No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Oxidized Low-Density Lipoprotein Is Present in Astrocytes Surrounding Cerebral Infarcts and Stimulates Astrocyte Interleukin-6 Secretion
Abstract
Ischemic injury to brain is associated with both disruption of the blood-brain barrier and increased oxidative stress. Given the neurotoxicity associated with exposure to oxidized low-density lipoprotein (oxLDL) in vitro, we tested the hypothesis that oxLDL may be present in parenchymal cells of cerebrum after infarction and that oxLDL may influence the pathophysiology of cerebral infarction. Our results showed that the subacute phase of cerebral infarction in patients was characterized by the appearance of oxLDL epitopes in astrocytes, but not neurons or microglia, in the perinecrotic zone. We further demonstrated that minimally oxLDL was most effectively internalized by primary cultures of rat astrocytes, and that exposure to minimal oxLDL stimulated astrocyte interleukin-6 secretion but did not alter nitric oxide production. These results demonstrate for the first time that oxLDL is present in brain parenchyma of patients with ischemic infarction and suggest a potential mechanism by which oxLDL may activate innate immunity and thereby indirectly influence neuronal survival.
... The inconsistent results might be explained by differences in sample size, patient selection, potential confounder, outcome assessment, and different measurement times of LDL-C. Several studies suggested that LDL got oxidized into oxLDL under oxidative stress [19], and oxLDL may contribute to exacerbate free-radical damage in the acute phase of AIS [20,[32][33][34][35]. Since AIS patients with reperfusion therapy could suffer from enhanced oxidative injury [12], focusing on these patients might clarify the role of LDL-C. ...
... Though the underlying mechanism of the association between ΔLDL-C and outcome remains unclear, it could be explained as follows: LDL-C gets oxidized into oxLDL under oxidative stress [19] and oxLDL is the major marker of oxidative stress [20,[32][33][34]. Previous studies found that high oxLDL is positively associated with poor functional outcomes in AIS patients [21][22][23]. ...
... Therefore, we speculated that the increased oxLDL level may be associated with decreased LDL-C level during hospitalization, the extent of decreased LDL-C may reflect the degree of increased oxLDL, which may indicate the severity of oxidative stress and contribute to poor functional outcomes. Although the specific mechanism of ΔLDL-C level during hospitalization remains unclear, during the oxidative challenge, LDL-C gets oxidized into oxLDL [19], which contributes to free-radical damage [20,[32][33][34][35] and poor outcome [21][22][23]. ...
Background
Low-density lipoprotein cholesterol (LDL-C) can increase cardiovascular risk. However, the association between LDL-C change and functional outcomes in acute ischemic stroke (AIS) patients who underwent reperfusion therapy remains unclear.
Methods
Patients who received reperfusion therapy were consecutively enrolled. LDL-C measurement was conducted at the emergency department immediately after admission and during hospitalization. The change of LDL-C level (ΔLDL-C) was calculated by subtracting the lowest LDL-C among all measurements during hospitalization from the admission LDL-C. Poor functional outcome was defined as modified Rankin Scale (mRS) > 2 at 90 days.
Results
A total of 432 patients were enrolled (mean age 69.2 ± 13.5 years, 54.6 % males). The mean LDL-C level at admission was 2.55 ± 0.93 mmol/L. The median ΔLDL-C level was 0.43 mmol/L (IQR 0.08–0.94 mmol/L). A total of 263 (60.9 %) patients had poor functional outcomes at 90 days. There was no significant association between admission LDL-C level and functional outcome (OR 0.99, 95 % CI 0.77–1.27, p = 0.904). ΔLDL-C level was positively associated with poor functional outcome (OR 1.80, 95 % CI 1,12-2.91, p = 0.016). When patients were divided into tertiles according to ΔLDL-C, those in the upper tertile (T3, 0.80–3.98 mmol/L) were positively associated with poor functional outcomes compared to patients in the lower tertile (T1, -0.91-0.13 mmol/L) (OR 2.56, 95 % CI 1.22–5.36, p = 0.013). The risk of poor functional outcome increased significantly with ΔLDL-C tertile ( P -trend = 0.010).
Conclusions
In AIS patients who underwent reperfusion therapy, the decrease in LDL-C level during hospitalization was significantly associated with poor functional outcomes at 90 days.
... The ischemic brain lesion might be the major source of increased IL-6 since, after stroke, IL-6 levels are greater in CSF than in serum [30]. Furthermore, it has been proved that microglia cells [36], neurons [37] and astrocytes [38,39] are able to produce IL-6. ...
... IL-6 is produced by T cells, B cells, monocytes, macrophages, fibroblasts, endothelial cells [46], microglial cells [36], neurons [46], and astrocytes [38,39]. A variety of proteins, such as IL-1 and TNF-α, enhance IL-6 production [46]. ...
The lack of a rapid and clinically accurate diagnostic tool remains a major obstacle to optimal care of patients with stroke. Cytokine changes in patients with acute stroke have been insufficiently studied. The purpose of this study is to delineate the relevance of IL-6 as a biochemical marker of stroke diagnosis, taking into account the genetic basis, and changes of the protein in serum and cerebrospinal fluid in relation to stroke development. Inflammation has an important role in ischemic cerebrovascular disease pathophysiology. Proinflammatory cytokines, such as IL-6, have been implicated in several mechanisms that might promote ischemic brain injury and an early neurological worsening. Cardiovascular diseases constitute one of the principal health problems in developing countries. Over the past few years, several studies have found evidence of the important role of inflammation in the ischemic cerebrovascular disease. The availability of a diagnostic biomarker panel for patients with stroke symptoms would be enormously valuable to complement clinical data and to precede radiological findings. IL-6 levels in cerebrospinal fluid and serum seem to reflect either the extent of tissue damage, or the accompanying clinical worsening. The -174 G/C functional polymorphism in the IL-6 gene might not be solely involved in disease susceptibility but also in linkage disequilibrium with other functional polymorphisms. Further studies are needed to solve this. Presently, the association between IL-6 genotype and stroke remains undetermined. Development of new neuroprotective therapies targeted to modulate cytokine-induced inflammation could be a promising way to prevent early deterioration in acute ischemic stroke.
... Clinical reports reveal that the patients with Alzheimer's disease exhibit an increased oxidation of lipoproteins potentially toxic to neurons in CNS (Montine et al. 1997). Furthermore, in patients with cerebral infarction, oxLDL is present in brain parenchyma and stimulates astrocytes to secrete interleukin-6 ( Shie et al. 2004), and may serve as an indicator to reflect the level of oxidative stress (Uno et al. 2005). However, little is known about the regulation of MMP-9 in astrocytes exposed to oxLDL, which plays a critical role in the progression of inflammatory diseases and remodeling processes in target tissues, including the CNS. ...
... In the CNS, oxLDL exhibits detrimental effects on brain cell functions, including induction of apoptosis, disruption of capillary homeostasis, and alteration of inflammatory protein activity in various brain cells (Keller et al. 1999;Kim et al. 2004;Lupo et al. 2005). It has been demonstrated that oxLDL is present in brain parenchyma after ischemic infarction (Shie et al. 2004). Moreover, oxLDL could induce MMP-9 expression in human THP-1 macrophages via a MEK/ERK pathway (Kang et al. 2007). ...
After ischemic injury to brain, disruption of the blood-brain barrier (BBB) raises the possibility of exposing the central nervous system (CNS) to oxidized low-density lipoprotein (oxLDL), a risk factor implicated in neurodegenerative diseases. Matrix metalloproteinases (MMPs), especially MMP-9, contribute to extracellular matrix (ECM) remodeling during the CNS diseases. However, the molecular mechanisms underlying oxLDL-induced MMP-9 expression in astrocytes remained unclear. Here, we reported that oxLDL induced MMP-9 expression via a PKC-delta/p42/p44 MAPK-dependent Elk-1 activation in rat brain astrocyte (RBA)-1 cells, revealed by gelatin zymography, RT-PCR, and Western blotting analyses. These responses were attenuated by pretreatment with pharmacological inhibitors and transfection with dominant negative mutants. Moreover, Elk-1-mediated MMP-9 gene transcription was confirmed by transfection with an Elk-1 binding site-mutated MMP-9 promoter construct (mt-Ets-MMP9), which blocked oxLDL-stimulated MMP-9 luciferase activity. Understanding the regulatory mechanisms by which oxLDL induced MMP-9 expression in astrocytes might provide a new therapeutic strategy of brain diseases.
... However, it has not been established whether CD36 contributes to injury in brain diseases associated with inflammation . Cerebral ischemia produces several CD36 ligands, including long-chain fatty acids, A, oxidized LDL, and thrombospondin-1 (Nihashi et al., 2001;Hayashi et al., 2003;Pilitsis et al., 2003;Uno et al., 2003;Shie et al., 2004). It is therefore conceivable that this receptor participates in the inflammatory response associated with brain ischemia and contributes to its deleterious effects. ...
... The ligands that interact with CD36 during ischemia-reperfusion and the signaling pathways downstream of CD36 remain to be defined. Some CD36 ligands, such as thrombospondin-1, oxidized LDL, A, and long-chain fatty acids, are produced in cerebral ischemic injury (Hayashi et al., 2003;Pilitsis et al., 2003;Uno et al., 2003;Shie et al., 2004). One or more of these factors produced during cerebral ischemia could activate CD36 and initiate subsequent signaling, leading to ROS production. ...
The class B scavenger receptor CD36 is involved in the cytotoxicity associated with inflammation, but its role in the inflammatory reaction that accompanies cerebral ischemia has not been examined. In this study, we investigated whether CD36 contributes to the brain damage produced by cerebral ischemia. The middle cerebral artery was transiently occluded in wild-type mice and in mice deficient in CD36. In wild-type mice, CD36 protein expression was increased in the ischemic brain, such that it was located predominantly in cells expressing the microglia/macrophage marker CD11b. The infarct produced by middle cerebral artery occlusion was 49% smaller in CD36-null mice than in wild-type controls, an effect associated with improved neurological function. The attenuation in brain injury in CD36 nulls could not be attributed to differences in cerebral blood flow during ischemia-reperfusion. However, the increase in reactive oxygen species (ROS) produced by cerebral ischemia was markedly attenuated in CD36-null mice in the early stage after reperfusion. The data unveil a previously unrecognized role of CD36 in ischemia-induced ROS production and brain injury. Modulation of CD36 signaling may provide a new strategy for the treatment of ischemic stroke.
... Ischemic brain injury is clearly associated with a disruption of the blood-brain barrier (8) raising the possibility of exposing CNS to plasma lipoproteins. The significant increase in levels of oxidized LDL (oxLDL) in plasma of patients supports a role of lipoprotein peroxidation in stroke (9,10). ...
... The physiological roles of lipoproteins in the human CNS are not completely elucidated. However, several studies have suggested a role of oxidized lipoproteins in the development of neurodegenerative diseases (10,30). The disruption of the blood-brain barrier after ischemic brain injury raises the possibility of exposing CNS to oxidized plasma lipoproteins (8). ...
Paraoxonase is high-density lipoprotein (HDL)-associated esterase/lactonase implicated to play a role in the antioxidant and anti-inflammatory properties exerted by HDL. Increasing evidence support a role of free radicals and oxidative stress in neuronal damage induced by ischemia-reperfusion. The aim of this study was to further investigate the relationship between lipoprotein oxidative damage and stroke.
We compared the paraoxonase activity and levels of lipid hydroperoxides in plasma isolated from healthy subjects (n=50) and from stroke patients (n=49). Moreover, the correlations between biochemical markers and the National Institute of Health Stroke Scale (NIHSS), which is widely used to study neurological severity, were evaluated.
Our results demonstrated, for the first time, that the activity of paraoxonase in plasma of stroke subjects was significantly lower than controls (p<0.001) and the levels of lipid hydroperoxides were significantly higher in plasma from patients (p<0.001). Moreover, using linear regression analysis, significant correlations between the activity of paraoxonase, lipid peroxidation and the severity of neurological deficit at admission were observed.
These results provide further evidence that oxidative stress and impairment of the antioxidant system may play a role in stroke. Antioxidant activity of plasma may be an important factor providing protection from neurological damage caused by stroke-associated oxidative stress.
... Moreover, hyperlipidemia exacerbates ischemic stroke outcomes via stimulating the expression of CD36 in the post-ischemic brain and peripheral macrophages [133]. FAT/CD36 and its ligands, including oxLDL, NEFA, Aβ, and thrombospondin-1, become available during ischemic stroke [134,135]. Infiltration of peripheral immune cells is the primary source of FAT/CD36 in the post-ischemic brain and contributes to stroke-induced brain damage [136,137]. Knockout of FAT/CD36 in mice decreased microglial activation and neutrophil infiltration associated with cerebral ischemia [13]. ...
The dysregulation of fat metabolism is involved in various disorders, including neurodegenerative, cardiovascular, and cancers. The uptake of long-chain fatty acids (LCFAs) with 14 or more carbons plays a pivotal role in cellular metabolic homeostasis. Therefore, the uptake and metabolism of LCFAs must constantly be in tune with the cellular, metabolic, and structural requirements of cells. Many metabolic diseases are thought to be driven by the abnormal flow of fatty acids either from the dietary origin and/or released from adipose stores. Cellular uptake and intracellular trafficking of fatty acids are facilitated ubiquitously with unique combinations of fatty acid transport proteins and cytoplasmic fatty acid-binding proteins in every tissue. Extensive data are emerging on the defective transporters and metabolism of LCFAs and their clinical implications. Uptake and metabolism of LCFAs are crucial for the brain's functional development and cardiovascular health and maintenance. In addition, data suggest fatty acid metabolic transporter can normalize activated inflammatory response by reprogramming lipid metabolism in cancers.
Here we review the current understanding of how LCFAs and their proteins contribute to the pathophysiology of three crucial diseases and the mechanisms involved in the processes.
... Oxidative modi ication of lowdensity lipoprotein (LDL) is thought to contribute to the production of oxygen derivedfree radicals [110]. Oxidative LDL (Ox-LDL) may be important in neurotoxicity in the brain [111]. It is well known that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor reduce the oxidizability of LDL [112]. ...
... Preliminary studies suggest that caveolae are likely to be involved in the potential transport of LDL from the blood to the brain. It has been demonstrated that oxidized LDL is present in brain parenchyma of patients with ischemic infarction and suggests a potential mechanism by which oxidized LDL may activate innate immunity and thereby indirectly influence neuronal survival (Shie et al., 2004). ...
PATHOPHYSIOLOGY OF ISCHEMIC DISORDERS: I- LDL CHOLESTEROL
AND ISCHEMIC STROKE
Nazish Iqbal Khan and Zahir Hussain*
Department of Physiology, University of Karachi, Karachi-75270, Pakistan.*E-Mail: zahussai@yahoo.ca
ABSTRACT
Blood lipids, particularly total and LDL cholesterol levels, are associated with all subtypes of brain infarction.
