Published online before print October 7, 2004, doi: 10.1161/01.ATV.0000147534.69062.dc
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© 2004 American Heart Association, Inc.
Brief Reviews |
Potential Role of Endotoxin as a Proinflammatory Mediator of Atherosclerosis
From the Department of Internal Medicine, Divisions of Cardiovascular Diseases (L.L.S., N.L.W.) and Infectious Diseases (G.M.D.), University of Iowa and The VA Medical Center, Iowa City, Iowa.
Correspondence to Dr Lynn L. Stoll, Department of Internal Medicine, Division of Cardiovascular Diseases, E 317C GH, University of Iowa, Iowa City, IA 52242. E-mail stolll{at}mail.medicine.uiowa.edu
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Abstract |
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Top
Abstract
IntroductionAtherosclerosis is increasingly recognized as a chronic inflammatory disease. Although a variety of inflammatory markers (ie, C-reactive protein) have been associated with atherosclerosis and its consequences, it is important to identify principal mediators of the inflammatory responses. One potentially important source of vascular inflammation in atherosclerosis is bacterial endotoxin. Mutations in Toll-like receptor 4 (TLR-4), an integral component of the endotoxin signaling complex, are fairly common in the Caucasian population and have recently been associated with reduced incidence of atherosclerosis and other cardiovascular diseases in some studies. Moreover, epidemiological studies suggest that endotoxemia at levels as low as 50 pg/mL constitutes a strong risk factor for the development of atherosclerosis. Endotoxin concentrations in this range may be produced by a variety of common subclinical Gram-negative infections. In this article, we outline the main elements of the endotoxin signaling receptor complex that initiates proinflammatory signaling (lipopolysaccharide binding protein [LBP], CD14, TLR-4, and MD-2) and discuss how changes in expression of these molecules may affect proatherogenic responses in the vessel wall. We also describe some of the proinflammatory effects of endotoxin that may be relevant to atherosclerosis, and discuss how serum lipoproteins, especially high-density lipoprotein, may modulate endotoxin-induced inflammatory responses. Further, we discuss recent findings suggesting that the lipid-lowering statins may have an additional protective role in blocking at least some of these proinflammatory signaling pathways. Finally, we discuss species diversity with regard to endotoxin signaling that should be considered when extrapolating experimental data from animal models to humans.
Bacterial endotoxin is a potential source of vascular inflammation and may be an important risk factor for atherosclerosis. Here, we discuss the endotoxin signaling pathway, vascular proinflammatory effects of endotoxin and their relevance to atherogenesis, interactions between endotoxin and serum lipoproteins, and possible immunomodulatory effects of statins.
Key Words: inflammation • vascular smooth muscle cells • endothelial cells • lipopolysaccharide • species specificity
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Introduction |
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In recent years, there has been an increasing recognition of the link between inflammation and atherosclerosis.1–8 Although markers of chronic inflammation, such as C-reactive protein, are clearly predictive of clinical atherosclerosis,9,10 the sources of inflammatory responses, and the mechanisms by which inflammation leads to vascular disease, remain to be elucidated. One potentially important source of inflammation is endotoxin (lipopolysaccharide [LPS]), a unique glycolipid that comprises most of the outer leaflet of the outer wall of Gram-negative bacteria (GNB).11–15 This complex molecule, found exclusively in GNB, consists of a highly variable carbohydrate portion and a unique lipid A region that is highly conserved across many GNB species. Gram-negative organisms colonize the human gastrointestinal, genitourinary, and respiratory tracts and generate endotoxin not only during overt infections but also in common subclinical or chronic conditions such as periodontitis, sinusitis, bronchitis, or diverticulitis.16–18
Even in apparently healthy individuals, endotoxin can be detected in human plasma. The Bruneck study provided the first epidemiological evidence that subclinical endotoxemia constitutes a strong risk factor for the development of carotid atherosclerosis, particularly among smokers.19 In this 5-year prospective study, in subjects without atherosclerosis at baseline, 
40% of newly developed carotid atherosclerosis was estimated to be attributable to chronic infection, making it a leading atherogenic risk predictor.20 Moreover, chronic infections conferred an increased risk of atherosclerosis development even in low-risk subjects, who lack conventional vascular risk factors. A variety of infections caused by GNB, including respiratory and urinary tract infections, were associated with an increased risk of atherosclerosis, whereas infection with viruses was not.20 In addition, plasma was collected from patients without atherosclerosis at the time of enrollment in the study and tested for its capacity to induce endothelial cell activation and transmigration of leukocytes. Increased plasma-induced endothelial cell activation was associated with an increased risk for the development of atherosclerotic lesions in patients during the 5-year study period.21 In animal studies, weekly injections of endotoxin accelerated the development of atherosclerotic lesions in rabbits on hypercholesterolemic diets22 and in apolipoprotein E-deficient mice.23 These observations support the hypothesis that chronic exposure to endotoxin may be pathogenically linked to atherosclerosis.
