Angiopoietin-like protein 3(ANGPTL3)de f i ciency and fam ilial combined hypolipidemia

Patrizia Tarugi,Stefano Bertolini,Sebastiano Calandra

1 Department of Life Sciences,University of Modena and Reggio Emilia,Modena 41125,Italy;

2 Department of Internal Medicine,University of Genova,Genova 16148,Italy;

3 Department of Biomedical,Metabolic and Neural Sciences,University of Modena and Reggio Emilia,Modena 41125,Italy.

Abstract Three members of the angiopoietin-like(ANGPTL)protein fam ily-ANGPTL3,ANGPTL4 and ANGPTL8-are important regulatorsof plasma lipoproteins.They inhibit theenzyme lipoprotein lipase,which playsakey role in the intravascular lipolysis of triglycerides present in some lipoprotein classes.This review focuses on the role of ANGPTL3 asemerged from the study of genetic variantsof Angptl3 gene in m iceand humans.Both lossof function genetic variantsand inactivation of Angptl3 gene in m ice areassociated w ith amarked reduction of plasma levelsof triglyceride and cholesterol and an increased activity of lipoprotein lipase and endothelial lipase.In humans w ith ANGPTL3 de f i ciency,caused by homozygous loss of function(LOF)variants of Angptl3 gene,the levels of all plasma lipoproteins are greatly reduced.This plasma lipid disorder referred to as fam ilial combined hypolipidemia(FHBL2)does not appear to be associated w ith distinct pathological manifestations.Heterozygous carriers of LOF variants have reduced plasma levels of total cholesterol and triglycerides and are at lower risk of developing atherosclerotic cardiovascular disease,as compared to non-carriers.These observations have paved the way to the development of strategies to reduce the plasma level of atherogenic lipoproteins in man by the inactivation of ANGPTL3,using either a speci f i c monoclonal antibody or anti-sense oligonucleotides.

Keywords:angiopoietin-like protein 3,ANGPTL3 de f i ciency,loss of function variants,FHBL2

Introduction

Angiopoietin-like proteins(ANGPTLs)represent a fam ily of eight secreted glycoproteins that show structural homology to angiopoietins and carry distinct physiological functions,including putative roles in lipid metabolism,expansion of stem cells,in f l ammation,tissue remodeling and angiogenesis[1-2].In recentyears,three ANGPTLs,ANGPTL3,ANGPTL4 and ANGPTL8,havebeen shown to play a role in lipid metabolism and in the regulation of plasma lipid levels[3-4].

Morespeci f i cally,ANGPTL 3,ANGPTL4 and ANGPTL8 share a common feature,being,to a variable extent,negative regulators of the activity of lipoprotein lipase(LPL),the key enzyme involved in the intravascular lipolysis of triglyceride(TG)present in some lipoprotein classes,such as chylom icrons and very low density lipoproteins(VLDL)[5-6].

The authors reported no con f l ict of interests.

This is an open access article under the Creative Commons Attribution(CC BY 4.0)license,which permits others to distribute,rem ix,adapt and build upon thiswork,for commercial use,provided the original work is properly cited.

This overview focuses on the role of ANGPTL3 in lipoprotein metabolism and the effect of its de f i ciency/inactivation in humans and animal models.

ANGPTL3 de f i ciency:discovery of monogenic combined hypolipidemia in mice

The link between ANGPTL3 and plasma lipoprotein metabolism emerged from the identi f i cation of the KK/San mousestrain,amutant strain derived from acolony of KK mice characterized by diabetes,obesity and hypertriglyceridemia.The KK/San mice,despite maintaining the phenotype of obesity and diabetes,had a marked decrease of plasma TG(＞90%),as compared with the colony of KK mice.This severe hypotriglyceridemiawasdueto amarked reduction of plasma VLDL.These micewerefound to be homozygousfor a loss-offunction(LOF)variant of the Angptl3 gene,causing the formation of a truncated Angptl3,and leading to a complete Angptl3 de f i ciency[7].The hypolipidemia observed in this mutant mouse strain was found to be associated with an increased activity of LPL[8].Since this observation,other studies conducted in Angptl3-/-mice not only con f i rmed that Angptl3 is an inhibitor of LPL,but also showed that complete Angptl3 de f i ciency is associated with a reduction of TG-containing lipoproteins(VLDL)and also with a reduction of cholesterol-carrying lipoproteins,such as LDL and HDL[9].On the other hand,overexpression of Angptl3 in mice increased plasma TG by inhibiting the activity of LPL[10].

