Cytochrome P450 — physiological key factor against cholesterol accumulation and the atherosclerotic vascular process

Abstract

In the early 1960s liver cytochrome P450 (P450) was known as an enzyme in drug metabolism. By the late 1970s, P450 induction was associated with elevation of plasma high‐density lipoprotein cholesterol and apolipoprotein AI indicating a reduced risk of atherosclerotic disease. Later on, 57 human P450 genes have been identified. One P450 enzyme participates in cholesterol synthesis, and several others catabolize it to oxysterols and other metabolites. Oxysterols are physiological ligands specific for liver X receptors (LXRs) in the activation of ATP‐binding cassette (ABC) transporter and other cholesterol‐lowering genes. Elevation of cholesterol leads to an endogenous induction of P450 and consequently to enhanced generation of oxysterols and activation of genes coding proteins which efflux cholesterol out of cells, transport it to the liver, catabolize and excrete cholesterol into bile, and prevent absorption of cholesterol in the intestine in the processes that maintain cellular cholesterol homeostasis and protect arteries from atherosclerosis. Peroxisome proliferator‐activated receptors (PPARs) co‐operate with LXRs and ABC transporters in cholesterol regulation. Secretion of oxysterol is a direct pathway for cellular cholesterol elimination. Several compounds induce P450 and other genes regulating cholesterol balance and prevent or regress atherosclerosis, whereas inhibition of P450 blocks oxidative reactions, promotes cholesterol accumulation, and enhances the atherosclerotic vascular process.

• ABC transporter
• apolipoprotein AI
• CYP
• cytochrome P450
• gene activation
• HDL
• induction
• LXR
• oxysterol
• PPAR

Abbreviations
ABC	=	ATP‐binding cassette
apo	=	apolipoprotein
ATP	=	adenosine 5′‐triphosphate
cAMP	=	cyclic adenosine monophosphate
CAR	=	constitutive androstane receptor
CYP	=	cytochrome
FXR	=	farnesoid X receptor
HDL	=	high‐density lipoprotein
HMG‐CoA	=	hydroxymethyl‐glutaryl coenzyme A
LDL	=	low‐density lipoprotein
LXR	=	liver X receptor
OSBP	=	oxysterol‐binding protein
ORP	=	OSBP‐related protein
P450	=	cytochrome P450
PPAR	=	peroxisome proliferator‐activated receptor
RXR	=	retinoid X receptor
SR‐B1	=	scavenger receptor B1
SREBP	=	sterol regulatory element binding protein
TZD	=	thiazolidinedione
VDR	=	vitamin D3 receptor

Introduction

Despite enormous research efforts and advances in treatment, atherosclerosis remains the leading cause of death in the industrialized world. Lipoproteins are intimately associated with the development of atherosclerosis. Persons with high plasma high‐density lipoprotein (HDL) cholesterol or apolipoprotein AI (apo AI), have a reduced risk of coronary heart disease (CHD) and prolonged life expectancy, whereas those with high low‐density lipoprotein (LDL) cholesterol or apo B have an increased risk of CHD and death.

Cytochrome P450 (P450, CYP), a cellular chromophore, was first named in 1961 because in spectral analysis the pigment had a peak at 450 nm 1,2. At that time it was thought to be one enzyme, by the mid 1960s it was connected with drug and steroid metabolism, and by the late 1970s with apo AI and HDL 3,4. Our original observations showed that apo AI and HDL cholesterol levels in plasma vary proportionately to microsomal P450 concentrations in the liver 3,5. They also revealed that the increase of apo AI and HDL cholesterol in persons treated with drugs inducing protein synthesis parallels the extent of P450 induction, and put forward the hypothesis that P450 and the induction phenomenon have beneficial exploitable effects against atherosclerosis 3,5. The decrease in plasma LDL cholesterol in proportion to the induction supported this possibility 6,7.

