Cholesterol, the most prominent family member of the sterols, is originally discovered as a major component in human gallstones by F. Pouletier de la Salle in 1769. M.E. Chevreul named the organic molecule “cholesterine” (chole for bile, stereos for solid) in 1815, later adjusted with the chemical suffix of –ol for the alcohol component [REF Review Olson 1998 1]. Over the past 100 years, cholesterol has been extensively studied and linked to a variety of pathologies and tightly regulated metabolic pathways. Structure of cholesterol consists, in its free form (free cholesterol; FC), of four linked hydrocarbon rings with on one side a hydrocarbon tail, opposing a hydroxyl group [REF]. The two ends create an amphipathic molecule with a hydrophobic and hydrophilic side. This structural phenotype is of great importance in animal cellular membranes formation. The hydrophilic hydroxyl group binds to the phospholipid heads in the cell membrane, turning the hydrophobic hydrocarbon tail towards the core of the membrane bilayer. This structural phenotype increases membrane fluidity and permeability, allowing the cell to change shape [REF bloch 1991 363-381]. The membrane FC/phospholipid ratio is thus essential for membrane rigidity any misbalance could influence cellular mobility and eventually induce cell death [REF Simons 2000 1721-6 2]. Mechanisms that are associated with the accumulation of membrane-bound FC induced cytotoxicity are intracellular cholesterol crystallization, toxic oxysterol formation [REF Björkhem I. 2002 3] and apoptotic signalling pathway activation [REF Tabas I. 1997 & 2002 4,5]. It is therefore that the majority of the cholesterol found in the body exists in its more stable, less cytotoxic, esterified form (cholesteryl esters (CE)) that take up about 2/3 of the serum cholesterol. Lecithin-cholesterol acyltransferase (LCAT) drives the esterification of a FC molecule in plasma, adding a single fatty acid to the hydroxyl group [REF 6 glomset 1968]. The conversion of un-esterified cholesterol towards CE enables cells to store and transport cholesterol, without the risk of FC induced cytotoxicity [REF]. Upon hydrolyzation by cholesteryl ester hydrolase, cholesterol and free fatty acids are regained for further biosynthesis [REF 36 goedeke].
Besides the eminent role in animal cellular membrane modulation, cholesterol influences a range of pathways i.a. as the precursor for hormone steroidogenesis [REF] and bile acids [REF], plays a significant role in transmembrane signalling [REF] and cellular proliferation [REF fernandez 7]. Despite the functional diversity between cholesterol using pathways, acquisition of cholesterol follows, for most mammalian cells, a comparable pattern. Cellular cholesterol is either de novo synthesized or derived from exogenous uptake from the circulation.
3 LIPID METABOLISM
3.1 DE NOVO SYNTHESIS OF CHOLESTEROL
De novo synthesis of cholesterol is mainly found in vertebrates and in low amounts in plants, (not in prokaryotes) [REF Behrman EJ, 2005 8] and derived via the mevalonate (MVA) pathway. The MVA is a fundamental metabolic network providing essential elements for normal cellular metabolism and executed in the endoplasmic reticulum (ER) and cytoplasm of a cell. Despite the presence of MVA pathway in almost all animal cells, the contribution per organ differs. The human brain generates vast amounts of de novo synthesized cholesterol, approximately 20% of the total cholesterol pool and primary FC, mainly found in myelin sheaths that insulate axons [REF dietschy turley 2004 9]. Moreover, the hepatic contribution to the cholesterol pool derived from de novo synthesis varies per species, hepatic cells in mice contribute approximately 40% to the whole cholesterol synthesis, while human liver cells adds only 10% to the total pool [REF Dietschy turley 2001 10 REF 30 Goedeke ].
The MVA-pathway is a highly controlled enzymatic process, resulting in the stepwise formation of FC [REF reviewed by 11 tricarico 2015 16067-16084]. The newly formed cellular cholesterol is either directly used as a precursor for metabolites (bile acids, steroids, water soluble vitamins, included in the membrane) or converted towards CE by acyl-Co A acyl transferase (ACAT) and either effluxed towards the plasma compartment or stored in lipid droplets [REF 12 35 goedeke]. The stored CE within lipid droplets can be converted into FC by hormone sensitive lipase (HSL)[REF]. Since appropriate cellular cholesterol levels are critical for normal cell metabolism, the regulation of intracellular cholesterol levels are tightly controlled by feedback mechanisms that operate at both transcriptional as well as post-transcriptional levels [REF goedeke 10.11]. Low cellular cholesterol triggers the MVA-pathway to upregulate the activation of the rate limiting enzymes i.a. 3-hydroxy-3methylgkutaryl (HMGCR) [REF] and receptor mediated exogenous uptake [REF]. High cellular cholesterol levels activate nuclear hormone receptors that in turn trigger transcription of cholesterol efflux related genes i.a. ABC transporters and inhibit HMGCR expression [REF].
