Download Special Review

Special Review
Membrane Microdomains and Vascular Biology
Emerging Role in Atherogenesis
R. Preston Mason, PhD; Robert F. Jacob, PhD
T
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
have been shown to play an important role in other disease
processes, including hypertension, Alzheimer’s disease, prion
disease, and viral infection.5
Pharmacological inhibition of 3-hydroxy-3-methylglutaryl
coenzyme A (HMG-CoA) reductase leads to potent stimulation of eNOS, independently of changes in extracellular
low-density lipoprotein (LDL) concentrations.6 By lowering
the levels of plasma membrane cholesterol and potentially
interacting with specific lipids, the HMG-CoA reductase
inhibitor atorvastatin was shown to attenuate the expression
of caveolin-1 and the abundance of caveolae in endothelial
cells. These effects were observed in the absence of changes
in cytosolic eNOS levels and 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.6 This finding provides insight into the
complex biochemical relationships between membrane cholesterol levels, microdomain abundance, and endothelial
function (Figure). Indeed, restoration of normal endotheliumderived NO production with a statin has important benefits
for treating the clinical manifestations of atherosclerosis. The
release of NO promotes vasodilatation while interfering with
various atherogenic pathways, such as platelet adhesion,
superoxide formation, expression of adhesion molecules, and
smooth muscle cell proliferation.7
The existence of a unique membrane microdomain in
vascular smooth muscle cells and macrophages that is causally related to cholesterol enrichment during atherosclerosis
has been recently characterized using biophysical approaches.
This microdomain consists exclusively of unesterified cholesterol and is found prominently in atherosclerotic tissues.
Small-angle x-ray diffraction analyses have been used to
directly characterize cholesterol monohydrate domains,
which are identified as highly ordered lipid structures that
have a consistent width of 34 Å and are contained within the
surrounding plasma membrane (Figure).8,9 These distinct
membrane regions consist of cholesterol in a tail-to-tail
orientation, because a single cholesterol monohydrate crystal
has a long-axis dimension of 17 Å.10 Cholesterol microdomains have a remarkably smaller intra-bilayer dimension as
compared with the overall smooth muscle cell plasma membrane, which has a typical molecular width of 54 to 60 Å. We
he classic model of cell plasma membrane organization
is that of a uniform, fluid environment in which protein
and lipid constituents diffuse rapidly in an unrestricted
fashion. Recent experimental evidence, however, reveals a far
more complex membrane organization consisting of microdomains assembled from lipid constituents that have distinct
biophysical characteristics. Such microdomains or “lipid
rafts” are typically detergent-resistant and highly enriched
with cholesterol and sphingolipids compared with the overall
membrane lipid bilayer environment.1 These defined regions
of lipid domains sequester proteins that mediate signal
transduction in a variety of cell types, including endothelial
cells and myocytes. Lipid rafts move or “float” as a coherent
structural unit within the liquid-disordered lipid bilayer and
can also cluster with other rafts to form larger platforms. The
basis for differences in fluidity between rafts and the surrounding membrane is the degree of hydrocarbon chain
saturation; domains are characterized by sphingolipids containing saturated fatty acids that attract unesterified cholesterol. The planar sterol nucleus of cholesterol further restricts
the mobility and rotation of sphingolipid acyl chains, resulting in limited random diffusion and an expanded molecular
width as compared with the rest of the membrane.
The plasma membrane caveola is a lipid raft subtype that is
the subject of intensive investigation. Caveolae typically
appear as microscopic, flask-shaped invaginations along the
membrane surface and are commonly found in endothelial
cells, adipocytes, and smooth muscle cells (Figure). The
principal protein component of caveolae is caveolin, a scaffolding protein that binds cholesterol efficiently and interacts
with various signaling macromolecules, including G proteins
and calcium regulating proteins.2 Caveolin is also a potent
inhibitor of endothelial nitric oxide synthase (eNOS) as it
binds directly to the enzyme, blocking access of the co-factor,
calcium/calmodulin.3 Caveolin may also regulate intracellular
and surface cholesterol levels.2 Genetically engineered animals that lack caveolin-1 protein, and thus caveolae, demonstrate marked defects in arterial relaxation, myogenic tone,
and exercise tolerance as a result of abnormalities in cell
signaling and NO metabolism.4 Conversely, the expression of
caveolin is markedly elevated under conditions of hypercholesterolemia because of enrichment of plasma membrane
cholesterol levels. In addition to atherosclerosis, lipid rafts
From the Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston (R.P.M.), and Elucida
Research, Beverly (R.P.M., R.F.J.), Mass.