Furthermore, many of the effects of elevated or modified low density lipoproteins on endothelial cells and endothelial cell processes could be expected to contribute to the development of atherosclerosis and therefore, to the association between lipids and atherosclerotic, particularly coronary and cerebrovascular disease. However, the extent to which “endothelial dysfunction” accounts for the known relationships between serum lipid concentrations, ischemic disease and endothelial progenitor cell (EPC) pathophysiology is yet to be established. On the basis of the mentioned and other reports, we hypothesize that higher levels of plasma LDL cholesterol not only directly impair endothelial cells, but also effect EPC number and function at the same time, thus influencing the endothelial repair process and disturbing the balance between the magnitude of injury and the capacity for repair, which leads to endothelial dysfunction and promotes the progression of stroke. Because EPCs have been implicated in various events requiring endothelialization, we further hypothesized that LDL-C could influence the action of EPCs and sensory neurons and that this could be important to know how LDL-C effects repair, injury and vasculoprotection . In order to corroborate this proposal, quantification is required for EPCs and DRG neurons treated with various concentrations of LDL-C and characterizing the underlying molecular mechanisms. The relative contributions of apoptotic and necrotic death to ischemia-induced neuronal loss may provide us information for understanding the underlying mechanisms. Conclusively, the aggressive management of risk factors, like cholesterol,
could have a significant and positive impact on the natural history of atherosclerotic cerebrovascular disease.
Key words: LDL cholesterol, ischemic stroke, pathophysiology
... Ischemic stroke induces the production of CD36 as well as its ligands, including oxLDL, long-chain fatty acids, Aβ and thrombospondin-1 [127,128]. Hyperlipidemia exacerbates ischemic stroke outcomes via stimulating the expression of CD36 in the post-ischemic brain as well as in peripheral macrophages [70]. In addition, CD36 facilitates the production of free radical production during the post-ischemic phase [128]. ...
Polyunsaturated fatty acids are required for the brain development and significantly impact aging and stroke. Due to the hydrophobicity of fatty acids, fatty acids transportation related proteins that include fatty acid binding proteins (FABPs), long chain acyl-coA synthase (ACS), fatty acid transportation proteins (FATPs), fatty acid translocase (FAT/CD36) and newly reported major facilitator superfamily domain-containing protein (Mfsd2a) play critical roles in the uptake of various fatty acids, especially polyunsaturated fatty acids. They are not only involved in neurodevelopment, but also have great impact on neurological disease, such as aging related dementia and stroke.
... During trauma, if LA was not metabolised by the peroxisomes, it would logically be available for oxidation to oxylipins including by the LOX12/15 enzyme, of which ALA followed by LA is the preferred substrate. Further astrocytes in ischaemia may take up plasma-oxidised LDL [97]. ...
Current metabolic considerations of “fuel” sources for brain energy, and substrate creation, generally focus on glucose and externally derived ketones. However, the healthy existence of Inuit, with a common Inuit CPT1A carnitine polymorphism that substantially inhibits mitochondrial uptake of long fats, who apparently were not in ketosis, with little access to glucose, suggests humans can fuel their brains largely from peroxisomally produced medium-chain fats (MCFs), ACoA and derivatives. The health, and ability to build and run a brain, of neonates nourished with breast milk, which is high in lipids and low in carbohydrates, whom are very rarely in significant measured ketosis, adds to evidence the brain can metabolise fats as a major energy source. Whilst brain lipid research primarily focuses on arachidonic (AA) and docosahexaenoic acids (DHA); alpha-linolenic (ALA) and linolenic acids (LA), as preferred peroxisomal beta-oxidation substrates, and to lesser extents palmitic (PA) and oleic acids (OA), likely have underappreciated but fundamental roles in the brain as the primary substrates for peroxisomal beta-oxidation, so indirect sources of MCFs, ACoA and downstream-derivatives, mitochondrial “fuels” for ATP production. Alternatively ACoA is a substrate for “endogenous” lipid manufacture within the BBB, and co-peroxisomal-product peroxide acts as a signalling agent. LA and ALA cross the blood-brain-barrier (BBB), but are not significantly present in brain structural-tissue, likely being largely metabolised through peroxisomal pathways in astrocytes to substrate or energy. LA and ALA also have wider brain roles, including the following: LA-/ALA-oxidised products in injured brain tissue moderate immune function; as preferred substrates for LOX12/15; LA oxylipins the HODEs are the primary endogenous activators of PPAR gamma related peroxisomal activity; PPAR gamma and peroxide promote iNOS activity; iNOS-based NO production inhibits catalase assisting microglial oxidative function, in excess causing oxidative damage; further PPAR gamma moderates microglial function. LA oxylipin 13HODE overactivation of the PPAR gamma-related peroxisomal pathways results in imbalances in brain lipid composition; loss of LA; increased denovo lipid and cholesterol production; increased desaturation by SCD1 so increased mono- and polyunsaturated Omega-7 and Omega-9 fats including mead acid; intracellular lipid deposition including of cholesterol, increased oxidative stress; cardiolipin lipid species changes and imbalances; and mitochondrial dysfunction; which changes link to diseases of cognitive impairment, including depression and Alzheimer’s. Further changes in the LA, OA and PA desaturase products, so the lipid-membrane content including of cardiolipin, will change mitochondrial energetics. Exposure to LA oxylipins in the absence of sufficient lipid protective antioxidant capacity, makes mitochondria more susceptible to damage, including reduced cytochrome C-related ATP production, and results in release from cardiolipin of damaging LA-based oxylipins, including HODEs and 4HNE. Conversely ALA has surprising beneficial effects on brain function. A single “subchronic” injection of ALA into the bloodstream before induction of a stroke in mice reduced post-infarct ischaemic damage. Multiple pre-stroke ALA treatments improved survival by a factor of 3 at ten days, increased neurogenesis, enhanced brain plasticity, and were significantly antidepressant. The first casualties of nutrient-depleted pre-oxidised Omega-3:6 imbalanced diets are likely loss of IQ, abstract thought and crucially empathy, and arguably accompanied by increased aggression and territoriality. What is the future for individuals, nations and more widely humanity, if increasing numbers of humans are more aggressive and territorial, have falling IQs and depleted capacity for abstract thought and empathy? ‘The greatness of humanity is not being human but humane’ Gandhi.
... LysoPC has also been demonstrated to be a mediator of inflammatory stress on brain microvascular endothelial cells [7] and to increase the permeability of endothelial cells [8]. Literature supports that oxLDL can be detected in the central nervous system (CNS) after blood-brain barrier (BBB) disruption [9]. ...
Background
The lipoprotein-associated phospholipase A2 inhibitor (Lp-PLA2), rilapladib (SB659032), is being evaluated as a potential treatment to slow the progression of Alzheimer's disease (AD).
Methods
One hundred twenty-four subjects with possible mild AD and with neuroimaging evidence of cerebrovascular disease were randomized to placebo or 250-mg rilapladib once daily, for 24 weeks, in addition to stable background acetylcholinesterase inhibitor and/or memantine. The study assessed the safety and tolerability of rilapladib and its effects on cognition, mechanistic, and disease-related biomarkers. Although the overall intent behind the study was to take a broad exploratory view of the data, two primary end points of interest (cerebrospinal fluid [CSF] amyloid beta peptide 1–42 [Aβ1–42] and CogState executive function/working memory [EF/WM] composite score at week 24) were prespecified in the analysis plan for inferential statistical analysis.
Results
Rilapladib was well tolerated with no significant safety concerns. A significant difference from placebo was observed for rilapladib on change from baseline in EF/WM (effect size, 0.45; P = .026). There was no significant difference between groups on the change from baseline in CSF Aβ1–42 (P = .133). Preliminary evidence of effects was detected on other mechanistic (albumin quotient) and disease-related biomarkers (tau/P-tau and neurofilament light chain).
Conclusion
These data provide initial evidence supporting Lp-PLA2 inhibition as a novel treatment for dementia.
Clinical Trial Registration
Clinicaltrials.gov identifier: NCT01428453.
... Aging and visceral fat accumulation play important roles in LDL atherogenicity including increased LDL oxidation and smaller LDL particle size, and oxidative stress activates inflammation and cytokine expression (Paik et al. 2012a, b;Chae et al. 2011;Hyun et al. 2008). Ox-LDL modifies IL-6 secretion (Shie et al. 2004;Shi et al. 2007) and stimulates Lp-PLA 2 expression in monocytes through the phosphatidylinositol 3-kinase and p38 mitogen-activating protein kinase pathways (Wang et al. 2010). Lp-PLA 2 is preferentially associated with small dense LDL (Tsimihodimos et al. 2002) and cleaves the oxidized phosphatidylcholine contained in the ox-LDL into two inflammatory mediators: lysoPC and oxidized free fatty acids (Macphee et al. 1999). ...
Controversy remains regarding whether there is an association between circulating lipoprotein-associated phospholipase A2 (Lp-PLA2), cytokines, and oxidative stress in healthy postmenopausal women. We investigated the influence of age on Lp-PLA2 activity in postmenopausal women not using hormone therapy and the relationship of Lp-PLA2 enzyme activity to serum cytokine levels and oxidative stress indices. Normal weight (n = 1284) and overweight/obese (n = 707) postmenopausal women not using hormone therapy were categorized into five age groups: 50-54, 55-59, 60-64, 65-69, and 70-89 years. Overweight-obese women showed higher plasma Lp-PLA2 activity, urinary 8-epi-prostaglandin F2α (8-epi-PGF2α), serum interleukin (IL)-6, and smaller LDL particles than normal-weight women after adjusting for age, years postmenopause, smoking, drinking, blood pressure, glucose, insulin, lipid profiles, BMI, and waist circumference. Overweight/obese women 70-89 years old showed higher Lp-PLA2 activity than those aged 50-54 years, whereas no significant difference in Lp-PLA2 activity existed across normal-weight female age groups. Overweight/obese women aged ≥65 years showed higher Lp-PLA2, oxidized LDL (ox-LDL), IL-6, and 8-epi-PGF2α than age-matched normal-weight controls. Overweight/obese women aged ≥70 years had higher ox-LDL levels than those aged 50-59, and overweight/obese women aged 65-89 showed higher IL-6 and 8-epi-PGF2α. There were strong positive correlations between Lp-PLA2 and ox-LDL (r = 0.385, P < 0.001), Lp-PLA2 and IL-6 (r = 0.293, P < 0.001), and ox-LDL and IL-6 (r = 0.303, P < 0.001) in overweight/obese women; however, these relationships were weak in normal-weight women. These results suggest that aging and obesity-related oxidative and inflammatory mediators are associated with Lp-PLA2 activity in overweight/obese postmenopausal women not using hormone therapy.
... CD36 expression occurs in a feed-forward manner in the presence of ligands [34][35][36]. With increased generation of CD36 ligands including long-chain fatty acids, oxidized low-density lipoprotein (oxLDL), and thrombospondins in stroke [37][38][39][40][41], CD36 elicits intracellular signaling and produces inflammatory cytokines and chemokines [17,[42][43][44]. Among them, monocyte chemoattractant protein-1 (MCP-1) and CCR2, a receptor for MCP-1, play an important role in attracting peripheral immune cells into the injury site [45][46][47][48]. ...
AimsThe pathology of stroke consists of multiple pro-death processes, and CD36 has been suggested as a multimodal target to reduce oxidative stress and inflammation in ischemic stroke. Using CD36-deficient mice and SS-31, a cell permeable tetrapeptide known to down-regulate CD36 pathways, the current study investigated whether targeting CD36 is effective in transient and permanent ischemic stroke.Methods
Wild-type or CD36-deficient mice were subjected to either 30-min transient or permanent focal ischemic stroke. In parallel, a cohort of mice subjected to either transient or permanent stroke received either vehicle or 5 mg/kg of SS-31. Monocyte chemoattractant protein-1 (MCP-1) and its receptor CCR2, mRNA levels, and infarct volume and percent hemispheric swelling were measured in the postischemic brain.ResultsCD36 deficiency or SS-31 treatment significantly attenuated MCP-1 or CCR2 mRNA up-regulation and injury size in the transient ischemic stroke. However, the approaches failed to show the protective effect in permanent ischemic stroke.Conclusion
The study revealed that targeting CD36 has a beneficial effect on transient but not permanent focal ischemic stroke. The study thus precludes a generalized strategy targeting CD36 in ischemic stroke and suggests careful consideration of types of stroke and associated pathology in developing stroke therapies.
... OxLDL has been shown to accumulate in astrocytes following cerebral infarcts and stimulates IL6 release from astrocytes in culture [53], while ACA have been shown to reduce viability of neuronal cultures [54] and damage to cerebral white and gray matter and inhibit astrocyte function [55,56]. ...
The majority of neurodegenerative (ND) and autoimmune diseases (AID) remain idiopathic. The contribution of environmental chemicals to the development of these disorders has become of great interest in recent years. A convergence of mechanism between of ND and AID development has also emerged. In the case of ND, including neurotoxicity, the focus of this review, work over the last two decade in the realm of biomarker development, indicates that the immune response provides a venue whereby humoral immunity, in the form of autoantibodies to nervous system specific proteins, or neuroantibodies (NAb), may provide, once validated, a sensitive high throughput surrogate biomarker of effect with the potential of predicting outcome in absence of overt neurotoxicity/neurodegeneration. In addition, NAb may prove to be a contributor to the progression of the nervous system pathology, as well as biomarker of stage and therapeutic efficacy. There is a compelling need for biomarkers of effect in light of the introduction of new chemicals, such as nanoengineered material, where potential neurotoxicity remains to be defined. Furthermore, the convergence of mechanisms associated with ND and AID draws attention to the neglected arena of angiogenesis in defining the link between environment, ND, and AID.
... Preliminary studies suggest that caveolae are likely to be involved in the potential transport of LDL from the blood to the brain. It has been demonstrated that oxidized LDL is present in brain parenchyma of patients with ischemic infarction and suggests a potential mechanism by which oxidized LDL may activate innate immunity and thereby indirectly influence neuronal survival (Shie et al., 2004). ...
PATHOPHYSIOLOGY OF ISCHEMIC DISORDERS: I- LDL CHOLESTEROL
AND ISCHEMIC STROKE
Nazish Iqbal Khan and Zahir Hussain*
Department of Physiology, University of Karachi, Karachi-75270, Pakistan.*E-Mail: zahussai@yahoo.ca
ABSTRACT
Blood lipids, particularly total and LDL cholesterol levels, are associated with all subtypes of brain infarction.