Furthermore, the MVA- pathway is best known as a target for Statins, an extensive prescribed drug that inhibits the rate limiting step; HMGcoA reductase. As a result of the HMGCOA reductase inhibition, cholesterol levels decrease in patients that suffer from hypercholesterolemia.
3.2 EXOGENOUS CHOLESTEROL
The second source for cellular cholesterol is exogenous mediated uptake. Exogenous cholesterol obtained via dietary uptake cover approximately 30% of the total cholesterol pool [REF Kapourchali 2016 13]. Nearly 50% of the total dietary cholesterol is absorbed, the remainder is excreted via feces [REF Clearfield 2003 Crouse 1978; Sudhop 2009 14–16]. Lipid absorption from the intestine is a complex functional collaboration along the whole digestive track; gastric, intestinal, biliary and pancreatic. In short, solubilisation of dietary lipids starts in the duodenum and proximal jejunum parts of the intestine where bile acid micelles hydrolyse CE into FC and fatty acids (FA). Micelles absorb the FC and FA and facilitate transport to the enterocytes of the small intestines were FA is synthesized into triacylglycerol to form triglycerides. Exogenous FC is converted into CE in the ER by ACAT [REF 17]. Due to the hydrophobic character of CE its transport throughout the body is facilitated by lipoproteins.
3.3 LIPOPROTEIN METABOLISM
Lipoproteins are spherical macromolecular particles consisting of a hydrophobic core and a hydrophilic shell. The lipoprotein shell contain a mono layer of phospholipids (PL), amphipathic molecules, FC and apolipoproteins [REF], enfolding the hydrophobic content of CE and triglycerides (TG) [REF]. Five lipoprotein classes are distinguished based on their buoyant density: Chylomicrons (CM), very low-density lipoprotein (VLDL), intermediate low-density lipoprotein (IDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL). The difference in lipid composition of the five lipoprotein classes is depicted in TABLE 1.Cholesterol, the most prominent family member of the sterols, is originally discovered as a major component in human gallstones by F. Pouletier de la Salle in 1769. M.E. Chevreul named the organic molecule “cholesterine” (chole for bile, stereos for solid) in 1815, later adjusted with the chemical suffix of –ol for the alcohol component [REF Review Olson 1998 1]. Over the past 100 years cholesterol has been extensively studied and linked to a variety of pathologies and tightly regulated metabolic pathways. Structure of cholesterol consists, in its free form (free cholesterol; FC), of four linked hydrocarbon rings with on one side a hydrocarbon tail, opposing a hydroxyl group [REF]. The two ends create an amphipathic molecule with a hydrophobic and hydrophilic side. This structural phenotype is of great importance in animal cellular membranes formation. The hydrophilic hydroxyl group binds to the phospholipid heads in the cell membrane, turning the hydrophobic hydrocarbon tail towards the core of the membrane bilayer. This structural phenotype increases membrane fluidity and permeability, allowing the cell to change shape [REF bloch 1991 363-381]. The membrane FC/phospholipid ratio is thus essential for membrane rigidity any misbalance could influence cellular mobility and eventually induce cell death [REF Simons 2000 1721-6 2]. Mechanisms that are associated with the accumulation of membrane bound FC induced cytotoxicity are intracellular cholesterol crystallization, toxic oxysterol formation [REF Björkhem I. 2002 3] and apoptotic signalling pathway activation [REF Tabas I. 1997 & 2002 4,5]. It is therefore that the majority of the cholesterol found in the body exists in its more stable, less cytotoxic, esterified form (cholesteryl esters (CE)) that take up about 2/3 of the serum cholesterol. Lecithin-cholesterol acyltransferase (LCAT) drives the esterification of a FC molecule in plasma, adding a single fatty acid to the hydroxyl group [REF 6 glomset 1968]. The conversion of un-esterified cholesterol towards CE enables cells to store and transport cholesterol, without the risk of FC induced cytotoxicity [REF]. Upon hydrolyzation by cholesteryl ester hydrolase, cholesterol and free fatty acids are regained for further biosynthesis [REF 36 goedeke].