Improved analytical methods in 1980s and thereafter have revealed 57 P450 genes which code enzymes catalysing numerous reactions in important life processes in man 2. One P450 enzyme participates in cholesterol synthesis, and several others catabolize cholesterol to oxysterols and other metabolites. Studies in the mid 1990s identify oxysterols as signalling molecules which, through the activation of nuclear receptors, induce the transcription of genes that regulate cholesterol balance in the body 8,9 and as metabolites for cholesterol elimination via direct secretion 10–12. Several compounds induce P450 and other genes coding proteins in lipid metabolism including apo AI, the major protein constituent of HDL important in cholesterol transport. This review focuses on P450 enzymes and gene activation together with nuclear receptors and transport proteins in maintaining cholesterol homeostasis and protecting arteries from atherosclerosis.

Key messages

• Cytochrome P450 enzymes are essential in the synthesis, metabolism, and elimination of cholesterol and affect the development of atherosclerosis in many ways.

• Elevated cholesterol upregulates P450 that turns on mechanisms eliminating surplus cholesterol in the process of maintaining cholesterol homeostasis and atheroprotection.

• Several compounds induce P450 and other genes that regulate cholesterol balance and prevent or regress atherosclerosis, whereas inhibition of P450 suppresses oxidative reactions and promotes atherogenesis.

P450, lipids, and apolipoproteins—earlier studies

Apo AI and HDL pick up cholesterol that cells transfer to their outer membranes and shuttle it to the liver for biliary excretion and disposal 13–15. The original studies in the 1970s connecting P450, apo AI, and HDL led to further investigations clarifying the effects of P450 and gene activation on lipid and protein metabolism (reviewed in 16). The studies linked plasma apo AI and HDL cholesterol with liver protein and phospholipid concentrations and P450 induction 17, and subsequently the P450 inducers were found to enhance 18–23 and P450 inhibitors 22,23 to prevent apo AI synthesis. Studies in the 1990s on transgenic animals further emphasized the significance of apo AI in atheroprotection by showing that overexpression of human apo AI gene raises HDL cholesterol, enhances reverse cholesterol transport and inhibits atherogenesis 24, and protects from diet‐induced atherosclerosis 25. In humans, an infusion of recombinant proapolipoprotein AI was found to promote faecal cholesterol excretion, implying a stimulation of reverse cholesterol transport 26.

P450 enzymes, oxysterols, and bile acids

Cholesterol is essential for normal cell function, but when accumulated in excess, it has deleterious effects. This is particularly true for the cells of the arterial wall during the process of atherosclerosis. Highly sophisticated regulatory systems take care to eliminate excess cholesterol out of the cells. P450 enzymes have a physiological key role in this process in catabolizing cholesterol to oxysterols and other metabolites and turning on the cholesterol‐lowering mechanisms 8–12,27 (Tables I and II).

Table I. Cytochrome P450 enzymes in the synthesis and metabolism of major oxysterols, bile acids and cholesterol.

CYP7A1	cholesterol hydroxylase: 7α‐hydroxycholesterol synthesis
CYP8B1	12α‐hydroxylase of 7α‐hydroxycholesterol: bile acid synthesis
CYP27A1	cholesterol hydroxylase: 27‐hydroxycholesterol synthesis
CYP7B1	7α‐hydroxylase of 27‐hydroxycholesterol: bile acid synthesis
CYP46A1	cholesterol hydroxylase: 24S‐hydroxycholesterol synthesis
CYP39A1	7α‐hydroxylase of 24S‐hydroxycholesterol: bile acid synthesis
CYP3A4	cholesterol hydroxylase: 4β‐hydroxycholesterol synthesis, bile acid synthesis, detoxification of bile acids
CYP51A1	lanosterol 14α‐demethylase: cholesterol synthesis

Table II. Effects of P450‐inducing and ‐inhibiting compounds on plasma HDL cholesterol (HDL‐C) and apo AI levels, apo AI synthesis (‐S), ABCA1 and/or ABCG1 activity (‐A), development of atherosclerosis^a and morbidity/mortality ^b.