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Endotoxin and Vascular Dysfunction |
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Septicemia caused by GNB is frequently accompanied by systemic vascular collapse, disseminated intravascular coagulation, and vascular leak syndromes, all of which are thought to arise from endothelial cell injury and/or dysfunction.24–26 In Gram-negative septicemia, plasma endotoxin is markedly elevated and has been implicated as the causative agent responsible for these processes. In inflammatory cells such as monocytes and macrophages, and in cultured endothelial and smooth muscle cells, levels of endotoxin equivalent to those observed during sepsis evoke numerous proinflammatory responses, including upregulation of adhesion molecule expression; increased production of cytokines, reactive oxygen species (ROS) and reactive nitrogen species, prostaglandins, and tissue factor; loss of monolayer integrity and barrier function; and apoptosis.27 Although these observations provide proof of principle that endotoxemia can elicit vascular inflammation and endothelial dysfunction, it is important to point out that endotoxin levels observed during sepsis are far greater than the low-level endotoxemia that has been associated with atherosclerosis.
Data from the Bruneck study indicate that blood endotoxin levels in an ambient population of 516 apparently healthy volunteers, with no clinical evidence of infection, ranged from 6 to 209 pg/mL, with a median of 14.3. Individuals with levels of 50 pg/mL or greater were identified to have an increased risk for development of atherosclerosis. Levels of endotoxin in this range induce inflammatory responses in human monocytes and macrophages. In addition, recent data from our laboratory indicate that vascular smooth muscle cells and intact human blood vessels also exhibit profound responsiveness (cytokine release, superoxide production, and monocyte adhesion) to very low levels of endotoxin.28,29 These findings establish the biological plausibility of low level endotoxemia as a mediator of vascular inflammation in atherosclerosis. Moreover, they suggest that in addition to tissue-resident inflammatory cells, vascular smooth muscle cells likely contribute significantly to the inflammation induced by low-level endotoxin in patients who are at risk for atherosclerosis.
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Signaling by Endotoxin |
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Activation of Cellular Signaling Pathways
Studies with mononuclear cells have identified several factors that play an important role in endotoxin signaling. Initiation of signaling at the membrane involves a number of proteins, including LPS binding protein (LBP), CD14, Toll-like receptor-4 (TLR-4), and MD-2. Together, these proteins initiate a signaling pathway that leads to activation of a series of downstream protein kinases, the most proximal of which are myeloid differentiation factor 88 (MyD88) and IL-1 receptor-associated kinase (IRAK-1), ultimately resulting in activation of nuclear factor-kappa B. Based on these studies, we propose the following model for potential interactions between endotoxin and vascular cells (Figure ).
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) attracted to inflammatory loci. Statins block proinflammatory signaling by inhibiting the endotoxin/TLR-4 pathway.
LBP
LPB is a 60-kDa lipid/phospholipid binding and transfer protein with fairly broad specificity.30,31 Its primary role with regard to endotoxin is believed to be the extraction of endotoxin monomers from the bacterial membrane or from aggregates of circulating endotoxin, and the subsequent delivery of these molecules to CD14, resulting in target cell activation, or to lipoproteins, leading to hepatic clearance.32,33 In particular, high-density lipoprotein (HDL) is believed to play a major role in clearance of circulating endotoxin (see Endotoxin and Lipoproteins).32,34–37
CD14
CD14 is a pattern-recognition receptor that plays a central immunomodulatory role in proinflammatory signaling in response to a variety of ligands, including endotoxin and other bacterial products from both Gram-negative and Gram-positive bacteria.38,39 Monocytes and neutrophils respond to endotoxin via membrane-bound CD14 (mCD14), a 55-kDa GPI-anchored protein.40 In cells that are deficient in mCD14, overexpression of this protein markedly enhances their sensitivity to endotoxin, indicating that the level of mCD14 can be an important determinant of endotoxin-induced cellular activation.38,41 Of note, a human CD14 promoter polymorphism has been shown to be associated with reduced incidence of myocardial infarction in a population of low-risk patients, although the effects of this polymorphism on CD14 expression and on LPS responsiveness were not determined.42 More recently, in a population-based study, levels of soluble CD14 (sCD14) were positively correlated with aortic stiffness and carotid plaque formation,43 thus supporting a proatherosclerotic effect of CD14 in humans.