Correspondence to R. Preston Mason, PhD, 100 Cummings Center, Suite 135L, Beverly, MA 01915. E-mail [email protected]
(Circulation. 2003;107:2270-2273.)
© 2003 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/01.CIR.0000062607.02451.B6
2270
Mason and Jacob
Membrane Lipid Rafts and Atherogenesis
2271
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Schematic diagram of changes in lipid raft structure and cell function during cholesterol enrichment and atherosclerosis. Subtypes of
lipid rafts enriched with sphingolipid (blue lipids) and cholesterol (red lipids) include caveolae1 that contain caveolin protein (in green)
and detergent-resistant membrane domains.2 The Figure shows that with cholesterol enrichment, separate cholesterol crystalline membrane domains3 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 nitric oxide synthase (eNOS). Loss of normal membrane structure and function with cholesterol enrichment is also associated with
disruptions in calcium regulation and redox potential.
have also observed that certain oxidized derivatives of cholesterol form microdomains in a manner dependent on their
3-dimensional structure and intermolecular packing constraints.11 The stability of membrane cholesterol domains is
dependent on numerous factors, including temperature, membrane cholesterol-to-phospholipid mole ratio, composition of
the surrounding phospholipid acyl chains (eg, degree of
saturation), and the extent of lipid peroxidation.8,11,12
Alterations of Vascular Cell Membrane
Structure in Atherosclerosis
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
neighboring phospholipid molecules. The unesterified cholesterol molecule is oriented in the membrane such that the
long axis of the highly planar sterol lies parallel to adjacent
phospholipid acyl chains, thereby increasing the order parameter associated with the acyl chain region of the membrane.
By restricting the random motion of membrane lipids and the
mean cross-sectional area occupied by neighboring phospholipid acyl chains, cholesterol has a pronounced condensing
effect on biological membranes. Investigators have proposed
that certain proteins preferentially localize to cholesterolenriched regions of the membrane (eg, caveolae), as the
microenvironment created by cholesterol enrichment may be
essential for maintaining the tertiary structure and function of
restricted proteins.1
Abnormal accumulation of cholesterol in vascular cells
during atherosclerosis, however, has deleterious effects on
membrane function, including ion transport and signal transduction mechanisms. In endothelial cells, excessive membrane cholesterol incorporation during hyperlipidemia may
interfere with the active transport of amino acids, such as
L-arginine. As a result, activation of eNOS leads to overproduction of superoxide, the alternative product of NO synthase
when quantities of L-arginine or essential cofactors are
insufficient.13 By modulating the physicochemical properties
of membrane lipids, cholesterol enrichment disrupts the
function of other transport proteins, including voltagesensitive ion channels. In single-channel electrophysiological
recordings of calcium-activated K⫹ channels, cholesterol
enrichment caused the ion channel pore to favor the closed
state, as a result of increased intra-bilayer structural stress and
lateral elastic stress energy.14
In vascular smooth muscle cells obtained from atherosclerotic plaque, calcium transport mechanisms and basal intracellular calcium levels are disrupted by increased membrane
cholesterol content.15 These changes have important consequences for atherosclerosis, because calcium participates
directly in signal transduction pathways that promote smooth
muscle cell proliferation and migration, among other effects.
2272
Circulation
May 6, 2003
Collectively, these observations provide compelling experimental evidence supporting the concept that membrane cholesterol levels are carefully regulated within a certain physiological range to facilitate normal activity of constituent
proteins. Normal cholesterol levels are also necessary in the
formation of lipid rafts, such as caveolae and detergent-resistant membrane domains. An excess amount of cholesterol
results in adverse consequences for vascular biology.
Membrane Cholesterol Domains Precede
Formation of Crystals
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Unstable atherosclerotic lesions are characterized by large
extracellular lipid deposits consisting of cholesterol (both free
and esterified), phospholipids, and lesser amounts of triacylglycerol and fatty acids.16 The lipid core is bordered on its
luminal side by a fibrous cap and on its abluminal side by the
plaque base. 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.17
Unesterified cholesterol in the plaque is associated with
membrane phospholipid or extracellular crystals, a prominent
feature of atherosclerotic lesions in humans and animals.