Besides the eminent role in animal cellular membrane modulation, cholesterol influences a range of pathways i.a. as the precursor for hormone steroidogenesis [REF] and bile acids [REF], plays a significant role in transmembrane signalling [REF] and cellular proliferation [REF fernandez 7]. Despite the functional diversity between cholesterol using pathways, acquisition of cholesterol follows, for most mammalian cells, a comparable pattern. Cellular cholesterol is either de novo synthesized or derived from exogenous uptake from the circulation.
3 LIPID METABOLISM
3.1 DE NOVO SYNTHESIS OF CHOLESTEROL
De novo synthesis of cholesterol is mainly found in vertebrates and in low amounts in plants, (not in prokaryotes) [REF Behrman EJ, 2005 8] and derived via the mevalonate (MVA) pathway. The MVA is a fundamental metabolic network providing essential elements for normal cellular metabolism and executed in the endoplasmic reticulum (ER) and cytoplasm of a cell. Despite the presence of MVA pathway in almost all animal cells, the contribution per organ differs. The human brain generates vast amounts of de novo synthesized cholesterol, approximately 20% of the total cholesterol pool and primary FC, mainly found in myelin sheaths that insulate axons [REF dietschy turley 2004 9]. Moreover, the hepatic contribution to the cholesterol pool derived from de novo synthesis varies per species, hepatic cells in mice contribute approximately 40% to the whole cholesterol synthesis, while human liver cells adds only 10% to the total pool [REF Dietschy turley 2001 10 REF 30 Goedeke ].
The MVA-pathway is a highly controlled enzymatic process, resulting in the stepwise formation of FC [REF reviewed by 11 tricarico 2015 16067-16084]. The newly formed cellular cholesterol is either directly used as a precursor for metabolites (bile acids, steroids, water soluble vitamins, included in the membrane) or converted towards CE by acyl-Co A acyl transferase (ACAT) and either effluxed towards the plasma compartment or stored in lipid droplets [REF 12 35 goedeke]. The stored CE within lipid droplets can be converted into FC by hormone sensitive lipase (HSL)[REF]. Since appropriate cellular cholesterol levels are critical for normal cell metabolism, the regulation of intracellular cholesterol levels are tightly controlled by feedback mechanisms that operate at both transcriptional as well as post-transcriptional levels [REF goedeke 10.11]. Low cellular cholesterol triggers the MVA-pathway to upregulate the activation of the rate limiting enzymes i.a. 3-hydroxy-3methylgkutaryl (HMGCR) [REF] and receptor mediated exogenous uptake [REF]. High cellular cholesterol levels activate nuclear hormone receptors that in turn trigger transcription of cholesterol efflux related genes i.a. ABC transporters and inhibit HMGCR expression [REF].
Furthermore, the MVA- pathway is best known as a target for Statins, an extensive prescribed drug that inhibits the rate limiting step; HMGcoA reductase. As a result of the HMGCOA reductase inhibition, cholesterol levels decrease in patients that suffer from hypercholesterolemia.
3.2 EXOGENOUS CHOLESTEROL
The second source for cellular cholesterol is exogenous mediated uptake. Exogenous cholesterol obtained via dietary uptake cover approximately 30% of the total cholesterol pool [REF Kapourchali 2016 13]. Nearly 50% of the total dietary cholesterol is absorbed, the remainder is excreted via feces [REF Clearfield 2003 Crouse 1978; Sudhop 2009 14–16]. Lipid absorption from the intestine is a complex functional collaboration along the whole digestive track; gastric, intestinal, biliary and pancreatic. In short, solubilisation of dietary lipids starts in the duodenum and proximal jejunum parts of the intestine where bile acid micelles hydrolyse CE into FC and fatty acids (FA). Micelles absorb the FC and FA and facilitate transport to the enterocytes of the small intestines were FA is synthesized into triacylglycerol to form triglycerides. Exogenous FC is converted into CE in the ER by ACAT [REF 17]. Due to the hydrophobic character of CE its transport throughout the body is facilitated by lipoproteins.
