in type 2 diabetics[END_REF].

As previously mentioned, the expression of ApoCIII is increased by insulin deficiency, and insulin resistance and hyperglycemia induce ApoCIII transcription [START_REF] Chen | Transcriptional regulation of the apoC-III gene by insulin in diabetic mice: correlation with changes in plasma triglyceride levels[END_REF][START_REF] Caron | Transcriptional activation of apolipoprotein CIII expression by glucose may contribute to diabetic dyslipidemia[END_REF]. In addition, mice overexpressing ApoCIII are more susceptible to highfat diet (HFD)-induced diabetes (106). In humans, elevated levels of circulating ApoCIII are associated with insulin resistance [START_REF] Altomonte | Foxo1 mediates insulin action on apoC-III and triglyceride metabolism[END_REF] and, consequently, T2DM. In fact, many observational studies have reported a positive correlation between circulating ApoCIII concentrations and the incidence of both type 1 and T2DM (107-110). However, despite many of these associations are apparently independent from plasma TG levels and other confounding factors, the cross-sectional nature of these investigations does not permit to give definite conclusions and it is still not clear if ApoCIII is itself the driver of hyperglycemia and diabetes onset (111). Nevertheless, findings from a study involving a multi-ethnic population of 4579 individuals showed that the association might probably be determined primarily by TG-related mechanisms (112). In fact, increasedtotal ApoCIII concentrations were associated with a higher rate of diabetes whereas ApoCIII-defined HDL subspecies had an opposite effect. These results were also supported by in vivo study in which overexpression of the APOC3 gene resulted in elevated circulating triacylglycerol levels and increased susceptibility to hepatic insulin resistance and fatty liver disease (113).

These results have been also confirmed in a randomized, double blind, placebo-controlled trial performed on 15 adult patients with T2DM (HbA1c >7.5% [58 mmol/mol]) and HTG (TG >200 and <500 mg/dL) that have been treated with volanesorsen (114). In this study, the action of volanesorsen was not only associated with the reduction of plasma ApoCIII and TG and increased HDL-C levels but also with the improvement of glucose disposal and insulin sensitivity (114).

Recently, Bozzetto et al (115) evaluated if ApoCIII concentrations associate with differences in body mass index (BMI) or glycemic response during oral glucose tolerance test in young and healthy monozygotic (MZ) twins. Despite they did not find any relation between ApoCIII and acquired differences in BMI, they showed that ApoCIII levels were associated with early dysregulation of glucose metabolism independently of obesity and genetics.

Other lines of evidence suggest that ApoCIII may influence β-cell function. In rats, increased ApoCIII concentrations in pancreatic islets promoted a local inflammatory milieu, with consequent β-cell failure (117). Moreover, in vitro and animal studies suggested a role of ApoCIII in inducing islet insulin resistance (118). Insulin resistance and β-cell failure are the major defects in T2DM. Preventing insulin resistance at the islet level is thus crucial to preserve β-cell mass, especially during the late phase of T2DM, where β cells are progressively lost. Overall, these studies support the notion that the regulation of APOC3 expression pathway may be involved in the crosstalk between glucose and lipid metabolism (119). Therefore, studying ApoCIII helps in understanding TG swings in chronic inflammatory states (such as T2DM) but whether its inhibition translates into a potential benefit still remains to be addressed.

The role of ApoCIII in hepatic steatosis

ApoCIII was also suggested as a risk factor for hepatic steatosis. However, the physiological function of ApoCIII in lipid metabolism of the hepatocyte is still unclear and matter of debate.

Duivenvoorden et al. (120) showed that APOC3 knockout mice are more susceptible to develop diet-induced obesity and NAFLD. Moreover, in order to gain mechanistic insight about the pivotal role of ApoCIII protein in liver, Cheng et al (121) fed APOC3-transgenic and wild-type mice with a high fructose or high fat diet. Then, these authors tried to determine the effect of APOC3 on hepatic TG synthesis and secretion, inflammation and the progression of NAFLD. They provide compelling evidence that while APOC3 is clearly culpable for HTG, it does not play a pivotal role in the pathogenesis of NAFLD in high fat-fed APOC3-tg mice (121). Apparently, also studies in humans do not support a role of ApoCIII in favoring NAFLD (122-127).

Although Petersen et al. (122) reported that subjects with the common genetic variants within the APOC3 promoter (-455T > C rs2854116 and -482C > T rs2854117) are at increased risk of developing NAFLD, Kozlitina et al. (123) found no association between these two APOC3 variants and NAFLD in humans. This latter observation was supported by three other independent clinical studies showing that carriers of neither one nor both APOC3 alleles are associated with NAFLD (124-126). Finally, results of a large metanalysis (127) involving seven clinical trials have clearly disproved the association between polymorphisms in APOC3 and the risk of NAFLD.

Moreover, given the role of ApoCIII in facilitating the intracellular VLDL-TG assembly and secretion (4), this protein is probably an ill target for therapeutic intervention of NAFLD. In a study on chow-and WD-fed C67BL/6 and apoC3-/mice treated with the ASO against ApoCIII, authors found that the secretion of TG by the liver was apparently unaffected by the treatment. In this study,authors observed a non-statistically significant increaseof liver TG levels in this mouse model [START_REF] Graham | Antisense oligonucleotide inhibition of apolipoprotein C-III reduces plasma triglycerides in rodents, nonhuman primates, and humans[END_REF].