Familial combined hypolipidemia in humans

In humans,a lipoprotein phenotype similar to that observed in KK/San mice was f i rstly described by Musunuru et al.in a large family originally thought to be affected by familial hypobetalipoproteinemia(FHBL1)(OMIM#615558),in view of the low levels of plasma LDL-C[11].Two siblings of this family showed extremely low levels of total cholesterol(TC)and LDL-C and low levels of TG and HDL-C.Exome sequencing revealed that thesesiblingswere compound heterozygous for two LOF variants of ANGPTL3[p.(E129*)/p.(S17*)],expected to cause a complete de f i ciency of ANGPTL3.This novel lipoprotein phenotype was designated Familial Combined Hypolipidemia(FHBL2,OMIM#605019)[11].The genetic screening in the family led to the identi f i cation of 13 heterozygotes.These individuals showed intermediate plasma levels of LDL-C and TG between compound heterozygous and non-carrier family members with a gene-dosage effect.This was not the case for plasma HDL-Clevelsthat were similar in heterozygouscarriers and in non-carriers.The nonsense variant p.(S17*)of ANGPTL3 was also found in acohort of hypolipidemic individualsliving in adistrictof Central Italy,wherethe resequencing of Angptl3 gene in nine families and in a large cohort of individuals of the local population(352 individuals)led to theidenti f i cation of 62 carriersof this variant(8 homozygotes and 54 heterozygotes)[12].In this survey homozygotes had undetectable plasma levels of ANGPTL3,low TG and cholesterol levels and a striking reduction of all lipoprotein classes(VLDL,LDL and HDL);heterozygotes had a 50%reduction in circulating ANGPTL3 and reduced levels of TC and HDL-C,as compared to non-carriers and levels of LDL-C and TG similar to controls[12].Furthermore,p.(S17*)homozygotes had signi f i cantly higher LPL activity and mass as compared to controls and lower plasma level of free fatty acids,which suggested areduced lipolysisin adiposetissue[13].Other individuals with familial combined hypolipidemia due to different LOF variants of Angptl3 gene were identi f i ed in Italian and Spanish families,as well as in a cohort of subjects with primary hypocholesterolemia[14-16].In a pooled analysis of carriers of LOF variants of Angptl3 gene,Minicocci et al.evaluated the biochemical characteristics of 115 individuals carrying different LOF variants of Angptl3(including 14 homozygotes,8 compound heterozygotes and 94 heterozygotes)[17].These investigators showed that,as compared to controls,the carriers of two LOF alleles(homozygotes/compound heterozygotes)as well as carriers of a single LOF allele(simple heterozygotes)showed a signi f i cant reduction of all plasma lipoproteins.

From aclinical point of view,carriersof two Angptl3 LOF alleles identi f i ed so far in family studies did not show a distinct pathological phenotype.More speci f ically,they did not show clinical manifestations of premature atherosclerosis or increased risk of ischemic heart disease,which might have been expected in view of the lifelong exposure to low levels of HDL-C(a known clinical predictor of risk of atherosclerotic cardiovascular disease)[18].Minicocci et al.evaluated the vascular status in a group of FHBL2 subjects(7 homozygotes and 59 heterozygotes)carrying the Angptl3 LOF mutation p.(S17*)[19].They found that FHBL2 individualsdid not show signi f i cant changes in carotid intima-media thickness(a surrogate marker for atherosclerosis)with respectto controls.Theseobservations suggest that,despite the presence of low HDL-C levels,FHBL2 subjects are protected from developing premature atherosclerosis by the concomitant reduction of the levels of atherogenic lipoproteins such as VLDL and LDL.

Another key issueregarding theclinical phenotypein FHBL2 concerns the presence of fatty liver disease,a condition frequently encountered in individuals with FHBL1.The latter individuals,who have persistently low levels of TG and LDL-C,resulting from APOB gene LOFvariantswhich impair thehepatic secretion of VLDL,usually develop fatty liver of variable severity[20].Carefully conducted clinical studieshaveshown that in FHBL2 there was no increased prevalence of fatty liver or chronic liver disease with respect to controls[21].