3.3 LIPOPROTEIN METABOLISM
Lipoproteins are spherical macromolecular particles consisting of a hydrophobic core and a hydrophilic shell. The lipoprotein shell contain a mono layer of phospholipids (PL), amphipathic molecules, FC and apolipoproteins [REF], enfolding the hydrophobic content of CE and triglycerides (TG) [REF]. Five lipoprotein classes are distinguished based on their buoyant density: Chylomicrons (CM), very low-density lipoprotein (VLDL), intermediate low-density lipoprotein (IDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL). The difference in lipid composition of the five lipoprotein classes is depicted in TABLE 1.
Chylomicrons are essential in the transport of exogenous cholesterol from the intestines towards the liver. Within the ER of enterocytes nascent chylomicron particles are formed as a result of lipidation of one APOB48 molecule with cellular CE, TG and phospholipids, alongside apolipoproteins [TABLE 1]. The major apolipoproteins classes are de novo synthesized by intestine and liver [REF] and located in the membrane of lipoproteins. The amphipathic apolipoproteins serve in the membrane as enzymatic cofactors and receptor ligands, regulating lipoprotein metabolism [REF 18]. The function and presence of apolipoproteins differ per lipoprotein class.
Once the chylomicrons enter the circulation via the lymphatic system, circulating APOC’s are acquired. APOC’s in the membrane of CM’s serve as a substrate for lipoprotein lipase (LPL) that is present on the endothelial cells of adipose tissue and skeletal muscle and hydrolyse the TG content for energy storage [REF goldberg 1996 19]. Upon hydrolysis, superfluous membrane phospholipids are transferred by the phospholipid transfer protein (PLTP) towards HDL. PLTP, a plasma glycoprotein and a family member of the lipopolysaccharide (LPS)-binding proteins [REF XC Jiang 1999 20], is involved in the metabolism of both the APOB lipoproteins as well as HDL. Deficiency in PLTP expression results in a marked decrease in plasma levels of APOB containing lipoproteins [REF 21] as well as HDL [REF 20].
In the circulation chylomicrons exchange APOE and APOC’s at the expense of APOA-1 and APOA-IV with HDL, resulting in a smaller TG poor and APO enriched remnant particles [REF patrick]. Furthermore, chylomicron exchanges TG for HDL-CE, achieved via cholesteryl ester transfer protein (CETP), which is present in humans not in mice [REF Ha 1981;Jiao 1990 22,23]. Hepatic clearance of the remaining remnants commences with sequestration in the space of Disse via an APOE dependant route. Synthesized in many tissues but predominately the liver, APOE is a constituent apolipoprotein of CM, VLVL and HDL, an essential for lipid transport between tissues since it binds with a high affinity to the LDLr. The liver subsequently converts the remnant content either into bile acids or reuses the content for VLDL metabolism.
3.3.2 VLDL/LDL
Hepatic metabolism of VLDL is a highly controlled mechanism, facilitating endogenous produced cholesterol transport. Within the ER membrane of hepatocytes a single copy of APOB100 is lipidated with triglycerides and de novo and/or exogenous cholesterol, subsequently supplemented with new synthesized APO E and C’s [REF gibbons 1990;268- 1-13 Spring 1992 ; 267 14839-45 Tiwari S, Siddiqi SA. 2012 May;32(5):1079-86]. The needed VLDL TGs are obtained by the liver either from de novo synthesized fatty acids (FA), a sterol family member derived via the MVA pathway [REF Cornforth 2002 24], extracted from the circulation as nonesterified FAs, or recycled from lipoprotein remnants cleared by hepatic receptors [REF Gibbons 2003 25]. Since hepatic VLDL metabolism is dependent on the availability of TG’s, the de novo synthesized APOB100 undergo degradation when they are not lipidated [REF]. Once the VLDL particles enter the circulation, an interaction with LPL in the endothelial cells reduces the TG content similar to CM degradation. The remaining VLDL remnant is deprived from TGs, intermediate-density lipoproteins (IDL), and is either removed from the circulation through hepatic clearance via the LDLr or converted by LPL and hepatic lipase (HL) into low-density lipoproteins (LDL). The LDL particle maintains the APOB100 molecule and is subjected to LDLr mediated internalization and degradation of the particle.
Subsequently, LDL derived FC is either reused for endogenous lipoprotein metabolism or excreted via the bile.
