1 Reverse cholesterol transport dysfunction is a feature of familial 2 hypercholesterolemia 3 Joan Carles Escolà-Gil1,2,3† orcid.org/0000-0001-9021-2485, Noemí Rotllan 1,2 (https://orcid.org/0000-0002-0587- 4 8045) , Josep Julve1,2,3† (http://orcid.org/0000-0002-6531-2246), Francisco Blanco-Vaca2,3,4 5 (https://orcid.org/0000-0001-7380-5385) 6 7 1 Institut de Recerca de l'Hospital de la Santa Creu i Sant Pau, Institut d’Investigacions 8 Biomèdiques (IIB) Sant Pau, Barcelona, Spain 9 2 CIBER de Diabetes y Enfermedades Metabólicas Asociadas, CIBERDEM, Madrid, 10 Spain 11 3 Departament de Bioquímica i Biologia Molecular, i Universitat Autònoma de Barcelona, 12 Barcelona, Spain 13 4 Servei de Bioquímica, Hospital de la Santa Creu i Sant Pau, Institut d’Investigacions 14 Biomèdiques (IIB) Sant Pau, Barcelona, Spain 15 16 Address correspondence to: Joan Carles Escolà Gil (jescola@antpau.cat) 17 or Josep Julve (jjulve@santpau.cat) 18 19 Other author e-mails: 20 fblancova@santpau.cat, nrotllan@santpau.cat 21 22 Key words: HDL, LDL receptor, macrophage, autosomal dominant 23 hypercholesterolemia 1 24 25 Structured abstract 26 Purpose of review: We seek to establish whether high-density lipoprotein HDL 27 metabolism and reverse cholesterol transport (RCT) impairment is an intrinsic feature of 28 familial hypercholesterolemia (FH). 29 Recent findings: RCT from macrophages (m-RCT), a vascular cell type of major 30 influence on atherosclerosis, is impaired in FH due to defective low-density lipoprotein 31 receptor (LDLR) function via both the HDL- and LDL-mediated pathways. Potential 32 mechanisms include impaired HDL metabolism, which is linked to increased LDL levels, 33 as well as the increased transport of cellular unesterified cholesterol to LDL, which 34 presents a defective catabolism. 35 Summary: RCT dysfunction is a consistent finding in the literature and thus an intrinsic 36 feature of mutation-positive FH linked to decreased HDL levels as well as impaired HDL 37 remodeling and LDLR function. It remains to be explored whether these alterations are 38 also present in less well-characterized forms of FH, such as cases with no identified 39 mutations, and whether they are fully corrected by current standard treatments. 2 40 ABBREVIATIONS 41 ABC, ATP binding cassette transporter 42 APO, apolipoprotein 43 CE, cholesterol ester 44 CEC, cholesterol efflux capacity 45 CETP, cholesteryl ester transfer protein 46 CVD, cardiovascular disease 47 FH, familial hypercholesterolemia 48 HDL, high-density lipoprotein 49 HDL-c, HDL cholesterol 50 LCAT, lecithin:cholesterol acyltransferase 51 LDL, low-density lipoprotein 52 LDL-c, LDL cholesterol 53 LDLR, LDL receptor 54 LXR, liver X receptor 55 miRNA, micro RNA 56 m-RCT, macrophage-specific reverse cholesterol transport 57 PCSK9 proprotein convertase subtilisin/kexin type 9 PLTP, phospholipid transfer protein 58 RCT, reverse cholesterol transport 59 SR-BI, scavenger receptor BI 60 SREBP, sterol response element-binding protein 61 TICE, transintestinal cholesterol excretion 62 UC, unesterified cholesterol 3 63 VLDL, very low-density lipoprotein 64 4 65 Introduction 66 Familial hypercholesterolemia (FH) is classically defined as an autosomal codominant 67 disease characterized by elevated plasma low-density lipoprotein (LDL) cholesterol 68 (LDL-c) and a high risk of premature cardiovascular disease (CVD) [1]. It is mainly due 69 to the loss of function variants in the LDL receptor gene (LDLR). FH cases with no 70 detected LDLR mutations might be due to pathogenic variants in other genes encoding 71 proteins that interact with the LDLR, such as the LDLR ligand, apolipoprotein B-100 72 (APOB), and proprotein convertase subtilisin/kexin type 9 (PCSK9). Recently, some 73 studies using different weighted LDL-c gene scores showed that 20 to 80% of mutation- 74 negative FH patients exhibited a high score, suggesting potential forms of polygenic FH 75 [2, 3]. 76 Recent reviews have critically addressed the role of high-density lipoprotein (HDL) in 77 atherosclerosis development, highlighting the potential rapid movement of unesterified 78 cholesterol (UC) from cells and triglyceride-rich particles to HDL as well as from HDL to 79 LDL or tissues [4, 5]. There is also evidence indicating that HDL remodeling, 80 metabolism, and function, including its ability to induce macrophage cholesterol efflux, 81 are impaired in the monogenic forms of FH [6-10]. Whether this impairment is intrinsic to 82 the disease and influences the entire reverse cholesterol transport (RCT) pathway and 83 cardiovascular risk and is corrected by current treatments is not yet well-established. 84 85 5 86 HDL content and remodeling alterations in FH 87 The HDL particle composition is determined by the presence of a diversity of proteins, 88 enzymes, lipids, and microRNAs (miRNAs) that confer specific functions to HDL. The 89 physicochemical composition of HDL may vary and determine the particle shape, 90 density, size, charge, and biological activity. Indeed, HDL composition is a key 91 atheroprotective determinant. Decreased levels of HDL-cholesterol (HDL-C) have been 92 consistently observed in both heterozygous FH and homozygous FH in association with 93 HDL structural and functional abnormalities [6-10]. 