	HDL‐C	Apo AI	Apo AI‐S	ABCA1/ABCG1‐A	Atherosclerosis	Morbidity/mortality
P450‐inducers:
Fibrate	↑	↑	↑	↑	↓	↓
Statin	↑	↑	↑	↑	↓	↓
Cholestyramine	↑	↑	↑		↓	↓
TZD	↑	↑	↑	↑	↓	↓
Anticonvulsant	↑	↑	↑		↓
Alcohol	↑	↑	↑	↑	↓	↓
P450 inhibitors:
Carbon monoxide	↓^c	↓^c			↑	↑
Ketoconazole	↓	↓	↓	↓
Interferon‐γ	↓	↓	↓	↓	↑	↑

^a Biochemical or histological study: promotion 109,113,115; inhibition 125,126. Clinical angiographic or ultrasonographic study: retardation of progression or prevention/regression by P450 inducers 118–124,127,128.

^b Morbidity and/or mortality from CHD and/or other cardiovascular disease, and/or all‐cause mortality 112,130–138.

^c Decrease with exposure to CO 110 or environmental tobacco smoke 111.

Oxysterols are biologically active signalling molecules which as specific ligand activators for liver X receptors (LXRs) induce transcription of genes regulating cholesterol homeostasis in the body, i.e. its efflux, transport, excretion, and absorption 8,9,28. The conversion of cholesterol to oxysterol is necessary for transcriptional activity; neither free cholesterol nor cholesterol esters have been identified as ligands for LXRs 29. Most oxysterols are produced through the actions of P450 enzymes that are particularly prevalent in the liver 30. Major oxysterols present in circulation, such as 7α‐hydroxycholesterol, 27‐hydroxycholesterol, 24S‐hydroxycholesterol, and 4β‐hydroxycholesterol, are generated by CYP7A1, CYP27A1, CYP46A1, and CYP3A4 enzymes, respectively 31. P450 enzymes metabolize oxysterols further to bile acids via two principal pathways: the classic or neutral pathway, and alternative or acidic pathway 30.

CYP7A1, cholesterol 7α‐hydroxylase, is the first and rate‐limiting hepatic enzyme in the neutral, quantitatively most significant pathway for bile acid synthesis and plays a major role in maintaining cholesterol balance. 7α‐Hydroxycholesterol is an intermediate in the synthesis of primary bile acids, cholic acid, and chenodeoxycholic. CYP8B1, sterol 12α‐hydroxylase, is essential for cholate synthesis 2.

CYP27A1, mitochondrial sterol 27‐hydroxylase, is found in almost all tissues and cell types including macrophages. 27‐Hydroxycholesterol is a ligand for liver X receptor (LXR) in the activation of genes coding ATP‐binding cassette (ABC) transporters which efflux cholesterol from macrophages and other cells 32, and it contributes to cholesterol elimination via direct secretion 12. The enzyme could also independently of ABC transporters enhance the efflux of non‐oxidized cholesterol to extracellular acceptors 33. 27‐Hydroxycholesterol is further hydroxylated by microsomal oxysterol 7α‐hydroxylase, CYP7B1, and CYP7A1 34. This pathway produces mainly chenodeoxycholic acid.

CYP46A1, a P450 enzyme in neuronal cells of central nervous system, plays an active role in cholesterol metabolism in the brain 35,36. The enzyme generates 24S‐hydroxycholesterol which can readily pass the blood brain barrier, and the efflux into the circulation represents a pathway of cholesterol turnover in the brain and a part of reverse cholesterol transport 37. Hepatic CYP39A, another oxysterol 7α‐hydroxylase, converts it to bile acid 38. 24S‐hydroxycholesterol also is a potent activator of LXR 9, and could hence through transcriptional mechanisms regulate cholesterol balance in brain cells 39,40. It has also been found to increase ABCA1 expression and apo AI‐dependent cholesterol efflux from cultured brain endothelial cells 41. Elevated plasma 24S‐hydroxycholesterol levels have been measured in early stages of Alzheimer's disease, possibly reflecting on‐going demyelinization 42, and, in contrast, reduced levels in the advanced Alzheimer's disease, probably reflecting the loss of neuronal cells containing CYP46A1 35.

