Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation. 2004;109:II-34-II-41
doi: 10.1161/01.CIR.0000129503.62747.03
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mason, R. P.
Right arrow Articles by Jacob, R. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mason, R. P.
Right arrow Articles by Jacob, R. F.

(Circulation. 2004;109:II-34 – II-41.)
© 2004 American Heart Association, Inc.

Vascular Effects of Statins

Effects of HMG-CoA Reductase Inhibitors on Endothelial Function

Role of Microdomains and Oxidative Stress

R. Preston Mason, PhD; Mary F. Walter, PhD; Robert F. Jacob, PhD

From the Cardiovascular Division, Department of Medicine (R.P.M.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass; and the Cardiovascular Research Group (R.P.M., M.F.W., R.F.J.), Elucida Research LLC, Beverly, Mass.

Correspondence to R. Preston Mason, PhD, 100 Cummings Center, Suite 135L, Beverly, MA 01915. E-mail rpmason{at}elucidaresearch.com

Abstract

Certain pleiotropic activities reported for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are related to reductions in cellular cholesterol biosynthesis and isoprenoid levels. In endothelial cells, these metabolic changes contribute to favorable effects on nitric oxide (NO) bioavailability. Given the essential role of NO in preserving vascular structure and function, this effect of statins is of considerable therapeutic importance. Statins have been demonstrated to restore endothelial NO production by several mechanisms, including upregulating endothelial NO synthase (eNOS) protein expression and blocking formation of reactive oxygen species. In this article, we will discuss additional ways in which statins restore endothelial NO production and improve endothelial function. (1) Statins modulate membrane microdomain formation, resulting in reduced expression of proteins that specifically inhibit eNOS activation. (2) Statins reduce sterol biosynthesis, thus interfering with the formation of pathologic microdomains, including cholesterol crystalline structures. This observation has important implications for plaque stabilization, as these microdomains contribute to cholesterol crystal formation and endothelial apoptosis. Finally, (3) statins improve endothelial function by interfering with oxidative stress pathways through both enzymatic and nonenzymatic mechanisms. The relationships between membrane microdomains, cholesterol biosynthesis, and endothelial function will be discussed.


Key Words: atherosclerosis • endothelial function • microdomain • nitric oxide • statins

Vascular Plasma Membranes Are Structurally Heterogeneous and Consist of Discrete Microdomains

The endothelial cell plasma membrane is composed of liquid-ordered microdomains assembled from lipid constituents; these have distinct biophysical characteristics and limited random movement.1–3 Such microdomains, or "lipid rafts," are typically detergent-resistant and highly enriched with cholesterol and sphingolipids, as compared with the overall membrane lipid bilayer.1,3 These regions of ordered lipid account for local sequestration of proteins that mediate signal transduction in a variety of cell types, including endothelial and vascular smooth muscle cells (SMCs). This new model of the membrane as a mosaic structure consisting of microdomains has displaced an earlier perspective on the membrane in which lipid and protein constituents diffuse in a random and unrestricted fashion. In certain disease conditions, such as hyperlipidemia, the composition and abundance of certain membrane microdomains are altered, contributing to mechanisms of atherogenesis in vascular cells (Figure 1).



View larger version (50K):
[in this window]
[in a new window]
  		Figure 1. Schematic illustration of vessel wall and endothelial cell membrane changes during hyperlipidemia. Atherosclerotic changes in the vessel wall include the infiltration of native and oxidized LDL-C, adherence of monocytes, platelet aggregation, foam cell formation, and cholesterol crystal accumulation in the intima. Additionally, there are changes in endothelial cell membrane organization, such as formation of separate cholesterol-rich domains and an increase in the number of flask-shaped caveolae. The principal protein component of caveolae is the scaffolding protein caveolin, which regulates endothelial nitric oxide synthase (eNOS) activity. These changes are attenuated in the presence of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which reduce serum levels of LDL-C and block cholesterol and isoprenoid biosynthetic pathways.

A lipid raft subtype that has been the subject of intensive investigation is plasmalemmal caveolae. Caveolae are morphologically characterized as flask-shaped invaginations along the membrane surface (Figure 1). They are richly expressed in endothelial cells, fibroblasts, adipocytes, macrophages, and SMCs.4 In macrophages, caveolae have recently been shown to have a key role in cholesterol homeostasis, an important function during atherosclerosis.5 The principal component of caveolae is the protein caveolin, a scaffolding element that efficiently binds cholesterol and interacts with various signaling macromolecules, including G proteins and calcium-regulating proteins.4,6–8

Beyond its role in trafficking lipids between various intracellular compartments, caveolin also functions as a specific inhibitor of endothelial nitric oxide synthase (eNOS).8 By blocking access of eNOS to its cofactor, calcium/calmodulin, caveolin regulates the production of nitric oxide (NO) in the endothelium.9 In genetically engineered animals that specifically lack caveolin protein and caveolae microdomains, phenotypic changes included decreases in arterial dilation, myogenic tone, and contractility.10 Conversely, the expression of caveolin is markedly elevated under conditions of hypercholesterolemia, due to excessive cholesterol enrichment of the plasma membrane.11 High levels of caveolae are associated with a reduction in endothelial NO synthesis (Figure 1), which contributes to increased levels of superoxide and loss of NO-mediated benefits, including inhibition of platelet aggregation, SMC proliferation, and leukocyte adhesion.