94 In an early study of heterozygous FH patients, HDL particles were found to be smaller 95 than those of control subjects [11]. Patients with type IIa hypercholesterolemia 96 displayed reduced HDL3 production and enhanced fractional catabolic rates 97 concomitantly with decreased apolipoprotein (APO) A1 (APOA1) levels [9, 12, 13]. 98 Additionally, FH patients exhibited elevated concentrations of small nascent preβ1-HDL 99 particles [9, 14, 15] but reduced levels of large HDL2 particles when compared to 100 normolipidemic subjects [9]. Other studies—but not all [8]—have demonstrated 101 hypoalphalipoproteinemia due to increased catabolic rates in FH [16, 17]. Interestingly, 102 elevations in plasma APOE have been reported in FH patients [13, 18], and the APOE 103 genotype might influence the plasma HDL-C levels in these patients [19]. Another 104 apolipoprotein differentially expressed in FH is APOL1, which has been proposed as a 105 predictor of CVD events and mortality [20]. Additionally, a decrease in 106 lecithin:cholesterol acyltransferase (LCAT) content in FH patients suggested significant 107 modifications of HDL atheroprotective properties [20]. Indeed, the HDL3 particles of FH 108 patients have been reported to have diminished antioxidant and anti-inflammatory 6 109 functions [12]. HDL particles from FH patients are enriched with cholesteryl esters (CE), 110 depleted in phospholipids, and have an increased sphingomyelin/phosphatidylcholine 111 ratio [9]. In a recent study from our group, different lipid transfer proteins and enzymes 112 associated with HDL remodeling were evaluated in non-treated FH patients with an 113 identified LDLR mutation and compared to normolipidemic patients similar in age [14]. 114 The adult FH patients had lower levels of APOA1 and HDL-C but higher HDL APOA2 115 and APOE content. Interestingly, cholesteryl ester transfer protein (CETP) and 116 phospholipid transfer protein (PLTP) activities were found to be higher in the non- 117 treated FH patients along with reduced LCAT activity [14]. Therefore, these changes 118 might explain, at least in part, the increased content of preβ-HDL particles as well as the 119 decrease in the amount of mature HDL particles in FH [14]. All these changes were also 120 found in LDLR-deficient mice as well as in human APOB100 transgenic mice (it is 121 noteworthy that mice do not express CETP) [21]. The latter mouse model also exhibited 122 hypercholesterolemia due to elevated LDL but also a functional LDLR, indicating that 123 hypercholesterolemia might be directly linked to altered HDL remodeling independently 124 of CETP and LDLR. Although the mechanisms involved in these biochemical changes 125 are largely unknown, they could be related to the accumulation of cholesterol- 126 derivatives activating the liver X receptor (LXR), which may increase CETP and PTLP 127 gene expression [22, 23]. We suggest that increased CETP may not promote RCT in 128 cases with reduced LDLR function, and increased PTPL-mediated remodeling may not 129 be effective in generating more mature HDL particles in the context of low LCAT action. 130 131 7 132 HDL- and plasma-mediated macrophage cholesterol efflux is impaired in FH 133 The cholesterol efflux capacity (CEC) of HDL represents the first step in the reverse 134 cholesterol transport (RCT) pathway. This functional property of HDL has been 135 associated with atherosclerotic cardiovascular disease independently of HDL-C [24, 25]. 136 As previously mentioned, compelling evidence supports the notion that an altered HDL 137 metabolism and remodeling underlies a defective CEC mediated by HDL in FH patients. 138 Indeed, HDL2 isolated from the plasma of FH patients presented a lesser CEC from 139 cholesterol-loaded macrophages, which was mediated through both scavenger receptor 140 class B type I (SR-BI) and ATP-binding cassette (ABC) transporter G1 (ABCG1), when 141 compared to non-FH subjects [9]. In this same study, HDL3-mediated, SR-BI-dependent 142 (but not ABCG1) CEC was also reduced [9]. In line with these findings, FH-derived 143 HDL3 with a high triglyceride content also showed a reduced CEC from lipid-loaded 144 macrophages in an independent study [26]. The reduced concentrations of HDL2 and 145 HDL3 [9, 12], along with the altered activities of remodeling enzymes and lipid transfer 146 proteins [14], may conceivably explain the lower CEC rates promoted by APOB- 147 depleted plasmas or serums in both treated and untreated FH patients [14, 27]. It is 148 noteworthy that CEC of APOB-depleted serum was inversely and independently 149 associated with the carotid intima-media thickness of FH patients treated with a lipid- 150 lowering therapy, although this association was weaker following adjustment for HDL-C 151 and APOA1 levels [27]. We also reported a significant reduction in the amount of 152 macrophage-derived cholesterol accumulated in HDL when the CEC of FH plasmas 153 was evaluated [21]. Taken together, these observations identify the significant 8 154 alteration of HDL-mediated CEC as a feature of FH patients with signs of atherosclerotic 155 cardiovascular disease—even after adjustment for classical risk factors. 