Considering all these data, more studies are warranted to definitively establish if ApoCIII is a predisposing factor for exacerbating the development of NAFLD in humans and if its inhibition is associated with adverse outcomes.

ApoCIII as a therapeutic target

Previously summarised evidence allowed identifying ApoCIII as a therapeutic target to treat HTG and, potentially, T2DM, and to control ASCVD risk. At present, clinical trials evaluating the benefit of reducing ApoCIII synthesis in HTG are under way.

We have recently summarized the available evidence on the efficacy and safety of a recently developed, secondgeneration ASO directed towards APOC3 mRNA synthesis, named volanesorsen, that has been used to treat HTG (128). Preclinical and clinical studies have highlighted an unprecedented efficacy of this silencing technology in reducing TG even in the more resistant form of HTG as familial chylomicronemia syndrome (FCS) [START_REF] Graham | Antisense oligonucleotide inhibition of apolipoprotein C-III reduces plasma triglycerides in rodents, nonhuman primates, and humans[END_REF][START_REF] Witztum | Volanesorsen and Triglyceride Levels in Familial Chylomicronemia Syndrome[END_REF]. The administration of volanesorsen subcutaneously has led to a significant reduction of TG levels, with 77% of FCS subjects achieving TG below 750 mg/dl as compared with only the 10% in the placebo group [START_REF] Witztum | Volanesorsen and Triglyceride Levels in Familial Chylomicronemia Syndrome[END_REF]. Nevertheless, in a recent published meta-analysis on 3 randomized controlled clinical studies including a total of 156 patients (with diabetes, FCS or with moderately high levels of TG) (129), authors showed that the benefit of volanesorsen was also extended to HDL-C (+ 40%), VLDL-C (- 73%), ApoB-48 (- 65%), ApoCIII (- 75%), and VLDL ApoCIII (- 84%). This dose-dependent decrease in ApoCIII obtained with volanesorsen might be particularly interesting if considering the role of ApoCIII as risk factor for ASCVD (130).

However, despite these promising data, Phase III trials have raised some concerns especially with regards to the severe thrombocytopenia side effect [START_REF] Witztum | Volanesorsen and Triglyceride Levels in Familial Chylomicronemia Syndrome[END_REF]. As previous mentioned, the decrease in platelet count was an unexpected result and an interesting debate is still ongoing to establish the possible causes of this side effect [START_REF] Khetarpal | Volanesorsen, Familial Chylomicronemia Syndrome, and Thrombocytopenia[END_REF][START_REF] Witztum | Familial Chylomicronemia Syndrome, and Thrombocytopenia[END_REF]128).

Considering the potential severity of side effects, the benefit/risk ratio of volanesorsen has been considered positive only by the European Medical Agency (EMA) so far, leading to the authorization of volanesorsen for the treatment of FCS patients with high risk of acute pancreatitis for whom an alternative drug does not currently exist (128,131,132).

Noteworthy, new targeting approaches of ASOs, using a triantennary N-acetyl galactosamine (GalNAc3) modified ASOs that targets the asialoglycoprotein receptor (ASGPR) in hepatocytes, has been recently developed. This new ASO, AKCEA-APOCIII-LRx, was recently tested and results have shown similar efficacy than volanesorsen, with 20-30-fold lower dosing, thus minimizing systemic exposure (133). As volanesorsen, the benefit of the administration of AKCEA-APOCIII-LRx was also associated with significant reduction in total cholesterol, apoB, non-HDL-C, VLDL , and increases in HDL-C (133).

Nevertheless, AKCEA-APOCIII-LRx was also well tolerated with one injection site reaction of mild erythema, and no flu-like reactions, liver or renal safety signals and, more importantly, no thrombocytopenia events (133).

Further studies are needed to clarify the efficacy and safety of pharmacological inhibition of ApoCIII in treating not only hypertriglyceridemia but also ASCVD. In this context, the ongoing trial with AKCEA-APOCIII-LRx might probably help answering to this question (134).

Conclusions

The "ApoCIII saga" is a clear example of how the translational research approach in metabolic disease has the potential to influence quality of life and health-span beyond the boundaries of traditional disease-based research. From the analysis of this big plethora of data, it emerges how the role of ApoCIII in humans is more complex than initially thought.

Investigations using anti-ApoCIII ASO in LPL deficient states have been crucial in illustrating that ApoCIII regulates TRL catabolism beyond the modulation of LPL function. In addition, novel evidences have highlighted how ApoCIII may act in other pathways involved in the network between lipids, glucose metabolism, endothelial cells dysfunction, inflammation and coagulation cascade. Even though other investigations are needed to better clarify this complex interplay, the available evidence may clearly expand the role of ApoCIII in cardiometabolic diseases. In this context, the development of new drugs aimed at inhibiting ApoCIII-synthesis is emerging as a very promising strategy in controlling severe HTG states. It remains to be established if this approach may be useful also in controlling the residual risk associated with atherosclerosis and other metabolic disorders.

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors.