Under normal conditions, eNOS is associated with cholesterol-enriched caveolae in endothelial cells, where its activity can be carefully regulated.12 However, in hyperlipidemia, lipoprotein particles modulate the activity and subcellular distribution of eNOS. Incubation of endothelial cells with low-density lipoprotein (LDL), particularly oxidized LDL (ox-LDL), causes an increase in binding of eNOS to CD36, which attenuates its activity and causes displacement of the protein from endothelial caveolae.13,14 In endothelial cells, CD36 and scavenger receptor BI (SR-BI) cofractionate with caveolae and also coimmunoprecipitate with caveolin-1. Ox-LDL also perturbs the levels of cholesterol associated with endothelial caveolae. This deleterious relationship between ox-LDL levels, eNOS activity, and cellular localization can be reversed with binding of high-density lipoprotein (HDL) to SR-BI.14 HDL replaces cholesterol that is depleted from caveolae after exposure to ox-LDL. In mice aorta, HDL binding to SR-BI causes a pronounced increase in NO production, an effect that cannot be reproduced in homozygous null SR-BI knockout mice.15

In addition to lipid rafts enriched in cholesterol and sphingolipid, a distinct type of membrane domain exists in vascular and macrophage foam cells that is causally related to cholesterol enrichment during atherosclerosis.16,17 This type of microdomain consists exclusively of free cholesterol and is prominent in cell membranes derived from atherosclerotic plaque.17 Direct evidence for the existence of such cholesterol microdomains in atherosclerotic cell membranes comes from our small-angle x-ray diffraction analyses, which have identified a highly ordered lipid structure with a molecular width of 34 Å (Figure 2).17,19 These membrane regions formed under conditions of hyperlipidemia consist exclusively of two cholesterol monohydrate molecules in a tail-to-tail orientation, based on the fact that a single cholesterol monohydrate crystal has a long dimension of 17 Å (Figure 3).17–21 We have also observed that oxidized derivatives of cholesterol form microdomains in a manner dependent on their 3-dimensional structure.21 By interfering with cholesterol biosynthesis, and reducing the relative concentration of cholesterol in the membrane, statins attenuate the formation of these crystalline-like membrane domains.



View larger version (40K):
[in this window]
[in a new window]
  		Figure 2. Schematic diagram of changes in lipid raft structure and cell function during cholesterol enrichment and atherosclerosis. Subtypes of lipid rafts enriched with sphingolipid (blue) and cholesterol (red) include caveolae (1) that contain caveolin protein (green) and detergent-resistant membrane domains (2). The figure shows that with cholesterol enrichment, separate cholesterol crystalline membrane domains (3) form and precede the development of extracellular crystals (4). Cholesterol crystals contribute to mechanisms of cell injury and death, including apoptosis. With cholesterol enrichment, there is an increase in the number of caveolae, leading to inhibition of endothelial nitric oxide synthase (eNOS) activity along with the vascular benefits of nitric oxide. Loss of normal membrane structure and function with cholesterol enrichment is also associated with disruptions in calcium regulation and redox potential. Reprinted with permission from Mason RP et al.19



View larger version (70K):
[in this window]
[in a new window]
  		Figure 3. A schematic model of cholesterol domain formation in atherosclerotic vascular cell membranes during hyperlipidemia. In the disease state, cholesterol microdomains with a molecular width of 34 Å are observed in the same plane as the overall lipid bilayer. Reprinted with permission from Mason RP et al.18

In normal cell plasma membranes, the ratio of cholesterol to phospholipid ranges between 0.5 and 1.0, under physiological conditions. The only mammalian cell type that contains cholesterol microdomains under physiological conditions is the fiber cell of the human ocular lens.22,23 The plasma membrane of the fiber cell contains molar ratios of sterol to phospholipid as high as 3:1,22 resulting in cholesterol tail-to-tail bilayer structures similar to those observed in atherosclerotic vascular cell membranes and macrophage foam cells.23,24 Certain drugs that inhibit cholesterol biosynthesis have been shown to induce cataract development in animal models as a result of reducing essential levels of lens membrane cholesterol.25–27

Cholesterol as an Important Determinant of Cell Membrane Structure and Function
Unesterified or free cholesterol is a major constituent of the vascular cell plasma membrane, where it regulates lipid bilayer dynamics and structure by modulating the packing of phospholipid molecules.28–31 The amount of free cholesterol in the cell is controlled by extracellular sources of cholesterol (serum LDL levels) and intracellular biosynthesis of sterols. Free cholesterol molecules partition into the plasma membrane where they assume an orientation with the long axis of the sterol lying parallel to the phospholipid acyl chains, increasing order in the upper acyl chain region of the membrane while decreasing constraints at the terminal methyl groups.28,32 In contrast, esterified cholesterol does not readily associate with the plasma membrane. By restricting the random motion of membrane lipids and the mean cross-sectional area occupied by neighboring phospholipid acyl chains, free cholesterol has a pronounced condensing effect on biologic membranes.33 Investigators have proposed that certain proteins localize preferentially to cholesterol-rich regions of the membrane, as the microenvironment created by cholesterol enrichment appears to be essential for protein folding and tertiary structure.34 It has also been hypothesized that proteins associated with areas enriched in cholesterol have specific roles in signal transduction, cell adhesion, motility, and the sorting and trafficking of membrane components.35–38