156 Several large prospective studies have measured CEC promoted by either APOB- 157 depleted serum or plasma as a surrogate of HDL functionality related to the risk of 158 atherosclerotic cardiovascular disease [24, 25, 28]. However, the CEC of APOB- 159 depleted serum does not always correlate with that of the whole serum, which is likely 160 because the latter contains more physiological cholesterol acceptors [29]. Significant 161 evidence indicates that LDL also contributes to macrophage CEC [30]. We recently 162 reported that CEC induced from the plasma of untreated FH patients (with an identified 163 LDLR mutation) was significantly impaired compared to that of normolipidemic 164 volunteers. Further, our results also indicated that isolated LDL particles were major 165 acceptors of the macrophage-derived radiolabeled cholesterol in both normolipidemic 166 and FH plasmas [21]. Importantly, most of the transferred cholesterol in the LDL was 167 unesterified, thereby indicating the marginal role of CETP and LCAT in this process 168 [21]. Overall, these observations reveal that LDL enhances the efflux of cholesterol 169 directly and indirectly by acting as a sink for cholesterol released from cells by HDL, 170 which is in line with previous observations [30]. However, the transport of cholesterol 171 from HDL to LDL and its slow return into the circulation for LCAT esterification (if the 172 LDL is not previously removed from the circulation by the cell LDLR, as is often the case 173 in FH) may not be representative of the exchanges that happen at the arterial intima 174 level. Indeed, at this location, LDL is trapped by interactions with proteoglycans, and the 175 UC transferred into LDL would likely accumulate at the intima lesion sites [30]. 9 176 CETP could also be driving CE transfer from HDL to LDL in the plasma of FH patients, 177 promoting the formation and vascular accumulation of atherogenic LDL [31]. Very low- 178 density lipoproteins (VLDL) lipolysis, albumin, and a number of enzymes and transfer 179 proteins activities may also be relevant factors associated with plasma CEC [32, 33]. 180 While VLDL do not show significant CEC [21], the transfer of UC from triglyceride-rich 181 lipoproteins to HDL during lipolysis could compete with that from macrophages [4]. 182 Taken together, plasma elevations of LDL-C in FH subjects would appear directly 183 related to the existence of dysfunctional HDL, which are characterized by both an 184 altered remodeling and impaired CEC from macrophages. However, such a decreased 185 cellular cholesterol efflux could also be in part due to a lower macrophage transporter 186 activity. Indeed, ABCA1 expression has been shown to be downregulated in the 187 monocytes of FH patients with genetic defects in LDLR [34]. In this regard, compelling 188 evidence suggests that these transporters also can be regulated by microRNAs 189 (miRNA). Also, many studies over the past years have identified miRNAs as important 190 regulators of HDL metabolism [35, 36]. Furthermore, it has been shown that miRNAs 191 can be transported in the plasma and delivered to recipient cells by HDL, thus directly 192 influencing gene expression [37]. The most abundant miRNAs associated with HDL in 193 FH patients are miR-223, -105, and -106a [37]. The incubation of human cultured 194 hepatocytes (Huh7 cells) with HDL isolated from FH patients, that contained increased 195 miR-223 and miR-105 levels, induced downregulation of several of their putative target 196 genes [37]. Furthermore, in silico target prediction identified mRNAs that were 197 conserved putative target of 22 differentially abundant miRNAS on FH HDL [38, 39]. In 198 addition to its anti-inflammatory role, miR223 may influence systemic and hepatic 10 199 cholesterol metabolism. Indeed, miR233 overexpression attenuates SR-BI protein 200 expression and liver HDL-C uptake [40]. Consistently, the genetic ablation of miR-223 201 enhances hepatic SR-BI expression and HDL-C uptake [41]. Interestingly, miR-223 also 202 prevents cholesterol biosynthesis through directly repressing the sterol enzyme, 3- 203 hydroxy-3-methylglutaryl-coA synthase 1 (HGMCS1), expression [41]. Moreover, the 204 overexpression of miR-223 also enhances ABCA1 expression in Huh7 cells, and thus 205 promotes cholesterol efflux through the repression of transcription factor Sp3. Overall, 206 these studies highlight the role of miR-223 in regulating cholesterol homeostasis, albeit 207 further studies are needed to specifically establish its role in regulating FH macrophage 208 transporters. 209 On the other hand, several studies measuring circulating miRNAs in FH children have 210 shown that miR-33a/b and miR-200c were upregulated [42, 43]. However, whether 211 these circulating miRNAs were associated with HDL has not been reported [44-46]. Like 212 many intronic miRNAs, miR-33a is co-transcribed with its host gene sterol response 213 element-binding protein (SREBP) 2, which targets genes involved in cholesterol export, 214 including ABCA1. 