Disclosure Statement: LD has received personal fees for public speaking, consultancy or grant support from Amryt Pharmaceuticals, Akcea Therapeutics, Pfizer, Amgen and Sanofi; MA has received research grant support from Amryth Pharmaceutical, Amgen, IONIS, Akcea Therapeutics, Pfizer and Sanofi; has served as a consultant for Amgen, Aegerion, Akcea Therapeutics, Regeneron, Sanofi and Alfasigma and received lecturing fees from Amgen, Amryth Pharmaceutical, Pfizer, Sanofi and AlfaSigma; AG has received personal fees for public speaking or consultancy support from Akcea Therapeutics, AMGEN, MSD, Mylan, Novartis, Sanofi and Regeneron, Unilever; EB has received honoraria from AMGEN, MSD, Sanofi and Regeneron, Unilever, Danone, Aegerion, Chiesi, Rottapharm-MEDA, Servier, Ionis-pharmaceuticals, AKCEA and MYLAN. ADC do not have any potential conflict of interest.

Contributions: LD and ADC wrote original draft, made substantial contributions to conception, acquisition of materials essential and prepared tables and figures; AG, EB and MA revised critically for important intellectual content and edited the text. All authors revised and accepted the final version of the manuscript. Plasma concentration of TG is a function of the relative equilibrium between production and clearance of TRL particles [START_REF] Ogami | Promoter elements and factors required for hepatic and intestinal transcription of the human ApoCIII gene[END_REF]. These processes are directly related to the balance between de novo synthesis of TRL by the liver (VLDL), intestinal absorption of fat from the diet (CM), lipolysis in peripheral tissues (LPL) and hepatic clearance, i.e., impaired clearance, or increased production, or both (CM remnants, VLDL and VLDL remnants). Fluctuations in either production or clearance rates may lead to accumulation of TG ultimately leading to HTG. During LPL-driven hydrolysis of TGs contained in VLDL and CM, there is a transfer of ApoCIII from VLDL to HDL particles and then back to other neo-secreted VLDLs [START_REF] Jong | Role of ApoCs in lipoprotein metabolism: functional differences between ApoC1, ApoC2, and ApoC3[END_REF][START_REF] Chan | An ABC of apolipoprotein C-III: a clinically useful new cardiovascular risk factor?[END_REF]. ApoCIII influences TRL metabolism through different pathways: A) by promoting the assembly and production of VLDLs at hepatic level, B) by inhibiting the activity of LPL and C) by interfering with ApoE-mediated hepatic uptake of VLDL particles.

Positive ( ) ad negative ( ) effects were reported. ChREBP, Carbohydrate-responsive element-binding protein; HNF4a, hepatic nuclear factor-4 alpha; FOXO1, forkhead box O1 protein, PGC1-β, Peroxisome proliferator-activated receptor gamma coactivator 1-beta; PPAR-a, Peroxisome proliferator-activated receptor alfa.

Effect on APOC3

expression Factors Mechanism of action

Finally, with the

  aim to investigate the role of ApoCIII in restenosis and clarify how smooth muscle cells respond to TRLs with or without ApoCIII, Li et al (79) studied various ApoCIII transgenic and knockout mice undergoing endothelial denudation to model restenosis. They showed that ApoCIII-enriched TRL, via Akt signalling pathway, promoted SMC proliferation, VCAM-1 expression, and reactive oxygen species (ROS) production, thus worsening atherogenesis and coronary stenosis in mice with severe HTG (79).

Figure 1 .

 1 Figure 1. Apolipoprotein CIII (ApoCIII) and Triglyceride (TG)-Rich Lipoproteins (TRL) Metabolism. Plasma concentration of TG is a function of the relative equilibrium between production and clearance of TRL particles

Figure 1 .

 1 Figure 1. Apolipoprotein CIII (ApoCIII) and Triglyceride (TG)-Rich Lipoproteins (TRLs) Metabolism.

  .Ference et al (105), evaluating 63 cohort or case-control studies conducted in North America or Europe between 1948 and 2017, found similar lower risk of CHD per unit difference in ApoB, after comparing patients having LPL-triglyceridelowering and LDL-C-lowering variants (105). Therefore, the clinical benefit of lowering TG and LDL-C levels may be proportional to the absolute change in ApoB (105).The association between ApoCIII or ApoCIII-containing lipoproteins and ASCVD risk has been studied in several retrospective and prospective study. Pechlaner et al (100) directly measured apolipoproteins by mass spectrometry in the Brunech Study and compared their associations with incident ASCVD. They found that the apolipoproteins most significantly associated with incident cardiovascular disease were ApoCII, ApoE and ApoCIII (HR=1.38; 95% CI 1.17-1.63). These associations were independent of HDL and non-HDL-C and were extended to the risk of stroke and myocardial infarction.Plasma ApoCIII levels were measured in all baseline samples included in a prospective nested case-control study among participants of the European Prospective Investigation of Cancer (EPIC)-Norfolk cohort (101). Despite ApoCIII levels were significantly associated with the risk of CAD (HR=1.91; 95% CI 1.48-2.48), results from the mediation analysis found that the elevated levels of remnant lipoproteins, small dense LDL, and low-grade inflammation may explain this association. In a recent prospective study, it was reported that the risk for both fatal and nonfatal myocardial infarction was significantly increased in patients with an increased fraction of VLDL or LDL containing ApoCIII (102). As reported above, in two prospective studies, HDL subgroups (containing ApoCIII and without ApoCIII) were differentially associated with CVD,

Table 1 . Modulators of APOC3 gene expression

 1 Table 1 summarizes the main factors that up -( ) or down -( ) regulate APOC3 gene expression.


Yann Guiguen

Jeremy Pasquier

Alexis Fostier

Julien Bobe
email: julien.bobe@inrae.fr

Evolution of sex hormone binding globulins reveals early gene duplication at the root of vertebrates

Keywords: evolution, Sex hormone-binding globulin, teleosts, vertebrates, gene expression

Phylogeny, synteny and expression analyses shed new light on Shbg evolution in vertebrates.