Furthermore, many of the effects of elevated or modified low density lipoproteins on endothelial cells and endothelial cell processes could be expected to contribute to the development of atherosclerosis and therefore, to the association between lipids and atherosclerotic, particularly coronary and cerebrovascular disease. However, the extent to which “endothelial dysfunction” accounts for the known relationships between serum lipid concentrations, ischemic disease and endothelial progenitor cell (EPC) pathophysiology is yet to be established. On the basis of the mentioned and other reports, we hypothesize that higher levels of plasma LDL cholesterol not only directly impair endothelial cells, but also effect EPC number and function at the same time, thus influencing the endothelial repair process and disturbing the balance between the magnitude of injury and the capacity for repair, which leads to endothelial dysfunction and promotes the progression of stroke. Because EPCs have been implicated in various events requiring endothelialization, we further hypothesized that LDL-C could influence the action of EPCs and sensory neurons and that this could be important to know how LDL-C effects repair, injury and vasculoprotection . In order to corroborate this proposal, quantification is required for EPCs and DRG neurons treated with various concentrations of LDL-C and characterizing the underlying molecular mechanisms. The relative contributions of apoptotic and necrotic death to ischemia-induced neuronal loss may provide us information for understanding the underlying mechanisms. Conclusively, the aggressive management of risk factors, like cholesterol,
could have a significant and positive impact on the natural history of atherosclerotic cerebrovascular disease.
Key words: LDL cholesterol, ischemic stroke, pathophysiology
... In CNS, oxLDL exhibits detrimental effects on brain cell functions, including induction of apoptosis, disruption of capillary homeostasis, and alteration of inflammatory protein activity in various brain cells [93]. Furthermore, in patients with cerebral infarction, oxLDL is present in brain parenchyma and stimulates astrocytes to secrete interleukin-6 [94] and may serve as an indicator to reflect the level of oxidative stress [95]. In brain astrocytes, oxLDL can induce MMP-9 expression and cell migration, which plays a critical role in the progression of inflammatory diseases and remodeling processes in target tissues, including CNS [29,96]. ...
Reactive oxygen species (ROS), a redox signal, are produced by various enzymatic reactions and chemical processes, which are essential for many physiological functions and act as second messengers. However, accumulating evidence has implicated the pathogenesis of several human diseases including neurodegenerative disorders related to increased oxidative stress. Under pathological conditions, increasing ROS production can regulate the expression of diverse inflammatory mediators during brain injury. Elevated levels of several proinflammatory factors including cytokines, peptides, pathogenic structures, and peroxidants in the central nervous system (CNS) have been detected in patients with neurodegenerative diseases such as Alzheimer's disease (AD). These proinflammatory factors act as potent stimuli in brain inflammation through upregulation of diverse inflammatory genes, including matrix metalloproteinases (MMPs), cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), and adhesion molecules. To date, the intracellular signaling mechanisms underlying the expression of target proteins regulated by these factors are elusive. In this review, we discuss the mechanisms underlying the intracellular signaling pathways, especially ROS, involved in the expression of several inflammatory proteins induced by proinflammatory factors in brain resident cells. Understanding redox signaling transduction mechanisms involved in the expression of target proteins and genes may provide useful therapeutic strategies for brain injury, inflammation, and neurodegenerative diseases.
... Reduced free radical production and injury size in CD36 KO mice further substantiate the role of CD36 in stroke-induced brain injury. Several CD36 ligands including fibrillar β amyloid (fAβ), modified/oxidized low density lipoproteins (mLDL, oxLDL), TSPs, long chain fatty acids, and membrane components of apoptotic cells are elevated following stroke4445464748. Depending on prevalent clinical co-morbid conditions, differential sets of CD36 ligands may prevail and affect vascular integrity following stroke. ...
Stroke pathology involves multifactorial pro-death responses, including inflammation, oxidative stress, vascular dysfunction, and activation of necrotic and apoptotic pathways. The interruption of a single specific pathway in defined stroke model systems has not been sufficient to address the multifactorial nature of stroke-induced injuries in the human population. CD36 is a class B scavenger receptor that functions in regulating normal physiological and pathological functions. CD36 pathways are activated by several distinct ligands. Convergence of these pathways results in inflammatory responses and endothelial dysfunction, which may be an underlying cause of cardio- and cerebrovascular diseases. The current review describes receptor CD36-ligand interactions relevant to endothelial function and discusses how targeting CD36 may have therapeutic utility in stroke.
... Thus, the overproduction of oxLDL can induce cell insults and tissue dysfunction. oxLDL may be present in brain tissues where it participates in neurodegeneration associated with oxidative stress-related cerebral diseases (Kivatinitz et al., 1997; Shie et al., 2004; Kim et al., 2006). Exposure of rat embryonic neurons or motor neuron-like cells to oxLDL, but not LDL, increased reactive oxygen species (ROS) and caspase activities and finally induced cell death (Keller et al., 1999aKeller et al., , 1999b). ...
Cerebral endothelial cells (CECs) are crucial components of the blood-brain barrier. Oxidized low-density lipoprotein (oxLDL) can induce cell injuries. In this study, we attempted to evaluate the effects of oxLDL on mouse CECs and its possible mechanisms. Mouse CECs were isolated from brain tissues and identified by immunocytochemical staining of vimentin and Factor VIII. oxLDL was prepared from LDL oxidation by copper sulfate. Exposure of mouse CECs to oxLDL decreased cell viability in concentration- and time-dependent manners. oxLDL time-dependently caused shrinkage of cell morphologies. Administration of oxLDL to CECs induced DNA fragmentation in concentration- and time-dependent manners. Analysis of the cell cycle revealed that oxLDL concentration- and time-dependently increased the proportion of CECs which underwent apoptosis. Analysis of confocal microscopy and immunoblot revealed that oxLDL significantly increased cellular and mitochondrial Bax levels as well as the translocation of this proapoptotic protein from the cytoplasm to mitochondria. In parallel with the increase in the levels and translocation of Bax, oxLDL time-dependently decreased the mitochondrial membrane potential. Exposure of mouse CECs to oxLDL decreased the amounts of mitochondrial cytochrome c, but enhanced cytosolic cytochrome c levels. The amounts of intracellular reactive oxygen species were significantly augmented after oxLDL administration. Sequentially, oxLDL increased activities of caspase-9, -3, and -6 in time-dependent manners. Pretreatment with Z-VEID-FMK, an inhibitor of caspase-6, significantly decreased caspase-6 activity and the oxLDL-induced DNA fragmentation and cell apoptosis. This study showed that oxLDL induces apoptotic insults to CECs via signal-transducing events, including enhancing Bax translocation, mitochondrial dysfunction, cytochrome c release, increases in intracellular reactive oxygen species, and cascade activation of caspase-9, -3, and -6. Therefore, oxLDL can damage the blood-brain barrier through induction of CEC apoptosis via a Bax-mitochondria-caspase protease pathway.
... In multiple sclerosis, astrocytes play a key role in maintaining autoreactive responses and pathological plaque formation [40,41] . In stroke, activated astrocytes contribute to opening of the blood brain barrier [42] , as well as secrete various neurotoxic substances that contribute to post infarct neural damage [43,44]. Vargas et al compared brain autopsy samples from 11 autistic children with 7 age-matched controls. ...
Autism spectrum disorders (ASD) are a group of neurodevelopmental conditions whose incidence is reaching epidemic proportions, afflicting approximately 1 in 166 children. Autistic disorder, or autism is the most common form of ASD. Although several neurophysiological alterations have been associated with autism, immune abnormalities and neural hypoperfusion appear to be broadly consistent. These appear to be causative since correlation of altered inflammatory responses, and hypoperfusion with symptology is reported. Mesenchymal stem cells (MSC) are in late phases of clinical development for treatment of graft versus host disease and Crohn's Disease, two conditions of immune dysregulation. Cord blood CD34+ cells are known to be potent angiogenic stimulators, having demonstrated positive effects in not only peripheral ischemia, but also in models of cerebral ischemia. Additionally, anecdotal clinical cases have reported responses in autistic children receiving cord blood CD34+ cells. We propose the combined use of MSC and cord blood CD34+cells may be useful in the treatment of autism.
... On the other hand, cerebral ischemia is well known to produce numerous species of modified lipids (Adibhatla et al., 2006 ). For example, modified low-density lipoprotein (LDL), such as oxidized LDL, is formed after cerebral ischemia and could activate CD36 (Uno et al., 2003; Shie et al., 2004; Vibo et al., 2007). In addition, hypoxia has been reported to produce diacylglyceride, a CD36 ligand that is involved in inflammatory signaling via toll-like receptors 2/6 (TLR2/6; see below) (Bruder et al., 2005; Hoebe et al., 2005). ...
CD36, a class-B scavenger receptor involved in multiple functions, including inflammatory signaling, may also contribute to ischemic brain injury through yet unidentified mechanisms. We investigated whether CD36 participates in the molecular events underlying the inflammatory reaction that accompanies cerebral ischemia and may contribute to the tissue damage. We found that activation of nuclear factor-kappaB, a transcription factor that coordinates postischemic gene expression, is attenuated in CD36-null mice subjected to middle cerebral artery occlusion. The infiltration of neutrophils and the glial reaction induced by cerebral ischemia were suppressed. Treatment with an inhibitor of inducible nitric oxide synthase, an enzyme that contributes to the tissue damage, reduced ischemic brain injury in wild-type mice, but not in CD36 nulls. In contrast to cerebral ischemia, the molecular and cellular inflammatory changes induced by intracerebroventricular injection of interleukin-1beta were not attenuated in CD36-null mice. The findings unveil a novel role of CD36 in early molecular events leading to nuclear factor-kappaB activation and postischemic inflammation. Inhibition of CD36 signaling may be a valuable therapeutic approach to counteract the deleterious effects of postischemic inflammation.
Introduction
SNW domain containing 1 (SNW1), as a splicing factor to regulate the activity of transcription factors, has been reported to be involved in multiple disease processes, including neuroblastoma. Whereas, the latent function and concrete mechanism of SNW1 in brain microvascular endothelial cells (BMECs) have not been clarified.
Material and methods
BMECs were induced by oxidized low-density lipoprotein (ox-LDL), and high fat (HF)-fed rats were established. After SNW1 knockdown or NLR family pyrin domain containing 3 (NLRP3) overexpression, SNW1 and NLRP3 expressions were monitored via RT-qPCR, Western blot, or immunohistochemistry assays. Also, cell viability, apoptosis, and cholesterol efflux were determined via CCK-8, flow cytometry, and related kits; IL-18 and IL-3 levels were also certified by ELISA kits; and NLRP3 inflammasomes and cholesterol efflux-related proteins were identified by Western blot in vitro and in vivo.
Results
We discovered that ox-LDL or HF-feeding significantly elevated SNW1 and NLRP3 expressions, and prominently induced BMECs injury in BMECs or rat brain tissues. Subsequently, our data confirmed that SNW1 knockdown markedly accelerated cholesterol efflux and viability, and prevented apoptosis and NLRP3 inflammasomes, which also could be reversed by NLRP3 overexpression in ox-LDL-induced BMECs. In addition, we showed that SNW1 knockdown could signally induce cholesterol efflux and repress NLRP3 inflammasome activation in HF-fed rats.
Conclusions
We demonstrated that SNW1 knockdown has a great protection effect on the dysfunction of BMECs by inhibiting NLRP3. So, SNW1 might be a therapeutic target for BMECs injury.
Lipoprotein structure and metabolism are reviewed. The roles of lipoproteins, apolipoproteins, and tocopherols in the nervous system are described. Deficiency of the B apolipoproteins (abetalipoproteinemia, familial hypobetalipoproteinemia, and chylomicron retention disease) impairs the transport of lipids and antioxidant tocopherols resulting in oxidative damage. Defects involving impaired generation of high-density lipoproteins (Tangier disease and lecithin:cholesterol acyltransferase deficiency) impact multiple activities in the central nervous system and peripheral nerves. Emerging genomic determinants are identified and the pathophysiologic roles of individual lipid and protein molecular species, often pleiotropic in nature, are described.
Background and aims:
Oxidized low-density lipoprotein (oxLDL) has a defined role in the genesis and development of atherosclerosis, however, whether it is related to severity of neurological deficits is rarely reported. The aim of our study was to investigate the potential association between oxLDL and the National Institutes of Health Stroke Scale (NIHSS) score among patients with acute ischemic stroke.
Methods:
Between January 2014 and October 2014, we recruited 4111 patients with acute ischemic stroke (AIS), who were admitted within 7 days-43 hospitals in China, and participated in the SOS-Stroke Study. We collected detailed clinical data and then tested the relationship between oxLDL and the NIHSS score using a multivariate linear regression analysis.
Results:
After adjusting for age, gender, ethnicity, marriage and other confounding variables, the elevated NIHSS score was significantly associated with increased oxLDL levels, and each 1-μg/dL elevation in oxLDL concentration resulted in an increase of 0.027 in the NIHSS score.
Conclusions:
A positive correlation was found between plasma levels of oxLDL and the NIHSS score in patients with acute ischemic stroke. Higher plasma levels of oxLDL potentially suggest a worse prognosis in AIS patients.
Stroke is a major leading cause of death and disability in the human population. The pathology of stroke-induced brain injury involves multifactorial pro-death processes. Among them, inflammation is an important contributor to stroke pathology as indicated by the close association between excessive inflammation and exacerbation of the disease process. Considerable experimental evidence indicates that disease outcome is modulated by several factors including predisposing clinical conditions. Stroke compromises vascular permeability and leads to breakdown of the blood–brain barrier. While the pathology primarily occurs in the CNS, the presence of peripheral immune cells in the infarcted area suggests their potential role in post-ischemic inflammation. Given recent advances highlighting the heterogeneity of peripheral immune cells and diversity of their function, we review neuroimmune interaction in the setting of acute cerebral ischemia, post-ischemic inflammation, and the trafficking of peripheral immune cells to inflamed tissue, with specific focus on the involvement of the class B scavenger receptor, CD36. We discuss CD36 expression and functions, the contribution of the receptor to stroke pathology, its relevance to peripheral inflammatory conditions, and potential strategies to target the CD36-associated neuroinflammatory pathway.
Alzheimer’s disease (AD) is the most common cause of dementia. It is characterized by a progressive loss of higher cognitive functions. The strongest risk factor for sporadic AD is old age, which also coincides with the increased occurrence of cerebrovascular lesions (Table 1). Additional risk factors associated with AD vascular alterations include some forms of heart disease, atherosclerosis, high plasma cholesterol, increased fat intake, history of hypertension, diabetes mellitus, head injury, stroke, systemic infiammation and ApoE e4 (1,2). The ApoE genotype is the prime susceptibility factor for sporadic AD (3). The ApoE genotype has been linked to an increased risk and reduced age of onset of AD. The ApoE has also been associated with VaD, mild cognitive impairment (MCI) (4), impaired recovery after brain trauma, amyotrophic lateral sclerosis and Pick’s disease and, more recently, also with the risk and age of onset of Parkinson’s disease (5). The ApoE4 was found to interfere with cognitive functions in non-diseased adult men carrying ApoE4 in comparison with non-carriers (6,7). Memory deteriorates in ApoE4 carriers before the symptomatic occurrence of MCI, prior to age 60 (8). It remains to be clarified how ApoE4 affects cognitive functioning and neurodegenerative processes. Nevertheless, the ApoE genotype may be a leading aspect in the modification process of cerebrovascular functions during ageing and the increase of the predisposition to the pathogenesis of AD (9).
The role played by immune mechanisms in intracerebral hemorrhage (ICH) was evaluated based on the studies of thymus structure and cell composition in rats showing different degree of tolerance to emotional stress. A new experimental ICH model is proposed. It is concluded that both stereotype and autoimmune mechanisms are involved in the development of ICH.
