AN ODYSSEY OF PLAQUE TO STROKE: A LIPID PERSPECTIVE

Rao Muralikrishna Adibhatla 1-4* and James F. Hatcher 1

1
Department of Neurological Surgery, 2 Cardiovascular Research Center, 3 Neuroscience Training Program;
University of Wisconsin, Madison, WI; 4 Veterans Administration Hospital, Madison, WI

Address correspondence to: Dr. Rao Muralikrishna Adibhatla, Department of Neurological Surgery, H4-
330, Clinical Science Center, 600 Highland Avenue, University of Wisconsin-Madison, Madison, WI 53792-
3232, Phone: 608-263-1791, Fax: 608-263-1409, Email: adibhatl@neurosurg.wisc.edu

Stroke or “brain attack”: a problem of vast clinical significance

Stroke results from interruption of blood flow to a region of the brain, which severely impairs
the energy supply. The majority of strokes result from either thrombolic or embolic occlusion of
primarily the middle cerebral artery. Thrombolic stroke results from the formation of a clot or
thrombus in a cerebral artery that blocks blood flow at the site of formation. Embolic stroke occurs
when a cerebral artery is blocked by a clot that formed elsewhere and was carried to the brain through
the circulation.
Inflammation poses as one the high risk factors in the development and rupture of
atherosclerotic plaques, one of the primary causes of ischemic stroke [1, 2] and also aggravates brain
injury after stroke [3]. Inflammation is the first response of the immune system to infection or
irritation. Cytokines are low molecular weight, soluble proteins that are produced and serve as
chemical messengers for regulating the innate and adaptive immune systems.
Atherosclerosis is a risk factor for stroke
Atherosclerosis is defined by the accumulation in the intima of medium to large arteries of
mainly LDL-derived lipids along with apolipoprotein B-100 (apoB100), resulting in plaque formation and
disturbance of blood flow. LDL is the major carrier of cholesterol in the circulation and is composed of
one apoB100 together with phosphatidylcholine (PC), sphingomyelin and unesterified cholesterol
(500:200:400 molecules respectively) constituting a surface film surrounding a core of cholesteryl
esters and triacylglycerols [4].
A complex endothelial injury and dysfunction induced by a variety of factors such as
homocysteine, toxins (smoking), mechanical forces (shear stress), infectious agents (such as Chlamydia
pneumoniae) and oxidized LDL results in an inflammatory response that is instrumental in the
formation and rupture of atherosclerotic plaques [1, 2]. Increased levels of TNF-α and interleukin-1 up-