In the membrane, cholesterol tends to coalesce into discrete clusters at cholesterol to phospholipid mole ratios in excess of 0.339 and to form separate domains at ratios in excess of 1.0.40 Theoretic and model membrane studies have demonstrated that the systematic addition of cholesterol to biologic membranes can eventually yield such sterol domains.17,39–45 At cholesterol levels of >50 mol % (compared with total phospholipid), an immiscible cholesterol monohydrate phase is formed with a reproducible unit-cell periodicity of 34 Å, in coexistence with the surrounding liquid crystalline lipid bilayer,43 consistent with that observed in atherosclerotic tissues. This measurement corresponds to a tail-to-tail arrangement of the sterol, as proposed in other model membrane studies using a variety of techniques.17,46–48

Alterations in Vascular Cell Membrane Function Due to Cholesterol Enrichment

Under conditions of hyperlipidemia, elevated levels of LDL (and ox-LDL) interfere with the capacity of endothelial cells to generate NO (Figure 4).49 Instead, activation of eNOS leads to overproduction of superoxide from oxygen, the alternative product of NO synthase when quantities of L-arginine are insufficient.49 High levels of LDL and ox-LDL also cause displacement of eNOS from caveolae and CD36 binding. By modulating physicochemical properties of membrane lipids, cholesterol enrichment has been associated with disruption of L-arginine active transport and various membrane proteins, including voltage-sensitive calcium channels50 and ion pumps.51 Cholesterol reduces the mean molecular volume and lateral mobility of the membrane lipid bilayer, thereby increasing the energy required for conformational changes of integral membrane proteins. These effects of cholesterol enrichment on the activity of membrane proteins have been observed in erythrocytes,52 vascular cells,53 and renal cells.54



View larger version (18K):
[in this window]
[in a new window]
  		Figure 4. The effect of native low-density lipoprotein (n-LDL) and oxidized LDL (ox-LDL) treatment on ionophore (A23187)-stimulated nitric oxide (NO) production in aortic endothelial cells, measured with an amperometric probe. Relative to baseline levels, the release of NO was reduced as a function of increasing LDL, and especially ox-LDL, concentrations (1-hour incubation time). {dagger}P<0.005 n-LDL versus control; {ddagger}P<0.005 ox-LDL versus control. Probability values are for the significance of ox-LDL and n-LDL effects on NO levels (*P<0.005). Data are expressed as mean±SD. Reprinted with permission from Vergnani L et al.49

In vascular SMCs obtained from atherosclerotic plaque, calcium transport mechanisms and basal intracellular calcium levels are disrupted as a result of increased membrane cholesterol content.55 These changes have important consequences for atherosclerosis, as calcium participates directly in signal transduction pathways that promote SMC proliferation and migration, among other changes. In single channel electrophysiological recordings of calcium-activated K+ channels, we have observed that cholesterol enrichment favors the closed state of the ion channel pore, as a result of increased intrabilayer structural stress and lateral elastic stress energy.56 Collectively, these observations provide compelling experimental evidence for the concept that membrane cholesterol levels are carefully regulated within a certain physiological range to facilitate the normal activity of constituent proteins. When the amount of cholesterol exceeds these normal levels, there are broad and adverse consequences for vascular biology, leading to mechanisms of cell injury and death.

Evidence for Cholesterol Crystalline Membrane Domains in Atherosclerotic Vascular Membranes

Unstable atherosclerotic lesions are characterized by large extracellular lipid deposits consisting of both free and esterified cholesterol, phospholipid, and lesser amounts of triacylglycerol and fatty acids.57,58 Disruption in the integrity of the collagen-rich fibrous cap exposes elements of the blood to the thrombogenic lipid core, resulting in rapid thrombus formation.59 Free cholesterol in the plaque is associated with either membrane phospholipid or extracellular crystals and is a prominent feature of human and animal lesions.60,61 Although noncrystalline membrane cholesterol can readily exchange from the plaque with plasma lipoprotein particles, cholesterol in a crystalline state appears to be inert.57

In mouse macrophage cells, formation of free cholesterol crystals is enhanced with an acyl-coenzyme A:cholesterol acyltransferase inhibitor, as esterified cholesterol hydrolysis leads to free cholesterol accumulation.62 Microscopic cholesterol crystals form and extend out from the membrane with various morphologies that include plates, needles, and helices, as observed by scanning electron microscopy.16 Preventing crystal formation is an important goal, as cholesterol in this state does not respond well to pharmacological interventions that promote lesion regression.57,63 By modulating the chemical (eg, degree of acyl chain saturation and oxidation) and physical (eg, temperature) properties of the membrane, cholesterol crystalline domain formation may be slowed or blocked, thereby preventing subsequent extracellular crystal development. In models of atherosclerosis, systematic changes in the cholesterol content of vascular cell membranes have been measured and correlated with cholesterol microdomains.16 Under atherosclerotic-like conditions, prominent cholesterol domains with a unit-cell periodicity of 34 Å could be observed in SMC plasma membranes as free cholesterol levels in the membrane increased as a function of elevated serum cholesterol levels.16

Recent findings indicate a role for membrane lipid peroxidation in endothelial dysfunction and membrane microdomain formation. Oxidation of LDL and cellular membranes is a free radical reaction by which molecular oxygen is incorporated into constituent polyunsaturated fatty acids (PUFA) to yield lipid hydroperoxides. The first step in the reaction is the abstraction of a hydrogen atom from the bisallylic methylene group of PUFA to yield a lipid carbon radical. The lipid carbon radical can undergo resonance stabilization to form a radical with conjugated double bonds, which reacts with molecular oxygen to form lipid hydroperoxides.64 An elevation in reactive oxygen species (ROS) represents an essential component of atherosclerotic plaque development that contributes to nonenzymatic formation of isoprostanes from arachidonic acid.65–68 An important source of ROS are reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases that are activated by atherogenic factors, including angiotensin II and thrombin.69 During atherosclerosis, NADPH is expressed in vascular SMCs, leading to increased levels of ROS.69,70 At elevated levels, ROS cause endothelial dysfunction, react with NO to form peroxynitrite, and trigger a variety of inflammatory processes. ROS may additionally contribute to mechanisms of cell injury and death through promotion of cholesterol microdomains, resulting in extracellular crystal formation.