The barrier between blood and CSF contributes to homeostasis of the CNS and protects it from potentially harmful substances present in the blood. Lipoproteins present in the CSF are clearly distinct from their plasma counterparts. Human CSF lipoproteins contain mainly Apo AI and Apo E, the former deriving mostly from plasma after crossing the blood-cerebrospinal fluid barrier, the latter being also produced by CNS. Apo AI and E containing lipoproteins in the brain are key players in transport and delivery of lipids, cholesterol homeostasis, and are also involved in CNS remodeling mechanisms. On the other hand, the isoform apo E4 represents the most important genetic risk factor for sporadic and familial late-onset Alzheimer's disease and is involved in brain injury and neurodegenerative diseases. Apo B containing lipoproteins are not produced by CNS and the characterization of normal human CSF lipoproteins did not allowed the isolation of low density lipoproteins. Lipoprotein(a) is a low density lipoprotein-like particle with the unique protein apo(a), which is characterized by a dimensional polymorphism. Lipoprotein(a) is a well known risk factor for athero-thrombosis. The pathological role of Lipoprotein(a) is strictly associated with its plasma concentrations and the size of apo(a) isoforms, with inverse relation. The pathophysiology of Lipoprotein(a) in cardio and cerebrovascular system is widely studied. Recently, we demonstrated that, in neuroinflammatory and neurodegenerative disorders, Lipoprotein(a) can cross a dysfunctional blood-CSF barrier and be found in the CSF. This chapter focuses on the physiological presence of the lipoproteins in CNS, on the pathological aspects deriving from their isoforms, and in particular on the anomalous presence in CSF of Lipoprotein(a).
Lipoprotein disorders are reviewed including lipoprotein structure and metabolism, transport of dietary lipids, apolipoproteins in the nervous system, tocopherols, apoprotein B, disorders of high-density lipoproteins, and the description of a patient who has no expression of apolipoprotein E.
Frontotemporal dementia (FTD) is a neurodegenerative disease with devastating changes in behavioral performance and social function. Mutations in the progranulin gene (GRN) are one of the most common causes of inherited FTD due to reduced progranulin expression or activity, including in brain where it is expressed primarily by neurons and microglia. Thus, efforts aimed at enhancing progranulin levels might be a promising therapeutic strategy. Bone marrow (BM)-derived cells are able to engraft in the brain and adopt a microglial phenotype under myeloablative irradiation conditioning. This ability makes BM-derived cells a potential cellular vehicle for transferring therapeutic molecules to the central nervous system. Here, we utilized BM cells from Grn(+/+) (wild type or wt) mice labeled with green fluorescence protein for delivery of progranulin to progranulin-deficient (Grn(-/-)) mice. Our results showed that wt bone marrow transplantation (BMT) partially reconstituted progranulin in the periphery and in cerebral cortex of Grn(-/-) mice. We demonstrated a pro-inflammatory effect in vivo and in ex vivo preparations of cerebral cortex of Grn(-/-) mice that was partially to fully reversed 5 months after BMT. Our findings suggest that BMT can be administered as a stem cell-based approach to prevent or to treat neurodegenerative diseases.Laboratory Investigation advance online publication, 8 September 2014; doi:10.1038/labinvest.2014.113.
IntroductionFormation of the Neural TubeOrigin of Neurons and GliaMorphology of Glial Polarity in Adult CNSAstrocyte Spacing and Boundary LayersOrigin and Molecular Basis of Cell PolarityFunctional Polarity of Astrocytes and Other Ependymoglial Derivatives„Secretory Functions of AstrocytesInduction of BBB Properties in Brain EndotheliumAstrocyte-Endothelial SignalingConclusion
References
Stroke is a heterogeneous syndrome caused by multiple disease mechanisms, resulting in a disruption of cerebral blood flow
with subsequent tissue damage. Stroke remains the third leading cause of death and the leading cause of disability in adults.
In 25–40% of patients with ischemic stroke, neurological symptoms progress during the initial hours. Early clinical deterioration
results in increased mortality and functional disability. The molecular mechanisms underlying early clinical worsening are
still not well clarified. There is considerable evidence that reactive oxygen species (ROS), reactive nitrogen species (RNS),
and oxidative state are important mediators to tissue injury in cerebral ischemia. In normal conditions, free radical levels
are controlled by cellular endogenous antioxidants. In fact, oxidative stress occurs when there is an impairment or inability
to balance antioxidant production with ROS and RNS levels. The brain is highly susceptible to oxidative stress because of
its high consumption of body oxygen (~20%) to produce energy and free radicals, which in turn can cause damage to the main
components of cells (DNA, lipids, proteins). In this chapter we focus our attention on the evidence of oxidative stress in
ischemic stroke; particular attention is given to the current knowledge about the biomarkers of oxidative stress that can
possibly be used to monitor the severity and outcome of stroke.
KeywordsStroke-Free radicals-Reactive nitrogen species-Reactive oxygen species.
Class A scavenger receptor (SR-A) is primarily expressed in microglia/macrophages and plays an important role in immune responses. However, whether SR-A can influence microglia/macrophage polarization in cerebral ischemic injury is not known. To this end we monitored the phenotypic alteration of microglia/macrophages in an animal model of cerebral ischemia injury. SR-A was up-regulated in mouse brains 24h after permanent occlusion of middle cerebral artery (MCAO). SR-A-deficient mice displayed reduced infarct size and improved neurological function compared with wild-type mice littermate controls. Furthermore, a decrease in inflammatory F4/80(+)CD11b(+)CD45(high)CD11c(+) microglia/macrophages and attenuated nuclear factor-kappaB (NF-κB) activation was found in ischemic brains in the SR-A null mice. This was accompanied by alleviation of classically activated M1 macrophage markers and preservation of alternatively activated M2 macrophage markers. These data suggest that SR-A contributes to cerebral ischemic injury by pivoting the phenotype of microglia/macrophages to a skewed M1 polarization.
A delipid extracorporeal lipoprotein filter (DELP) system has been used to treat patients with stroke and has shown favorable prognosis. However, the mechanism for the neuronal functional recovery remains unclear. This study aimed to investigate the neuronal histological assessment, and the levels of C-reactive protein (CRP), tumor necrosis factor alpha (TNF-α), and oxidized low-density lipoprotein (oxLDL) after ischemic stroke following DELP treatment. Hyperlipidemic rabbits underwent middle cerebral artery occlusion. After 30 min, the animals received an extracorporeal apheresis treatment with a DELP filter. Total cholesterol (TC), triglyceride, and low-density lipoprotein (LDL) of the plasma were measured. The levels of CRP, TNF-α, and oxLDL in brain tissue were also measured by enzyme-linked immunosorbent assay. Hematoxylin and eosin staining, cresyl violet staining, neuron-specific enolase (NSE) and glial fibrillary acidic protein immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed. DELP apheresis reduced TC and LDL by >30%. The number of neurons at day 7 (P < 0.01) and the integrated optical density of NSE at day 1 (P < 0.05) and day 7 (P < 0.01) were significantly increased in the DELP group. TUNEL-positive cells were significantly decreased (P < 0.05). Astrocytes were moderately activated, and this activation persisted up to 7 days. Gliosis was not found in the DELP group. After treatment, the level of CRP declined at day 1 (P < 0.05); TNF-α and oxLDL declined at day 7 (P < 0.05). DELP apheresis decreased neuronal apoptosis, reduced inflammatory markers and oxidative stress in cerebral ischemia, and improved neuronal survival.
The relationship between acrolein (CH(2) =CH-CHO) and brain infarction is the focus of this review. It has been found that acrolein is produced mainly within cells from polyamines by polyamine oxidases (PAOs), especially from spermine by spermine oxidase during cell damage, and that acrolein is more toxic than reactive oxygen species (ROS) in a cell culture system. Thus, the possibility that acrolein and PAOs are good biochemical markers of stroke was tested because there are no other reliable biochemical markers at the early stage of stroke. Levels of protein-conjugated acrolein (PC-Acro) and PAOs (acrolein-producing enzymes) were significantly increased in the plasma of stroke patients. The multiplied value of PC-Acro by PAOs was nearly parallel with the size of stroke. Furthermore, when the combined measurements of PC-Acro, interleukin-6 (IL-6) and C-reactive protein (CRP) were evaluated along with age using a receiver operating characteristic (ROC) curve, even silent brain infarction (SBI), which is a small brain infarction, was indicated with approximately 84% sensitivity and specificity. These findings clearly indicate that acrolein is strongly correlated with cell damage during brain infarction.
Upregulation of matrix metalloproteinases (MMPs), especially MMP-9, by oxidized low-density lipoprotein (oxLDL) is implicated in many inflammatory diseases including brain injury. However, the signaling mechanisms underlying oxLDL-induced MMP-9 expression in astrocytes largely remain unknown. Here we report that oxLDL induces expression of proMMP-9 via a MAPK-dependent AP-1 activation in rat brain astrocyte (RBA)-1 cells. Results revealed by gelatin zymography, RT-PCR, and Western blotting analyses showed that oxLDL-induced proMMP-9 gene expression was mediated through Akt, JNK1/2, and p42/p44 MAPK phosphorylation in RBA-1 cells. These responses were attenuated by inhibitors of PI3K (LY294002), JNK (SP600125), and p42/p44 MAPK (PD98059), or transfection with dominant negative mutants and short hairpin RNA. Moreover, we demonstrated that AP-1 (i.e., c-Fos/c-Jun) is crucial for oxLDL-induced proMMP-9 expression which was attenuated by pretreatment with AP-1 inhibitor (curcumin). The regulation of MMP-9 gene transcription by AP-1 was confirmed by oxLDL-stimulated MMP-9 luciferase activity which was totally lost in cells transfected with the AP-1 binding site-mutated MMP-9 promoter construct (mt-AP1-MMP-9). These results suggested that oxLDL-induced proMMP-9 expression is mediated through PI3K/Akt, JNK1/2, and p42/p44 MAPK leading to AP-1 activation. Understanding the regulatory mechanisms underlying oxLDL-induced MMP-9 expression in astrocytes might provide a new therapeutic strategy of brain injuries and diseases.
Neuroinflammation mediated by microglial activation appears to play an essential role in the pathogenesis of Parkinson disease; however, the mechanisms by which microglia are activated are not fully understood. Thus, we first evaluated the effects of two parkinsonian toxicants, manganese ethylene bisdithiocarbamate (Mn-EBDC) and 1-methyl-4-phenylpyridine (MPP+), on microglial activation as well as associated dopaminergic (DAergic) neurotoxicity in primary cell culture systems. The results demonstrated that, when rat primary mesencephalic neuron-enriched or neuron-microglia mixed cultures were treated with Mn-EBDC at 2-8 microm or MPP+ at 0.25-5 microm, respectively, for 7 days, both toxicants were capable of inducing DAergic neurodegeneration as well as activating microglia via a mechanism secondary to DAergic neurodegeneration. Furthermore activated microglia subsequently enhanced DAergic neurotoxicity induced by Mn-EBDC or MPP+. Detailed scrutiny of neuron-microglia interactions identified a fraction of the conditioned media derived from a DAergic cell line treated with Mn-EBDC or MPP+ that potently activated microglia. To further define potential mediators leading to microglial activation secondary to neurodegeneration, we utilized a quantitative proteomic technique termed SILAC (for stable isotope labeling by amino acids in cell culture) to compare the protein profiles of MPP+-treated cellular fraction that mediated microglial activation as compared with controls. The search revealed numerous novel proteins that are potentially important in neurodegeneration-mediated microglial activation, a process believed to be critical in Parkinson disease progression.
Free radical production is increased in ischemic and hemorrhagic stroke, leading to oxidative stress that contributes to brain damage. The measurement of oxidative stress in stroke would be extremely important for a better understanding of its pathophysiology and for identifying subgroups of patients that might receive targeted therapeutic intervention. Since direct measurement of free radicals and oxidized molecules in the brain is difficult in humans, several biological substances have been investigated as potential peripheral markers. Among lipid peroxidation products, malondialdehyde, despite its relevant methodological limitations, is correlated with the size of ischemic stroke and clinical outcome, while F2-isoprostanes appear to be promising, but they have not been adequately evaluated. 8-Hydroxy-2-deoxyguanosine has been extensively investigated as markers of oxidative DNA damage but no study has been done in stroke patients. Also enzymatic and nonenzymatic antioxidants have been proposed as indirect markers. Among them ascorbic acid, alpha-tocopherol, uric acid, and superoxide dismutase are related to brain damage and clinical outcome. After a critical evaluation of the literature, we conclude that, while an ideal biomarker is not yet available, the balance between antioxidants and by-products of oxidative stress in the organism might be the best approach for the evaluation of oxidative stress in stroke patients.
Although the pathogenesis of Parkinson's disease (PD) remains unknown, it appears that microglial activation is associated with enhanced neurodegeneration in animal models of PD as well as in PD patients. Experimentally, C57BL/6 and SWR/J mice demonstrate striking differences in the extent of dopaminergic (DAergic) neurodegeneration induced by a parkinsonian toxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The purpose of this study was to determine whether differences in microglial activation between these two strains of mice could provide insight into the variability seen in toxicant induced neuronal death, and subsequently to use a high-throughput proteomic method, combining stable isotope labeling with amino acids in cell culture (SILAC) with liquid chromatography and tandem mass spectrometry, to compare the microglial proteomes of C57BL/6 and SWR/J mice after stimulation with a classical microglial activator, lipopolysaccharide (LPS). We found that DAergic neurotoxicity induced by LPS in a primary neuron-microglia coculture was twofold greater with microglia isolated from the brains of C57BL/6 mice compared with that of SWR/J mice. Upon proteomic analysis we found that, out of over 1,000 proteins identified and quantified, 400 displayed a significant difference in their relative abundance between these two murine strains. Several proteins, which had relatively higher levels in C57BL/6 mice, have previously been implicated in LPS-mediated microglial activation, including those involved in the COX-2 pathway and in prostaglandin E-2 (PGE(2)) production. To validate our proteomic results we confirmed the increased expression level of iNOS in C57BL/6 vs. SWR/J microglia with semiquantitative Western blot. Further analysis of our proteomic discovery data will likely reveal numerous novel proteins involved in inflammation-mediated neurotoxicity in PD.