Although noncrystalline membrane cholesterol can readily
exchange from the plaque with plasma lipoprotein particles,
cholesterol in a crystalline state appears to be inert and
resistant to reverse transport mechanisms.16
In mouse macrophage cells, formation of free cholesterol
crystals is enhanced with an acyl-CoA-cholesterol acyl transferase inhibitor, as esterified cholesterol hydrolysis leads to
free cholesterol accumulation.9 Microscopic cholesterol crystals form and extend out from the subcellular sites with
various morphologies that include plates, needles, and helices, as observed by scanning electron microscopy approaches.9 Interestingly, formation of membrane cholesterol microdomains, as measured by x-ray diffraction approaches,
precedes any evidence of extracellular cholesterol crystals.
This observation suggests that membrane microdomains may
represent a key nucleating site for extracellular cholesterol
crystals. Preventing crystal formation is an important goal, as
these rigid macromolecules contribute to mechanisms of cell
injury and death, including apoptosis.9,11 Because cholesterol
in this state does not respond well to pharmacological
interventions that promote lesion regression, early intervention is essential. Cholesterol crystalline domain formation
may be slowed or blocked by modulating the chemical (eg,
degree of acyl chain saturation and oxidation) and physical
(eg, temperature) properties of the membrane, thereby slowing or even preventing subsequent extracellular crystal
development.
In models of atherosclerosis, systematic changes in the
cholesterol content of vascular cell membranes have been
measured and shown to correlate with the development of
cholesterol microdomains.8 Cholesterol enrichment had remarkably consistent effects on the molecular dimensions and
lipid organization of plasma membranes derived from an
intact animal model and from those obtained from smooth
muscle cells grown in vitro. Under atherosclerotic-like conditions, prominent cholesterol domains were observed in
smooth muscle cell plasma membranes as free cholesterol
levels in the membrane increased in parallel with serum LDL
levels.8 In both model and biological membranes, oxidized
cholesterol derivatives also formed domains within the membrane lipid bilayer.11 The development of such cholesterol
domains is associated with extracellular crystal formation and
cellular apoptosis and appears to be highly dependent on
sterol 3-dimensional structure.
Cholesterol Influences Drug Availability to
Cellular Receptor Sites
Numerous cardiovascular drugs bind to protein receptors (eg,
ion channels) embedded in vascular cell plasma membranes
after intercalation and diffusion of the drug through the
surrounding lipid bilayer. Calcium channel blockers (CCBs),
in particular, modulate excitation-contraction mechanisms in
smooth muscle cells by regulating the transmembrane influx
of calcium ions through voltage-sensitive ion channels. CCB
receptor binding affinity and kinetics can be precisely calculated from their membrane concentration in equilibrium with
the L-type calcium channel protein receptor.18 The available
volume in the plasma membrane is an important determinant
of the concentration of drug available for receptor binding, a
parameter highly influenced by membrane cholesterol content in an inverse fashion. This relationship between membrane cholesterol content and drug interactions is dependent,
in turn, on the lipophilicity and other physico-chemical
properties of the compound.19 Reduction in membrane available free volume, and hence drug-membrane partitioning, can
also be accomplished by changes in temperature and degree
of acyl chain saturation of the phospholipid constituents.19
Dihydropyridine-type CCBs with high affinity for the
membrane lipid bilayer under normal and even cholesterolenriched conditions are characterized by favorable pharmacokinetics, including a slow onset and long duration of
activity. These agents, referred to as third-generation CCBs,
have effects on membrane function that may help to elucidate
differences in their clinical benefit among patients with
coronary artery disease as compared with older, less lipophilic members of this class.20 In prospective, randomized
trials, highly lipophilic CCBs have been shown to reduce
cardiovascular events in patients with documented coronary
artery disease as compared with placebo or less lipophilic
CCBs.21,22 These pharmacological and clinical observations
suggest that the activity of cardiovascular drugs that target
receptor sites in vascular membranes may be highly influenced by the concentration and organization of cholesterol in
the lipid bilayer as a function of hyperlipidemia.
Conclusion
The use of high-resolution imaging techniques, such as x-ray
diffraction and electron microscopy, has provided direct
physical evidence for structurally distinct microdomains in
the cell plasma membrane. These lipid rafts, which include
detergent-resistant membranes and caveolae, consist of lipids
with unique lateral diffusion properties and molecular dimensions. Among other functions, lipid domains provide sites for
sequestering proteins that mediate signal transduction and
NO metabolism in vascular cells. Using small-angle x-ray
scattering methods, we have characterized a novel microdo-
Mason and Jacob
main that is causally linked to atherogenesis and consists of
unesterified cholesterol molecules organized into crystallinelike structures. These cholesterol domains disrupt cellular
function, influence drug access to membrane receptors, and
serve as potential nucleating sites for the formation of
extracellular crystals, a hallmark feature of the advanced
plaque. Pharmacological agents that interfere with the stability of membrane cholesterol microdomains may have therapeutic benefit by facilitating normal removal of cholesterol
by reverse transport mechanisms. Collectively, these findings
support an emerging model of the cell plasma membrane that
is remarkably complex with respect to function, structure, and
molecular organization.