3.3.3 HDL
HDL biogenesis is a complex interaction of membrane bound and circulating plasma proteins and can be diverted into five major processes [REF Zannis 2004]. (1) Production and secretion of APOA1 by either the liver of intestine [REF Zannis 1985]. Whereas intestinal APOA1 enters the circulation via CM’s and is rapidly transferred towards HDL during hydrolysis [REF]. Hepatic derived APOA1 is the origin of nascent pre-β HDL particles Consequently targeted APOA deficiency in mice result in 83% lowering of the HDL fraction and subsequent phenotypes [REF reviewed by Hoekstra and van Eck 26] (2) Via an ABCA1 dependant pathway, hepatic APOA1 incorporates cellular phospholipids leading to the formation of lipid poor pre-βHDL particles [REF]. (3) Once released in the circulation, lipid poor pre-βHDL particles take up excess amounts of FC from peripheral cells via ABCA1/G1 mediated efflux to form unesterified cholesterol enriched discoidal particles. The pivotal role of ABCA1 in biosynthesis of HDL is demonstrated in ABCA1 deficient patients (Tangier disease) and knockout mice, where the inadequate transport of cholesterol towards the lipoprotein results in the hypercatabolism of lipid poor nascent HDL particles [REF27,28]. (4) Subsequently, esterification of HDL-FC initiated by LCAT in the plasma leads to the maturation into spherical HDL3 particles [REF Zannis 2006]. Next, HDL3 are converted into larger HDL2 particles via a PLTP driven acquisition of phospholipids, along with the attraction of apolipoproteins released upon lipolysis (via HL) of VLDL. (5) Circulating HDL2 is transported back to the liver where scavenger receptor class B type I (SR-BI) mediates selective uptake of FC and CE without internalizing or degradation of the HDL particle [REF 1 25 MvE]. The most important property of SR-BI is considered its ability to act as the HDL receptor [REF 29,30], mediating bidirectional FC flux. In vivo deficiency of SR-BI showed FC accumulation in HDL particles, resulting in an enlarged particle [REF] associated with impaired serum decay and hepatic uptake of [3HCEt]-HDL [REF 31]. The process of extrahepatic uptake of CE and subsequent transport towards the liver is called reverse cholesterol transport (RCT), which is important in lowering accumulation of cholesterol in extrahepatic tissue.
HDL-cholesterol (HDL-C) can be cleared from the circulation via alternative routes. First, HDL particles can be enriched with APOE obtained via either extrahepatic tissue or from the circulation. APOE on HDL enables removal from the circulation via hepatic LDLr or LRP1 whole particle mediated uptake [REF]. Second cholesterol clearing route is, as previously noted, via the ability of HDL2 to transfer CE towards VLDL and LDL through a CETP mediated exchange. Hence, CETP expression is not present in rodents, excluding this pathway in our mice models.
Glucocorticoids (GC) are a class of corticosteroids, a family member of the steroid hormones, and are synthesized within the adrenal cortex (zona fasciculata). Steroidogenesis of GC’s is regulated by dynamic circadian rhythms and upon stress induced hypothalamic-pituitary-adrenal (HPA) axis activation [REF 32]. Furthermore, a range of processes are under the influence of GC’s including stress response and inflammation regulation, combined regulate the “Fight or flight” response and reduce the impact of a stressor induced septic shock [REF]. These characteristics make synthetic produced, as well as natural occurring GC’s, an interesting therapeutic treatment for a variety of inflammatory conditions. The use of GC’s since the 1940 treats symptoms of chronic inflammatory conditions like rheumatoid arthritis [REF Buttgereit 2012 26–29.], asthma, skin infections, ocular infections, multiple sclerosis or as an immunosuppressant for patients following organ transplantation [REF]. It is estimated that at any one time, ~1% of the UK adult population receives an oral GC therapy (REF van staa 2000 105-11].