Low density lipoprotein (LDL) is readily oxidized under certain conditions, resulting in the formation of oxidized LDL (oxLDL). Despite numerous in vitro reports that reveal the pathogenic role of oxidative stress, anti-oxidative strategies have underperformed in the clinic. In this study, we examine the role of oxLDL in brain inflammatory responses using cultured rat brain microglia. We demonstrate that oxLDL inhibits lipopolysaccharide (LPS)-induced inflammatory responses in these cells. It also decreases LPS-induced expression of inducible nitric oxide synthase and production of nitric oxide, and reduces LPS-induced secretion of tumor necrosis factor-alpha and monocyte chemoattractant protein-1. Oxysterols, known components of oxLDL and endogenous agonists of liver X receptor, can simulate the inhibitory effects of oxLDL in LPS-activated microglia. In addition, their inhibitory effects were mimicked by liver X receptor (LXR) agonists and potentiated by a retinoid X receptor agonist, suggesting these molecules heterodimerize to function as oxysterol receptors. Taken together, our results demonstrate that oxLDL inhibits LPS-induced inflammatory responses in brain microglia and that these inhibitory effects are mediated by oxysterols and, at least in part, by the nuclear receptor LXR. Our results suggest an additional mechanism of action for oxidative stress that acts indirectly via modulation of inflammatory responses. Although further studies are needed, these results answer in part the question of why anti-oxidative strategies have not been successful in clinical situations. Moreover, as brain inflammation participates in the initiation and progression of several neurodegenerative disorders, the present data provide information that should prove a useful guide for designing therapeutic strategies to combat oxidative brain diseases.
Lipoprotein(a) [Lp(a)] is a recognized pathogenic particle in human plasma, but its presence in the cerebrospinal fluid and its possible role in the central nervous system have not been documented. We tested the hypothesis that apolipoprotein(a) [apo(a)], free or as a component of the Lp(a) particle, can cross the blood-cerebrospinal fluid barrier and be found in the cerebrospinal fluid of patients affected by neurologic pathologies.
We studied paired cerebrospinal fluid/serum samples from 77 patients with inflammatory (n=20) or noninflammatory (n=34) blood-cerebrospinal fluid barrier dysfunction and without blood-cerebrospinal fluid barrier dysfunction (n=23). We used ELISA to measure Lp(a) concentrations and Western blot and immunodetection to analyze apo(a) isoforms in native and reducing conditions.
Entire Lp(a) with either small or large apo(a) isoforms was present in the cerebrospinal fluid of patients with blood-cerebrospinal fluid barrier dysfunction, regardless of its pathogenesis. Multiple linear regression analysis showed that both serum Lp(a) concentration (P=0.003) and cerebrospinal fluid/serum albumin ratio (P<0.001) were predictors of the Lp(a) concentration in cerebrospinal fluid.
Our results demonstrate that Lp(a) can cross a dysfunctional blood-cerebrospinal fluid barrier. The unusual presence of Lp(a) in the cerebrospinal fluid could extend some of its known pathogenic effects to the central nervous system.
Oxidative stress is implicated in the pathogenesis of ischemia/reperfusion injury. Recently, we demonstrated that activation of CD36, a class B scavenger receptor, mediates free radical production and tissue injury in cerebral ischemia (1). Oxidized low density lipoproteins (oxLDL) are among the ligands that bind to CD36 and are elevated in acute cerebral infarction. SS31 is a cell-permeable antioxidant peptide that reduces intracellular free radicals and inhibits LDL oxidation/lipid peroxidation (2). The current study was designed to investigate whether treatment with SS31 normalizes ischemia-induced redox changes and attenuates CD36-mediated tissue injury. C57BL/6 mice were subjected to transient middle cerebral artery occlusion (MCAO). Redox status and infarct volume were measured in animals treated with either saline or SS31. Oxidative stress induced by ischemia/reperfusion profoundly depleted glutathione (GSH) concentrations in the ipsilateral cortex and striatum. Treating mice with SS31 immediately after reperfusion significantly attenuated ischemia-induced GSH depletion in the cortex and reduced infarct size. By contrast, the protective effect of SS31 was absent in CD36 knock-out mice, indicating that SS31 is acting through inhibition of CD36. Treating C57BL/6 mice with SS31 reduced CD36 expression in postischemic brain and mouse peritoneal macrophages (MPM). Further in vitro studies revealed that SS31 attenuated oxLDL-induced CD36 expression and foam cell formation in MPM. These in vivo and in vitro studies indicate that the down-regulation of CD36 by novel class antioxidant peptides may be a useful strategy to treat ischemic stroke victims.
The goal of our study was to investigate the associations of oxidized LDL (apoB100 aldehyde-modified form) and acute phase proteins (fibrinogen, CRP) with acute ischemic stroke severity and outcome.
The study included 61 ischemic stroke patients and 64 controls. Strokes were subtyped according to TOAST criteria, the severity and outcome of stroke (at 1 year) were measured.
The mean triglyceride, fibrinogen, CRP and glucose values were significantly higher among cases. The median oxLDL value for patients with large artery atherosclerosis (LAA) type of stroke was significantly higher than for other subtypes. The oxLDL values did not correlate with age, stroke severity and outcome.
Inflammatory markers (fibrinogen and CRP) predicted the stroke severity and outcome whereas elevation of oxLDL levels did not. Our data refer to possibility that there may exist some links between the LAA subtype of stroke and elevated oxLDL (apoB100 aldehyde-modified form).
The role of circulating, oxidized low-density lipoprotein and interleukin-6 levels in acute ischemic stroke considering the primary-vessel disease was investigated. The study consisted of 28 patients with acute ischemic stroke and 23 control subjects. Patients were subdivided into large-vessel (n = 12) and small-vessel (n =16) disease stroke groups according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria. The means of oxidized low-density lipoprotein and interleukin-6 levels of patients with acute ischemic stroke were higher than controls (P < .01, P < .05). Mean oxidized low-density lipoprotein level was higher in the large-vessel disease group than in the small-vessel disease group (P < .01). The mean of inteleukin-6 levels was higher in the small-vessel disease group (P < .01). The results of the present study showed that oxidative stress promotes large-vessel disease rather than small-vessel disease stroke, and inflammation may play important an role in the development of small-vessel disease stroke.
Laminins are extracellular matrix glycoproteins with multiple functions in the central nervous system, including maintenance of the blood-brain barrier. Because ischemic brain damage results in rapid degradation of extracellular matrix, we used immunocytochemistry on rat central nervous system after permanent focal ischemia to identify laminins involved in pathophysiology of stroke. At 24 hr after stroke, laminin-1 is transiently expressed by neurons inside the ischemic core, but from 2-3 days to 28 days it is expressed only in basement membrane structures. During the first 24 hr, alpha1, alpha5, beta1, and gamma1 laminins are transiently expressed in neurons within the ischemic core as an acute reaction of the brain to ischemia. Rapid induction of gamma1 laminin but no other laminin in reactive astrocytes surrounding the ischemic core is clear at 24 hr, and importantly, expression of gamma1 laminin in astrocytes surrounding the ischemic core intensifies during the first days and persists up to 28 days after stroke. At 2-3 days, gamma1 laminin immunoreactive barrier of reactive astrocytes is already fully formed, isolating the ischemic area from the healthy brain. Similar to gamma1 laminin, its KDI domain localizes in reactive astrocytes isolating the ischemic core. Results indicate that gamma1 laminin and its KDI domain are rapidly induced in glial cells after stroke and their expression persists, forming a molecular barrier between the healthy and the damaged brain. Thus, gamma1 laminin is involved in pathology of stroke and is likely to serve a protective function, considering its potent neuroprotective role after spinal cord injury and in neurodegenerative disorders.
The expression of interleukin-6 (IL-6) mRNA in the focal ischemic rat cortex was studied by means of Northern hybridization. IL-6 mRNA was induced after permanent occlusion of the middle cerebral artery, reached a significant level at 3 h, and peaked at 12 h, i.e., ~ 10-fold increase in the ischemic zone compared with the nonischemic cortex or sham-operated controls. The increased IL-6 mRNA was elevated for at least 24 h. Low levels of IL-6 mRNA were detected in sham-operated rats or in the contralateral nonischemic cortex. The expression of c-fos and zif268 mRNAs, two early response genes, was rapid (increased by 1 h postischemia) and transient (returned to basal levels by 24 and 12 h, respectively), clearly having different kinetic patterns from that of IL-6 mRNA. The early response kinetic pattern of c-fos and zif268 mRNAs in focal ischemia suggests their transcriptional regulatory roles in response to ischemic insult, while the delayed induction pattern of IL-6 mRNA suggests a role for this pleiotropic cytokine in the inflammatory response to the focal ischemic damage of the brain.Keywords: c-fos; Focal brain ischemia; Inflammation; Interleukin-6; Stroke; zif268
Exposure of plasma from apolipoprotein E gene knockout (apoE-/-) and control (CBA or C57BL/6J) mice plasma to a constant rate of aqueous peroxyl radicals (ROO.) resulted in the depletion of ascorbate, urate and alpha-tocopherol (alpha-TOH), with substantial and little lipid peroxidation, respectively. Alpha-TOH levels were 3-times higher in plasma from apoE-/- than control mice and its addition enhanced the oxidizability of control mouse plasma. In apoE-/- mouse plasma, alpha-TOH was associated primarily with very low density lipoprotein (VLDL), whereas in plasma from control mice, the vitamin was located largely in high density lipoproteins. Oxidation of isolated lipoproteins by ROO. resulted in the accumulation of lipid hydroperoxides to an extent that reflected the plasma concentration and alpha-TOH content of the different lipoprotein fractions. Oxidation of 'plasma' reconstituted from components of apoE-/- mice and/or human plasma showed that human and apoE-/- mouse lipoproteins peroxidized with similar kinetics, although the initiation of lipid peroxidation was greater in the presence of mouse than human lipoprotein-deficient plasma. Also, the chain length of lipid peroxidation in apoE-/- mouse plasma after ascorbate depletion appeared to be independent of the rate of ROO. generation. Together, these results show that the ROO. induced peroxidation of plasma lipoproteins in atherogenesis-susceptible apoE-/- mice exhibits some, though not all, features of tocopherol-mediated peroxidation (TMP). Therefore, apoE-/- mice may represent a suitable animal model to test a role for TMP in atherogenesis and the prevention of this disease by anti-TMP agents.
Dopamine receptors in the central nervous system can be studied by measuring the specific binding of [3H]dopamine, [3H]haloperidol, d-[3H]LSD, [3H]dihydroergocryptine or [3H]apomorphine. The receptors are stereoselectively blocked by +)-butaclamol, a neuroleptic. All neuroleptics inhibit the specific binding of [3H]haloperidol in relation to their clinical potencies. The radioligand that desorbs most slowly from the receptor is [3H]apomorphine, thus making it a reliable ligand for dopamine receptors. Dopamine agonists that compete for [3H]apomorphine binding do so at concentrations that correlate with their potency in stimulating striatal adenylate cyclase. Structure-activity analysis, using [3H]apomorphine, confirms that the active dopamine-mimetic conformation is the beta rotamer of dopamine. Prolonged exposure in vitro of caudate homogenate to high concentrations of dopamine leads to increased binding of [3H]apomorphine or [3H]haloperidol, suggesting receptor "sensitization." Chronic haloperidol treatment of rats leads to an increased number of dopamine/neuroleptic receptors in the striatum, but a decrease in the pituitary.
This study was undertaken to determine if apolipoprotein (apo) E-containing lipoproteins and their receptors could provide a system for lipid transport and cholesterol homeostasis in the brain, as they do in other tissues. To accomplish this goal, the lipoproteins in human and canine cerebrospinal fluid (CSF) were characterized, and rat brain and monkey brain were examined for the presence of apoB,E(LDL) receptors. Apolipoprotein E and apoA-I were present in human and canine CSF, but apoB could not be detected. Apo-lipoprotein E and apoA-I were both present on lipoproteins with a density of approximately 1.09 to 1.15 g/ml. In human CSF, the lipoproteins were primarily spherical (approximately 140 A), whereas in canine CSF the lipoproteins were a mixture of discs (200 x 65 A) and spheres (approximately 130 A). Apolipoproteins E and A-I were contained primarily in separate populations of lipoproteins. Although the apoE of CSF was more highly sialylated than plasma apoE, the apoE-containing lipoproteins in canine CSF competed as effectively as canine plasma apoE HDLc for binding of 125I-LDL to the apoB,E(LDL) receptors on human fibroblasts. The presence of apoB,E(LDL) receptors in both rat and monkey brain was demonstrated by immunocytochemistry. Astrocytes abutting on the arachnoid space and pial cells of the arachnoid itself, both of which contact CSF, expressed apoB,E(LDL) receptors. Relatively few receptors were present in the cells of the gray matter of the cortex. Receptors were more prominent on the astrocytes of white matter and in the cells of the brain stem. The expression of apoB,E(LDL) receptors by brain cells and the presence of apoE- and apoA-I-containing lipoproteins in CSF suggest that the central nervous system has a mechanism for lipid transport and cholesterol homeostasis similar to that of other tissues.
F2-isoprostanes are prostaglandin F2-like compounds that are known to be formed in vivo by free radical oxidation of arachidonyl-containing lipids, and their plasma levels have been suggested as indicators of in vivo oxidative stress. As oxidation of LDL, a likely causal factor in atherosclerosis, involves lipid peroxidation, we investigated whether F2-isoprostanes are formed in plasma and LDL exposed to oxidative stress, and how F2-isoprostane formation is related to endogenous antioxidant status. In plasma exposed to aqueous peroxyl radicals, lipid hydroperoxides and esterified F2-isoprostanes were formed simultaneously after endogenous ascorbate and ubiquinol-10 had been exhausted, despite the continued presence of urate, alpha-tocopherol, beta-carotene, and lycopene. In isolated LDL exposed to aqueous peroxyl radicals or Cu2+, consumption of endogenous ubiquinol-10 and alpha-tocopherol was followed by rapid formation and subsequent breakdown of lipid hydroperoxides and esterified F2-isoprostanes, and a continuous increase in LDL's electronegativity, indicative of atherogenic modification. In Cu(2+)-exposed LDL, the decrease in esterified F2-isoprostane levels was paralleled by the appearance of free F2-isoprostanes, suggesting that hydrolysis by an LDL-associated activity had occurred. Our data suggest that F2-isoprostanes are useful markers of LDL oxidation in vivo. As F2-isoprostanes are potent vasoconstrictors and can modulate platelet aggregation, their formation in LDL demonstrated here may also have important implications for the etiology of cardiovascular disease.
Lipoproteins labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) are widely used to visualize LDL-and scavenger-receptor activity in cultured cells. The purpose of this study was to evaluate a new single-step fluorometric assay with high sensitivity for the quantitative determination of the LDL- or scavenger-receptor activity in adherent and non-adherent cells. We used an aqueous solution of 1 g/l SDS dissolved in 0.1 M NaOH to lyse the cells after incubation with DiI-LDL or DiI-acetylated LDL. This allows for the first time the determination of fluorescence intensity and cell protein in the same sample without prior lipid extraction of the fluorochrome. Fluorescence of the cell lysates was determined in microtiter plates with excitation-emission set at 520 and 580 nm, respectively. This rapid method demonstrates high specificity for determining the LDL- and scavenger-receptor activity in cultured cells (e.g., human skin fibroblasts from patients with and without familial hypercholesterolemia; human U-937 monocyte and murine P388 D1 macrophage cell lines). The validity of our fluorescence assay is demonstrated by comparison of cellular uptake and metabolism of lipoproteins labeled with both, DiI and 125iodine. The rapidity and accuracy of this assay allows its routine application for studying receptor-mediated lipoprotein uptake in various cell types.