• Shbg diversity originates from a duplication event at the root of vertebrate evolution. • This duplication was followed by many independent losses of Shbg paralogs in vertebrates.

• Shbg paralogs have acquired different tissue expression patterns.

Introduction

Sex hormone-binding globulin (Shbg) is mainly known as a blood-protein carrier involved in the transport of sex steroids in the plasma and in the regulation of their bioavailability to target organs. Shbg proteins present sequence similarities with the other LamG domaincontaining proteins growth arrest-specific 6 (Gas6) and protein S alpha (Pros1) [START_REF] Joseph | Sequence and functional relationships between androgen-binding protein/sex hormone-binding globulin and its homologs protein S, Gas6, laminin, and agrin[END_REF][START_REF] Joseph | Sex hormone-binding globulin, androgen-binding protein, and vitamin K-dependent protein S are homologous to laminin A, merosin, and Drosophila crumbs protein[END_REF]. By transporting and regulating androgens and estrogens access to the gonads, Shbg plays important roles in vertebrates reproduction [START_REF] Hammond | Diverse roles for sex hormone-binding globulin in reproduction[END_REF]. The Shbg protein was originally identified in the beta-globulin fraction of the human serum [START_REF] Rosner | Partial purification and preliminary characterization of estrogen-binding globulins from human plasma[END_REF] and has previously been known as Androgen-binding protein (Abp). Shbg genes and Shbg proteins have been characterized in a variety of tetrapod species with the notable exception of birds (Westphal, 1986;Wingfield et al., 1984). In aquatic vertebrates, Shbg was originally found in the plasma of an elasmobranch, the skate (Raja radiata) [START_REF] Freeman | Sex hormone binding proteins. II. Isolation from serum of an elasmobranch (Raja radiata)[END_REF], and of a teleost, the rainbow trout (Oncorhynchus mykiss) [START_REF] Fostier | Binding of steroids by plasma of a teleost: the rainbow trout, Salmo gairdneri[END_REF]. Since that moment on, Shbg have been subsequently identified and studied in many fish species (see for review [START_REF] Bobe | Diversity and biological significance of sex hormonebinding globulin in fish, an evolutionary perspective[END_REF]). Two different shbg genes i.e., shbga and shbgb, have been characterized in teleosts with shbga being the ortholog of the mammalian Shbg, which has been conserved from chondrichthyes to tetrapods and shbgb that has only been reported up to now in salmonids. In contrast to shbga that is mainly expressed in the liver [START_REF] Bobe | A novel, functional, and highly divergent sex hormone-binding globulin that may participate in the local control of ovarian functions in salmonids[END_REF], the shbgb transcript was mainly found in the ovary, suggesting a local mediation of the sex steroids effects by the Shbgb protein [START_REF] Bobe | A novel, functional, and highly divergent sex hormone-binding globulin that may participate in the local control of ovarian functions in salmonids[END_REF]. These two salmonid Shbg proteins share very low identity percentages, with for instance 26% identity between Shbga and Shgbb at the amino acid level in the rainbow trout [START_REF] Bobe | Diversity and biological significance of sex hormonebinding globulin in fish, an evolutionary perspective[END_REF][START_REF] Bobe | A novel, functional, and highly divergent sex hormone-binding globulin that may participate in the local control of ovarian functions in salmonids[END_REF]. In comparison to other vertebrates and teleost fishes, the salmonid ancestor has experienced an additional (4R) whole genome duplication known as the salmonid specific whole genome duplication, or SaGD [START_REF] Berthelot | The rainbow trout genome provides novel insights into evolution after whole-genome duplication in vertebrates[END_REF]. For this reason, and because no shbgb gene had ever been reported in a nonsalmonid species, it was first hypothesized that shbga and shbgb were ohnologous genes resulting from the SaGD [START_REF] Bobe | A novel, functional, and highly divergent sex hormone-binding globulin that may participate in the local control of ovarian functions in salmonids[END_REF]. This hypothesis was subsequently challenged in another study and the possibility of an ancient duplication followed by a lineage-specific retention in salmonids was suggested (Miguel-Queralt et al., 2009).

The evolutionary history of Shbg genes in vertebrates thus remained unclear and deserved further investigations. Using the increasing amount of genomic and transcriptomic data available for many vertebrate species we revisited the evolutionary history of Shbg genes.

The transcriptomes of 24 actinopterygian species (including 22 teleosts) and vertebrate genomes were included in the analysis, which led to the identification of previously noncharacterized Shbgb genes in several non-salmonid vertebrate lineages. Using phylogenomic analyses, we identified several Shbgb orthologs in a variety of non-salmonid vertebrate species, including teleosts, non-teleost actinopterygians, amphibians and one agnatha. Combined with synteny reconstruction analysis, we demonstrated that Shbg diversity results from a duplication event much older than the SaGD. To gain new information on the functional evolution of shbg genes, we also used quantitative PCR and next generation sequencing approaches, to characterize the expression profiles of shbga and shbgb transcripts for several actinopterygian species. This showed that the paralogous shbg genes have acquired different expression profiles with shbgb having a predominant gonadal expression contrasting with a predominant liver expression of shbga.