LOF variants of Angptl3 gene identi f i ed in population studies

Early genome wide association studies(GWAS)had shown that common and rare variants of Angptl3 gene wereassociated with variations in plasma levels of TG,TC,LDL-C and HDL-C[22-23].In addition,resequencing of Angptl3 in some population studies had shown that some LOF variants were associated with reduced levels of plasma TG[24].By sequencing the Angptl3 gene in the DiscovEHR study participants and in four other population cohorts,Dewey et al.recently identi f i ed 400 subjects heterozygous for 13 different LOF Angptl3 variants,with an estimated allele frequency of 1 in 237[25].These heterozygous carriers had a signi f i cant reduction of TG,LDL-C and HDL-C levelsand a50%reduction of circulating ANGPTL3 as compared to non-carriers.In addition,the LOFvariants of Angptl3 were found to be associated with a 39%reduction of coronary artery disease(CAD)[25].

In another recent study,Angptl3 sequence data from case-control studies and a population-based cohort study led to the identi f i cation of 23 LOF variants[26].These LOF variants were present in 130 of 40,112 participants(1 in 309 individuals).Furthermore,in heterozygous carriers of an Angptl3 LOF variant,selected from a cohort of more than 20,000 individuals of the Myocardial Infarction Genetics Consortium studies,the plasma levels of TC,LDL-C and TG were reduced by 11%,12%and 17%,respectively,with no signi f i cant changes in HDL-C as compared to noncarriers.In addition,the authors determined the relationship between Angptl3 LOF variants and the risk of CAD.They found that heterozygous carriers of LOF variants in Angptl3 had a 34%decreased risk of CAD.Thisreduced CADrisk wasassociated with lower levels of circulating ANGPTL3[26].Collectively,these two large surveys strongly indicate that partial ANGPTL3 de f i ciency dueto heterozygosity for LOF Angptl3 variantsresultsin areduced risk of CADascompared to non-carriers,suggesting that ANGPTL3 may beanovel target to reducethelevel of atherogenic lipoproteins[25-26].An updated list of LOF variants reported so far is shown in Supplementary Table 1(available online).

ANGPTL3 and lipoprotein metabolism

ANGPTL3 is synthesized in the liver as a precursor protein,which is converted into the mature form by proteolytic cleavage by several hepatic pro-protein convertases(such as Furin,PCSK2,PCSK4,PACE4,PCSK5 and PCSK7).The cleavage process yields the active N-terminal fragment,which has ef f i cient LPL inhibitory activity;this is supported by the observation that the deletion of ANGPTL3 amino terminal region causes the total loss of its inhibitory activity[8,27].The cleavage process of ANGPTL3 appears to be facilitated by ANGPTL8.ANGPTL8 is secreted by the liver into the circulation where it interacts with ANGPTL3 for cleavage and forms a complex with the N-terminal fragment of ANGPTL3.The complex,as well as the free N-terminal fragment of ANGPTL3,inhibits LPL[28].There is evidence in mice that ANGPTL8 has an LPL inhibitory motif,which is inactive as it is not accessibleto theenzyme.Theformation of thecomplex ANGPTL8-ANGPTL3 induces structural changes in ANGPTL8,which exposestheinhibitory motif for LPL inhibition.Thus,ANGPTL8 is inactive“per se”and requires ANGPTL3 to acquire LPL inhibitory activity.It has been demonstrated that the major ability of the ANGPTL8/ANGPTL3 complex to inhibit LPL depends on the active LPL inhibitory motif of ANGPTL8[29].This is supported by the observations that the LPL inhibition by the ANGPTL3/ANGPTL8 complex could not be reversed by an anti-ANGPTL3 blocking antibody[29].

Somegenetic variantsof ANGPTL8 affecting plasma lipidshavebeen reported.Quagliarini etal.found that a common variant c.175C＞T,p.Arg59Trp(rs2278426)was associated with lower plasma LDL-C and HDL-C levelsin Hispanicsand African Americansof the Dallas Heart Study[28].In a genome wide association study(including mostly individualsof European descent),the p.Arg59Trp variant was found to be associated with a reduction of both LDL-C and HDL-C,but not to be associated with reduction of plasma triglycerides[28].Furthermore,a low frequency variant of ANGPTL8[c.361C＞T,p.Gln121*(rs145464906)]was identi f i ed in a survey of a large group of individuals by Peloso et al.[30].Heterozygous carriers of this variant had higher HDL-C,lower TG and not signi f i cantly lower LDL-C levels as compared to non-carriers.It is conceivable to assume that the truncated ANGPTL8 generated by this variant is unable to form the ANGPTL8/ANGPTL3 complex,which isknown to exert astrong inhibition on LPL activity[31].