Free radicals have been implicated in the pathogenesis of an increasing number of diseases. Lipids, which undergo peroxidation, are major targets of free radical attack. We report the discovery of a pathway of lipid peroxidation that forms a series of isomers in vivo that are characterized by a substituted tetrahydrofuran ring structure, termed isofurans (IsoFs). We have proposed two distinct pathways by which IsoFs can be formed based on 18O2 and H2 18O labeling studies. Measurement of F2-isoprostanes (IsoPs), prostaglandin F2-like compounds formed nonenzymatically as products of lipid peroxidation, is considered one of the most reliable approaches for assessing oxidative stress status in vivo. However, one limitation with this approach is that the formation of IsoPs becomes limited at high oxygen tension. In contrast, the formation of IsoFs becomes increasingly favored as oxygen tension increases. IsoFs are present at readily detectable levels in normal fluids and tissues, and levels increase dramatically in CCl4-treated rats, an animal model of oxidant injury. The ratio of IsoFs to IsoPs in major organs varies according to normal steady-state tissue oxygenation. In addition, IsoFs show a marked increase early in the course of hyperoxia-induced lung injury, whereas IsoPs do not significantly increase. We propose that combined measurement of IsoFs and IsoPs should provide a more reliable index of oxidant stress severity than quantification of either alone because of the opposing modulation of the two pathways by oxygen tension, which can vary widely in different organs and disease states.
Cultured cells are able to oxidize low-density lipoproteins (LDL) and oxidized LDL (oxLDL), which are present in atherosclerosis areas, exhibit a variety of biological properties potentially involved in atherogenesis. This review is focused on the toxicity of oxLDL, more precisely on the toxic compounds generated during LDL oxidation, the features and the mechanisms of cell death (apoptosis or necrosis) induced by oxLDL. After internalization, toxic oxidized lipids, namely lipid peroxides, oxysterols and aldehydes, induce modifications of cell proteins, elicit oxidative stress, lipid peroxidation and alter various signaling pathways and gene expression. These events may participate in the toxic effect, and converge to trigger an intense, delayed and sustained calcium peak which elicits either apoptosis or necrosis processes. OxLDL-induced apoptosis involves both mitochondrial and death-receptor (Fas/FasL) apoptotic pathways, thereby activating the classical caspase cascade and subsequent biochemical and morphological apoptotic features. When apoptosis is blocked by overexpression of Bcl-2, oxLDL trigger necrosis through a calcium-dependent pathway. Apoptosis occurring in atherosclerotic areas is potentially involved in endothelial cell lining defects, necrotic core formation and plaque rupture or erosion which may trigger atherothrombotic events. However, the precise role of oxLDL in apoptosis/necrosis occurring in vivo in atherosclerotic plaques remains to be clarified.
The cerebral vascular supply is constructed to protect the cerebral hemispheres and brainstem from the consequences of blood flow cessation. Reversal of blood flow around local obstructions is a feature of the microvascular beds of the striatum and cerebral cortex. Cerebral capillaries of these beds consist of endothelial cells, basal lamina, and astrocyte end-feet that sit in close apposition. The interaction of astrocytes with neurons indicates the close relationship, of microvessels to neurons, These relationships are altered when blood flow ceases in the supplying artery. Increased endothelial cell permeability and endocytoses load to edema formation, and matrix degradation is associated with hemorrhage, Autoregulation is lost, Ischemia initiates leukocyte adhesion receptor expression, which is promoted by cytokine generation from the neuropil and activated monocytes. "Preactivation" may further augment the inflammatory responses to ischemia, The activation of cerebral microvessels by ischemia is heterogeneous, involving alterations in integrin-matrix interactions, leukocyte-endothelial cell adhesion, permeability changes, and the "no-reflow'' phenomenon due to platelet activation, fibrin formation, and leukocyte adhesion. Ischemia produces swelling of the microvascular endothelium, and rapid detachment and swelling of the astrocyte end-feet. Ischemic injury targets the microvasculature, where the inflammatory responses are initiated and contribute to tissue injury.
Cerebrovascular diseases are one of the major causes of death and morbidity in the entire world. In this paper, we reviewed the initial approach of the patients with ischemic stroke, the most common form of cerebrovascular disease. Treatment with the recombinant tissue plasminogen activator remains the most beneficial proven intervention for emergency treatment of stroke, but just a few patients can receive it. New interventions such as mechanical recanalization, intra-arterial thrombolytic agents and neuroprotective agents are under investigation.
Low-density lipoprotein (LDL) exists within the brain and is highly vulnerable to oxidative modifications. Once formed, oxidized LDL (oxLDL) is capable of eliciting cytotoxicity, differentiation, and inflammation in nonneuronal cells. Although oxLDL has been studied primarily for its role in the development of atherosclerosis, recent studies have identified a possible role for it in neurological disorders associated with oxidative stress. In the present study application of oxLDL, but not LDL, resulted in a dose- and time-dependent death of cultured rat embryonic neurons. Studies using pharmacological inhibitors implicate the involvement of calcium, reactive oxygen species, and caspases in oxLDL-induced neuronal death. Coapplication of oxLDL with either amyloid β-peptide or glutamate, agents that enhance oxidative stress, resulted in increased neuronal death. Taken together, these data demonstrate that oxLDL induces neuronal death and implicate a possible role for oxLDL in conditions associated with increased levels of reactive oxygen species, including Alzheimer's disease.
Low-density lipoprotein (LDL) may contribute to the pathogenesis of glomerulosclerosis by stimulating a mesangial cell inflammatory response. Interleukin-6 (IL-6) is a marker of active inflammation and ongoing glomerular injury. Therefore, we investigated the effects of native and oxidized LDL on human mesangial cell production of IL-6 and a possible modulation of this inflammatory response by lovastatin, which has been shown to ameliorate experimental glomerulosclerosis. Human mesangial cells were exposed for 6 or 24 h to culture medium containing either native LDL alone or a LDL mixture containing 5 or 20% oxidized LDL. We found that native LDL stimulated 6 h mRNA expression and secretion of IL-6. This effect was further enhanced, in a dose-related manner, when mesangial cells were exposed to increasing concentrations of oxidized LDL. Lovastatin markedly inhibited mesangial cell expression of IL-6 mRNA and reduced IL-6 secretion. The inhibitory effects of lovastatin were overridden at least partially by exogenous mevalonate. We conclude that LDL, and particularly oxidized LDL, might contribute to the pathogenesis of glomerular disease by modulating the inflammatory response of human mesangial cells, as assessed by the stimulation of IL-6 expression. Moreover, this inflammatory response can be prevented by lovastatin, providing a potential direct anti-inflammatory mechanism by which HMG-CoA reductase inhibitors may attenuate lipid-induced glomerular injury.
Oxidized LDL (OxLDL) competes with oxidatively damaged and apoptotic cells for binding to mouse peritoneal macrophages, implying the presence of one or more common domains. However, the nature of the ligands involved has not been determined. Studies in this laboratory over the last several years provide evidence that oxidized phospholipids, present in OxLDL and also in the membrane of apoptotic cells, represent one such ligand. These oxidized phospholipids, either in the lipid phase of OxLDL or becoming attached covalently to apoprotein B during LDL oxidation, have been shown to play a major role in the binding of OxLDL to CD36 and to SR-B1 expressed in transfected cells. The lipid and protein moieties compete with each other to some extent, indicating that they are binding to at least one common site. A monoclonal antibody selected because of its reactivity with OxLDL proved to be an antibody against oxidized phospholipids (but not native phospholipids). This antibody (EO6) blocked the uptake of OxLDL by CD36 and by SR-B1 in transfected cells by as much as 80%; it also inhibited macrophage phagocytosis of apoptotic cells by about 40%. Thus, the persistence of receptors for OxLDL during evolution is probably accounted for by their role in recognition of ligands on the surfaces of oxidatively damaged or apoptotic cells. This has important implications in biology generally and specifically in atherogenesis, because apoptosis is a prominent feature of late lesions.
Brain regional oxidative damage is thought to be a central mechanism in the pathogenesis of Alzheimer’s disease (AD). Recent
studies of cerebrospinal fluid (CSF) have suggested that increased lipid peroxidation of CSF and CSF lipoproteins also may
occur in AD patients. In the present study, we determined the susceptibility of human CSF to ex vivo lipid peroxidation and tested the hypothesis that oxidized CSF lipoproteins may be neurotoxic. Whole CSF or a CSF lipoprotein
fraction (d<1.210 g/mL) was oxidized with 2,2′-azobis(2-amidino-propane)dihydrochloride (AAPH), a hydrophilic free-radical generator.
Kinetics of CSF lipid peroxidation were followed by a standard fluorescence product accumulation assay. Oxidation of AD CSF
yielded significantly shorter fluorescent lag times than controls, indicating reduced antioxidant capacity. Electrophoretic
mobilities of CSF apolipoproteins were specifically reduced upon oxidation of CSF with AAPH, suggesting that lipoproteins
are primary targets of CSF lipid peroxidation. Cultured neuronal cells were exposed to physiological concentrations of isolated
CSF lipoproteins oxidized with increasing concentrations of AAPH; the resulting neurotoxicity showed a significant linear
AAPH concentration-response relationship. These results suggest that oxidized CSF lipoproteins may contribute to the pathogenesis
of neurodegeneration in AD.
The antioxidant and neuroprotective potential of the glutathione peroxidase mimic ebselen has been investigated in experimental stroke. Intravenous ebselen (1 mg/kg/h) or vehicle infusion was started 45 min before permanent middle cerebral artery occlusion in the rat, and continued until the end of the experiment. The topography and extent of oxidative damage to the brain was assessed immunohistochemically using an antibody for DNA damage that identified hydroxylated products of 2'-deoxyguanosine (8-OHdG/8-oxodGuo) and an antibody for lipid peroxidation that identified the 4-hydroxynonenal histidine adduct (4-HNE). Ischemic damage was mapped and evaluated with standard histopathology. In the vehicle-treated rats immunopositive staining for both 8-oxodGuo and 4-HNE extended beyond the boundary of ischemic damage. In ebselen-treated rats, the extent of tissue immunopositive for 8-oxodGuo, and 4-HNE was less than that demonstrating ischemic damage confirming the antioxidant mechanism of action in vivo. In addition, ebselen treatment induced a 28% reduction in cortical ischemic damage (p <.02).
Central nervous system(CNS) has nearly a quarter of total body cholesterol, although its weight is only 2% of the whole body. In the adult brain, cholesterol synthesis is extremely low, and a half life of cholesterol is about 5 years. In the cerebrospinal fluid(CSF), the only lipoprotein is high-density lipoprotein (HDL). CSF-HDL is larger than plasma HDL, and rich in apolipoprotein E (apoE). In CNS, neurons and glia cells express several lipoprotein receptors and ATP-binding cassette (ABC) transporters. These lipoprotein receptors can bind to and take up CSF-HDL for cholesterol recycling. Recent research has focused on investigating the role of CSF-HDL and lipoprotein receptors in the pathogenesis of Alzheimer disease.
The presence of apolipoproteins A-I, E, C-II, and C-III and the absence of apolipoprotein B was demonstrated in human cerebrospinal fluid. The concentration of apolipoproteins was measured by electroimmunoassay. Apolipoproteins E, C-II, and C-III were present in cerebrospinal fluid at 3--5% of their concentration in plasma; the cerebrospinal fluid level of apolipoprotein A-I was 0.4%. Most of the cerebrospinal fluid apolipoproteins were present in the rho less than 1.21 g/ml lipoprotein fraction. The major apolipoporteins of cerebrospinal fluid are E and A-I. The possible mechanism of transfer and the physiological and pathophysiological role of apolipoproteins in cerebrospinal fluid are postulated.
We have previously demonstrated that stroke influences systemic immune responses. The aim of the present study was to investigate patterns of local inflammatory response as a consequence of acute stroke.
Thirty stroke patients were studied prospectively on days 0 to 3, 7 to 9, 21 to 26, and after day 90 with clinical evaluations, radiological assessments, and analysis of serum and cerebrospinal fluid cytokine levels.
Significantly increased levels of interleukin-6 (IL-6) in cerebrospinal fluid (P < .001) were observed in virtually all patients studied compared with healthy control subjects. This increase was observed during the whole observation period but was significantly more pronounced within the first days after stroke onset, with a peak level on days 2 and 3. This initial increase was significantly correlated (r = .65, P = .002) with the volume of infarct measured by MRI 2 to 3 months later. Serum levels of IL-6 in stroke patients were significantly lower than cerebrospinal fluid levels of IL-6 (P = .013) and did not display any significant correlation to the size of the brain lesion. Also, increase in intrathecal but not systemic production of IL-1 beta was observed early during the stroke. Only minor increases of cerebrospinal fluid interferon-gamma levels were observed in two patients.
Our study demonstrates an intrathecal production of IL-6 and IL-1 beta in patients with stroke, supporting the notion of localized inflammatory response to acute brain lesion. In addition, the significant correlation between early intrathecal production of IL-6 and the subsequent size of the brain lesion can be used as a prognostic tool, predicting the size of the brain damage before it is possible to accurately visualize it with radiological methods.
To investigate either the role oxidized LDL plays in atherosclerosis or structural changes on the surface of oxidized LDL, monoclonal antibodies (mAbs) are an important tool. After immunizing mice with Cu(2+)-oxidized LDL (oxLDL) and fusion of splenocytes, hybridoma supernatants were screened and cloned. Two mAbs, OB/04 and OB/09 (IgG and IgM), were further characterized. In solid-phase fluorescence immunoassays and Western blot analysis both mAbs reacted with oxLDL, LDL oxidized by a free radical-generating azo compound, or oxVLDL but not with native LDL, acetylated LDL, oxHDL3, azo-oxidized HDL3, or HDL3 modified with malondialdehyde (MDA). In competitive immunoassays with LDL modified by oxidized fatty acid-derived aldehydes, mAb OB/09 strongly reacted with MDA-LDL or MDA-VLDL and LDL modified with 4-hydroxyhexenal followed by 4-hydroxynonenal but not with 4-hydroxyoctenal or hepta-2,4-dienal. mAb OB/04 had a weak affinity for LDL after modification with these aldehydes except for MDA-LDL. LDL modified with arachidonic acid oxidation products (AAOPs) was also recognized by this mAb. However, albumin modified either by the aldehydes applied or by AAOPs did not react with either mAb. Thus, the data indicate that each of the mAbs recognizes a different epitope that is expressed only on apoB-containing lipoproteins upon oxidative modification. An immunostaining with mAb OB/04 was obtained in areas rich in macrophages and in connective tissue of a human atherosclerotic lesion.
Death induced by oxidized low density lipoproteins (oxLDL) to embryonic CNS neuronal and neuroblastoma cells was investigated. Cell damage and viability were evaluated by LDH leakage and the MTT method, respectively. Dose- and time-dependent degeneration of neurons occurred after oxLDL (1-100 microg/ml) treatment but was absent after native low density lipoproteins (LDL). This degeneration was mediated, in part, by apoptosis because increased TUNEL and Hoechst dye-positive staining was observed. These effects occurred in the absence of microglia. However, DNA degradation was not detected. The cytotoxicity was attenuated by pre-treatment with antioxidants. These results suggest that oxidation by oxLDL may be important in neurocytotoxicity in the brain.