1
regulate expression of adhesion molecules and promote monocyte recruitment into developing
atherosclerotic lesions. Expression of macrophage matrix metalloproteinase 9 (MMP-9) degrades
extracellular matrix components including the fibrous cap of atheromatous plaques, leading to plaque
rupture.
Two critical events involved in atherogenesis involve accumulation and oxidation of LDL in the
arterial intima and recruitment of monocytes to the developing lesion. After diffusion through the
endothelial cell junctions into the arterial intima, LDL can be retained through interaction of apoB100
and matrix proteoglycans. While the exact mechanisms governing LDL accumulation remain to be
elucidated [5], evidence indicates that LDL uptake and retention are increased at plaque sites, which
may involve degradation or binding to cellular and matrix components. Once in the arterial intima, LDL
can be oxidized to OxLDL through oxidation of polyunsaturated fatty acids of LDL lipids, particularly PC
[6, 7].
A second critical event in atherosclerosis is an inflammatory response that triggers expression
of adhesion molecules in the arterial endothelium, stimulating adhesion of monocytes to the
endothelium. Monocytes penetrate into the arterial intima, differentiate into macrophages and
eventually become foam cells by binding and endocytosing OxLDL through CD36 scavenging receptors.
Oxidized phospholipids bearing the PC headgroup as a ligand on OxLDL [8] mediate uptake by
macrophage scavenging receptors such as CD36 [9]. Absence of CD36 protected against atherosclerosis
in ApoE null mice; however no additional benefit was observed in CD36/scavenger receptor A dual
knockout mice [10]. The macrophage foam cells generate reactive oxygen species (ROS), produce TNF-
α and interleukin-1, and MMP-9 that promote atherosclerosis, degrade the fibrous cap, and eventually
lead to plaque rupture [4]. Plaque rupture triggers formation of a blood clot, which on destabilization
releases an embolus into the blood stream, which can lodge in a cerebral artery and induce an
ischemic stroke [11-13]. Atherosclerotic carotid plaques from patients symptomatic of stroke had
higher expression of OxLDL, lipoprotein-phospholipase A2 (Lp-PLA2)/PAF acetylhydrolase, lyso-PC,
macrophage content and MMP-2 compared to carotid plaques from asymptomatic patients [14].
ROS and lipid peroxidation: Oxidized PC (OxPC) and 4-hydroxynonenal (HNE) as inflammatory
markers.
Atherosclerosis or cardiomyopathy is associated with increased production of ROS in
mitochondria and respiratory chain dysfunction [15, 16]. Dysfunctional mitochondria can lead to
impaired vascular cell growth, function and apoptosis leading to plaque rupture. ROS-mediated
peroxidation of fatty acids in phospholipids results in an oxidized phospholipid such as OxPC [17] with a
fatty acid containing an aldehyde residue, and an aldehyde byproduct such as HNE. These reactive
aldehydes exhibit cytotoxicity by binding to cellular proteins. The presence of OxPC on the apoptotic
cell surface has been characterized by EO6 monoclonal antibodies that exclusively bind to OxPC and
OxPC-protein adducts [18]. OxPC may enhance pro-inflammatory signals and also serve as a marker of
inflammation and apoptosis [17, 19, 20]. HNE can also serve as inflammatory marker and contributes
to foam cell formation through increased expression of scavenging receptors [21]. Formation of HNE
and OxPC were also demonstrated in animal models of stroke [22, 23].
Lipoprotein-PLA2 (Lp-PLA2)/platelet activating factor (PAF) acetylhydrolase
Lp-PLA2, also known as plasma PAF acetylhydrolase [24], is found in blood circulation and is
associated with apoB-100 of LDL and atherosclerotic plaques [25]. Higher levels of Lp-PLA2 correlated
with coronary heart disease, stroke and dementia [14, 26]. The enzyme is best known for its PAF
acetylhydrolase activity and also hydrolyzes oxidized phospholipids such as OxPC of LDL to generate
2
oxidized fatty acids and lyso-PC [25]. Local coronary lyso-PC formation is associated with endothelial
dysfunction and supports the role of Lp-PLA2 in vascular inflammation and atherosclerosis in humans
[27]. Lp-PLA2 also has an anti-inflammatory function arising through hydrolysis of PAF.
Atherosclerosis, stroke and group IIA secretory PLA2 (sPLA2)
sPLA2, also known as inflammatory PLA2, has been found in human atherosclerotic lesions [28]
and is implicated in atherosclerosis [29-31] and stroke [32, 33]. It has been suggested that sPLA2 IIA
regulates collagen deposition in the plaque and fibrotic cap development [29, 34]. sPLA2 also releases
lyso-PC via its catalytic action and these two play a crucial role in the development of atherosclerosis
[30]. sPLA2 IIA is induced by TNF-α and IL-1, after stroke and plays a critical role in the pathogenesis of
CNS injuries and disorders [32].
Sphingomyelinase activity of LDL: A link between atherosclerosis and ceramide
Sphingomyelinase activity (hydrolyzes sphingomyelin to ceramide) is present in LDL and may be
intrinsic to apoB-100. Ceramide is elevated in atherosclerotic plaques as well as in LDL isolated from
these lesions. Ceramide plays an important role in aggregation of LDL within the arterial wall, a critical
step in the initiation of atherosclerosis [35].
What is awaiting the future?
Specific antibody treatment holds promise for reducing onset and progression of
atherosclerosis. Mice immunized against phosphorylcholine showed 3-fold increase in anti-
phosphorylcholine and -OxLDL antibodies compared with controls. The extent of atherosclerosis was
reduced by >40% in phosphorylcholine immunized mice and serum from these immunized mice also
reduced macrophage-derived foam cell formation in the presence of OxLDL in vitro [36] suggesting
these immunizations drive a specific immune response that reduces foam cell formation in vitro and
atherosclerosis in vivo. As always, with all this excitement a word of caution is lurking: some OxLDL
antibodies had exactly opposite effect of increasing the uptake of OxLDL by macrophages and
accelerating atherosclerosis in mouse model [37]. Auto-antibodies against OxLDL antibodies may block
their protective effects [38].
Acknowledgements
This work was supported by grants from NIH/NINDS (NS42008), American Heart Association
Greater Midwest Affiliate Grant-in-Aid (0655757Z), UW-School of Medicine and Public Health Research
Committee (161-PRJ13MX), UW-School of Medicine and Public Health, UW-Graduate school and UW-
Neurological Surgery Department and laboratory resources provided by William S. Middleton VA
Hospital (to RMA).

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