Themechanism of ANGPTL3-mediated inhibition of LPL isstill notfully understood.Enzymekinetic studies in cell free system showed that ANGPTL3 binds and reversibly inhibits the catalytic activity of LPL without affecting the LPL self-inactivation rate[32].In the presence of cells,ANGPTL3 binds LPL attached to thecell surface,promotesthedissociation of full-length LPL from the cells and induces the cleavage of the enzymeby protease(PACE4 and Furin).Thistranslates into the irreversible inactivation of the enzyme[33].

In addition,ANGPTL3 hasan inhibitory effecton the activity of the endothelial lipase(EL),an extracellular lipase,which carries mainly a phospholipase activity acting predominantly on HDL,and increases the catabolism of HDL particles.Angptl3 de f i cient mice show low plasma levels of HDL-C,accompanied by increased phospholipase activity[34].

Treatment of mice with a monoclonal antibody against ANGPTL3 signi f i cantly reduced plasma HDLC levels in wild-type but not in endothelial lipase de f i cient mice[35](Fig.1).

ANGPTL3 and metabolic conditions associated with hypertriglyceridemia

Angptl3 gene expression and ANGPTL3 plasma levels may show marked changes in some metabolic disorders in humans or experimental manipulations in rodents which are characterized by marked elevation of the level of plasma triglycerides.These disorders include conditions such as diabetes,obesity,hypothyroidism,and leptin de f i ciency etc.,as reported below.

ANGPTL3 and diabetes

In vitro and in vivo studieshaveindicated that insulin acts as a negative regulator for ANGPTL3 production.In rat and human hepatomacells,theamount of Angptl3 mRNA and secreted ANGPTL3 protein decreased in a dose dependent fashion in the presence of insulin.In striking contrast,Angptl3 gene expression and plasma protein level were increased in insulin-de f i cient streptozotocin-treated mice[36].Inukai et al.con f i rmed that thelevel of Angptl3 mRNAwasincreased in theliver of streptozotocin diabetic miceand thiseffectwasreversed by administration of insulin[37].In addition,thelevel of Angptl3 mRNA and protein was increased more than 3 fold in type 2 diabetic obese mice(db/db mice)[37].

Haridas et al.investigated the in vivo effect of insulin on circulating ANGPTL3 in humans[38].They found that in vivo 6-hours euglycemic hyperinsulinemia decreased plasma ANGPTL3(and ANGPTL8)at 3 and 6 hours.They also found that in immortalized human hepatocytes,insulin decreased Angptl3 gene expression and ANGPTL3 secretion into the medium[38].Therefore,also in humans,insulin decreases plasma ANGPTL3 by decreasing Angptl3 expression in the liver.These combined results indicate that the expression of ANGPTL3 is increased in both insulin de f i cient and insulin resistant diabetic states,suggesting that increased plasma ANGPTL3 contributesto diabetic hypertriglyceridemia.

ANGPTL3 and leptin de f i ciency

Fig.1 Effectsof ANGPTL3 de f i ciency.ANGPTL3 de f i ciency increasestheactivity of lipoprotein lipase(LPL)and endothelial lipase(EL).The activation of LPL results in an enhanced catabolism of TG-rich lipoproteins which translates into a reduction of plasma triglyceride.The activation of EL results in an increased hydrolysis of HDL phospholipids and a decreased plasmalevel of HDL-cholesterol(HDL-C).In mouse models and in human hepatocytes,ANGPTL3 de f i ciency reduces ApoB-100 secretion and increases LDL/VLDL uptake by the liver,thus contributing to thelow LDL-Clevels.ANGPTL3 de f i ciency in humansreduces VLDL-apoB production rateand increases LDL-apoBfractional catabolic rate.In adipose tissue,ANGPTL3 de f i ciency suppresses lipolysis,resulting in a decreased release of free fatty acids(FFA)into the circulation.