215 216 11 217 m-RCT is impaired in FH caused by LDLR-mediated function 218 The entire RCT transport from cells, such as macrophages, loaded with radiolabeled 219 cholesterol to feces, also called macrophage-specific RCT (m-RCT), has been 220 assessed in experimental animals. Genetically modified mice resembling a human 221 monogenic FH mutation, usually in homozygosis, have been used in these experiments. 222 We recently found that [3H]cholesterol derived from labeled macrophages injected into 223 the peritoneal cavity of FH mouse models (i.e., LDLR-deficient mice or PCSK9- 224 overexpressing mice) was rapidly transferred to the hypercholesterolemic plasma and 225 mostly associated with LDL. However, the fecal excretion of macrophage-derived 226 cholesterol was significantly impaired in these mice, thereby indicating that that LDLR 227 was essential in supporting the last step of the m-RCT route [21]. In line with these 228 findings, a reduction in the liver and adrenal gland uptake of radiolabeled CE in HDL 229 was previously noted in LDLR-deficient mice [47]. More importantly, both radiolabeled 230 LDL- and HDL-cholesterol showed an impaired clearance in LDLR-deficient mice, and 231 this was concomitant with a lower transfer of cholesterol from both lipoproteins to the 232 feces [21]. Although these mice were homozygous lacking LDLR, there is no reason to 233 believe that the defective m-RCT would not occur in heterozygous animals—albeit 234 presumably at a lower scale. In contrast, the m-RCT rate remained unchanged in 235 human APOB100 transgenic mice with fully functional LDLR despite increased levels of 236 plasma APOB-containing lipoproteins and a higher accumulation of macrophage- 237 derived cholesterol in the LDL fraction [21]. Since the APOB100 transgenic mice 238 presented with HDL remodeling impairment, it can be suggested that LDLR is needed to 239 maintain m-RCT rate. 12 240 Overall, these findings strongly indicate that monogenic FH due to LDLR functionality 241 impairment in mice does present with defective m-RCT. FH patients may present a 242 more intense impairment, because their CETP activity could enhance the transfer of 243 HDL-CE (formed by the LCAT action) to LDL and be subjected to a slower transfer to 244 the liver. Indeed, CETP enhanced the amount of macrophage-derived cholesterol in 245 LDL and the overall flux of [3H]cholesterol to the feces in mice, but failed at promoting 246 the m-RCT rate in the absence of LDLR [48]. 247 It should be noted that LDL provides a significant amount of cholesterol for 248 transintestinal cholesterol excretion (TICE) in human and mouse jejunal explants at its 249 basolateral side [49]. Interestingly, TICE was increased in PCSK9-deficient mice 250 whereas decreased upon an acute injection of PCSK9 [49]. However, TICE tended to 251 be higher in LDLR-deficient mice [49], suggesting the activation of alternative 252 compensatory mechanism(s) in conditions of chronic LDLR deficiency. 253 254 Experimental and clinical therapeutic strategies 255 Early reports demonstrated that LDL-apheresis was highly efficient in reducing not only 256 LDL-C in severe FH patients but also large APOE-containing HDL and preβ-HDL 257 particles [50]. This transitorily reduced the ability of these plasmas to induce 258 macrophage cholesterol efflux [51]. In line with these findings, a recent report found that 259 HDL from FH patients had increased malondialdehyde-APOA1 adducts, which was in 260 close association with a defective CEC induced by APOB-depleted serum; this 261 functional HDL alteration was not improved by LDL-apheresis [52]. Interestingly, 262 dicarbonyl scavenging with 2-hydroxybenzylamine was able to prevent atherosclerosis 13 263 and foam cell formation by improving HDL CEC in LDLR-deficient mice [52]. Several 264 therapeutic approaches have also failed to induce macrophage cholesterol efflux in FH 265 patients. Indeed, lomitapide treatment was found to reduce the amount of large-buoyant 266 HDL and preβ-HDL particles in homozygous FH patients, and, consistently, the ABCA1- 267 mediated CEC of APOB-depleted serum was impaired after this treatment [53]. 268 Furthermore, a recent study reported that an antibody to PCSK9, evolocumab, did not 269 affect either plasma HDL subclasses nor macrophage cholesterol efflux [54]. However, 270 this study was conducted in only three FH patients who had been under apheresis 271 treatment for 11 years [54]. It should be noted that human PCSK9 reduced ABCA1- 272 dependent macrophage cholesterol efflux to APOA1 induced by the activation of the 273 LXR/RXR pathway; this effect was fully abrogated by an anti-PCSK9 antibody or LDLR 274 deficiency [55]. Non-treated FH children displayed alterations in HDL, such as an 275 increase in HDL3-C and large HDL with respect to healthy controls. Interestingly, the 276 smaller HDL particles were enriched in CE and had lower UC and phospholipid content 277 [56]. The amount of very large HDL was normalized in statin-treated FH children [56], 278 but whether this affected macrophage cholesterol efflux is unknown. In this context, we 279 found that statin treatment was not able to normalize the ability of adolescent FH 280 plasmas to induce macrophage cholesterol efflux (unpublished data). However, the 281 lipoprotein distribution of macrophage-derived radiolabeled cholesterol was in part 282 