Although it is generally believed that endothelial cells lack mCD14 and respond to endotoxin primarily through sCD14,44,45 a recent report by Jersmann et al indicates that mCD14 is expressed in primary and first-passage human umbilical vein endothelial cells and suggests that mCD14 expression may be lost during subsequent passaging of cells.46 In addition, we recently reported that human coronary artery smooth muscle cells (HCASMC) express functional mCD14.29 Furthermore, mCD14 produced by tissue macrophages can be converted to sCD14.45,47 Collectively, these findings suggest that expression of CD14 in the vascular wall of humans could contribute to the inflammatory milieu within atherosclerotic lesions.
LBP/CD14 Ratio
LBP and sCD14 are present in normal serum at levels of 5 to 15 µg/mL and 2 to 3 µg/mL, respectively.48,49 Patients with chronic low-grade infections such as periodontitis appear to have chronically elevated levels of sCD14,50 and increases in circulating sCD14 are correlated with increased mortality in bacteremia.51 However, during the acute inflammatory response, whereas sCD14 levels increase slightly (
2-fold), LBP levels increase by 10- to 30-fold.49 The increased LBP/sCD14 ratio associated with the acute phase response is believed to play a role in limiting the endotoxin-mediated inflammatory response by downregulating cellular activation and enhancing endotoxin clearance.51 In support of this hypothesis, intraperitoneal injection of LBP protected mice from LPS-induced cytokine release and hepatic failure, resulting in significantly decreased mortality.52
Chronic subacute infections implicated in atherosclerosis are associated with a lower LBP/sCD14 ratio, suggesting that the capacity of LPS to activate vascular cells might be relatively greater in these conditions.45,47 Recently, our laboratory reported that LPS-induced activation of human coronary artery endothelial cells (HCAEC) and HCASMC is modulated in a biphasic manner by the LBP/sCD14 ratio; maximal stimulation was observed at the low LBP/sCD14 ratios (maximal 1 LBP:5 sCD14), whereas ratios >1:1 (corresponding to those seen in acute inflammation) were inhibitory.29
The TLR Signaling Pathway
Because CD14 lacks a transmembrane and cytoplasmic domain, the downstream signaling pathway requires one or more accessory proteins. These proteins include members of the pattern recognition receptor family known as Toll-like receptors (TLRs), which specifically recognize pathogen-associated molecules, including endotoxin. These receptors are part of the innate immune system and are transmembrane proteins containing extracellular domains rich in leucine-repeat motifs, and a cytosolic domain homologous to the signaling domain of the IL-1 receptor. Recent studies suggest that the endotoxin–CD14 complex engages a Toll-like receptor (TLR-4).53,54 The secreted accessory protein MD-2, which appears to bind to both TLR-4 and to endotoxin, is also a critical element in this receptor complex.55–58 TLR-4 was recently shown to be expressed and to mediate the effects of endotoxin in human dermal microvascular endothelial cells.59 TLR-4 expression has been detected in murine and human atherosclerotic plaques, preferentially localized to macrophages and perhaps endothelial cells.60,61 Our laboratory has demonstrated expression of TLR-4 in intact human saphenous vein and coronary artery, as well as cultured HCAEC and HCASMC.28,29 In human coronary artery and saphenous vein, TLR-4 immunostaining was detected throughout the vessel wall, rather than being restricted to a specific location or cell type.
TLR-4 Polymorphisms and Cardiovascular Disease
Recently, mutations in human TLR-4, particularly the Asp299Gly polymorphism, which is relatively common in the Caucasian population, have been shown to be associated with a reduced incidence of atherosclerosis and other cardiovascular diseases,62–64 as well as acute coronary events and decreased plasma fibrinogen and soluble vascular cell adhesion molecule-1 levels.65 Subjects with the Asp299Gly allele also had lower levels of proinflammatory cytokines, acute-phase reactants, and soluble adhesion molecules.64 Two other population studies, however, were not able to detect an association between TLR-4 polymorphisms and cardiovascular disease.66,67 These inconsistent findings may be because of differences in study design, such as inclusion criteria and methods used to detect and quantify atherosclerosis.
Studies in TLR-4–deficient mice have also shown smaller infarctions after coronary ligation and reperfusion, reduced myocardial ischemia-reperfusion injury, and decreased lipid peroxide levels.68 These findings suggest that TLR-4–mediated inflammatory responses may contribute to ischemic myocardial damage, although Wright et al reported that there was no difference in the magnitude of aortic root atherosclerosis in TLR-4/apoE double knockout mice as compared with control apoE knockout mice.69 However, because of unique characteristics of the murine innate immune system, findings in mice may not be readily extrapolated to humans.