Effects of HMG-CoA Reductase Inhibitors on Cholesterol Microdomains, Endothelial Function, and Oxidative Stress

HMG-CoA reductase inhibitors (statins) provide therapeutic benefit by restoring normal endothelial NO synthesis under disease conditions (eg, hyperlipidemia). Beyond serum LDL reduction, statins enhance the release of NO through various mechanisms, such as increasing the expression of eNOS and interfering with superoxide formation. Restoration of NO is essential, as the bioavailability of this molecule is dramatically reduced under conditions of hyperlipidemia, resulting in a loss in vasodilatory and cardioprotective benefits.71 Interest has focused on how changes in cellular lipids and oxidative stress levels can restore NO production by the endothelium. The release of NO promotes vasodilation while interfering with various atherogenic pathways, such as platelet adhesion, superoxide formation, expression of adhesion molecules, and SMC proliferation.71,72 Given the central role of endothelial NO production in preserving vascular function and inhibiting various proatherogenic pathways, the effects of statins on NO metabolism are of considerable importance.

Statins have been shown to improve NO synthesis by mechanisms unrelated to changes in serum LDL levels, including upregulation of eNOS expression73 and reduced superoxide formation.74,75 Additionally, statins stimulate endothelial NO production through a dramatic reduction in plasma membrane caveolin levels76 (Figure 1). By interfering with cholesterol biosynthesis and lowering plasma membrane cholesterol levels, atorvastatin was shown to attenuate the expression of caveolin-1 (Figure 5).76 Reduced caveolin-1 levels resulted in pronounced increases in eNOS activation and NO production, in a highly dose-dependent fashion (Figure 6). 76 These effects on NO metabolism were reversed with mevalonate. When incubated with increasing amounts of extracellular LDL, atorvastatin also promoted the agonist-induced association of eNOS and the chaperone Hsp90, resulting in potent eNOS activation.76 This finding indicates a novel effect of a statin on endothelial function by modulating plasma membrane microdomains and caveolin expression.



View larger version (30K):
[in this window]
[in a new window]
  		Figure 5. This figure shows the dose-dependent effect of atorvastatin on reducing caveolin-1 (CAV-1) levels while simultaneously increasing endothelial nitric oxide synthase (eNOS) expression at various concentrations of low-density–lipoprotein cholesterol (LDL-C). A, Immunoblotting analyses of CAV-1 (left) and eNOS (right) abundance are shown. B, Densitometric analyses (n =3 to 4) of caveolin immunoblots (IB) as illustrated in A in arbitrary units (au). Reprinted with permission from Feron O et al.76



View larger version (19K):
[in this window]
[in a new window]
  		Figure 6. This figure shows the effects of atorvastatin on basal and agonist-stimulated nitric oxide (NO) metabolite (NOx) release from endothelial cells. A, Measurements of basal NOx production after treatment with atorvastatin and/or LDL-C. B, Effects of atorvastatin (100 nM) on ionophore (A23187)-induced NO release, measured with an amperometric probe. Data are expressed as percent of L-NAME–inhibitable NOx production (A) or NO release (B) determined in the absence of LDL-C and atorvastatin treatments. Values are mean±SEM. *P<0.01 versus corresponding values in the absence of atorvastatin treatment. Reprinted with permission from Feron O et al.76

Statins may also enhance the normal production of endothelial NO by reducing membrane cholesterol levels, thereby restoring normal transport of L-arginine, the substrate for eNOS. Previous studies indicate that L-arginine uptake through the amino acid transporter in endothelial-cell plasma membranes may be impaired under conditions of atherosclerosis when membrane unesterified cholesterol levels are elevated.49 Changes in the membrane microenvironment alter the active transport properties of membrane-bound proteins, such as cationic amino acid transporters. Thus, statins restore NO production by improving the uptake of this key amino acid as membrane cholesterol levels are returned to normal levels.

Under atherosclerotic conditions, higher ratios of cholesterol to phospholipid leads to the formation of discrete crystalline-like domains that alter the physicochemical properties of the membrane and serve as potential nucleating sites for extracellular crystals (Figure 1). Inhibition of endothelial-cell cholesterol biosynthesis reduces the amount of free cholesterol available for forming such domains. Statins may also interfere with cholesterol crystal development by reducing levels of unesterified cholesterol in the vascular cell plasma membrane.