CYP3A4, the predominant hepatic P450 enzyme, which is responsible for biotransformation of approximately half of commercially available drugs, also influences cholesterol and bile acid metabolism. It catabolizes cholesterol to 4β‐hydroxycholesterol, a major oxysterol found in circulation, particularly in persons treated with P450 inducers such as phenobarbital, phenytoin, or carbamazepine 43.

CYP51A1 gene, which is ubiquitously expressed, codes lanosterol‐14α‐demethylase, the only P450 enzyme in the biosynthesis of cholesterol. It demethylates lanosterol to cholesterol in a reaction that produces oxysterols inhibiting hydroxymethyl‐glutaryl coenzyme A (HMG‐CoA) reductase and sterol synthesis 44. One major intermediate in cholesterol synthesis, squalene, is in a shunt pathway metabolized to 24S,25‐epoxycholesterol, an efficient activator of LXR that may act as a signalling molecule in the feedback regulation of cholesterol synthesis 9.

Oxysterol binding protein (OSBP) and related proteins form a cytosolic family of OSBP‐related protein (ORPs) including 12 members 45,46. Several ORPs bind intracellular oxysterols which are mainly generated by P450 enzymes. Recent studies with yeast have identified ORPs as non‐vesicular transport proteins moving oxysterols among intracellular compartments 47, but it is still unclear whether this could apply to mammals. It is believed that ORPs act as sterol sensors co‐ordinating cellular lipid metabolism and membrane trafficking 46.

Oxysterols, LXRs, PPARs, and ABC transporters

LXRs, LXRα and LXRβ, are nuclear receptors which are activated by physiological concentrations of several oxysterols 28,48. Oxysterols are the natural ligands for LXRs to induce multiple genes in the regulation of cholesterol balance 8,9,32. LXR upregulates ABC transporters such as ABCA1, ABCG1, ABCG4, ABCG5 and ABCG8 acting in intracellular cholesterol trafficking.

ABCA1 has been identified as the key protein responsible for cellular lipid efflux 49–52. It transfers cholesterol and phospholipid across cellular membranes, where they are removed by apo AI 13. Mice deficient in LXRs accumulate foam cells in multiple peripheral tissues 53,54, and upregulation of ABCA1 results in protection against atherosclerosis 55. The significance of ABCA1 is demonstrated in Tangier disease which is characterized by a defective ABCA1 gene and lack of cholesterol and phospholipid efflux, accumulation of cholesterol esters in tissues enriched with macrophages, and premature onset of atherosclerosis 49–52. Liver ABCA1 has a major role in maintaining circulating HDL cholesterol levels 56,57, and intestinal ABCA1 contributes to it 58. Intestinal ABCA1 also regulates the absorption of dietary cholesterol by directing it from the enterocytes to intestinal lumen 59.

ABCG1, which is expressed in numerous tissues, and ABCG4 expressed in the brain, are transporters which have been found to efflux cholesterol to HDL2 and HDL3 14,15. ABCG5 and ABCG8 are transporters required for biliary and intestinal excretion of cholesterol 60, and in the case of impaired biliary excretion enterocytes can compensate 61. Mutations in the genes encoding ABCG5 and ABCG8 cause sitosterolaemia, a rare recessive disorder of sterol metabolism, that is characterized by hypercholesterolaemia accompanied by xanthomas and premature coronary atherosclerosis 62.