Like all steroid hormones, synthesis of GC’s requires the ubiquitous substrate, cholesterol. Within the mitochondria of the zona fasciculata of the adrenal a stepwise enzyme controlled pathway leads to the production of GC’s. Most enzymes in the steroidogenesis pathway are either from the cytochrome P450 or hydroxystreoid dehydrogenases (HSDs) family and function unidirectional. Free cholesterol, derived from either de novo synthesis or endogenous receptor mediated uptake, is transported towards the mitochondria and bind to the streroidogenic acute regulatory protein (StAR) that mediates the cholesterol movement into the mitochondria. Here, P450scc, encoded by CYP11A1, initiates the first step in steroidogenesis, conversion of cholesterol into pregnenolone. This rate limiting step is crucial for the final output, as shown in mice, both a decline in substrate as well as inhibition of CYP11A1 results in a lower maximal GC output [REF APOA1, SR-BI KO]. Next, as summarized in Figure XX, the conversion of pregnenolone leads to the formation of the glucocorticoids; cortisol in humans and corticosterone in mice [REF]
Steroidogenesis of GC’s is initiated upon stressor induced activation of the HPA axis, subsequently followed by the release of pituitary gland derived adenocorticotropic hormone (ACTH) [REF]. ACTH binds to the G¬s-coupled melanocortin-2 receptor, present on adrenal cortex cell membrane, resulting in an instant increase of cytoplasmic adenosine monophosphate (cAMP) [REF]. The main function of adrenal cAMP is controlling the expression of CYP11A1 (P450ssc) thereby regulating the steroidogenesis. Beside this crucial role, cAMP is involved in various other pathways associated with the steroidogenesis, including, stimulation of HMGCOA reductase synthesis for de novo cholesterol production, increasing genetic expression of genes involved in the receptor mediated cholesterol uptake route (SR-BI / LDLr), and stimulating expression of HSL and inhibiting expression of ACAT, thereby increasing the availability of FC for the synthesis.
Action of GC’s is transduced via its binding to the GC receptor (GCr), present on the cellular membrane, this initiates translocation of the GCr towards the nucleus where it triggers genomic mechanisms reviewed by Kadmiel et al [REF 33]. Any imbalance in the plasma levels of GC could result in pathological disorders known as respectively, Addison’s disease (low plasma GC) or Cushing’s syndrome (high plasma GC) [REF]
Because GC’s are potent molecules that influence a variety of pathways, sufficient feedback-mechanisms are required. One of with is the direct inhibitory role by GC’s on the expression of corticotropin-releasing factor (CRF) in the brain, supressing the stimulation of ACTH synthesis [REF].
5 ATHEROSCLEROSIS
Despite the importance in mammalian physiology imbalance in the circulating cholesterol levels implicated in many diseases, such as cancer [REF Montero 2008], diabetes mellitus type 2 [REF Cho 2009], and Alzheimer’s disease (AD) [REF arispe 2002 Shobab 2005]. Among the cholesterol associated diseases, cardiovascular diseases (CVD) are the most frequent cause of death in western society [REF ZL37]. The underlying pathology driving CVD is atherosclerosis, an ongoing process of thickening of the vessel wall leading to deprivation of oxygen and nutritions in distally located tissues [REF]. Atherosclerosis is characterized as a chronic inflammatory disease, driven by high cholesterol levels. The development of atherosclerotic lesions starts with the infiltration of LDL particles into the vascular wall driven by physical forces (hypertension), chemical insults (hyperglycemia) or genetic alterations [REF R Ross 1999 115-126]. Within the endothelial wall LDL particles become oxidized through either non-enzymatically or enzymatically pathways [REF]. In addition, it has been proposed that the interaction with endothelial cells, smooth muscle cells or (monocyte-derived) macrophages could drive the oxidation of the LDL particle (oxLDL) [REF Yoshida 2010 1875-1882]. Furthermore, the general hypothesis is that oxidation of LDL particles is not possible in the circulation due to strong anti-oxidant defences present in plasma and on the lipoproteins [REF Yoshida 2010].
Modified LDL particles activate the expression of adhesion molecules (selectins P- and E , intercellular adhesionmolecule-1; ICAM and vascular cell adhesion molecule-1; VCAM) (REF Glass andWitztum, 2001; Mestas and Ley, 2008). Subsequently monocytes react to the inflammatory trigger and migrate into the subendothelial space via diapedesis. Upon entering in the subenodthelial space, monocytes differentiate into macrophages and internalize the modified LDL particles via a scavenger receptor mediated uptake (Scavenger receptor A and CD36) [REF]. To maintain cellular cholesterol homeostasis and avoid cellular cholesterol induced toxicity, transporters ABCA1 and ABCG1 efflux excess cholesterol towards HDL particles via the RCT, as described above [REF]. As soon as cholesterol influx exceeds efflux, macrophages and dendritic cells turn in immobile lipid loaded cells with a “foamy” appearance (foam cells). The formation and accumulation of foam cells in the sub-endothelial space are the hallmark for lesion initiation, called fatty streaks [REF 43 ZL]. Lesion progression is a dynamic process including cell proliferation and migration, as well as cell death and presumably migration of cells [REF ZL 44]. Where the early lesions contain primarily foam cells, the content of more advanced lesions are characterized by a variety of cell types accompanied by a small necrotic core. In more advanced lesions, smooth muscle cell migration is triggered by the inflammatory response from the media into the intima. Here, SMC’s start proliferating and produce a fibrous cap covering the plaque [REF 46 45 ZL]. Over time the plaque advances, narrowing the vessel lumen and blocking the blood flow. Rupturing of the fibrous cap exposes the lesion core to the circulation, resulting in activation of blood coagulation and thrombus formation, which causes most acute coronary syndromes [REF].