Statins also reduce LDL oxidation, a key initiator of endothelial dysfunction. Statins interfere with LDL oxidation by several mechanisms: (1) blocking isoprenylation of rac 1, an important component of NADPH oxidase complex;74,77 (2) reducing expression of NADPH oxidase subunits;75 and (3) reducing serum levels of LDL available for oxidation. Additionally, atorvastatin was shown to reduce levels of a free radical initiator by enhancing catalase expression, independently of LDL changes.75 By reducing sources of oxidative stress, statins increase the bioavailability of NO by preserving eNOS activity and endothelial function while reducing the loss of NO through its reaction with superoxide.

Certain statins have demonstrated antioxidant effects by direct scavenging of free radical molecules, independently of their effects on lipid metabolism.75,78–80 A review of these studies, however, indicates that suprapharmacological (micromolar) concentrations of statin were required to produce this activity in these experimental models, and thus, the physiological relevance is uncertain. By contrast, we have observed that the active o-hydroxy metabolite of atorvastatin is capable of increasing the resistance of LDL to oxidative modification at pharmacological levels (nanomolar). This is a relevant observation, as the majority of atorvastatin in the plasma is in the form of active hydroxy metabolites. The dose-dependent effect of the o-hydroxy metabolite on human LDL oxidation at pharmacological concentrations in vitro is demonstrated in Figure 7. This distinct antioxidant mechanism is specifically attributed to the o-hydroxy moiety, as it is capable of quenching free radical reactions by proton-donation mechanisms after partitioning into the lipid environment. In support of this hypothesis, the activity of the atorvastatin active hydroxy metabolite could not be reproduced by the parent atorvastatin compound or other statins that lack this chemical feature, including lovastatin, pravastatin, rosuvastatin, and simvastatin. A similar protective effect of the atorvastatin metabolite against LDL oxidation was observed in other experimental models.80 As a further control, measurement of oxygen radical absorbance capacity for atorvastatin o-hydroxy metabolite was shown to be 10-fold greater than for Trolox, a water-soluble analog of vitamin E with well-characterized antioxidant properties.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 7. Concentration-dependent effect of atorvastatin o-hydroxy active metabolite on formation of malondialdehyde (MDA) in human LDL (100 µg protein/mL) after initiation with CuSO4 at 37°C. Measurement of MDA, a product of LDL oxidation, was calculated from the reaction of this aldehyde with two molecules of thiobarbituric acid in an acid medium to form a stable chromogen, referred to as thiobarbituric acid reactive substances (TBARS), with an absorbance of 532 nm. The inhibition of LDL oxidation with atorvastatin metabolite was compared with vehicle (control). *P<0.05, different from vehicle-treated samples.

Conclusion

Statins have an effect on endothelial function and NO production, independent of changes in serum LDL levels. By enhancing the expression of eNOS, interfering with oxidative stress pathways, and reducing expression of caveolin, statins enhance NO bioavailability. These pleiotropic activities of statins are related to reductions in cellular cholesterol biosynthesis and isoprenylation reactions. Additional effects beyond lipid metabolism for certain statins may be attributed to distinct lipophilic properties and physicochemical properties. Collectively, these findings support a beneficial effect for statins on endothelial function that leads to improved vascular compliance while interfering with atherosclerotic pathways that lead to plaque vulnerability in coronary artery disease.

Acknowledgments

The authors acknowledge funding support for these studies from the National Heart, Lung, and Blood Institute, the American Heart Association, and National Eye Institute. The authors acknowledge additional funding from Pfizer Inc.