THE NATIONAL INSTITUTE ON AGING AND REAGAN INSTITUTE WORKING GROUP ON DIAGNOSTIC CRITERIA FOR THE NEUROPATHOLOGICAL ASSESSMENT OF ALZHEIMER'S DISEASE.Consensus recommendations for the postmortem diagnosis of Alzheimer's disease. NEUROBIOL AGING18(S4) S1-S2, 1997.—This report summarizes the consensus recommenda- tions of a panel of neuropathologists from the United States and Europe to improve the postmortem diagnostic criteria for Alzheimer's disease. The recommendations followed from a two-day workshop sponsored by the National Institute on Aging (NIA) and the Ronald and Nancy Reagan Institute of the Alzheimer's Association to reassess the original NIA criteria for the postmortem diagnosis of Alzheimer's disease published in 1985 (2). The consensus recommendations for improving the neuropathological criteria for the postmortem diagnosis of Alzheimer's disease are reported here, and the "position papers" by members of the Working Group that accompany this report elaborate on the research findings and concepts upon which these recommendations were based. Further, commentaries by other experts in the field also are included here to provide additional perspectives on these recommendations. Finally, it is anticipated that future meetings of the Working Group will reassess these recommendations and the implementation of postmortem diagnostic criteria for Alzheimer's disease. © 1997 Elsevier Science Inc.
Alzheimer's disease (AD) is the most common form of dementia in the United States and has been associated with APOE genotype. Apolipoprotein (apo) E along with apoAI serve as the major apolipoproteins in the central nervous system; however, we are unaware of any study addressing lipoprotein metabolism in AD. We tested the hypothesis that lipoprotein metabolism is altered in patients with AD by isolating and characterizing ventricular fluid (VF) lipoproteins obtained during a rapid autopsy protocol from patients with AD and age-matched nondemented control patients. Our results demonstrated abnormalities in the protein and lipid constituents of VF lipoproteins from AD patients. Apolipoprotein concentration was reduced by half in AD patients relative to controls; however, there was no selective reduction in apoE or apoAI. In addition, cholesteryl ester fatty acids, but not phospholipid fatty acids, from AD patients demonstrated a significant reduction in some polyunsaturated fatty acids (18:2 and 22:6) and an enrichment in 18:0. None of these changes were directly related to APOE genotype. Our data indicate that VF lipoprotein composition is altered, at least terminally, in AD patients, and that these changes are not associated with APOE. These findings suggest that altered VF lipoprotein metabolism may be a component of AD pathogenesis.
Two major risk factors for late-onset familial and sporadic Alzheimer disease (AD), a leading cause of dementia worldwide, are increasing age and inheritance of the epsilon4 allele of the apolipoprotein E gene (APOE4). Several isoform-specific effects of apoE have been proposed; however, the mechanisms by which apoE isoforms influence the pathogenesis of AD are unknown. Also associated with AD is increased lipid peroxidation in the regions of the brain most damaged by disease. 4-hydroxynonenal (HNE), the most potent neurotoxic product of lipid peroxidation, is thought to be deleterious to cells through reactions with protein nucleophiles. We tested the hypothesis that accumulation of the most common forms of HNE-protein adducts, borohydride-reducible adducts, is associated with AD and examined whether there was a relationship to APOE. Our results demonstrated that reducible HNE adducts were increased in the hippocampus, entorhinal cortex, and temporal cortex of patients with AD. Furthermore, our data showed that the pattern of reducible HNE adduct accumulation was related to APOE genotype; AD patients homozygous for APOE4 had pyramidal neuron cytoplasmic accumulation of reducible HNE adducts, while AD APOE3 homozygotes had both pyramidal neuron and astrocyte accumulation of reducible HNE adducts. This is in contrast to our previous observations that a distinct HNE protein adduct, the pyrrole adduct, accumulates on neurofibrillary tangles in AD patients. We conclude that APOE genotype influences the cellular distribution of increased reducible HNE adduct accumulation in AD.
Astrocytes are thought to control extracellular glutamate concentrations ([Glu]o) in the brain, thereby protecting neurons from excitotoxic injury. We investigated the effects of metabotropic glutamate receptor (mGluR) agonists on glutamate transport and [Glu]o in primary hippocampal astrocytic cultures. Acute or chronic exposure of astrocytes to the mGluR agonist trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) or its active isomer 1S,3R-ACPD reduced [Glu]o in a time- and dose-dependent manner (44.5 +/- 3.6% reductions of [Glu]o in astrocytes from P0-P10 rats and 65.9 +/- 4.1% from rats P20 by 100 microM 1S,3R-ACPD, EC50 approximately 5 microM). 1S,3R-ACPD effects developed slowly (median effective at approximately 60 min) and persisted for several hours after agonist removal. ACPD-pretreated astrocytes established lower steady-state [Glu]o levels. ACPD effects persisted in the presence of the glutamate uptake inhibitors D,L-threo-beta-hydroxyaspartate (THA) and L-trans-pyrrolidine-2,4-dicarboxylate (PDC) but were impaired by disruption of the transmembrane Na+, K+, or H+ gradients. In addition, 1S,3R-ACPD had no effects on intracellular glutamate content and did not directly block glutamate transport. Furthermore, ACPD effects could be mimicked by glutamate per se and several other compounds presumed to be mGluR agonists, although (S)-3,5-dihydroxyphenylglycine (DHPG), (2S,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV), and L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) were without effect. These data suggest that glutamate and certain mGluR agonists may regulate [Glu]o by modulating the transmembrane equilibrium of glutamate transport, especially by attenuating glutamate release.
To quantify F2-isoprostane levels in CSF obtained from the lumbar cistern of patients with AD, ALS, and controls.
Studies of human postmortem tissue and experimental models have suggested a role for oxidative damage in the pathogenesis of several neurodegenerative diseases, especially AD and ALS. F2-isoprostanes are exclusive products of free-radical-mediated peroxidation of arachidonic acid that have been widely used as quantitative biomarkers of lipid peroxidation in vivo in humans. Recently, we showed that F2-isoprostane concentrations are significantly elevated in CSF obtained postmortem from the lateral ventricles of patients with definite AD compared with controls.
F2-isoprostanes were quantified by gas chromatography/negative ion chemical ionization mass spectrometry.
CSF F2-isoprostanes were increased significantly in patients with probable AD, but not in ALS patients, compared with controls.
Increased CSF F2-isoprostanes are not an inevitable consequence of neurodegeneration and suggest that increased brain oxidative damage may occur early in the course of AD.
Low-density lipoprotein (LDL) exists within the brain and is highly vulnerable to oxidative modifications. Once formed, oxidized LDL (oxLDL) is capable of eliciting cytotoxicity, differentiation, and inflammation in nonneuronal cells. Although oxLDL has been studied primarily for its role in the development of atherosclerosis, recent studies have identified a possible role for it in neurological disorders associated with oxidative stress. In the present study application of oxLDL, but not LDL, resulted in a dose- and time-dependent death of cultured rat embryonic neurons. Studies using pharmacological inhibitors implicate the involvement of calcium, reactive oxygen species, and caspases in oxLDL-induced neuronal death. Coapplication of oxLDL with either amyloid beta-peptide or glutamate, agents that enhance oxidative stress, resulted in increased neuronal death. Taken together, these data demonstrate that oxLDL induces neuronal death and implicate a possible role for oxLDL in conditions associated with increased levels of reactive oxygen species, including Alzheimer's disease.
High-density lipoprotein (HDL) exists within the brain and is highly vulnerable to oxidative modifications. The focus of the present study was to determine the effect of HDL and oxidized HDL (oxHDL) upon neurons, astrocytes, and microglia. Administration of highly oxidized HDL, but not native, minimally, or moderately modified HDL resulted in a dose- and time-dependent increase in oxidative stress and death of cultured rat embryonic neurons. Astrocyte and microglia cultures treated with highly oxidized HDL displayed increased reactive oxygen species formation but no toxicity. Application of oxHDL exacerbated oxidative stress and neuron death induced by beta-amyloid peptide. Studies using pharmacological inhibitors implicate the involvement of calcium and reactive oxygen species in oxHDL-induced neuronal loss. Neural cells expressing increased levels of BCL-2 had decreased levels of oxidative stress and neuron death following exposure to oxHDL. Together, these data demonstrate that oxHDL increases oxidative stress in neurons, astrocytes, and microglia which ultimately culminate in neuron death.
The cerebral vascular supply is constructed to protect the cerebral hemispheres and brainstem from the consequences of blood flow cessation. Reversal of blood flow around local obstructions is a feature of the microvascular beds of the striatum and cerebral cortex. Cerebral capillaries of these beds consist of endothelial cells, basal lamina, and astrocyte end-feet that sit in close apposition. The interaction of astrocytes with neurons indicates the close relationship of microvessels to neurons. These relationships are altered when blood flow ceases in the supplying artery. Increased endothelial cell permeability and endocytoses lead to edema formation, and matrix degradation is associated with hemorrhage. Autoregulation is lost. Ischemia initiates leukocyte adhesion receptor expression, which is promoted by cytokine generation from the neuropil and activated monocytes. "Preactivation" may further augment the inflammatory responses to ischemia. The activation of cerebral microvessels by ischemia is heterogeneous, involving alterations in integrin-matrix interactions, leukocyte-endothelial cell adhesion, permeability changes, and the "no-reflow" phenomenon due to platelet activation, fibrin formation, and leukocyte adhesion. Ischemia produces swelling of the microvascular endothelium, and rapid detachment and swelling of the astrocyte end-feet. Ischemic injury targets the microvasculature, where the inflammatory responses are initiated and contribute to tissue injury.
Interleukin-6 (IL-6) appears to be involved in the inflammatory response associated with central nervous system (CNS) ischemia. Although IL-6 levels increase after stroke, it is not known whether IL-6 directly influences CNS ischemic injury. In this study, we used a focal reversible stroke model to investigate whether mice lacking IL-6 were protected against acute ischemic injury.
We bred IL-6-deficient C57 black mice (I-129 IL-6 KO back-crossed with C57), including homozygous knockouts (IL-6 -/-), heterozygous littermates (IL-6 +/-), and normal littermates (IL-6 +/+). The status of all animals was confirmed by DNA sampling and polymerase chain reaction analysis. Reversible middle cerebral artery occlusion was produced by advancing a silicone-coated 8-0 filament into the internal carotid artery for 2 hours (experiment 1) or 45 minutes (experiment 2). At 24 hours, animals were evaluated on a 28-point clinical scale, blood and cerebrospinal fluid were obtained, and the brains were evaluated for infarct volume and IL-6 mRNA levels.
In experiment 1 (severe ischemia), no differences were seen in lesion size or neurological function between the groups: lesion volume was IL-6 -/- (n=15), 57+/-13 mm(3); IL-6 +/- (n=15), 58+/-23 mm(3); and IL-6 +/+ (n=15), 58+/-18 mm(3) (P=NS). ELISA testing confirmed very low to absent levels of IL-6 in the serum and cerebrospinal fluid of knockout animals. Brain mRNA levels of the other proinflammatory cytokines, including tumor necrosis factor-alpha, IL-1beta, and IL-1 receptor antagonist, were 50% lower in IL-6-deficient ischemic animals than in normal animals. In experiment 2 (mild ischemia), no differences were seen in lesion size or neurological function between the groups: lesion volume was IL-6 -/- (n=10), 16+/-8 mm(3); IL-6 +/- (n=10), 14+/-4 mm(3); and IL-6 +/+ (n=10), 19+/-12 mm(3) (P=NS).
In this study, infarct size and neurological function at 24 hours were not different in animals deficient in IL-6 after transient CNS ischemia. This suggests that IL-6 does not have a direct influence on acute ischemic injury. Further study investigating the role of IL-6 on long-term recovery after stroke is in progress.
With the advent of thrombolytic therapy for acute stroke, reperfusion-associated mechanisms of tissue injury have assumed greater importance. In this experimental study, we used several MRI techniques to monitor the dynamics of secondary ischemic damage, blood-brain barrier (BBB) disturbances, and the development of vasogenic edema during the reperfusion phase after focal cerebral ischemia in rats.
Nineteen Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion of 30 minutes, 60 minutes, or 2.5 hours with the suture occlusion model. MRI, including diffusion-weighted imaging (DWI), T2-weighted imaging, perfusion-weighted imaging, and T1-weighted imaging, was performed 5 to 15 minutes before reperfusion, as well as 0.5, 1.5, and 2.5 hours and 1, 2, and 7 days after withdrawal of the suture. Final infarct size was determined histologically at 7 days.
In the 30-minute ischemia group (and partially also after 60 minutes), DWI abnormalities reversed transiently during the early reperfusion period but recurred after 1 day, probably due to secondary ischemic damage. After 2.5 hours of ischemia, DWI abnormalities no longer reversed, and signal intensity on both DWI and T2-weighted images increased rapidly in the previously ischemic region due to BBB damage (enhancement on postcontrast T1-weighted images) and edema formation. Early BBB damage during reperfusion was found to be predictive of relatively pronounced edema at subacute time points and was probably related to the increased mortality rates in this experimental group (3 of 7).
Reperfusion after short periods of ischemia (30 to 60 minutes) appears to be mainly complicated by secondary ischemic damage as shown by the delayed recurrence of the DWI lesions, whereas BBB damage associated with vasogenic edema becomes a dominant factor with longer occlusion times (2.5 hours).
Interest in cerebrospinal fluid (CSF) lipoproteins has been stimulated by the association of certain alleles of the human apolipoprotein E gene (APOE) with an increased risk of Alzheimer's disease (AD), and because apolipoprotein E (apoE) is one of the major apolipoproteins in CSF. CSF lipoproteins (d < 1.210 g/ml fraction) are distinct from their plasma counterparts, and in AD patients CSF may contain novel particles. The protein concentration of CSF lipoproteins is reduced in AD patients. Moreover, the molecular distribution of apoE- and apoAII-containing apolipoproteins in CSF is dictated by APOE. The lipid composition suggests that CSF lipoproteins from AD patients may have undergone increased free radical-mediated damage; experimental data support the possibility that this may occur both before and after lipoprotein assembly. Finally, human CSF lipoproteins oxidized ex vivo are neurotoxic to neuronal cells in culture and disrupt microtubule structure, an activity not observed with oxidized bovine CSF lipoproteins. CSF lipoproteins may represent a means whereby apoE influences the outcome of free radical-mediated damage to brain.