The PPARs, PPARα, PPARγ, and PPARδ, are ligand‐activated transcription factors that affect lipid metabolism and the development of atherosclerosis in many ways. They mediate induction of apo AI synthesis and co‐operate with LXR and ABC transporters to efflux cholesterol from macrophages to extracellular acceptors 48,63,64.

All members of the apolipoprotein E, CI, CII, CIV gene cluster respond to LXR activation and have been shown to serve in the ABCA1‐mediated cholesterol efflux 28,65. Apo AIV, synthesized in the intestine and to a lesser extent in the liver, is also a direct target gene of LXRs and may contribute to the antiatherogenic effects of LXR activation 66,67. In addition, LXR increases the expression of lipoprotein demodelling enzymes such as cholesterol ester transfer protein, phospholipid transfer protein and lipoprotein lipase 28, and SREBPs (sterol regulatory element binding proteins) which mediate the synthesis of cholesterol, fatty acids, and triglycerides 68,69, and liver scavenger receptor B1 (SR‐B1), that mediates the selective uptake of HDL associated cholesterol esters to liver 70. SREBP‐1c enhances preferentially transcription of genes in fatty acid synthesis and may cause hypertriglyceridaemia, a significant adverse effect of LXR agonists. SR‐B1 is also expressed by macrophages and may, in addition to ABC transporters, contribute to cholesterol efflux 71.

P450 enzymes, bile acids, and nuclear receptors

The liver has an essential function to eliminate cholesterol from the body, owing to its ability to synthesize bile acids and transport cholesterol into bile. Bile acids generated by P450 enzymes activate nuclear receptors in the regulation of bile acid balance, such as farnesoid X receptor (FXR), pregnane X receptor (PXR), vitamin D3 receptor (VDR) and constitutive androstane receptor (CAR). This chapter is a short summary of P450 enzymes and bile acid regulation; for further details, see recent reviews 72,73.

FXR has a key role in the regulation of bile acid levels 72. It is expressed particularly in the liver, and also intestine, kidneys, and adrenals, and forms a heterodimer with retinoid X receptor (RXR). Accumulation of bile acids stimulates the FXR‐mediated suppression of CYP7A1and CYP8B1, key enzymes in bile acid synthesis. FXR has been found to inhibit the expression of CYP7A1 also by inducing intestinal fibroblast growth factor 19 (FGF‐19) 72,73. FXR also induces expression of CYP3A4 that reduces the toxicity of bile acids by oxidizing them 73, bile acid transporters ABCB11 and ABCC2, and intestinal bile acid binding protein, IBABP, a mediator in the enterohepatic circulation of bile acids 72,73.

PXR and VDR, which form heterodimers with RXR, are sensors of secondary bile acids and affect their metabolism in the liver and intestine 72,73. They mediate the expression of CYP3A4 gene in the metabolism of toxic bile acids into less toxic derivatives 72,74. PXR can also downregulate CYP7A1 in response to increased levels of intracellular bile acids 72. CAR which is known for its role in xenobiotic metabolism also ameliorates the effects of hyperbilirubinaemia and toxic bile acids 75.

Induction of genes in cholesterol metabolism by drugs and other compounds

Several drugs and other compounds affecting lipid and protein metabolism are P450‐inducers. They include drugs indicated for dyslipidaemias, e.g. fibrates such as gemfibrozil, fenofibrate, and bezafibrate 16,76,77, statins such as simvastatin, atorvastatin, fluvastatin, lovastatin 78,79, and pitavastatin 80, and cholestyramine 16, antidiabetic thiazolidinediones (TZDs), pioglitazone, and rosiglitazone 81, anticonvulsants such as phenobarbital, phenytoin, and carbamazepine 16, steroid hormones dexamethasone and prednisone 16, alcohol 23,82,83, adenosine 2A receptor agonists 84, retinoids such as 9‐cis‐retinoid acid, and all‐trans retinoic acid 85.