References

  1. Brown DA, London E. Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J Biol Chem. 2000; 275: 17221–17224.[Free Full Text]
  2. Simons K, Ehehalt R. Cholesterol, lipid rafts, and disease. J Clin Invest. 2002; 110: 597–603.[CrossRef][Medline] [Order article via Infotrieve]
  3. Ostermeyer AG, Beckrich BT, Ivarson KA, Grove KE, Brown DA. Glycosphingolipids are not essential for formation of detergent-resistant membrane rafts in melanoma cells. Methyl-ß-cyclodextrin does not affect cell surface transport of a GPI-anchored protein. J Biol Chem. 1999; 274: 34459–34466.[Abstract/Free Full Text]
  4. Smart EJ, Graf GA, McNiven MA, et al. Caveolins, liquid-ordered domains, and signal transduction. Mol Cell Biol. 1999; 19: 7289–7304.[Free Full Text]
  5. Gargalovic P, Dory L. Caveolins and macrophage lipid metabolism. J Lipid Res. 2003; 44: 11–21.[Abstract/Free Full Text]
  6. Edidin M. Lipid microdomains in cell surface membranes. Curr Opin Struct Biol. 1997; 7: 528–532.[CrossRef][Medline] [Order article via Infotrieve]
  7. Feron O, Belhassen L, Kobzik L, et al. Endothelial nitric oxide synthase targeting to caveolae. J Biol Chem. 1996; 271: 22810–22814.[Abstract/Free Full Text]
  8. Feron O, Saldana F, Michel JB, et al. The endothelial nitric-oxide synthase-caveolin regulatory cycle. J Biol Chem. 1998; 273: 3125–3128.[Abstract/Free Full Text]
  9. Ju H, Zou R, Venema VJ, et al. Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity. J Biol Chem. 1997; 272: 18522–18525.[Abstract/Free Full Text]
  10. Drab M, Verkade P, Elger M, et al. Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science. 2001; 293: 2449–2452.[Abstract/Free Full Text]
  11. Feron O, Dessy C, Moniotte S, et al. Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide synthase. J Clin Invest. 1999; 103: 897–905.[Medline] [Order article via Infotrieve]
  12. Shaul PW, Smart EJ, Robinson LJ, et al. Acylation targets endothelial nitric-oxide synthase to plasmalemmal caveolae. J Biol Chem. 1996; 271: 6518–6522.[Abstract/Free Full Text]
  13. Blair A, Shaul PW, Yuhanna IS, et al. Oxidized low-density lipoprotein displaces endothelial nitric-oxide synthase (eNOS) from plasmalemmal caveolae and impairs eNOS activation. J Biol Chem. 1999; 274: 32512–32519.[Abstract/Free Full Text]
  14. Uittenbogaard A, Shaul PW, Yuhanna IS, et al. High-density lipoprotein prevents oxidized low density lipoprotein-induced inhibition of endothelial nitric-oxide synthase localization and activation in caveolae. J Biol Chem. 2000; 275: 11278–11283.[Abstract/Free Full Text]
  15. Yuhanna IS, Zhu Y, Cox BE, et al. High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase. Nat Med. 2001; 7: 853–857.[CrossRef][Medline] [Order article via Infotrieve]
  16. Kellner-Weibel G, Yancey PG, Jerome WG, et al. Crystallization of free cholesterol in model macrophage foam cells. Arterioscler Thromb Vasc Biol. 1999; 19: 1891–1898.[Abstract/Free Full Text]
  17. Tulenko TN, Chen M, Mason PE, et al. Physical effects of cholesterol on arterial smooth muscle membranes: evidence of immiscible cholesterol domains and alterations in bilayer width during atherogenesis. J Lipid Res. 1998; 39: 947–956.[Abstract/Free Full Text]
  18. Mason RP, Tulenko TN, Jacob RF. Direct evidence for cholesterol crystalline domains in biological membranes: role in human pathobiology. Biochimica et Biophysica Acta. 2003; 1610: 198–207.[Medline] [Order article via Infotrieve]
  19. Mason RP, Jacob RF. Membrane microdomains and vascular biology. Emerging role in atherogenesis. Circulation. 2003; 107: 2270–2273.[Free Full Text]
  20. Craven BM. Crystal structure of cholesterol monohydrate. Nature. 1976; 260: 727–729.[CrossRef][Medline] [Order article via Infotrieve]
  21. Phillips JE, Geng Y-J, Mason RP. 7-Ketocholesterol forms crystalline domains in model membranes and murine aortic smooth muscle cells. Atherosclerosis. 2001; 159: 125–135.[CrossRef][Medline] [Order article via Infotrieve]
  22. Cenedella RJ. Cholesterol and cataracts. Surv Ophthalmol. 1996; 40: 320–337.[CrossRef][Medline] [Order article via Infotrieve]
  23. Jacob RF, Cenedella RJ, Mason RP. Direct evidence for immiscible cholesterol domains in human ocular lens fiber cell plasma membranes. J Biol Chem. 1999; 274: 31613–31618.[Abstract/Free Full Text]
  24. Jacob RF, Cenedella RJ, Mason RP. Evidence for distinct cholesterol domains in fiber cell membranes from cataractous human lenses. J Biol Chem. 2001; 276: 13573–13578.[Abstract/Free Full Text]
  25. Cenedella RJ, Bierkamper GG. Mechanism of cataract production by 3-ß(2-diethylaminoethoxy) androst-5-en-17-one hydrochloride, U18666A: an inhibitor of cholesterol biosynthesis. Exp Eye Res. 1979; 28: 673–688.[CrossRef][Medline] [Order article via Infotrieve]
  26. Cenedella RJ. Source of cholesterol for the ocular lens, studied with U18666A: a cataract-producing inhibitor of lipid metabolism. Exp Eye Res. 1983; 37: 33–43.[CrossRef][Medline] [Order article via Infotrieve]
  27. Cenedella RJ, Kuszak JR, Al-Ghoul KJ, Qin S, Sexton PS. Discordant expression of the sterol pathway in lens underlies simvastatin-induced cataracts in Chbb: Thom rats. J Lipid Res. 2003; 44: 198–211.[Abstract/Free Full Text]