Material and Methods

Genomic and transcriptomic databases

Phylogenetic and synteny analyses

Amino-acid sequences of 126 predicted Shbg (a, b), Gas6 and Pros1 proteins were first aligned using ClustalW [START_REF] Thompson | CLUSTAL W: improving the sensitivity of[END_REF], then alignments were manually adjusted, to improve the quality of the multiple sequence alignments. The JTT (Jones, Taylor and Thornton) protein substitution matrix of the resulting alignment was determined using ProtTest software [START_REF] Darriba | ProtTest 3: fast selection of best-fit models of protein evolution[END_REF]. Phylogenetic analysis of the proteins presenting LamG domains (i.e. Shbga, Shbgb, Gas6 and Pros1) was performed using the neighbour joining (NJM) method (MEGA 5.1 software), with 1000 bootstrap replicates [START_REF] Tamura | MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF]. Trees were edited online with iTOL [START_REF] Letunic | Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees[END_REF] and exported as Scalable Vector Graphics.

Synteny maps of the conserved genomic regions in human, Xenopus, coelacanth, spotted gar and zebrafish were constructed based on information available within the Genomicus [START_REF] Muffato | Genomicus: a database and a browser to study gene synteny in modern and ancestral genomes[END_REF]

RNA-seq shbga and shbgb tissue expression in holosteans and teleosts.

RNA-seq and de novo assembly were performed for all studied species as previously described [START_REF] Berthelot | The rainbow trout genome provides novel insights into evolution after whole-genome duplication in vertebrates[END_REF][START_REF] Braasch | The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons[END_REF][START_REF] Pasquier | Gene evolution and gene expression after whole genome duplication in fish: the PhyloFish database[END_REF]. In order to study the expression patterns and levels of shbg transcripts for each actinopterygian species with two shbg genes, we mapped RNA-seq reads on the corresponding shbg coding sequence (CDS) using BWA-Bowtie [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF] with stringent mapping parameters (maximum number of allowed mismatches -aln 2). Mapped reads were counted using SAMtools [START_REF] Li | The Sequence Alignment/Map format and SAMtools[END_REF] idxstat command, with a minimum alignment quality value (q 30) to discard ambiguous mapping reads. For each species, the numbers of mapped reads were then normalized for each shbg gene across the eleven tissues using the reads per kilo base per million mapped reads (RPKM) normalization. All RNA-seq data are available here:

(http://phylofish.sigenae.org/index.html)

Quantitative PCR analysis (QPCR).

QPCR was performed using the RNA collections of the PhyloFish RNA-seq project as previously described [START_REF] Braasch | The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons[END_REF][START_REF] Pasquier | Gene evolution and gene expression after whole genome duplication in fish: the PhyloFish database[END_REF]. Briefly, tissues were sampled from the same female individual and testis from a male individual, when possible. In some species and depending on the tissues, RNA samples from different individuals were pooled to obtain sufficient amounts of RNA. Total RNA was extracted using Tri-Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's instructions. Reverse transcription (RT) was performed using 1 μg of RNA for each sample with M-MLV reverse transcriptase and random hexamers (Promega, Madison, WI, USA).

Briefly, RNA and dNTPs were denatured for 6 min at 70°C, chilled on ice for 5 min before the RT master mix was added. RT was performed at 37°C for 1 h and 15 min followed by a 15min incubation step at 70°C. Control reactions were run without reverse transcriptase and used as negative control in the real-time PCR study. Quantitative RT-PCR (QPCR) experiments were performed sing an Applied Biosystems StepOne Plus. RT products, including control reactions, were diluted to 1/25, and 4 μl were used for each PCR. All QPCR were performed in triplicates. QPCR was performed using a real-time PCR kit provided with a Fast-SYBR Green fluorophore (Applied Biosystems) with 200 nM of each primer in order to keep PCR efficiency between 80% and 100% for all target shbg genes. The relative abundance of target cDNA within a sample set was calculated from serially diluted cDNA pool (standard curve) using Applied Biosystem StepOne V.2.0 software. After amplification, a fusion curve was obtained to validate the amplification of a single PCR product. The fusion curves obtained showed that each primer pair used was specific of a single shbg transcript.

The negative control reactions were used to estimate background level. Genes were considered significantly expressed when measured level was significantly above background at p < 0.05 and within the range of the standard curve. For each studied tissue, cDNA originating from three individual fish were pooled and subsequently used for real-time PCR.

Before further analysis, real-time PCR data were collected using the same detection threshold for all studied genes. Data were subsequently normalized using the ΔΔCt method to 18S transcript abundance in samples diluted to 1:2,000.

Clustering analysis

Expression profiles originating from either QPCR and RNA-seq were represented using supervised clustering methods [START_REF] Eisen | Cluster analysis and display of genome-wide expression patterns[END_REF]. Hierarchical clustering was processed using centroïd linkage clustering, that uses the average value of all points in a cluster as a reference to calculate distance of other points, with Pearson's uncentered correlation as similarity metric on data that were normalized and median-centered using the Cluster program [START_REF] Eisen | Cluster analysis and display of genome-wide expression patterns[END_REF]. Results (colorized matrix) of hierarchical clustering analyses were visualized using the Java TreeView program [START_REF] Saldanha | Java Treeview--extensible visualization of microarray data[END_REF].