increased in HDL following statin treatment, whereas that of LDL was reduced 283 concomitantly (unpublished data). Of note, rosuvastatin—but not atorvastatin—induced 284 ABCA1-dependent macrophage cholesterol efflux and promoted m-RCT in wild-type 285 mice [57]. Clearly, more studies are needed to clarify the potential of PCSK9 inhibitors 14 286 and statins in regulating HDL-mediated macrophage cholesterol efflux and their impact 287 on the entire RCT pathway in FH. 288 Another therapeutic approach is the injection of recombinant HDL particles, such as 289 CER-001, which has been shown to enhance macrophage cholesterol efflux, fecal 290 cholesterol excretion, and atherosclerosis regression in LDLR-deficient mice [58]. CER- 291 001 reduced the mean vessel wall area measured by magnetic resonance imaging in 292 homozygous FH patients [59], thereby indicating that targeting HDL-mediated 293 cholesterol efflux may represent a successful strategy for regressing atherosclerotic 294 plaque. However, CER-001 did not favorably influence the carotid atherosclerosis of 295 patients with HDL deficiencies despite significant elevations in plasma CEC after CER- 296 001 infusion [60]; whether the activity of this compound in patients is disease-dependent 297 remains to be seen. 298 299 Conclusions 300 There is a notable consensus that points to an altered HDL remodeling and composition 301 and impaired m-RCT in FH. This impairment can be captured, at least in part, in 302 cholesterol efflux experiments in FH patients and the m-RCT experiments in genetically 303 modified mice. These functional alterations have been reported at different steps of the 304 RCT pathway (summarized in Figure 1) and seem to be especially dependent on the 305 existence of increased LDL and LDLR function. Whether this FH feature is critical for 306 atherosclerosis development and is ameliorated by current standard treatments for the 307 disease needs to be further investigated. Although the latter could be anticipated 308 considering the potential of current treatments to achieve important reductions in LDL- 15 309 C, the data currently available would support a rather incomplete improvement in HDL 310 and CEC in FH patients treated with statins. Future research should also compare the 311 effect of different pharmacological treatments in CEC in both mutation-detected FH and 312 mutation-negative FH cases. 313 314 Sources of Funding 315 Thais Works was partly funded by the Instituto de Salud Carlos III and FEDER "Una 316 manera de hacer Europa" grants PI18/00164 (to F.B.-V.), PI17/00232 (to J. J.), and 317 PI19/00136 (to J.C.E-G), and Miguel Servet Type 2 contract (CPII18/00004 to J.J.); 318 grant 12/C/2015 from La Fundació la Marató TV3 (to F.B-V); Ministerio de Ciencia, 319 Innovación y Universidades, Subprograma Ramón y Cajal (RUC-20172879 to N.R.) and 320 Red de Investigación "Enfermedades Metabólicas y Cáncer" (RED2018-102799-T to 321 J.J). CIBERDEM is an Instituto de Salud Carlos III project. 322 323 Disclosures 324 All authors have reported that they have no relationships relevant to the contents of this 325 paper to disclose. 326 327 Human and Animal Rights and Informed Consent 328 This article does not contain any studies with human or animal subjects. 329 330 16 331 Figure legend 332 333 Figure 1. Macrophage reverse cholesterol transport (RCT) pathway is impaired in 334 familiar hypercholesterolemia (FH). The functional alterations of the HDL-mediated RCT 335 pathway detected at the different steps are based on results from human studies and 336 FH mouse models. The first step of reverse unesterified cholesterol (UC) transport is 337 initiated in macrophage foam cells. FH patients display increased cholesteryl ester 338 transfer protein (CETP) and phospholipid transfer protein (PLTP) activities but reduced 339 lecithin-cholesterol acyltransferase (LCAT) activity. These changes are associated with 340 lower α-HDL cholesterol and APOA1 levels along with significant alterations in the α- 341 HDL composition and higher levels of nascent preβ-HDL particles. UC efflux from 342 macrophages to HDL particles, promoted by both the transmembrane cholesterol ATP 343 binding cassette transporters (ABC), A1 and G1, is impaired in FH patients. A significant 344 part of macrophage-derived UC present on the surface of preβ-HDL and α-HDL 345 particles and can be rapidly transferred to LDL into the circulation. This process appears 346 to be independent of CETP. It should be noted that, based on whether cholesterol efflux 347 from macrophages takes place in the arterial intima, LDL could be trapped by 348 interactions with proteoglycans, and the UC transferred into LDL at this location would 349 be finally accumulated at the intima lesion sites. Circulating CETP in FH patients can 350 also drive the transfer of esterified cholesterol (EC) from HDL toward the core of LDL. In 351 experimental models of FH, LDLs, carrying their load of macrophage-derived 352 cholesterol, cannot be correctly internalized by the hepatic LDL receptor (LDLR). 