Recent studies suggest that TLR-4, like other TLRs, can potentially respond to alternative ligands, such as heat-shock protein 60, the EDA domain of fibronectin, and hyaluronan.70,71. Studies with putative alternative ligands must be interpreted cautiously, however, because the synthetic products used in these studies may be contaminated with endotoxin. Of particular interest to the atherosclerosis field, saturated fatty acids were demonstrated to induce proinflammatory activation of RAW 264.7 cells (a monocyte–macrophage cell line) through a TLR-4–dependent pathway.72,73 It has been known for some time that the biological activity of endotoxin is critically dependent on its lipid A moiety. Lipid A is a ß,1-6–linked disaccharide of glucosamine, acylated with R-3-hydroxylaurate or myristate, and phosphorylated at positions 1 and 4'. These saturated fatty acids are 3-O-acylated by lauric acid, myristic acid, or palmitic acid. The acyl-linked saturated fatty acids appear to confer bioactivity to endotoxin, because their hydrolysis by acyloxyacyl hydrolase results in loss of bioactivity.15 Together, these observations suggest a potential link between saturated fatty acids, TLR-4, and inflammation that may be pertinent to mechanisms of atherosclerosis.
Alternative Pathway
Although the LBP/CD14/MD-2/TLR-4 pathway has been extensively documented in many model systems, there is evidence that alternate signaling pathways may be operative at higher than physiologically relevant endotoxin concentrations in some cells. Endotoxin concentrations >100 ng/mL have been shown in numerous studies to activate host cells by mechanisms independent of the CD14–TLR-4 pathway.38,74–77 For example, Perera et al found a CD14-independent signaling pathway in macrophages from CD14 knockout mice.78 In this model, cytokine release in response to low doses of LPS (1 or 10 ng/mL) showed an absolute requirement for CD14, either the membrane-bound or soluble form. Conversely, at concentrations of 1 µg/mL or higher, LPS increased release of tumor necrosis factor (TNF)-
and IL-1ß in CD14 knockout mice to levels that were comparable to or greater than those seen in wild-type mice. Because many published studies report using concentrations of 1 µg/mL or higher, caution should be used in extrapolating the results of these studies to a more physiological setting, where concentrations in the pg/mL or very low ng/mL level are to be expected.19,25
Chlamydia pneumoniae and Endotoxin Signaling
Although Chlamydia pneumoniae has recently been identified in atherosclerotic lesions79–82 and has been implicated as a risk factor for atherosclerosis by some researchers,83–85 its role as a causal agent for atherosclerosis is still unclear. Chlamydia pneumoniae is an obligate intracellular pathogen with a complicated life cycle. Chlamydial endotoxin is only weakly active; Escherichia coli LPS is at least 10- to 100-fold more potent.86–89 This is probably because of the highly hydrophobic nature of the lipid A of C. pneumoniae, which contains unusual long fatty acid tails (
20 carbon atoms), and because of the presence of only 5 fatty acid chains (compared with 6 in E. coli, Salmonella spp., and Neisseria spp.) in Chlamydial endotoxin.86,90–92 Although endotoxin has been shown to be responsible for the immunostimulatory activity of Chlamydia trachomatis,93 endotoxin-stimulated bioactivity appears to be only a minor component of C. pneumoniae proinflammatory activity.88,94,95 These latter studies suggest that bacterial components other than endotoxin (eg, heat-shock protein 60 or other outer membrane proteins), perhaps acting intracellularly, may play a causal role in atherogenesis.89,96–98
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Endotoxin and Lipoproteins |
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Serum lipoproteins, particularly HDL, are believed to play a major role in clearance of circulating endotoxin.32,34–37,99 Endotoxin bound to lipoproteins is preferentially shunted to hepatocytes for clearance, rather than hepatic macrophages, and is ultimately excreted in bile.32 Low-density lipoprotein (LDL) is generally believed to be much less effective than HDL in removing endotoxin from the blood. Further, the relative importance of LDL in endotoxin clearance, which would presumably be antiatherogenic, is probably outweighed by the proatherogenic contributions of its oxidized lipids and other LDL components. This would be particularly true, for example, in studies of apoE–/– mice on a high-fat diet, which causes extremely high LDL levels. In one study using human volunteers, a 4-hour infusion of reconstituted HDL beginning 3.5 hours before a subsequent endotoxin challenge (4 ng/kg) markedly reduced the LPS-induced release of TNF-
