COMMENTARIES

An Odyssey of Plaque to Stroke: A Lipid Perspective

Rao Muralikrishna Adibhatla¹-⁴ and James F. Hatcher¹, ¹Department of Neurological Surgery, ²Cardiovascular Research Center, ³Neuroscience Training Program, University of Wisconsin, Madison, WI; ⁴Veterans Administration Hospital, Madison, WI Please 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
Tel: (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-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 an 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 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 in the Future?

Specific antibody treatment holds promise for reducing onset and progression of atherosclerosis. Mice immunized against phosphorylcholine showed a 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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