353 Hepatic cholesterol is ultimately secreted into the bile and to the intestine by ABCG5/G8 354 as UC, thereby completing the hepatobiliary RCT route. Overall, under the genetic 17 355 absence of the LDLR, or under conditions leading to its dysfunctionality, the rate of this 356 macrophage-derived UC reverse transport to feces is decreased. Although LDLR- 357 deficiency does not show clear effects on transintestinal cholesterol export (TICE), the 358 acute injection of PCSK9 may regulate TICE in vivo. 359 18 360 References 361 Papers of particular interest or published recently, have been highlighted as: 362 • Of importance 363 •• Of major importance 364 1. Nordestgaard BG, Chapman MJ, Humphries SE, et al: Familial hypercholesterolaemia is 365 underdiagnosed and undertreated in the general population: guidance for clinicians to prevent 366 coronary heart disease: consensus statement of the European Atherosclerosis Society. Eur Heart 367 J 2013, 34(45):3478-3490a. 368 2. Talmud PJ, Shah S, Whittall R, et al: Use of low-density lipoprotein cholesterol gene score to 369 distinguish patients with polygenic and monogenic familial hypercholesterolaemia: a case- 370 control study. Lancet 2013, 381(9874):1293-1301. 371 3. • Martin-Campos JM, Ruiz-Nogales S, Ibarretxe D, et al: Polygenic Markers in Patients Diagnosed 372 of Autosomal Dominant Hypercholesterolemia in Catalonia: Distribution of Weighted LDL-c- 373 Raising SNP Scores and Refinement of Variant Selection. Biomedicines 2020, 8(9). 374 In line with previous findings of different groups, the increased LDL genetic score based on selected 375 SNPs in mutation-negative FH patients suggest the existence of polygenic forms of the disease 376 4. •• Kontush A: HDL and Reverse Remnant-Cholesterol Transport (RRT): Relevance to Cardiovascular 377 Disease. Trends Mol Med 2020, 26(12):1086-1100. 378 This review proposes a potential novel pathway that explains the U-shaped relationship between 379 plasma HDL-C levels and cardiovascular disease via the impairment of the transfer of 380 unesterified cholesterol from triglyceride-rich lipoproteins to HDL 381 5. •• Rosales C, Gillard BK, Xu B, Gotto AM, Jr., Pownall HJ: Revisiting Reverse Cholesterol Transport in 382 the Context of High-Density Lipoprotein Free Cholesterol Bioavailability. Methodist Debakey 383 Cardiovasc J 2019, 15(1):47-54. 384 This review hypothesizes that an increased bioavailability of unesferified cholesterol in dysfunctional 385 HDL would promote the excess transfer of this molecule to cells, thus constituting a potential 386 proatherogenic mechanism 387 6. Jansen AC, van Aalst-Cohen ES, Tanck MW, et al: The contribution of classical risk factors to 388 cardiovascular disease in familial hypercholesterolaemia: data in 2400 patients. J Intern Med 389 2004, 256(6):482-490. 390 7. van Aalst-Cohen ES, Jansen AC, Boekholdt SM, et al: Genetic determinants of plasma HDL- 391 cholesterol levels in familial hypercholesterolemia. Eur J Hum Genet 2005, 13(10):1137-1142. 392 8. Inazu A, Koizumi J, Mabuchi H, Kajinami K, Takeda R: Enhanced cholesteryl ester transfer protein 393 activities and abnormalities of high density lipoproteins in familial hypercholesterolemia. Horm 394 Metab Res 1992, 24(6):284-288. 395 9. Bellanger N, Orsoni A, Julia Z, et al: Atheroprotective reverse cholesterol transport pathway is 396 defective in familial hypercholesterolemia. Arterioscler Thromb Vasc Biol 2011, 31(7):1675- 397 1681. 398 10. • Badimon L, Padro T, Cubedo J: Protein changes in non-LDL-lipoproteins in familial 399 hypercholesterolemia: implications in cardiovascular disease manifestation and outcome. Curr 400 Opin Lipidol 2017, 28(5):427-433. 401 This review focuses on the identification of HDL protein changes that might contribute to the 402 increased cardiovascular risk of familial hypercholesterolemia 19 403 11. Hogue JC, Lamarche B, Gaudet D, et al: Association of heterozygous familial 404 hypercholesterolemia with smaller HDL particle size. Atherosclerosis 2007, 190(2):429-435. 405 12. Hussein H, Saheb S, Couturier M, et al: Small, dense high-density lipoprotein 3 particles exhibit 406 defective antioxidative and anti-inflammatory function in familial hypercholesterolemia: Partial 407 correction by low-density lipoprotein apheresis. J Clin Lipidol 2016, 10(1):124-133. 408 13. Koizumi J, Inazu A, Fujita H, et al: Removal of apolipoprotein E-enriched high density lipoprotein 409 by LDL-apheresis in familial hypercholesterolaemia: a possible activation of the reverse 410 cholesterol transport system. Atherosclerosis 1988, 74(1-2):1-8. 411 14. •• Cedo L, Plana N, Metso J, et al: Altered HDL Remodeling and Functionality in Familial 412 Hypercholesterolemia. J Am Coll Cardiol 2018, 71(4):466-468. 413 Short communication showing impairment in lipid transfer proteins and enzymes regulating HDL 414 remodeling and cholesterol efflux in untreated heterozygous patients with an LDLR identified 415 mutation 416 15. Miida T, Nakamura Y, Okada M: Development of coronary atherosclerosis in asymptomatic 417 heterozygous patients with familial hypercholesterolemia. J Cardiol 1996, 28(2):71-77. 418 16. Frenais R, Ouguerram K, Maugeais C, et al: Apolipoprotein A-I kinetics in heterozygous familial 419 hypercholesterolemia: a stable isotope study. J Lipid Res 1999, 40(8):1506-1511. 