The role of leptin on the expression and plasma level of Angptl3 has emerged from early studies in mice.Shimamura et al.found that:(1)Angptl3 mRNA expression and plasma Angptl3 levels were increased in both leptin resistant C57Bl/J6db/dbmice and in leptin de f i cient C57Bl/J6ob/obmice;(2)elevation of ANGPTL3 in plasma was associated with elevation of plasma TG;(3)leptin administration to leptin de f i cient ob/ob mice reversed Angptl3 expression and ANGPTL3 plasma levels and induced a normalization of plasma TG[36].

The role leptin on ANGPTL3 expression and plasmalevel in humans was recently documented in a study[39]conducted in patients with generalized lipodystrophy, a leptin deficient condition associated with hypertriglyceridemia[40].These patients were found to have increased plasma levels of ANGPTL3(but not plasma levels of ANGPTL4),suggesting the possibility that hypertriglyceridemia might be the result of ANGPTL3 mediated inhibition of LPL[39].After metreleptin treatment,the plasma level of leptin increased whereas the plasma level of ANGPTL3 and TG showed a signi f i cant decrease[39].Thus,the fi nding of elevated ANGPTL3 levels in patients with lipodystrophy and their reduction following leptin treatment is consistent with the results obtained in leptin de f i cient mice and suggests that ANGPTL3 may contribute to hypertriglyceridemia in leptin de f i cient states.

ANGPTL3 and hypothyroidism

Studies in humans with hypothyroidism and elevated plasma TG have shown that the administration of T4 induced a reduction of plasma VLDL cholesterol and triglyceride,associated with an increased LPL activity[41].On the other hand,LPL activity has been found reduced in hypothyroidism and increased in hyperthyroid state[41].Furthermore,the administration of selective agonists of thyroid hormone receptor beta(TRβ)resulted in a selective decrease of VLDL-TG in rodents[42].The study by Fugier et al.showed that thyroid hormone down-regulates Angptl3(but has no effect on Angptl4)in hypothyroid rats[43].Using thyroid hormone receptor de f i cient mice,these investigators showed that thyroid hormone down-regulates Angptl3 expression in a TRβ-dependent manner[43].Therefore,decreased Angptl3 expression and reduced ANGPTL3 secretion would result in increased LPL activity and a more rapid removal of plasma TG in hypothyroid patients after thyroid hormone administration.

ANGPTL3 and LXR activation

LXR(Liver X Receptor)is a nuclear receptor that formsaheterodimer with RXR(retinoid X receptor)and activates the transcription of several genes involved in lipid metabolism.The compound T0901317 is a synthetic LXR ligand broadly used in the studies of LXR biology.Treatment of rodents with T0901317 causes an accumulation of TGin the liver and a marked increase of plasma TG[44].While the accumulation of TG in the liver was explained by the increased expression of hepatic fatty acid synthase mediated by sterol regulatory element binding protein 1(SREBP-1)[44],the mechanism of hypertriglyceridemia has not been fully clari f i ed.The involvement of Angptl3 in hypertriglyceridemia after LXR activation was f i rstly suggested by Inaba et al.who found that in human hepatoma cells LXR ligands induced an increased expression of Angptl3 gene and an increased secretion of ANGPTL3 protein[45].In addition T0901317 administration to C57BL/6J mice increased hepatic mRNA expression and plasmaconcentration of Angptl3,which were associated with a marked increase of plasma TG.By contrast,T0901317 administration to C57BL/6JAngptl3 de f i cient mice(C57BL/6J Angptl3hypl,which do not produce Angptl3 mRNA or Angptl3 protein)increased hepatic triglyceride content but failed to increase plasma TG[45].These results demonstrate that hypertriglyceridemia induced by LXR activation in mice was accounted for by LXR-mediated induction of Angptl3 expression.Similar conclusions were reached by Kaplan et al.on thebasisof in vivo aswell as in vitro studies[46].

ANGPTL3 and acute phase reaction

The acute phase response is characterized by elevation of plasma TG due to both hepatic overproduction of VLDL and defect in the clearance of TGrich lipoproteins,secondary to reduction of the LPL activity[47].Treatment of mice with lipopolysaccharide(LPS)(a model of Gram-negative infection and an inducer of acutephaseresponse)wasfound to induce a decreased expression of Angptl3 in the liver and an increased expression of Angptl4 in heart muscle and adiposetissue[48].Theseresultsindicatethat ANGPTL3 is not a positive acute phase protein and suggest that ANGPTL3 is not responsible for the reduction of LPL activity observed during acute phase response[48].