Results

Shbg gene evolution in vertebrates

In order to decipher phylogenetic relationships among Shbg sequences, a phylogenetic reconstruction of the evolution of Shbg was made based using the alignment of 126 vertebrate LamG domain-containing proteins. This phylogeny includes Shbg proteins, growth arrest specific 6 proteins (Gas6) and Vitamin K-dependent protein S (Pros1) and the tree was rooted using as outgroup the zebrafish Laminin subunit alpha 4 (lama4). This analysis (Fig. 1) shows that these vertebrate LamG domains proteins cluster into two major clades containing Shbg proteins on one side and the Gas6 and Pros1 proteins on the other side.

These Shbg and Gas6/Pros1 clades are both significantly supported with high bootstrap values (i.e. 75% and 100%, respectively). The Shbg clade contains two sub-clades both supported by significant bootstrap values. The Shbga cluster (100% bootstrap support), in red, contains all classical vertebrate Shbg proteins from chondrichthyes to teleosts with the notable exception of birds in which no Shbg proteins have been detected (see also Fig. S2).

The Shbgb cluster (93% bootstrap support), in blue, contains not only the salmonid Shbgb proteins, but also other vertebrate sequences outside the salmonid family including sequences from teleosts (European eel and silver arowana), non-teleost bony fishes (reedfish, spotted gar and bowfin), some amphibians and an agnatha, i.e., the sea lamprey (Fig. 1 and Fig. S1). The tree topology indicates that Shbgb proteins are not specific to the salmonids lineage and thus suggests a much more ancient origin of Shbgb genes in vertebrates than previously hypothesized [START_REF] Bobe | A novel, functional, and highly divergent sex hormone-binding globulin that may participate in the local control of ovarian functions in salmonids[END_REF].

To strengthen this phylogeny-based analysis of Shbg protein evolutionary history we carried out a synteny analysis in order to better support this hypothesis of an ancient origin Shbga and Shbgb genes. The synteny analysis first revealed that these two Shbg genes are located on two different syntenic chromosome regions in vertebrates (see Fig. 2A for the Shbga locus and Fig. 2B for the Shbgb locus). In contrast, Gas6 and Pros1 genes are located on the same syntenic chromosome region in most studied species, with the exception of primates (Fig. 2C). In vertebrates, Shbga, Shbgb, and Gas6/Pros1 are also located in regions containing other syntenic gene families. These neighboring genes are spread over four syntenic regions (Fig. 2A-2D) like for instance for the Atp-related genes (Atp1b2, Atp1b3, Atp4b) that are found in all four syntenic chromosome regions. In addition, Zbtbrelated genes (Ztb4, Zbt38, Zbt33) and Lamp-related genes (Lamp1, Lamp2, Lamp3) are present in three of the four different syntenic chromosome regions depending of the gene family. Altogether, these results strongly suggest that the diversity of the gene family present on these four syntenic chromosome regions probably results from early whole genome duplication events (VG1 and/or VG2) that occurred at the root of vertebrate evolution with a subsequent complex pattern of gene retention and gene losses.

Using the recently released salmonid genome resources, we also re-investigated the presence of additional copies of shbg genes in salmonids and confirmed that shbga and shbgb were both retained as single copies (Fig. 1) in rainbow trout, Atlantic salmon and coho salmon suggesting that no functional duplicated copies were retained after the salmonid whole genome duplication (SaGD). However, we also found an additional shbgb gene conserved in these three salmonid species (Fig. 3A), but with many stop codons in its deduced open reading frame (see example for rainbow trout in Fig. 3B), suggesting that this gene (ψ shbgb) was subsequently pseudogenized after the SaGD.

Expression patterns of shbga and shbgb

In all investigated actinopterygians, shbga was found to be mainly expressed in the liver, supporting a conserved role for this blood-secreted Shbg (Fig. 4). However, in the silver arowana a low shbga expression in the gonads is also detected in addition to the predominant liver expression (Fig. 4A and Fig. 4B). In contrast to shbga, expression of shbgb is not predominant in the liver in all investigated actinopterygians (Fig. 4A and Fig. 4B). In contrast, shbgb expression is predominantly detected in the gonads (ovary and/or testis) with the notable exception of the European eel.