5.1 MOUSE MODELS
Development of atherosclerosis is under the influence of a range of environmental as well as genetic modulating factors. Studying atherosclerosis is due to its chronicity and complexity difficult in unregulated human cohorts. The development of homozygous animal models provided a controlled setting that enables studying the mechanisms and processes driving the lesion progression and regression. The most extensively used animal in CVD research is the mouse, chosen for its low maintenance cost, quick reproduction and increased availability of models with an atherosclerotic phenotype [REF]. However, the lipoprotein metabolism of mice differs from man in crucial aspects, influencing the development of atherosclerosis (TABLE 2). The major difference in lipid metabolism between mice vs humans is the lipoprotein profile. In mice the predominant lipoprotein is HDL, whereas humans display a LDL phenotype [REF Miranda]. A possible reason for the lipoprotein difference is the absence of CETP expression in mice [REF 34 Barter 2003 160-7 ]. Second, an important dissimilarity is that the predominant cholesterol transporting lipoprotein in mice is facilitated by VLDL versus LDL in humans. In sum, mice do not develop atherosclerosis without major interference like, genetic modification or diet feeding.
5.1.1 APOE-/- MICE
One of the commonly used models for atherosclerosis is the total body APOE knockout (APOE KO) mouse developed by the group of Maeda in 1992 [REF]. APOE belongs to the class of lipid transporter proteins and is synthesized by many tissues and cell types including liver, brain and macrophages [REF]. The most profound role of the APOE ligand is facilitating binding to the hepatic LDL receptor by TG enriched lipoproteins [REF Mahley & Ji (HOEKSTRA17]. An additional key role for APOE is found in the brain and adrenals, where it facilitates intercellular cholesterol transport [REF]. The targeted mutation of the APOE gene in mice resulted in severe hypercholesterolemia driven by accumulation of APOB containing lipoproteins [REF 35,36]. The most outstanding phenotype of APOE KO mice is its spontaneous development of lesions upon feeding non-cholesterol containing diet. Early stage lesion formation is measured in young mice around 8 weeks of age, with a strong progressive development of the lesion between 12 and 38 weeks [REF t’hoen]. Feeding APOE -/- mice a high-fat, high-cholesterol diet increases plasma cholesterol levels and accelerates the lesion development [REF ZL 61 T35 39].
5.1.2 LDLr -/- MICE
A second common used model for atherosclerotic lesion formation is the low density lipoprotein receptor (LDLr) total body knockout mouse. The LDLr is a cell surface receptor expressed primarily on mammalian hepatic cells that binds and internalizes lipoproteins carrying APOE, such as LDL, thereby regulating the plasma cholesterol levels [REF Ishibashi 1993 TH].
The association between elevated LDL-C level and an increased CVD occurrence is reflected in patients with familial hypercholesterolemia, an autosomal disorder caused by mutations in the LDLr gene [REF]. Mice with a homozygous deficiency of the LDLr gene (LDLr-/-), display increased plasma cholesterol levels by 2 to 3-fold, however lack the spontaneous development of lesions. Feeding LDLr-/- mice either a high cholesterol diet (1% cholesterol 4.4% fat) or a western type diet (0.06% cholesterol and 21% fat) subsequently increases the plasma cholesterol levels by 8 – 16-fold. The total cholesterol increase is primarily caused by sharp augmented levels of the LDL-C fraction, which in turn induces lesion development over a period of XX weeks [REF].