Acknowledgments
This work was supported by the National Heart, Lung, and Blood
Institute of the National Institutes of Health (NIH), the American
Heart Association, and the National Eye Institute of the NIH.
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
References
1. Brown DA, London E. Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J Biol Chem. 2000;275:17221–17224.
2. Smart EJ, Graf GA, McNiven MA, et al. Caveolins, liquid-ordered
domains, and signal transduction. Mol Cell Biol. 1999;19:7289 –7304.
3. Feron O, Belhassen L, Kobzik L, et al. Endothelial nitric oxide synthase
targeting to caveolae. J Biol Chem. 1996;271:22810 –22814.
4. 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.
5. Simons K, Ehehalt R. Cholesterol, lipid rafts, and disease. J Clin Invest.
2002;110:597– 603.
6. Feron O, Dessy C, Desager JP, et al. Hydroxy-methyglutaryl-coenzyme A
reductase inhibition promotes endothelial nitric oxide synthase activation
through a decrease in caveolin abundance. Circulation. 2001;103:
113–118.
7. Harrison DG. Cellular and molecular mechanisms of endothelial cell
dysfunction. J Clin Invest. 1997;100:2153–2157.
8. Tulenko TN, Chen M, Mason PE, et al. Physical effects of cholesterol on
arterial smooth muscle membranes: evidence of immiscible cholesterol
Membrane Lipid Rafts and Atherogenesis
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
2273
domains and alterations in bilayer width during atherogenesis. J Lipid
Res. 1998;39:947–956.
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.
Craven BM. Crystal structure of cholesterol monohydrate. Nature. 1976;
260:727–729.
Phillips JE, Geng YJ, Mason RP. 7-Ketocholesterol forms crystallin
domains in model membranes and murine aortic smooth muscle cells.
Atherosclerosis. 2001;159:125–135.
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.
Vergani L, Hatrik S, Ricci F, et al. Effect of native and oxidized lowdensity lipoprotein on endothelial nitric oxide and superoxide production:
key role of L-arginine availability. Circulation. 2000;101:1261–1266.
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.
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.
Small DM. Progression and regression of atherosclerotic lesions. Arteriosclerosis. 1988;8:103–129.
Libby P. Molecular bases of the acute coronary syndromes. Circulation.
1995;91:2844 –2850.
Mason RP, Rhodes DG, Herbette LG. Reevaluating equilibrium and
kinetic binding parameters for lipophilic drugs based on a structural
model for drug interaction with biological membranes. J Med Chem.
1991;34:869 – 877.
Mason RP. Membrane interaction of calcium channel antagonists modulated by cholesterol: implications for drug activity. Biochem Pharmacol.
1993;45:2173–2183.
Mason RP. Mechanisms of plaque stabilization for the dihydropyridine
calcium channel blocker amlodipine: review of the evidence. Atherosclerosis. 2002;165:191–199.
Pitt B, Byington RP, Furberg CD, et al. Effect of amlodipine on the
progression of atherosclerosis and the occurrence of clinical events.
Circulation. 2000;102:1503–1510.
Borhani NO, Mercuri M, Borhani PA, et al. Final outcome results of the
Multicenter Isradipine Diuretic Atherosclerosis Study (MIDAS): a randomized controlled trial. JAMA. 1996;276:785–791.
KEY WORDS: atherosclerosis
䡲 vasculature
䡲
cholesterol
䡲
lipids
䡲
pharmacology
Membrane Microdomains and Vascular Biology: Emerging Role in Atherogenesis
R. Preston Mason and Robert F. Jacob
Circulation. 2003;107:2270-2273
doi: 10.1161/01.CIR.0000062607.02451.B6
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2003 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7322. Online ISSN: 1524-4539
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circ.ahajournals.org/content/107/17/2270
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial
Office. Once the online version of the published article for which permission is being requested is located,
click Request Permissions in the middle column of the Web page under Services. Further information about
this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation is online at:
http://circ.ahajournals.org//subscriptions/