Discussion

In this study, we aimed at investigating the diversity of the Shbg family in vertebrates and the evolutionary history of Shbg genes. To date, despite the recent discovery of a second shbg gene (i.e. shbgb) in salmonids, the origin and diversity of Shbg genes in vertebrates has remained controversial. Because salmonids experienced an additional whole genome duplication (SaGD) approximately 100 Mya [START_REF] Berthelot | The rainbow trout genome provides novel insights into evolution after whole-genome duplication in vertebrates[END_REF][START_REF] Macqueen | A well-constrained estimate for the timing of the salmonid whole genome duplication reveals major decoupling from species diversification[END_REF] compared to other teleost fish and as shbgb genes were initially found in salmonids and, never reported at that time in any non-salmonid species, shbga and shbgb have been first hypothesized to be the result of the SaGD [START_REF] Bobe | Diversity and biological significance of sex hormonebinding globulin in fish, an evolutionary perspective[END_REF][START_REF] Bobe | A novel, functional, and highly divergent sex hormone-binding globulin that may participate in the local control of ovarian functions in salmonids[END_REF]. However pairwise comparison of Shbga and Shbgb reveals a surprisingly low sequence identity (around 25% at the amino acid level) that was initially interpreted as Shbga and Shbgb being highly divergent SaGD paralog. However, this paralogy relationship was not supported by the phylogeny reconstruction [START_REF] Bobe | A novel, functional, and highly divergent sex hormone-binding globulin that may participate in the local control of ovarian functions in salmonids[END_REF] and this discrepancy was thus explained as being the result of a long-branch attraction artifact resulting from the dramatic divergence of these two sequences [START_REF] Bobe | Diversity and biological significance of sex hormonebinding globulin in fish, an evolutionary perspective[END_REF][START_REF] Bobe | A novel, functional, and highly divergent sex hormone-binding globulin that may participate in the local control of ovarian functions in salmonids[END_REF]. Based on the cloning of another salmonid shbgb gene in coho salmon, Oncorhynchus kisutch and the low sequence identity between salmonids Shbga and Shbgb, other authors (Miguel-Queralt et al., 2009), hypothesized that the shbgb gene could stemmed from a much more ancient duplication than the SaGD. In order to decipher the evolutionary history of Shbg genes we re-analyzed the phylogenetic relationships of Shbg genes, their local synteny context, and the evolution of the phylogenetically and structurally closely related genes i.e., Gas6 and Pros1 that also contain LamG domains and are often identified as potential members of the same family. The identification of new Shbgb genes in vertebrates, the Shbg phylogenetic tree topology and their local synteny relationships strongly suggest that Shbga and Shbgb genes result from a whole genome duplication event that occurred very early at the root on the vertebrate lineage. The presence of a single Shbga gene and a single Shbgb gene in amphibians, holosteans, polypteriformes, agnatha and some teleost fishes, suggests that this Shbg duplication stems at least from the second round of vertebrate genome duplication (VG2). It is however also possible that Shbga and Shbgb originate the first round of vertebrate genome duplication (VG1) followed by the loss of one duplicate of each gene before VG2.

Following this early duplication, these two Shgb paralogs have evolved through many different phylum-specific gene retentions and/or gene losses. Among them the case of birds is interesting as they not only lost their Shbgb gene like reported here for many other tetrapods, but also their Shbga gene that is found to be conserved in all other vertebrates.

This complete absence of Shbga in birds has been already reported and it was hypothesized that this specific steroid hormone-binding transport would then be performed by a corticosteroid-binding globulin (Wingfield et al., 1984). Similarly, no Shbg homologs were detected by homology searches in Chondrichthyes (data not shown) but their complete absence in this clade requires further in-depth analysis and additional genome information as Shbg-like sex steroid binding capacities exist in the serum of the Thorny skate [START_REF] Freeman | Sex hormone binding proteins. II. Isolation from serum of an elasmobranch (Raja radiata)[END_REF]. In tetrapods, Shbgb was only found in Amphibians along with Shbga. Shbgb was also found in holosteans (spotted gar and bowfin), polypteriformes (reedfish), agnatha (sea lamprey) and in a few teleost fish orders i.e., in Elopomorphs (European eel), Osteoglossiforms (silver arowana) and Salmoniforms even though the protein is frequently misannotated in GenBank (Fig. S1). Interestingly we did not find any retention of additional whole genome [SaGD and the teleost specific duplication (TGD)] paralogs for both shbga and shbgb gene with the exception of a pseudogenized SaGD shbgb paralog (ψ shbgb). This indicates that these extra whole genome duplications did no impact the repertoire of shbg genes with a maximum of one shbga and one shbgb functional copies in all investigated teleost clades. This systematic and independent losses of additional shbga and shbgb duplicated paralogs in teleosts may reflect an evolutionary constraint of maintaining a correct gene and protein dosage as it has been suggested in other organisms [START_REF] Conant | Dosage, duplication, and diploidization: clarifying the interplay of multiple models for duplicate gene evolution over time[END_REF][START_REF] Gout | Maintenance and Loss of Duplicated Genes by Dosage Subfunctionalization[END_REF].

In consistency with existing data in mammals, our expression data showed that shbga is predominantly expressed in the liver in the different teleost species studied here. This confirms what has previously been reported in various teleost species including zebrafish (Danio rerio) [START_REF] Miguel-Queralt | Molecular and functional characterization of sex hormone binding globulin in zebrafish[END_REF], rainbow trout (Oncorhynchus mykiss) [START_REF] Bobe | A novel, functional, and highly divergent sex hormone-binding globulin that may participate in the local control of ovarian functions in salmonids[END_REF], Coho salmon (Oncorhynchus kisutch) (Miguel-Queralt et al., 2009), pejerrey (Odontesthes bonariensis) [START_REF] González | Sex hormone binding globulin: Expression throughout early development and adult pejerrey fish, Odontesthes bonariensis[END_REF] and sea bass (Dicentrarchus labrax) [START_REF] Miguel-Queralt | Sex hormone-binding globulin expression in sea bass (Dicentrarchus labrax L.) throughout development and the reproductive season[END_REF]. In addition, this strong hepatic expression is also observed in spotted gar (Lepisosteus oculatus) and bowfin (Amia calva) as shown by both RNA-seq and QPCR data.