5.1.3 SR-BI -/- MICE
As noted above, the integral membrane glycoprotein; SR-BI, is a key player in the metabolism of the anti-atherogenic HDL particles [REF Rigotti 12610-12615]. SR-BI mediates bidirectional flux of FC, CE and PL between APOB containing lipoproteins and cells. Expression of SCARB1, the gene encoding for SR-BI, is primarily found in the liver, steroidogenic tissues and endothelial cells as well as numerous other organs [REF acton 1996]. Patients with mutations in the gene encoding for SR-BI have elevated levels of HDL [REF vergeer 2011 en de rest van het rijtje in paper]. To study the role of SR-BI on cholesterol metabolism and in particularly HDL and the RCT route in more detail, SR-BI-/- mice were developed [REF Krieger]. These total body SR-BI-/- mice showed a range of cholesterol uptake related pathologies including, reticulocytosis [REF 37, 38], reduced platelet counts [REF Kaplan 2010, Korporaal 2010, 38,39 Ouweneel unpublished?], increased serum oxidative stress levels [REF 40], reduced hepatic expression of ABCA1 and APOA1 [REF], as well as reduced maximal output of adrenal derived glucocorticoids [REF]. In addition, the profound phenotype of SR-BI knockout mice is the enlarged and increased levels of HDL particles. Paradoxically, the augmented amount of the ant-atherogenic HDL particles did not improve its anti-atherogenic properties, but resulted in an increased susceptibility to atherosclerosis development upon western type diet feeding (0.25% cholesterol and 15% fat) [REF van eck 2003 23699]. We hypothesized that this controversy is driven by the loss of functionality of the HDL particles, however further research on this topic is needed.
5.1.4 LESION REGRESSION MODELS
Over the years inhibition of lesion formation and progression has been extensively studied in a variety of animal models. The concept that the existing atherosclerotic lesion was capable to regress, dates back ~60 years [REF friedman 1957 586-588]. Since then several rodent and non-rodent models are developed to prove this concept. Many rodent models are based on the APOE -/- or LDLr-/- progression models (reviewed by J.E. Feig [REF 2014 13-23]). Increasing the efficacy of the anti-atherogenic RCT route by additional introduction of APOA1 has been one of the early successful models resulting in substantial lesion shrinkage and variations on this theme were successful [REF Tangirala et al., 1999; (Belalcazar et al., 2003 (Shah et al., 2001 (Tian et al., 2015; Wang et al., 2016 ). Including the reintroduction of human APOA1 in LDLr-/- mice raising the HDL levels and significantly regressed foam cell rich lesions [REF Tangirala 1999 1816-22]. The potential role of HDL in lesion regression was underlined by the infusion of APOA1/Milano/PC complex. Increasing the HDL levels in APOE-/- mice reduced the foam cell content of existing plaques within 48 hours.
In addition to increasing the HDL levels, murine models that allow lesion progression followed by a lowering of the APOB fraction, display lesion regression. Lowering of the APOB fraction can be initiated via reintroduction of bone marrow derived APOE in APOE knockout mice resulting in a normalized plasma levels [REF 41vd Stoep 2013 1594-602]. A more invasive model is the aortic arch transplantation where a lesion enriched segment of a hyperlipidemic APOE-/- aortic arch is transplanted into a normo-lipidemic recipient [REF Reis 2001]. Subsequent regression was achieved with both early and advanced lesions within 3 days or 9 weeks post transplantation [REF Llodra 2004 Trogan 2006, Trogan 2004].
Recently the Reversa mouse (LDLr-/-APOB100/100MTTPfl/flMX1Cre-/- -/-) was developed. A LDLr knockout mouse with hyperlipidemia, driven by APOB100 only containing lipoproteins. In this model the hyperlipidemia can be reversed by the expression of the MX1-Cre transgene that inactivates the gene encoding for MTTP [REF Feig 2011, Lieu 2003] resulting in the reduction of the plasma NON-HDL fraction. Lowering of the NON-HDL fraction in the presence of existing lesions resulted in the Reversa mouse reduction of the lipid content, increased presence of collagen accompanied by in an egression of CD68 positive macrophages from the plaque.
Most recently a less technically demanding or time consuming model has been developed using an antisense oligonucleotide (ASO) that is targeted to the LDLr mRNA, inducing hypercholesterolemia and subsequent lesion formation in C57BL/6 mice. By using sense oligonucleotides (SO’s) targeted to the LDLr the hypercholesterolemia is reversible, inducing lesion regresion [REF Basu 2018 560-567].
Although the mainstay for regression is lowering cholesterol levels, Ross et al [REF Ross 1999] described atherosclerosis as a chronic inflammatory disease, since immune cells and its response promote lesion progression in every stage. Modulation of immune cells and pathways to provoke lesion regression is a relative new therapeutic strategy that could be the novel focus for future research [REF foks 2013 4573-80].
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