**4. Phenotypes of humans and experimental animals having apoA-I mutations**

#### **4.1 Natural apoA-I mutations**

176 Dyslipidemia - From Prevention to Treatment

SR-BI is an 82 kDa membrane glycoprotein primarily expressed in the liver, steroidogenic tissues and endothelial cells but is also found in other tissues. The most important property of SR-BI is considered to be its ability to act as the HDL receptor (Acton et al., 1996). SR-BI mediates both selective uptake of cholesteryl esters and other lipids from HDL to cells (Acton et al., 1996; Stangl et al., 1999; Thuahnai et al., 2001), as well as efflux of unesterified cholesterol (Gu et al., 2000). Transgenic mice expressing SR-BI in the liver had greatly decreased apoA-I and HDL levels as well as increased clearance of VLDL and LDL (Ueda et al., 1999) and were protected from atherosclerosis (Arai et al., 1999). SR-BI deficient mice had decreased HDL cholesterol clearance (Out et al., 2004), two fold increased plasma cholesterol and presence of large size abnormal apolipoprotein E (apoE) enriched particles that were distributed in the HDL/IDL/LDL region (Rigotti et al., 1997). The SR-BI deficiency in the background of LDL receptor (LDLr) deficient or apoE deficient mice accelerated dramatically the development of atherosclerosis (Huszar et al., 2000; Trigatti et al., 1999). The double deficient mice for apoE and SR-BI developed occlusive coronary atherosclerosis, cardiac hypertrophy, myocardial infarctions, cardiac dysfunction and died prematurely (mean age of death ~6 weeks) (Braun et al., 2002; Trigatti et al., 1999). The SR-BI deficiency reduced greatly cholesteryl ester levels in the steroidogenic tissues that utilize HDL cholesterol for synthesis of steroid hormones (Ji et al., 1999). It also decreased secretion of biliary cholesterol by approximately 50% (Mardones et al., 2001; Rigotti et al., 1997). The SR-BI deficiency also caused defective maturation of oocytes and red blood cells due to accumulation of cholesterol in the plasma membrane of progenitor cells (Holm et al., 2002; Trigatti et al., 1999) and caused infertility in the female but not the male mice (Trigatti et al., 1999; Yesilaltay et al., 2006). Interactions of HDL with SR-BI in endothelial cells triggers signaling mechanisms discussed below that involve activation of endothelial nitric oxide synthase (eNOS) and release of NO that causes vasodilation (Mineo et al., 2003; Yuhanna et al., 2001). Human subjects have been identified with a P297S substitution in SR-BI. Heterozygote carriers for this mutation had increased HDL levels, decreased adrenal stereoidogenesis and

dysfunctional platelets but did not develop atherosclerosis (Vergeer et al., 2011).

women (Yates et al., 2011).

2007).

**3.5 Interactions of HDL with ABCG1** 

A [Gly2Ser]SR-BI substitution in humans is associated with decreased follicular progesterone levels in Caucasian women and non viable fetuses 42 days post embryo transfer. Another single nucleotide polymorphism (SNP) (rs10846744) was associated with gestational sacs and fetal heart beats and with poor fetal viability in African-American

ABCG1 is a 67 kDa protein which is a member of ABC family of half transporters. ABCG1 is expressed in the spleen, thymus, lung, brain, endothelial cells and other tissues (Savary et al., 1996) and promotes cholesterol efflux from cells to HDL but not to lipid free apoA-I (Nakamura et al., 2004; Vaughan and Oram, 2005). The absence of ABCG1 in mice causes cholesterol accumulation in various tissues (Kennedy et al., 2005) and selective deletion of both ABCA1 and ABCG1 genes in macrophages further increases cholesterol accumulation and results in severe atherosclerosis (Out et al., 2008; Yvan-Charvet et al.,

**3.4 Interactions of lipid-bound ApoA-I with SR-BI** 

Several apoA-I mutations have been described in the general population that are associated with low plasma HDL levels. Most of the mutations affect the interaction of apoA-I with LCAT. Eight mutations between residues 26 and 107 and one on residue 173, have been associated with amyloidosis and low HDL levels (Sorci-Thomas and Thomas, 2002; Zannis et al., 1993) and one mutation on residue 164 is associated with increased risk for ischemic heart disease and reduced life expectancy (Haase et al., 2011). The in vivo interactions of representative naturally occurring apoA-I mutants with LCAT were studied by adenovirusmediated gene transfer in apoA-I deficient mice. The mutants apoA-I(Leu141Arg)Pisa and apoA-I(Leu159Arg)FIN produced only small amounts of HDL that formed mostly preβ1 and small size α4 HDL particles. The apoA-I(Arg151Cys)Paris and apoA-I(arg160Leu)Oslo formed discoidal HDL particles. These studies indicated that apoA-I(Leu141Arg)Pisa and apoA-I(Leu159Arg)FIN mutation may inhibit an early step in the biogenesis of HDL due to insufficient esterification of the cholesterol of the preβ1-HDL particles by the endogenous LCAT. The LCAT insufficiency appears to result from depletion of the plasma LCAT mass (Koukos et al., 2007). A remarkable finding of these studies was that all the aberrant phenotypes were corrected by treatment with exogenous LCAT. This indicates that LCAT administration could be a potential therapeutic intervention to correct low-HDL conditions in humans that are caused by these and other unidentified mutations (Amar et al., 2009).