In contrast to shbga, data on the tissue distribution of shbgb remain scarce. The ovarian predominant expression of shbgb was originally reported in rainbow trout, in which the transcript could also be detected at lower levels in muscle and stomach [START_REF] Bobe | A novel, functional, and highly divergent sex hormone-binding globulin that may participate in the local control of ovarian functions in salmonids[END_REF].

Semi quantitative data in Coho salmon confirmed the expression of shbgb in the ovary and stomach and revealed its presence in gills (Miguel-Queralt et al., 2009). Here we show that shbgb is also predominant expressed in the ovary in brown trout, silver arowana and grayling. We also report a strong testicular expression of shbgb in the two holostean species, spotted gar and bowfin, that appears to be lost in teleosts. In addition, the shbgb gene does not exhibit any gonad predominant expression in European eel. Together, our data show that shbga and shbgb have a very specific expression patterns with a predominant expression in liver and gonads, respectively. This pattern appears to be conserved during evolution without any significant change following whole genome duplications events (TGD and SaGD), with the exception of European eel in which the gonad predominant expression of shbgb appears to be lost. Finally, the strong testicular expression of shbgb revealed in bowfin and spotted gar is not found in any teleost species suggesting a specific role of Shbgb in testicular physiology in holostean species.

The multiple independent losses of Shbgb across vertebrates, while Shbga, Gas6 and Pros1

have been conserved in almost all vertebrates, could reflect different adapative and reproductive strategies as Shbg have been shown to be important carrier proteins for the blood transport of sex steroids and for their delivery to target reproductive tissues [START_REF] Hammond | Diverse roles for sex hormone-binding globulin in reproduction[END_REF]. However, despite this discrepancy among species, the distinct roles of Shgba in hormone transport in the blood and of Shbgb in local hormone action in reproductive organs as well as the associated expression in liver and gonads, respectively, appears to be evolutionary conserved in species that have retained both genes despite a few intriguing species-specific exceptions.

Acknowledgement

This work was supported by the French national research Agency (ANR-10-GENM-017-PhyloFish).

Figures Figure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: Synteny maps of conserved genomic regions around LamG domains proteins (i.e. Shbga, Shbgb, Gas6 and Pros1) in human, Xenopus, coelacanth, spotted gar, Euroean eel, zebrafish and rainbow trout. Synteny maps are given for genomic regions around Shbga (A), Shbgb (B), Gas6 and Pros1 locus (C) and for a fourth region containing homologs of neighbouring Shbga, Shbgb, Gas6 and Pros1 genes (D). Genes are represented by blocks with an arrowed side indicating the gene orientation on chromosomes, linkage group or scaffolds. Gene location on chromosomes (Chr for Human and zebrafish), Linkage group (LG for spotted gar) and scaffolds (Ensembl reference or scaffold number) is given in Mb below each gene block. Genes belonging to the same Chr, LG or scaffolds are linked by a solid line.

Figure 3 :

 3 Figure 3: Multiple alignments plots of shbgb genes in salmonids. (A). Percentage of sequence identity of shbgb genes in Atlantic salmon, Salmon salar, and Coho salmon, Oncorhynchus kisutch compared to the rainbow trout, Oncorhynchus mykiss, shbgb gene on linkage group (LG) 24. In addition to the functional shbgb genes that were found in all salmonid species investigated, all these species have an additional shbgb homolog containing multiple stop codons and thus considered as a pseudogene (ψ shbgb). (B). Rainbow trout Shbgb and ψ Shbgb protein alignment showing that the ψ Shbgb contains multiple stop codons (red asterisks) and a large deletion in exon 4 of the Shbgb protein.

Figure 4 :

 4 Figure 4: Tissue expression profiles of shhbga and shbgb genes. (A). Heatmap (colorized matrix) of the hierarchical clustering of tissue RNA-Seq expression profiles (NGS values) of shbg genes in different Holostean (spotted gar and bowfin) and teleost species. A predominant expression of shbga is found in the liver contrasting with the predominant expression of shbgb in gonads (B). Heatmap (colorized matrix) of the hierarchical clustering of tissue expression profiles of shbg genes analyzed by quantitative PCR (QPCR values) in different Holostean (spotted gar and bowfin) and teleost species. Colorized matrixes highlight the high expressing tissues in yellow, the low expressing tissues in blue and the median expression in black (see color scale).

Figure 5 :

 5 Figure 5: Evolution of Shbg/shbg, Gas6/gas6, and Pros1/pros1 genes through successive whole genome duplications and independent gene losses. (A). Schematic representation of the evolution of LamG domains proteins including, Shbga (red squares), Shbgb (blue squares), Gas6 (purple squares) and Pros1 (green squares) in some jawed vertebrates' representative species. Whole genome duplications (black circles; VG1 and VG2: vertebrate genome duplications 1 and 2, TGD: teleost genome duplication, SaGD: salmonid genome duplication) are indicated at each duplication nodes. Gene losses are represented by square boxes with a cross inside. (B). Simplified representation of the evolution of Shbg proteins after whole genome duplications showing the systematic losses of one Shbga and Shbgb paralog after each duplication.

penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680. Westphal, U., 1986. Steroid-protein interactions II. Monogr. Endocrinol. 27, 1-603. Wingfield, J.C., Matt, K.S., Farner, D.S., 1984. Physiologic properties of steroid hormonebinding proteins in avian blood. Gen. Comp. Endocrinol. 53, 281-292. https://doi.org/10.1016/0016-6480(84)90254-5