Considerable evidence now points to an important role for the immune system in experimental models of atherosclerosis. We have reviewed the growing body of evidence that oxidation of LDL generates a wide variety of neoself determinants that lead to cellular and humoral immune responses. In particular, we have demonstrated that at least some of the oxidation-specific epitopes generated on the oxidized LDL particle, such as oxidized phospholipid epitopes, are also generated on apoptotic cells and are also present on the surface of some bacteria. Many of these same epitopes serve as important ligands mediating the binding and clearance of oxidatively damaged lipoprotein particles and apoptotic cells, and the innate immune response to these epitopes can be seen as a concerted response to effect their removal. In addition, other epitopes of OxLDL also undoubtedly play a role in the immune activation that characterizes the progressive atherosclerotic plaque. It will be of great importance to define the importance of the role of these responses and to understand which are beneficial and which deleterious. Such information could lead one day to novel therapeutic approaches to inhibit atherogenesis that take advantage of the ability to manipulate the immune response.
The mechanisms for clinical deterioration in patients with ischemic stroke are not completely understood. Several proinflammatory cytokines are released early after the onset of brain ischemia, but it is unknown whether inflammation predisposes to neurological deterioration. We assessed the implication of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha in early neurological worsening in ischemic stroke.
Two hundred thirty-one patients consecutively admitted with first-ever ischemic cerebral infarction within the first 24 hours from onset were included. Neurological worsening was defined when the Canadian Stroke Scale (CSS) score fell at least 1 point during the first 48 hours after admission. IL-6 and TNF-alpha were determined in plasma and cerebrospinal fluid (CSF; n=81) obtained on admission.
Eighty-three patients (35.9%) deteriorated within the first 48 hours. IL-6 in plasma (>21.5 pg/mL; OR 37.7, CI 11.9 to 118.8) or in CSF (>6.3 pg/mL; OR 13.1, CI 2.2 to 77.3) were independent factors for early clinical worsening, with multiple logistic regression. The association was statistically significant in all ischemic stroke subtypes as well as in subjects with cortical or subcortical infarctions. IL-6 in plasma was highly correlated with body temperature, glucose, fibrinogen, and infarct volume. CSF and plasma concentrations of TNF-alpha were also higher in patients who deteriorated, but the differences observed did not remain significant on multivariate analysis.
In addition to participating in the acute-phase response that follows focal cerebral ischemia, IL-6 levels on admission are associated with early clinical deterioration. The association between IL-6 and early neurological worsening prevails without regard to the initial size, topography, or mechanism of the ischemic infarction.
Cerebral spinal fluid (CSF) lipoproteins have become a focus of research since the observation that inheritance of particular alleles of the apolipoprotein E gene affects the risk of Alzheimer's disease (AD). There is evidence of increased lipid peroxidation in CSF lipoproteins from patients with AD, but the biological significance of this observation is not known. A characteristic of the AD brain is a disturbance of the neuronal microtubule organization. We have shown previously that 4-hydroxy-2(E)-nonenal, a major product of lipid peroxidation, causes disruption of neuronal microtubules and therefore tested whether oxidized CSF lipoproteins had the same effect. We exposed Neuro 2A cells to human CSF lipoproteins and analyzed the microtubule organization by immunofluorescence. In vitro oxidized human CSF lipoproteins caused disruption of the microtubule network, while their native (nonoxidized) counterparts did not. Microtubule disruption was observed after short exposures (1 h) and lipoprotein concentrations were present in CSF (20 microg/mL), conditions that did not result in loss of cell viability. Importantly, adult bovine CSF lipoproteins, oxidized under identical conditions, had no effect on the microtubule organization of Neuro 2A cells. Comparison of human and bovine CSF lipoproteins revealed similar oxidation-induced modifications of apolipoproteins E and A-I as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Fatty acid analysis revealed substantially lower amounts of unsaturated fatty acids in bovine CSF lipoproteins, when compared to their human counterparts. Our data therefore indicate that oxidized human CSF lipoproteins are detrimental to neuronal microtubules. This effect is species-specific, since equally oxidized bovine CSF lipoproteins left the neuronal microtubule organization unchanged.
Reactive oxygen species have been implicated in brain injury after ischemic stroke. These oxidants can react and damage the cellular macromolecules by virtue of the reactivity that leads to cell injury and necrosis. Oxidants are also mediators in signaling involving mitochondria, DNA repair enzymes, and transcription factors that may lead to apoptosis after cerebral ischemia. Transgenic or knockout mice with cell- or site-specific prooxidant and antioxidant enzymes provide useful tools in dissecting the events involving oxidative stress in signaling and damage in ischemic brain injury.
The purpose of this single-center study was to evaluate the long-term (> or =8 years) outcome of Palmaz-Schatz intracoronary stenting and to identify independent predictors of outcome. Although short-term results of Palmaz-Schatz intracoronary stenting have been promising, with a reduction in both angiographic restenosis and clinical cardiac events up to 3 years, longer-term follow-up has not been established. We analyzed clinical outcome in 426 consecutive patients at least 8 years after coronary stenting. Demographic, clinical, and procedural predictors of restenosis, survival, and event-free survival, defined as freedom from death, myocardial infarction (MI), and coronary revascularization (target stented site, target vessel, and any revascularization) were analyzed. Before discharge, 28 patients (6.6%) sustained at least 1 major cardiovascular event: 3 deaths (0.7%), 18 MIs (4.2%), and 17 repeat revascularizations. Surviving patients were followed for 8.9 years (interquartile range 8.4 to 9.4). After discharge, 59 patients (13.9%) died, 47 (11.1%) sustained an MI, and 188 (44.4%) underwent coronary revascularization. The 8-year event-free survival (freedom from death, freedom from death/MI/target-stented site revascularization, and freedom from death/MI/any coronary revascularization) was (mean +/- SE) 0.86 +/- 0.01, 0.62 +/- 0.03, and 0.47 +/- 0.02, respectively. Unstable angina, lower left ventricular ejection fraction, and saphenous vein graft stenting were found to be independent predictors of death during follow-up. Hypertension, unstable angina, multivessel disease, and multiple stent implantation were found to be independent predictors of the composite of death/MI/any coronary revascularization during follow-up. This study provided a useful assessment of very long-term outcome in survival, event-free survival, and predictors of major cardiac events 8 to 10 years after Palmaz-Schatz stent implantation.
A reactive intermediate generated by lipid peroxidation, 4-hydroxy-2-nonenal (HNE), has received considerable attention as a potential effector of oxidative damage and Abeta peptide-mediated neurotoxicity in Alzheimer disease (AD). However, little is known about aldo-keto oxidoreductases, a group of enzymes that constitute a major detoxifying pathway for HNE and related reactive aldehydes in human brain. We have determined the regional, cellular, and class distribution in human brain of the 4 major aldo-keto oxidoreductases that detoxify HNE: aldehyde dehydrogenase (ALDH): aldose reductase; aldehyde reductase: and alcohol dehydrogenase (ADH). Of these 4 enzymes, only ALDH and aldose reductase were expressed in cerebral cortex. hippocampus, basal ganglia, and midbrain: all 4 enzymes were present in cerebellum. In cerebrum and hippocampus, aldose reductase was localized to pyramidal neurons and mitochondrial class 2 ALDH was localized to glia and senile plaques. ALDH, but not aldose reductase, activity was significantly increased in temporal cortex from patients with AD compared to age-matched controls. These results suggest that in brain regions involved in AD, neurons and glia utilize different mechanisms to detoxify HNE, and that increased ALDH activity is a protective response of cerebral cortex to AD.
Astrocytes are the major glial cell within the central nervous system (CNS) and have a number of important physiological properties related to CNS homeostasis. The aspect of astrocyte biology addressed in this review article is the astrocyte as an immunocompetent cell within the brain. The capacity of astrocytes to express class II major histocompatibility complex (MHC) antigens and costimulatory molecules (B7 and CD40) that are critical for antigen presentation and T-cell activation are discussed. The functional role of astrocytes as immune effector cells and how this may influence aspects of inflammation and immune reactivity within the brain follows, emphasizing the involvement of astrocytes in promoting Th2 responses. The ability of astrocytes to produce a wide array of chemokines and cytokines is discussed, with an emphasis on the immunological properties of these mediators. The significance of astrocytic antigen presentation and chemokine/cytokine production to neurological diseases with an immunological component is described.
Evidence from a broad range of studies demonstrates that atherosclerosis is a chronic disease that, from its origins to its ultimate complications, involves inflammatory cells (T cells, monocytes, macrophages), inflammatory proteins (cytokines, chemokines), and inflammatory responses from vascular cells (endothelial cell expression of adhesion molecules). Investigators have identified a variety of proteins whose levels might predict cardiovascular risk. Of these candidates, C-reactive protein, tumor necrosis factor-alpha, and interleukin-6 have been most widely studied. There is also the prospect of inflammation as a therapeutic target, with investigators currently debating to what extent the decrease in cardiovascular risk seen with statins, angiotensin-converting enzyme inhibitors, and peroxisome proliferator-activated receptor ligands derives from changes in inflammatory parameters. These advances in basic and clinical science have placed us on a threshold of a new era in cardiovascular medicine.
Lipid peroxidation is one of the major outcomes of free radical-mediated injury that directly damages membranes and generates a number of secondary products, both from fission and endocyclization of oxygenated fatty acids that possess neurotoxic activity. Numerous studies have demonstrated increased lipid peroxidation in brain of patients with Alzheimer's disease (AD) compared with age-matched controls. These data include quantification of fission and endocyclized products such as 4-hydroxy-2-nonenal, acrolein, isoprostanes, and neuroprostanes. Immunohistochemical and biochemical studies have localized the majority of lipid peroxidation products to neurons. A few studies have consistently demonstrated increased cerebrospinal fluid (CSF) levels of isoprostanes in AD patients early in the course of their dementia, and one study has suggested that CSF isoprostanes may improve the laboratory diagnostic accuracy for AD. Similar analyses of control individuals over a wide range of ages indicate that brain lipid peroxidation is not a significant feature of usual aging. Quantification of isoprostanes in plasma and urine of AD patients has yielded inconsistent results. These results indicate that brain lipid peroxidation is a potential therapeutic target in probable AD patients, and that CSF isoprostanes may aid in the assessment of antioxidant experimental therapeutics and the laboratory diagnosis of AD.
Oxidative stress, inducible nitric oxide synthase (iNOS) and neutrophils all contribute to post-ischemic brain damage. This study has determined the time courses of these three phenomena after ischemia in parallel with histological and functional outcomes. Ischemia was produced in rats by occluding the left middle cerebral artery and both common carotid arteries for 20 min. Regional cerebral blood flow (rCBF) rapidly decreased to 20% of its preischemic value during occlusion and stabilized at 60% following reperfusion. The striatal infarction was maximal 15 h after reperfusion (50+/-3 mm(3)), whereas the cortical infarction reached its maximum at 48 h (183+/-10 mm(3)). This drastic decrease in rCBF followed by incomplete reperfusion and massive infarction is, thus, extremely severe. The cortical infarction was strongly correlated with the neurologic deficit and loss of body weight. Oxidative stress, evaluated by the decrease in glutathione concentrations, appeared in the striatum at 6 h after reperfusion and in the cortex at 15 h. Calcium-independent NOS activity, considered as inducible NOS activity, was significantly enhanced at 24 h in the striatum and at 48 h in the cortex. Myeloperoxidase activity, a marker of neutrophil infiltration, was significantly increased at 48 h in both the striatum and cortex. These time courses show that the delayed iNOS activity and neutrophil infiltration that occur after the maturation of infarction in severe ischemia may not contribute to ischemic brain damage. By contrast, early oxidative stress may well be implicated in cerebral injury.
Indicators of an acute phase response (APR) in acute ischemic stroke have been shown to correlate with infarct size and predict stroke recurrence. In this study, we examined how well the APR indicators predicted long-term stroke recovery compared with standard clinical predictors of recovery. Plasma levels of interleukin-6 (IL-6), fibrinogen, white blood cells (WBCs), and serum albumin were measured within 4+/-2 days of onset in 131 stroke patients who were free of apparent infections. Standard clinical predictors included initial National Institutes of Health Stroke Scale (NIHSS), infarct size on computed tomography (CT), and Glasgow scale. The individual correlations with 6-month Glasgow outcome were IL-6, 0.42; fibrinogen, 0.24; WBC, 0.35; albumin, 0.47; NIHSS, 0.53; infarct size, 0.19; and initial Glasgow, 0.57. (all P<.005). Multiple regression analysis yielded an adjusted R(2) of .31 for the APR indicators, compared with .38 for the clinical variables. These results indicate that the initial APR is highly correlated with 6-month stroke recovery and that this correlation approaches that observed with standard clinical predictors.
Pathology of ischemic cerebrovascular disease
- Tj Montine
- Hulette
- Wilkins
- Rengachary
Montine TJ, Hulette CM: Pathology of ischemic cerebrovascular disease. Neurosurgery. Edited by RR Wilkins, SS Rengachary. New York, McGraw-Hill, 1995, pp 2045–2051
CSF lipoproteins are more vulnerable to oxidation in Alzheimer's disease and are neurotoxic when oxidized ex vivo 6. Neely MD, Swift LL, Montine T: Human, but not bovine, oxidized cerebral spinal fluid lipoproteins disrupt neuronal microtubules
- Cn Bassett
- Md Neely
- Kr Sidell
- Wr Markesbery
- Ll Swift
- Montine
- Shie
Bassett CN, Neely MD, Sidell KR, Markesbery WR, Swift LL, Montine TJ: CSF lipoproteins are more vulnerable to oxidation in Alzheimer's disease and are neurotoxic when oxidized ex vivo. Lipids 1999, 34:1273–1280 1180 Shie et al AJP April 2004, Vol. 164, No. 4 6. Neely MD, Swift LL, Montine T: Human, but not bovine, oxidized cerebral spinal fluid lipoproteins disrupt neuronal microtubules. Lipids 2000, 35:1249 –1257
Increased cerebrospinal fluid F 2 -isoprostane concentration in probable Alzheimer's disease
- Tj Montine
- Mf Beal
- Me Cudkowicz
- O Rh Brown
- H Donnell
- Ra Margolin
- L Mcfarland
- Af Bachrach
- We Zackert
- Lj Roberts
- Morrow
Montine TJ, Beal MF, Cudkowicz ME, Brown RH, O'Donnell H, Margolin RA, McFarland L, Bachrach AF, Zackert WE, Roberts LJ, Morrow JD: Increased cerebrospinal fluid F 2 -isoprostane concentration in probable Alzheimer's disease. Neurology 1999, 52:562–565
Consensus recommendations for the postmortem diagnosis of Alzheimer disease from the National Insti-tute on Aging and the Reagan Institute Working Group on diagnostic criteria for the neuropathological assessment of Alzheimer disease
- Hyman Bt Trojanowski
- Jq
Hyman BT, Trojanowski JQ: Consensus recommendations for the postmortem diagnosis of Alzheimer disease from the National Insti-tute on Aging and the Reagan Institute Working Group on diagnostic criteria for the neuropathological assessment of Alzheimer disease. J Neuropathol Exp Neurol 1997, 56:1095–1097
Increased cerebrospinal fluid F2-isoprostane concentration in probable Alzheimer's disease
- Montine
Metabotropic glutamate receptor agonists reduce glutamate release from cultured astrocytes
- Ye