#### **4.2 Specific bioengineered mutations in ApoA-I may cause dyslipidemia**

Four bioengineered mutations in apoA-I have been studied by adenovirus-mediated gene transfer in apoA-I deficient mice. Mutants apoA-I[Δ(62-78)], apoA-I [Glu110Ala/Glu111Ala] and apoA-I[Asp89Ala/Glu90Ala/Glu92Ala], caused combined hyperlipidemia, characterized by elevated plasma cholesterol and severe hypertriglyceridemia (Chroni et al., 2004; Chroni et al., 2005b; Kateifides et al., 2011). An apoA-I[Δ89-99] mutant induced high plasma cholesterol, but did not affect plasma triglyceride levels (Chroni et al., 2005b).

Fig. 2. The pathway of HDL biogenesis and functions of HDL. a-e represent sites of possible disruption of the HDL biogenesis pathway. Different subpopulations of HDL that have been generated due to these defects may have different functions.

Pleiotropic Functions of HDL Lead to Protection from Atherosclerosis and Other Diseases 179

et al., 2003). A photoactive derivative of cholesterol binds in the transmembrane region of SR-BI indicating that this region serves as a cholesterol sensor on the plasma membrane (Assanasen et al., 2005). HDL and lysophospholipids that are components of HDL including sphingosylphosphorylcholine, sphingosine-1-phospate (S1P) and lysosulphatide cause eNOS dependent relaxation of mouse aortic rings via intracellular Ca2+ mobilization and eNOS phosphorylation mediated by Akt (Nofer et al., 2004). Another study however, indicated that interactions of HDL with SR-BI stimulate eNOS by increasing intracellular ceramide levels without affecting intracellular calcium levels and Akt phosphorylation (Li et al., 2002). The proposed role of HDL-associated estradiol in the stimulation of eNOS activity is unclear (Gong et al., 2003). 5' AMP-activated protein kinase (AMPK) may also play a role in the HDL-mediated phosphorylation of eNOS at multiple sites (Ser116, Ser635, and Ser1179) (Drew et al., 2004). It was suggested that activation by AMPK may involve physical interactions between the apoA-I component of HDL and eNOS. Such interactions may be possible following SR-BI mediated endocytosis of HDL (Silver et al., 2001). HDL also affected the signaling in endothelial cells by the bone morphogenetic protein 4 (BMP4) and increased expression of the activin-like kinase receptor 1 and 2 (Yao et al., 2008). This resulted in increased expression of vascular endothelial growth factor (VEGF) and matrix gla protein (MGP). VEGF promotes endothelial cell survival and MGP prevents vascular calcification and thus contribute to the maintenance, the integrity and the preservation of

Fig. 3. A) Schematic representation of SR-BI signaling that can lead to vasodilation, cell migration and inhibition of inflammation and apoptosis. B) Schematic representation of PKC-dependent SR-BI-dependent signalling pathway that promote cell proliferation (Drew

et al., 2004; Grewal et al., 2003; Kimura et al., 2003; Mineo et al., 2003).

the functions of the endothelium (Yao et al., 2008).

The systematic study of the functions of apoA-I by adenovirus mediated gene transfer as well as the phenotypes of naturally occurring apoA-I mutants identified the following five steps where the pathway of biogenesis and/or catabolism of HDL can be disrupted: a) Lack of synthesis of HDL due to mutations in ABCA1 or mutations in apoA-I that affect the ABCA1/apoA-I interaction, b) Failure to convert efficiently the lipidated pre-β HDL to discoidal HDL. This defect most likely results from fast catabolism of apoA-I following its lipidation by ABCA1, c) Accumulation of discoidal HDL. This phenotype has been generated by the mutations in the 149-160 region of apoA-I that affect LCAT activation, d) Accumulation of discoidal HDL and induction of hypercholesterolemia. This condition has been observed in the case of the apoA-I[Δ(89-99)] mutant, e) Induction of hypertriglyceridemia. This defect has been observed in the case of apoA-I[Δ(62-78)], apoA-I[Glu110Ala/Glu111Ala] and apoA-I[Asp89Ala/Glu90Ala/Glu92Ala] mutants (Fig. 2).