Fibrates are PPARα activators which enhance apo AI synthesis, raise plasma HDL cholesterol and apo AI, and reduce LDL cholesterol and triglyceride levels 20–22,86. They induce LXRα expression, upregulate LXRα‐dependent ABCA1 gene, promote cholesterol efflux to apo AI 63,64,87, and inhibit cholesterol absorption in the intestine 88. Ketoconazole, a specific inhibitor of P450, prevents fibrate‐caused P450 induction and suppresses oxysterol generation and the activation of PPARα, LXRα, ABCA1 89, and apo A1 22 genes.

Statins reduce plasma LDL cholesterol and raise HDL cholesterol and apo AI. Simvastatin and atorvastatin have been found to upregulate ABCA1 and ABG1 genes in cholesterol‐loaded macrophages and to promote cholesterol efflux to apo AI and HDL3 90, and pitavastatin to upregulate ABCA1 91 and apo AI 92 genes in HepG2 cells. Several statins are ligands for PXR and CAR receptors which, via induction of P450 enzymes 78,79,93, enhance oxysterol generation. The promoting effect of statins on cholesterol elimination has been linked with oxysterol generation, inhibition of HMG‐CoA reductase and induction of PPARα 92, PPARγ 94, LXRα 91, apo AI 80,92, ABCA1, and ABCG1 genes.

Cholestyramine is a CYP7A1 inducer which in addition to reducing LDL cholesterol stimulates apo AI synthesis and raises plasma HDL cholesterol 95 and apo AI 96.

TZDs are P450 inducers and PPARγ ligands 97,98 that increase apo AI synthesis and plasma HDL cholesterol 99, and through the activation of LXR, ABCA1, ABCG1, apo E, and SR‐B1 genes promote cholesterol efflux from macrophages 48,100,101. A PPARγ agonist may also via upregulation of CYP27A1 contribute to cholesterol removal from macrophages 102,103.

Anticonvulsants such as phenobarbital, phenytoin, and carbamazepine induce expression of P450 enzymes including CYP3A4, which is a target gene for nuclear receptors such as PXR 104, CAR 105, and VDR 106. Persons treated with these drugs show elevation of plasma HDL cholesterol and apo AI in proportion to P450 induction 3,17, and increased generation of oxysterols, particularly 4β‐hydroxycholesterol 43. This oxysterol activates LXR 8 and could hence affect cholesterol balance through transcriptional regulation. The inverse relation of LDL cholesterol to the extent of P450 induction 6 could reflect increased oxysterol generation and consequent downregulation of HMG‐CoA reductase, and upregulation of the LDL receptor gene.

Alcohol is a P450 inducer 23,82, which enhances apo AI synthesis 19,23, raises HDL cholesterol and apo AI in proportion to P450 induction 83, and promotes cyclic adenosine monophosphate (cAMP)‐stimulated and particularly ABCA1‐dependent cholesterol efflux from macrophages 107.

Adenosine_A2 receptor agonists have been found to upregulate 27‐hydroxylase and ABCA1 expression and thus have potential to enhance cholesterol elimination 84.

Vitamin A derivatives, retinoids exert their biological effects as ligands for RXR. They heterodimerize with many other nuclear receptors and upregulate genes involved in reverse cholesterol transport, including CYP27A1, LXRα, PPARγ, ABCA1, ABCG1, and apolipoproteins CI, CII, CIV, and E 102,108. Significant adverse effects, including hypertriglyceridaemia, have limited the clinical usage of retinoids.