420 17. Schaefer JR, Rader DJ, Ikewaki K, et al: In vivo metabolism of apolipoprotein A-I in a patient with 421 homozygous familial hypercholesterolemia. Arterioscler Thromb 1992, 12(7):843-848. 422 18. Gibson JC, Goldberg RB, Rubinstein A, et al: Plasma lipoprotein distribution of apolipoprotein E 423 in familial hypercholesterolemia. Arteriosclerosis 1987, 7(4):401-407. 424 19. Miltiadous G, Cariolou MA, Elisaf M: HDL cholesterol levels in patients with molecularly defined 425 familial hypercholesterolemia. Ann Clin Lab Sci 2002, 32(1):50-54. 426 20. Cubedo J, Padro T, Alonso R, Mata P, Badimon L: ApoL1 levels in high density lipoprotein and 427 cardiovascular event presentation in patients with familial hypercholesterolemia. J Lipid Res 428 2016, 57(6):1059-1073. 429 21. •• Cedo L, Metso J, Santos D, et al: LDL Receptor Regulates the Reverse Transport of Macrophage- 430 Derived Unesterified Cholesterol via Concerted Action of the HDL-LDL Axis: Insight From Mouse 431 Models. Circ Res 2020, 127(6):778-792. 432 In vivo demonstration of impaired m-RCT in mouse models of familial hypercholesterolemia due to 433 major LDLR dysfunction, but not in models with a similar phenotype but conserved LDLR 434 capacity, such as the apoB100 transgenic mice 435 22. Lakomy D, Rebe C, Sberna AL, et al: Liver X receptor-mediated induction of cholesteryl ester 436 transfer protein expression is selectively impaired in inflammatory macrophages. Arterioscler 437 Thromb Vasc Biol 2009, 29(11):1923-1929. 438 23. Laffitte BA, Joseph SB, Chen M, et al: The phospholipid transfer protein gene is a liver X receptor 439 target expressed by macrophages in atherosclerotic lesions. Mol Cell Biol 2003, 23(6):2182- 440 2191. 441 24. Rohatgi A, Khera A, Berry JD, et al: HDL cholesterol efflux capacity and incident cardiovascular 442 events. N Engl J Med 2014, 371(25):2383-2393. 443 25. Saleheen D, Scott R, Javad S, et al: Association of HDL cholesterol efflux capacity with incident 444 coronary heart disease events: a prospective case-control study. Lancet Diabetes Endocrinol 445 2015, 3(7):507-513. 446 26. Ottestad IO, Halvorsen B, Balstad TR, et al: Triglyceride-rich HDL3 from patients with familial 447 hypercholesterolemia are less able to inhibit cytokine release or to promote cholesterol efflux. J 448 Nutr 2006, 136(4):877-881. 20 449 27. Ogura M, Hori M, Harada-Shiba M: Association Between Cholesterol Efflux Capacity and 450 Atherosclerotic Cardiovascular Disease in Patients With Familial Hypercholesterolemia. 451 Arterioscler Thromb Vasc Biol 2016, 36(1):181-188. 452 28. •• Soria-Florido MT, Schroder H, Grau M, Fito M, Lassale C: High density lipoprotein functionality 453 and cardiovascular events and mortality: A systematic review and meta-analysis. Atherosclerosis 454 2020, 302:36-42. 455 Systematic review and meta-analysis showing that although increased HDL cholesterol efflux capacity 456 and antioxidant/anti-inflammatory capacities are associated with a lower risk of 457 cardiovascular disease, there is a need of larger prospective studies with predefined 458 standardized assays and outcomes. 459 29. Guerin M, Silvain J, Gall J, et al: Association of Serum Cholesterol Efflux Capacity With Mortality 460 in Patients With ST-Segment Elevation Myocardial Infarction. J Am Coll Cardiol 2018, 461 72(25):3259-3269. 462 30. •• von Eckardstein A: LDL Contributes to Reverse Cholesterol Transport. Circ Res 2020, 127(6):793- 463 795. 464 Insightful editorial comment on the role of LDL on CEC measurements and on the potential 465 significance of unesterified cholesterol transfer from HDL to LDL in atherogenesis 466 31. Guerin M, Dolphin PJ, Chapman MJ: Preferential cholesteryl ester acceptors among the LDL 467 subspecies of subjects with familial hypercholesterolemia. Arterioscler Thromb 1994, 14(5):679- 468 685. 469 32. •• Feng M, Darabi M, Tubeuf E, et al: Free cholesterol transfer to high-density lipoprotein (HDL) 470 upon triglyceride lipolysis underlies the U-shape relationship between HDL-cholesterol and 471 cardiovascular disease. Eur J Prev Cardiol 2020, 27(15):1606-1616. 472 Study revealing that unesterified cholesterol transfer to HDL is linked to triglyceride-rich lipoprotein 473 lipolysis and parallels the U-shape relationship between HDL-C and cardiovascular disease 474 33. Sankaranarayanan S, de la Llera-Moya M, Drazul-Schrader D, Phillips MC, Kellner-Weibel G, 475 Rothblat GH: Serum albumin acts as a shuttle to enhance cholesterol efflux from cells. J Lipid Res 476 2013, 54(3):671-676. 477 34. Mosig S, Rennert K, Buttner P, et al: Monocytes of patients with familial hypercholesterolemia 478 show alterations in cholesterol metabolism. BMC Med Genomics 2008, 1:60. 479 35. Canfran-Duque A, Lin CS, Goedeke L, Suarez Y, Fernandez-Hernando C: Micro-RNAs and High- 480 Density Lipoprotein Metabolism. Arterioscler Thromb Vasc Biol 2016, 36(6):1076-1084. 481 36. Rottiers V, Naar AM: MicroRNAs in metabolism and metabolic disorders. Nat Rev Mol Cell Biol 482 2012, 13(4):239-250. 483 37. Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, Remaley AT: MicroRNAs are transported 484 in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol 2011, 485 13(4):423-433. 