  28. Yeagle PL. Cholesterol and the cell membrane. Biochim Biophys Acta. 1985; 822: 267–287.[Medline] [Order article via Infotrieve]
  29. Chen M, Mason RP, Tulenko TN. Structural, compositional and functional alterations of arterial smooth muscle plasma membranes in atherosclerosis. FASEB J. 1991; 5: A531. Abstract 929.
  30. McIntosh TJ. The effect of cholesterol on the structure of phosphatidylcholine bilayers. Biochim Biophys Acta. 1978; 513: 43–58.[Medline] [Order article via Infotrieve]
  31. Léonard A, Dufourc EJ. Interactions of cholesterol with the membrane lipid matrix: a solid state NMR approach. Biochimie. 1991; 73: 1295–1302.[Medline] [Order article via Infotrieve]
  32. Franks NP. Structural analysis of hydrated egg lecithin and cholesterol bilayers. I. X-ray diffraction. J Mol Biol. 1976; 100: 345–358.[CrossRef][Medline] [Order article via Infotrieve]
  33. Shinitzky M, Inbar M. Microviscosity parameters and protein mobility in biological membranes. Biochim Biophys Acta. 1976; 433: 133–149.[Medline] [Order article via Infotrieve]
  34. Mukherjee S, Chattopadhyay A. Membrane organization at low cholesterol concentrations: a study using 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled cholesterol. Biochemistry. 1996; 35: 1311–1322.[CrossRef][Medline] [Order article via Infotrieve]
  35. Janes PW, Ley SC, Magee AI, et al. The role of lipid rafts in T cell antigen receptor (TCR) signalling. Semin Immunol. 2000; 12: 23–34.[CrossRef][Medline] [Order article via Infotrieve]
  36. Langlet C, Bernard A-M, Drevot P, et al. Membrane rafts and signaling by the multichain immune recognition receptors. Curr Opin Immunol. 2000; 12: 250–255.[CrossRef][Medline] [Order article via Infotrieve]
  37. Simons K, Ikonen E. Functional rafts in cell membranes. Nature. 1997; 387: 569–572.[CrossRef][Medline] [Order article via Infotrieve]
  38. Simons K, Ikonen E. How cells handle cholesterol. Science. 2000; 290: 1721–1726.[Abstract/Free Full Text]
  39. Engelman DM, Rothman JE. The planar organization of lecithin-cholesterol bilayers. J Biol Chem. 1972; 247: 3694–3697.[Abstract/Free Full Text]
  40. Houslay MD, Stanley KK. Dynamics of Biological Membranes: Influence on Synthesis, Structure and Function. New York, NY: John Wiley & Sons; 1982: 79.
  41. Bach D, Borochov N, Wachtel E. Phase separation of cholesterol in dimyristoyl phosphatidylserine cholesterol mixtures. Chem Phys Lipids. 1998; 92: 71–77.[CrossRef]
  42. Rice PA, McConnell HM. Critical shape transitions of monolayer lipid domains. Proc Natl Acad Sci U S A. 1989; 86: 6445–6448.[Abstract/Free Full Text]
  43. Ruocco MJ, Shipley GG. Interaction of cholesterol with galactocerebroside and galactocerebroside-phosphatidylcholine bilayer membranes. Biophys J. 1984; 46: 695–707.[Abstract/Free Full Text]
  44. Slotte JP. Lateral domain formation in mixed monolayers containing cholesterol and dipalmitoylphosphatidylcholine or N-palmitoylsphingomyelin. Biochim Biophys Acta. 1995; 1235: 419–427.[Medline] [Order article via Infotrieve]
  45. Slotte PJ. Effect of sterol structure on molecular interactions and lateral domain formation in monolayers containing dipalmitoyl phosphatidylcholine. Biochim Biophys Acta. 1995; 1237: 127–134.[Medline] [Order article via Infotrieve]
  46. Bloom M, Thewalt JL. Time and distance scales of membrane domain organization. Mol Membr Biol. 1995; 12: 9–13.[Medline] [Order article via Infotrieve]
  47. Hui SW. Geometry of domains and domain boundaries in monolayers and bilayers. Mol Membr Biol. 1995; 12: 45–50.[Medline] [Order article via Infotrieve]
  48. Tocanne JF. Detection of lipid domains in biological membranes. Comments Mol Cell Biophys. 1992; 8: 53–72.
  49. Vergnani L, Hatrik S, Ricci F, et al. Effect of native and oxidized low-density lipoprotein on endothelial nitric oxide and superoxide production: key role of L-arginine availability. Circulation. 2000; 101: 1261–1266.[Abstract/Free Full Text]
  50. Bialecki RA, Tulenko TN. Excess membrane cholesterol alters calcium channels in arterial smooth muscle. Am J Physiol. 1989; 257 (pt 1): C306–C314.
  51. Broderick R, Bialecki R, Tulenko TN. Cholesterol-induced changes in rabbit arterial smooth muscle sensitivity to adrenergic stimulation. Am J Physiol. 1989; 257 (pt 2): H170–H178.
  52. Giraud F, Claret M, Bruckdorfer KR, Chailley B. The effects of membrane lipid order and cholesterol on the internal and external cationic sites of the Na+-K+ pump in erythrocytes. Biochim Biophys Acta. 1981; 647: 249–258.[Medline] [Order article via Infotrieve]
  53. Lau Y-T. Cholesterol enrichment inhibits Na+/K+ pump in endothelial cells. Atherosclerosis. 1994; 110: 251–257.[CrossRef][Medline] [Order article via Infotrieve]
  54. Yeagle PL, Young J, Rice D. Effects of cholesterol on (Na+,K+)-ATPase ATP hydrolyzing activity in bovine kidney. Biochemistry. 1988; 27: 6449–6452.[CrossRef][Medline] [Order article via Infotrieve]
  55. Gleason MM, Medow MS, Tulenko TN. Excess membrane cholesterol alters calcium movements, cytosolic calcium levels, and membrane fluidity in arterial smooth muscle cells. Circ Res. 1991; 69: 216–227.[Abstract/Free Full Text]
  56. Chang HM, Reitstetter R, Mason RP, et al. Attenuation of channel kinetics and conductance by cholesterol: an interpretation using structural stress as a unifying concept. J Membr Biol. 1995; 143: 51–63.[Medline] [Order article via Infotrieve]