Mechanisms underlying the combined hypolipidemia in FHBL2

The increased LPL-mediated hydrolysis of TG in VLDL and chylomicrons explains the low levels of plasma TG in subjects with FHBL2.In addition to the enhanced clearance of TG-rich lipoproteins,the low level of circulating free fatty acid(FFA)present in mice and humanswith Angptl3 de f i ciency reducesthehepatic availability of FFA for the denovo synthesisof TGto be incorporated into VLDL[13].

The catabolism of chylomicrons has been investigated in 7 homozygotes and 31 heterozygotes for the ANGPTL3 nonsense mutation p.(S17*)[49].These subjects were investigated at fasting and at 6 hours after a fat rich meal.In homozygotes,the complete Angptl3 de f i ciency was associated with a highly reduced postprandial hypertriglyceridemia,probably due to an accelerated catabolism of intestinal derived TG-rich lipoproteins(chylomicrons)secondary to the increased LPL activity.Additionally,heterozygotes with partial Angptl3 de f i ciency displayed an attenuated postprandial lipemia,as compared to controls[49].

The mechanism for low LDL-C levels in Angptl3 de f i ciency is a matter of active investigation.In in vivo studies of lipoprotein metabolism in carriersof Angptl3 LOF variant showed a reduced VLDL-apoB(the main protein constituent of VLDL)production rate and an increased LDL-apoB fractional catabolic rate,with a gene-dosage effect,suggesting that ANGPTL3 regulates hepatic lipoprotein secretion and clearance[11].

Wang et al.reported that in mice with genetic de f i ciencies in key proteins involved in lipoprotein clearance(e.g.apoe-/-or ldlr-/-mice),the inactivation of ANGPTL3 with an ANGPTL3 monoclonal antibody reduced the hepatic production of VLDL-TG but not that of VLDL-apoB[50].The decrease in hepatic TG secretion in Angptl3 de f i cient mice is caused by a decreased supply of FFA from the circulation into the liver for hepatic de novo synthesis of TG.Shortage of TG is expected to decrease VLDL lipidation[50].This f i nding isin keeping with theobservation thatcarriersof Angptl3 LOFvariants and Angptl3 de f i cient mice have low plasma FFA levels due to the absence of ANGPTL3-stimulated lipolysis in adipose tissue[13].

Alternatively,thelow LDL plasma levels may bethe result of receptor-mediated catabolism of LDL.However,theobservation that Angptl3 de f i cientmicelacking functional proteins involved in LDL plasma clearance(e.g.theldlr-/-or apoe-/-mice)had areduction of plasma LDL-C similar to that of wild-type Angptl3 de f i cient mice,indicated that the reduction of LDL-C in ANGPTL3 de f i ciency is independent of the canonical receptor-mediated clearance pathways[50-51].It was suggested that Angptl3 inactivation in mice increases the clearance of VLDL remnants(which are largely converted to LDL in thecirculation),leading eventually to a reduced plasma level of LDL.

Recently,Xu et al.performed RNAi-mediated Angptl3 gene silencing in f i ve mouse models and in human hepatoma cells and validated the results by deleting Angptl3 gene in vitro using CRISPR/Cas9 genome editing[52].They found that hepatic Angptl3 silencing in multiple mouse models is suf f i cient to reduce plasma LDL-C levels.On the other hand,in human hepatoma cells,Angptl3 silencing and deletion reduced apoB secretion and increased LDL/VLDL uptake[52].These results are consistent with the in vivo turnover study conducted by Musurunu etal.in subjects with familial combined hypolipidemia,as mentioned above[11].

The low levels of plasma HDL-C present in FHBL2 subjects may be the result of the increased activity of endothelial lipase(EL),as found in mice[34].The reduction of plasma HDL-Cwasfound to beassociated with qualitative changes of HDL,which have also a reduced size[14,19].In addition,it was reported that the functionof the HDLin FHBL2isimpaired,asplasmaof subjects with complete de f i ciency of ANGPTL3 showed a reduced cell cholesterol ef f l ux capacity through various ef f l ux pathways[14].