486 38. Lewis BP, Burge CB, Bartel DP: Conserved seed pairing, often flanked by adenosines, indicates 487 that thousands of human genes are microRNA targets. Cell 2005, 120(1):15-20. 488 39. Friedman RC, Farh KK, Burge CB, Bartel DP: Most mammalian mRNAs are conserved targets of 489 microRNAs. Genome Res 2009, 19(1):92-105. 490 40. Wang L, Jia XJ, Jiang HJ, et al: MicroRNAs 185, 96, and 223 repress selective high-density 491 lipoprotein cholesterol uptake through posttranscriptional inhibition. Mol Cell Biol 2013, 492 33(10):1956-1964. 493 41. Vickers KC, Landstreet SR, Levin MG, et al: MicroRNA-223 coordinates cholesterol homeostasis. 494 Proc Natl Acad Sci U S A 2014, 111(40):14518-14523. 495 42. Martino F, Carlomosti F, Avitabile D, et al: Circulating miR-33a and miR-33b are up-regulated in 496 familial hypercholesterolaemia in paediatric age. Clin Sci (Lond) 2015, 129(11):963-972. 21 497 43. • D'Agostino M, Martino F, Sileno S, et al: Circulating miR-200c is up-regulated in paediatric 498 patients with familial hypercholesterolaemia and correlates with miR-33a/b levels: implication 499 of a ZEB1-dependent mechanism. Clin Sci (Lond) 2017, 131(18):2397-2408. 500 Study showing that circulating miR-200c is upregulated in pediatric FH, probably due to oxidative 501 stress and inflammation and via a miR-33a/b-ZEB1-dependent mechanism 502 44. Rayner KJ, Suarez Y, Davalos A, et al: MiR-33 contributes to the regulation of cholesterol 503 homeostasis. Science 2010, 328(5985):1570-1573. 504 45. Najafi-Shoushtari SH, Kristo F, Li Y, et al: MicroRNA-33 and the SREBP host genes cooperate to 505 control cholesterol homeostasis. Science 2010, 328(5985):1566-1569. 506 46. Marquart TJ, Allen RM, Ory DS, Baldan A: miR-33 links SREBP-2 induction to repression of sterol 507 transporters. Proc Natl Acad Sci U S A 2010, 107(27):12228-12232. 508 47. Rinninger F, Heine M, Singaraja R, et al: High density lipoprotein metabolism in low density 509 lipoprotein receptor-deficient mice. J Lipid Res 2014, 55(9):1914-1924. 510 48. Tanigawa H, Billheimer JT, Tohyama J, Zhang Y, Rothblat G, Rader DJ: Expression of cholesteryl 511 ester transfer protein in mice promotes macrophage reverse cholesterol transport. Circulation 512 2007, 116(11):1267-1273. 513 49. Le May C, Berger JM, Lespine A, et al: Transintestinal cholesterol excretion is an active metabolic 514 process modulated by PCSK9 and statin involving ABCB1. Arterioscler Thromb Vasc Biol 2013, 515 33(7):1484-1493. 516 50. Orsoni A, Saheb S, Levels JH, et al: LDL-apheresis depletes apoE-HDL and pre-beta1-HDL in 517 familial hypercholesterolemia: relevance to atheroprotection. J Lipid Res 2011, 52(12):2304- 518 2313. 519 51. Orsoni A, Villard EF, Bruckert E, et al: Impact of LDL apheresis on atheroprotective reverse 520 cholesterol transport pathway in familial hypercholesterolemia. J Lipid Res 2012, 53(4):767-775. 521 52. •• Tao H, Huang J, Yancey PG, et al: Scavenging of reactive dicarbonyls with 2-hydroxybenzylamine 522 reduces atherosclerosis in hypercholesterolemic Ldlr(-/-) mice. Nat Commun 2020, 11(1):4084. 523 Treatment of FH mice with reactive dicarbonyl scavengers reduced HDL oxidative modification and 524 increased HDL cholesterol efflux capacity; HDL from subjects with FH had increased MDA- 525 apoAI adducts and defective cholesterol efflux capacity. 526 53. Yahya R, Favari E, Calabresi L, et al: Lomitapide affects HDL composition and function. 527 Atherosclerosis 2016, 251:15-18. 528 54. Lappegard KT, Kjellmo CA, Ljunggren S, et al: Lipoprotein apheresis affects lipoprotein particle 529 subclasses more efficiently compared to the PCSK9 inhibitor evolocumab, a pilot study. Transfus 530 Apher Sci 2018, 57(1):91-96. 531 55. Adorni MP, Cipollari E, Favari E, et al: Inhibitory effect of PCSK9 on Abca1 protein expression and 532 cholesterol efflux in macrophages. Atherosclerosis 2017, 256:1-6. 533 56. •• Christensen JJ, Ulven SM, Retterstol K, et al: Comprehensive lipid and metabolite profiling of 534 children with and without familial hypercholesterolemia: A cross-sectional study. 535 Atherosclerosis 2017, 266:48-57. 536 Interesting study showing that the NMR plasma analysis of children uncovers major changes in FH HDL 537 57. Shimizu T, Miura S, Tanigawa H, et al: Rosuvastatin activates ATP-binding cassette transporter 538 A1-dependent efflux ex vivo and promotes reverse cholesterol transport in macrophage cells in 539 mice fed a high-fat diet. Arterioscler Thromb Vasc Biol 2014, 34(10):2246-2253. 540 58. Tardy C, Goffinet M, Boubekeur N, et al: CER-001, a HDL-mimetic, stimulates the reverse lipid 541 transport and atherosclerosis regression in high cholesterol diet-fed LDL-receptor deficient mice. 542 Atherosclerosis 2014, 232(1):110-118. 543 59. Hovingh GK, Smits LP, Stefanutti C, et al: The effect of an apolipoprotein A-I-containing high- 544 density lipoprotein-mimetic particle (CER-001) on carotid artery wall thickness in patients with 22 545 homozygous familial hypercholesterolemia: The Modifying Orphan Disease Evaluation (MODE) 546 study. Am Heart J 2015, 169(5):736-742 e731. 547 60. Zheng KH, Kaiser Y, van Olden CC, et al: No benefit of HDL mimetic CER-001 on carotid 548 atherosclerosis in patients with genetically determined very low HDL levels. Atherosclerosis 549 2020, 311:13-19. 550 23