  57. Small DM. Progression and regression of atherosclerotic lesions. Arteriosclerosis. 1988; 8: 103–129.[Abstract/Free Full Text]
  58. Felton CV, Cook D, Davies MJ, et al. Relation of plaque lipid composition and morphology to the stability of human aortic plaques. Arterioscler Thromb Vasc Biol. 1997; 17: 1337–1345.[Abstract/Free Full Text]
  59. Libby P. Molecular bases of the acute coronary syndromes. Circulation. 1995; 91: 2844–2850.[Free Full Text]
  60. Katz SS, Small DM. Isolation and partial characterization of the lipid phases of human atherosclerotic plaques. J Biol Chem. 1980; 255: 9753–9759.[Free Full Text]
  61. Guyton JR, Klemp KF. The lipid-rich core region of human atherosclerotic fibrous plaques. Am J Pathol. 1989; 134: 705–717.[Abstract]
  62. Kellner-Weibel G, Jerome WG, Small DM, et al. Effects of intracellular free cholesterol accumulation on macrophage viability: a model for foam cell death. Arterioscler Thromb Vasc Biol. 1998; 18: 423–431.[Abstract/Free Full Text]
  63. Katz SS, Small DM, Smith FR, et al. Cholesterol turnover in lipid phases of human atherosclerotic plaque. J Lipid Res. 1982; 23: 733–737.[Abstract]
  64. Porter NA. Chemistry of lipid peroxidation. Methods Enzymol. 1984; 105: 273–282.[Medline] [Order article via Infotrieve]
  65. Praticò D, Iuliano L, Mauriello A, et al. Localization of distinct F2-isoprostanes in human atherosclerotic lesions. J Clin Invest. 1997; 100: 2028–2034.[Medline] [Order article via Infotrieve]
  66. Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med. 1999; 340: 115–126.[Free Full Text]
  67. Lynch SM, Frei F, Morrow JD, et al. Vascular superoxide dismutase deficiency impairs endothelial vasodilator function through direct inactivation of nitric oxide and increased lipid peroxidation. Arterioscler Thromb Vasc Biol. 1997; 17: 2975–2981.[Abstract/Free Full Text]
  68. Reilly MP, Praticò D, Delanty N, et al. Increased formation of distinct F2-isoprostanes in hypercholesterolemia. Circulation. 1998; 98: 2822–2828.[Abstract/Free Full Text]
  69. Warnholtz A, Nickenig G, Schulz E, et al. Increased NADH-oxidase–mediated superoxide production in the early stages of atherosclerosis: evidence for involvement of the renin-angiotensin system. Circulation. 1999; 99: 2027–2033.[Abstract/Free Full Text]
  70. Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res. 2000; 86: 494–501.[Abstract/Free Full Text]
  71. Vane JR, Änggård EE, Botting RM. Regulatory functions of the vascular endothelium. N Engl J Med. 1990; 323: 27–36.[Medline] [Order article via Infotrieve]
  72. Harrison DG. Cellular and molecular mechanisms of endothelial cell dysfunction. J Clin Invest. 1997; 100: 2153–2157.[Medline] [Order article via Infotrieve]
  73. Laufs U, La Fata V, Plutzky J, et al. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation. 1998; 97: 1129–1135.[Abstract/Free Full Text]
  74. Wagner AH, Köhler T, Rückschloss U, Just I, Hecker M. Improvement of nitric oxide–dependent vasodilatation by HMG-CoA reductase inhibitors through attenuation of endothelial superoxide anion formation. Arterioscler Thromb Vasc Biol. 2000; 20: 61–69.[Abstract/Free Full Text]
  75. Wassmann S, Laufs U, Muller K, et al. Cellular antioxidant effects of atorvastatin in vitro and in vivo. Arterioscler Thromb Vasc Biol. 2002; 22: 300–305.[Abstract/Free Full Text]
  76. Feron O, Dessy C, Desager JP, et al. Hydroxy-methylglutaryl-coenzyme A reductase inhibition promotes endothelial nitric oxide synthase activation through a decrease in caveolin abundance. Circulation. 2001; 103: 113–118.[Abstract/Free Full Text]
  77. Bokoch GM, Prossnitz V. Isoprenoid metabolism is required for stimulation of the respiratory burst of oxidase of HL-60 cells. J Clin Invest. 1992; 89: 402–408.
  78. Yamamoto A, Hoshi K, Ichihara K. Fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, scavenges free radicals and inhibits lipid peroxidation in rat liver microsomes. Eur J Pharmacol. 1998; 361: 143–149.[CrossRef][Medline] [Order article via Infotrieve]
  79. Girona J, La Ville AE, Sola R, et al. Simvastatin decreases aldehyde production derived from lipoprotein oxidation. Am J Cardiol. 1999; 83: 846–851.[CrossRef][Medline] [Order article via Infotrieve]
  80. Aviram M, Rosenblat M, Bisgaier CL, et al. Atorvastatin and gemfibrozil metabolites, but not the parent drugs, are potent antioxidants against lipoprotein oxidation. Atherosclerosis. 1998; 138: 271–280.[CrossRef][Medline] [Order article via Infotrieve]



This article has been cited by other articles:


Home page
 JAMAHome page
S. P. Schulman, L. C. Becker, D. A. Kass, H. C. Champion, M. L. Terrin, S. Forman, K. V. Ernst, M. D. Kelemen, S. N. Townsend, A. Capriotti, et al.
L-Arginine Therapy in Acute Myocardial Infarction: The Vascular Interaction With Age in Myocardial Infarction (VINTAGE MI) Randomized Clinical Trial
JAMA, January 4, 2006; 295(1): 58 - 64.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mason, R. P.
Right arrow Articles by Jacob, R. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mason, R. P.
Right arrow Articles by Jacob, R. F.

Circulation Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 2004 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.