P450 enzymes, atherosclerosis, morbidity, and mortality

Both endogenous and exogenous P450 induction protects arteries from atherosclerosis and P450 inhibition has an opposite effect. Studies in the 1960s and 1970s demonstrated that carbon monoxide (CO) inactivates P450 by binding to its ferrous haem, blocks oxidative reactions 1, promotes cholesterol accumulation, and enhances the atherosclerotic process 109. More recent studies show that an exposure to CO 110 or environmental tobacco smoke 111 reduces HDL cholesterol and apo AI, and that cardiovascular events and deaths increase with the degree of CO exposure 112. Ketoconazole, another inhibitor of P450, suppresses oxysterol generation and the induction of LXRα, PPARα, ABCA1 89, and apo AI 22 genes. Interferon‐γ reduces the activity of CYP27A1 113 and other P450 enzymes 114, and ABCA1 115; it also impedes reverse cholesterol transport and promotes atherogenesis 113,115.

A genetic defect in P450 may also enhance the atherosclerotic process. Mutation in CYP27A1 gene causes a sterol storage disease, cerebrotendinous xanthomatosis (CTX), which is characterized by xanthomas in tendons and brain and premature atherosclerosis 116, and a defect in CYP7A1 gene could lead to hepatic cholesterol accumulation and hypercholesterolaemia, and enhance atherogenesis 117. A change in P450 activity and oxysterol generation also affects the LXR‐mediated induction of several proteins which participate in cholesterol transport, such as ABC transporters, apolipoproteins and lipoprotein‐modifying enzymes, and SREBPs.

Several P450‐inducing drugs and other compounds retard the progression or even regress coronary atherosclerosis (Table II). Positive angiographic results have been obtained with gemfibrozil 118, bezafibrate 119, fenofibrate 120, and with statins such as simvastatin 121 and lovastatin 122, and cholestyramine 123. Recently, also pioglitazone was found to reduce carotid intima‐media thickness as assessed by ultrasonography in type 2 diabetic subjects 124. Phenobarbital inhibits cholesterol accumulation in the arterial wall and the formation of atherosclerotic plaque 125,126, and moderate alcohol consumption associates with less carotid 127 and coronary atherosclerosis 128.

Several large‐scale trials have evaluated the effects of fibrates, statins, and other gene‐activating compounds on cardiovascular morbidity and mortality. Recent follow‐up data from the Helsinki Heart Study 129 revealed that gemfibrozil reduces CHD mortality and also all‐cause mortality in the subgroup of obese persons with high triglycerides 130. Bezafibrate has been found to reduce the incidence of coronary events 119, and fenofibrate that of non‐fatal myocardial infarctions 131. Statins also reduce coronary events, strokes, and in addition all‐cause mortality 132–135. A recent meta‐analysis including 14 randomized trials of statins showed that the decrease in total mortality reflects a reduction in coronary mortality 135. Cholestyramine reduces the risk of definite CHD death and/or non‐fatal myocardial infarction 136, and pioglitazone that of the composite of all‐cause mortality, non‐fatal myocardial infarction, and stroke in type 2 diabetics who have a high risk of macrovascular events 137. Moderate alcohol consumption decreases CHD morbidity and mortality and also total mortality, and increases the lifespan 138.

Discussion and conclusions

Studies in the past three decades have identified P450 enzymes as active participants in the control of cellular homeostasis, instead of the original belief of being one enzyme in the drug detoxication system. They respond to both endogenous and exogenous signals and generate new signalling molecules acting in important metabolic processes. Our studies originating from the 1970s evaluated the effects of P450 and gene activation on the fate of lipids and proteins in the atherosclerotic vascular process. Beneficial changes in lipid and apolipoprotein levels with P450 induction were seen in different patient groups, including for example diabetic 139, non‐diabetic, and epileptic subjects 3,16, and persons using alcohol regularly 83. Also healthy, sober subjects without any drug therapy show high plasma HDL cholesterol and HDL2 cholesterol together with high microsomal P450 activity in the liver 140, which was assessed by determining plasma elimination rate of orally taken antipyrine 7. Clinicopharmacological studies associate high P450 level and high hydroxylase activity in the liver 141, an exposure to two or three P450 inducers results in greater increase in P450 and HDL cholesterol 3,16 than the use of one inducer only, and HDL cholesterol levels are related to plasma concentrations of P450‐inducing agents 142. Persons undergoing P450‐inducing drug therapy show reduced LDL cholesterol and elevated HDL cholesterol levels together with a rapid antipyrine elimination rate 7. These investigations presented a novel mechanism to prevent and treat atherosclerotic disease, i.e. induction of P450 and other genes coding proteins that regulate the cholesterol balance and protect arteries from atherosclerosis 3,7,16,17, which has then been associated with interesting findings in different population 82,143,144, clinical 19,82,145,146 and experimental studies 18,23,147. The studies now reviewed identify P450s as enzymes which are upregulated by elevated cholesterol and gene‐activating compounds and which via cholesterol hydroxylation turn on mechanisms that eliminate excess cholesterol and counteract the atherosclerotic process, and as enzymes the inhibition of which suppresses oxidative reactions and enhances the development of atherosclerosis.