Plasma levels of ANGPTL3

In vivo,ANGPTL3 is cleaved by proprotein convertases to yield the biologically active N-terminal fragmentand an inactive C-terminal fragment.Thus,the formsof ANGPTL3 presentinplasmaarethefull length as well as the N-terminal and C-terminal fragments.To measuretheplasmalevel of ANGPTL3 two quantitative sandwich ELISA assays have been developed.The f i rst method used commercially available rabbit polyclonal anti-human ANGPTL3 antibody and biotin labelled anti-human ANGPTL3 rabbit polyclonal antibody as a capture and detection antibody respectively(Biovendor assay).This method is likely to detect the full-length ANGPTL3 protein as well as the cleaved forms of the protein depending on the epitope localization[53].The second method employed a rabbit polyclonal antibody raised against the N-terminal recombinant human ANGPTL3 as a capture antibody and a biotinylated sheep IgGraised againsthuman full-length recombinant ANGPTL3 as detection antibody.This method should detectthefull-lengthprotein butdoesnotdistinguishthe two cleaved forms[54].

Theconcentration of ANGPTL3 in plasmaof healthy subjects is highly variable,probably depending on the type of antibodies used and the different populations investigated.The mean plasma level of ANGPTL3 was found to be(470±122)ng/mL[35]and(764±291)ng/mL[55]in the Japanese population while in the Finnish population the mean concentration was found to be(368±168)ng/mL[54].The plasma level of ANGPTL3 has shown a positive correlation with plasma HDL-C and LDL-C[54-56].The correlation between plasma levels of ANGPTL3 and TG is still controversial;in one study a negative correlation was found[54]whereas in another study such acorrelation wasnotobserved[56].In subjectshomozygousor compound heterozygousfor LOF mutations in ANGPTL3,plasma ANGPTL3 was undetectablewhilein simpleheterozygotesitslevel was reduced by 50%-60%of thevaluesfound in thecontrol subjects[13,49,57].

ANGPTL3 as ther apeutic target

The marked reduction of the level of atherogenic lipoproteins(VLDL and LDL)observed in FHBL2 subjects suggested that the inactivation of ANGPTL3 may be used as a therapeutic strategy in the management of dyslipidemic conditions.Two different approaches have been used to inactivate ANGPTL3.The f i rst is based on the administration of a human monoclonal antibody against ANGPTL3.When administered to monkeys and dyslipidemic mice,this antibody induced a marked reduction of plasma TG,LDL-Cand HDL-C[35].Thetreatment of healthy human dyslipidemic volunteers with ANGPTL3-blocking antibody(evinacumab)wasfound toreduce TGand LDL-C by 76%and 23%,respectively.In dyslipidemic mice,evinacumab was found to reduce total cholesterol and TGlevels by 50%and 85%respectively,as well as the size of atherosclerotic plaques and their necrotic content,providing a proof of principle that the combined hypolipidemia associated with therapeutic inhibition of ANGPTL3 is anti-atherogenic[25].Evinacumab was also administered to nine patients with homozygous familial hypercholesterolemia due to complete de f i ciency of LDLR.Four-week treatment reduced LDL-C,TG and HDL-C by approximately 50%,47%and 36%,respectively,in addition to reductions in baseline levels already achieved with aggressive lipid-lowering therapy[58].

The second approach is based on the administration of antisense oligonucleotides(ASOs)targeting hepatic Angptl3 mRNA,which is expected to markedly reduce the level of ANGPTL3 protein.ASO treatment in mice reduced thelevelsof TGand LDL-Caswell asliver TG content and retarded the progression of atherosclerosis.In Phase 1 trial in humans,treatment for 6 weeks with multipledosesreduced theplasmaconcentrationsof TG by a maximum of 63%,LDL-C by a maximum of 32.9%,with no signi f i cant changes in HDL-C and without important side effects[59].

Conclusions

The discovery of ANGPTL3 de f i ciency in mice and humans has stimulated a series of studies which have clari f i ed the role of ANGPTL3 in lipoprotein metabolism.These investigations have suggested that ANGPTL3 may be a novel therapeutic target in the management of dyslipidemiasin humans.Theresultsof recent intervention trials aimed at inhibiting ANGPTL3 appear to support this hypothesis.