Important investigations in the 1990s discovering LXR and identifying oxysterols as ligands for it 8,9, and as metabolites for a direct removal of intracellular cholesterol 12, uncovered mechanisms for cholesterol elimination. Identification of the endogenous ligands and physiological functions for PPARs 48,63, FXR, and several other nuclear receptors 148 and ABC transporters 28 further clarified processes controlling cholesterol homeostasis. The liver is the principal site for the synthesis and elimination of lipoproteins circulating in plasma. The effect of liver structure and function on hepatic P450 and other enzymes, lipids and proteins has been evaluated in investigations including patients with different drug regimens and liver diseases, such as fatty liver, cirrhosis 3,5, and polycystic liver 149. Studies clarifying the effect of liver size on lipids and proteins associate P450 induction with a favourable change in plasma HDL cholesterol level, and the HDL/LDL profile proportionately to the increase in metabolically active liver mass as assessed by relative liver weight (liver weight/body weight) 150. The effect of liver size has been found important also in the evaluation of circulating 24S‐hydroxycholesterol levels that reflect the balance between cerebral production and hepatic metabolism of the oxysterol 151. A recent study showing that increased hepatic LXRα expression protects from atherosclerosis in susceptible mice which were fed a Western diet demonstrated that LXRα activation in the liver also has beneficial effects 152. The benefit of the LXRα activation was lost upon treatment with synthetic LXR agonist raising triglycerides due to activation of SREBP‐1c pathway. The study shows an important difference between endogenous oxysterols and pharmacological LXR agonists that promote lipogenesis and also emphasizes the need of new agonists without such effects.

The ability of different compounds to activate key proteins controlling cholesterol homeostasis and to attenuate the development of atherosclerosis has led to intensive search for new agents to prevent and treat atherosclerotic disease. Studies on LXR agonists in mouse models concluded that activation of LXR inhibits the development of atherosclerotic lesions, probably via direct actions of the LXR ligands on vascular gene expression 153–155. A recent study also showed that macrophage LXR expression is necessary for the antiatherogenic effects of LXR agonists and that an LXR agonist increases the expression of ABCA1 within pre‐existing atherosclerotic lesions 156. It also demonstrated that an LXR agonist not only inhibits the development but also induces regression of atherosclerotic lesions. Another recent study presented a direct proof that administration of a synthetic LXR agonist substantially increases reverse cholesterol transport of macrophage‐derived cholesterol in vivo157. It also established that LXR agonist promotes cholesterol efflux from macrophages into liver and ultimately into bile and faeces.

Acknowledgements

The author is grateful for excellent collaboration especially to Eero Sotaniemi (†), Olavi Pelkonen, Markku Savolainen, and Vilho Myllylä from the Medical Faculty, University of Oulu, Finland; Christian Ehnholm from the National Public Health Institute, Helsinki; and Heikki Vapaatalo from the Department of Pharmacology, University of Helsinki. The studies were supported by the Academy of Finland and the Paavo Nurmi Foundation, Finland.