, IL-6, and IL-8.100 Moreover, HDL alone, when given before the endotoxin challenge, produced a >60% reduction in monocyte mCD14 expression and reduced TNF- production by the isolated cells in response to subsequent incubation with endotoxin. In these experiments, no changes in sCD14 were seen, whereas serum LBP levels were reduced by 17%.100 Conversely, when rats were made hypolipidemic by administration of either 4-aminopyrolo-(3,4-D)pyrimide, which blocks hepatic secretion of lipoproteins, or estradiol, which increases expression of hepatic LDL receptors and lipoprotein clearance, LPS-induced mortality was significantly greater than in control animals.35 In both of these hypolipidemic animal models, LPS produced a 3- to 5-fold greater increase in serum TNF- levels compared with controls.35 These findings suggest that modulation of endotoxin-induced cellular activation could be one mechanism for the antiatherogenic effects of high HDL levels. However, given that the capacity of HDL to bind LPS is at least 10-fold higher than the LPS concentrations reported in septic patients, it is questionable whether even a 2-fold change in HDL levels would provide significant additional protection against endotoxin-induced inflammation.36 Among subjects in the Bruneck study who were identified as having chronic infections, HDL levels were reported as 56±13 mg/dL, whereas in the group who were free from such infections, reported HDL levels were 58±15 mg/dL.20 The authors cite a P value of 0.042 for these 2 groups (0.013 when corrected for age, sex, and social status); however, although these values may be statistically significant, their physiological relevance is questionable. |
Proinflammatory Effects of Endotoxin |
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Endotoxin elicits a variety of proinflammatory responses that may be pertinent to mechanisms of atherosclerosis. These responses are summarized in the Figure.
ROS and Inflammation
ROS play a key role in the pathogenesis of many chronic inflammatory disorders, including atherosclerosis.101 In endothelial cells, treatment with endotoxin resulted in upregulation of antioxidant enzyme activity, consistent with increases in oxidative stress;102–104 however, the enzymatic source of the ROS was not identified. In phagocytic cells, endotoxin potently induces ROS production by stimulating NADPH oxidase activity.105 It does so by promoting assembly via phosphorylation of p47phox and its subsequent translocation to the plasma membrane.106 A nonphagocytic NADPH oxidase that can be activated by cytokines has recently been identified as a major source of ROS in blood vessels.107–110 The vascular NADPH oxidase is expressed in the 3 major types of resident vascular cells (endothelial cells, smooth muscle cells, and fibroblasts), participates in induction of cytokine expression and smooth muscle cell growth and apoptosis, and may play an important role in the pathogenesis of hypertension as well as atherosclerosis.101,108,111–113 Very recently, Rice et al showed that LPS increased superoxide production in human saphenous vein explants; the response was dose-dependent and was found throughout the vessel wall, rather than being localized to a single cell type.28
Proinflammatory Cytokines
Of particular importance with regard to atherosclerosis is the induction of proinflammatory cytokines and chemokines, which recruit inflammatory cells to loci of inflammation.3,5,6,114–119 One of the earliest cytokines identified was macrophage inhibitory factor (MIF), which has since been shown to be expressed by monocytes/macrophages, T cells, B cells, as well as by endocrine and epithelial cells.120 Deletion of the gene for MIF was shown to protect animals from lethal endotoxemia.121 Moreover, MIF-deficient macrophages from these animals were shown to be hyporesponsive to LPS and to express lower levels of TLR-4. Thus, MIF may exert both autocrine and paracrine effects on TLR-4 expression. Endotoxin contributes either directly or indirectly to increased release of a large number of other immunomodulatory inflammatory cytokines and other effectors, including interferon-
, IL-1, IL-6, IL-8, TNF-, and granulocyte-macrophage colony-stimulating factor, along with platelet-activating factor and other bioactive arachidonic acid metabolites.14,122,123 Endotoxin also contributes to increased expression of the TNF receptor.124,125 This in turn leads to upregulation of numerous proinflammatory factors, including inducible nitric oxide synthase, chemokines, and adhesion molecules.
Chemotactic Cytokines (Chemokines)
Endotoxin directly upregulates expression of a number of chemokines that act as chemoattractants and activators of leukocytes. For the purposes of this review, we focus on two of these, MCP-1 and IL-8, both of which appear to play critical roles in atherosclerosis. MCP-1 is highly expressed in human atherosclerotic plaques and is believed to play a crucial role in monocyte recruitment into subendothelial lesions.114,126 Depletion of the MCP-1 receptor CCR2 markedly attenuated atherosclerotic lesions by inhibiting macrophage accumulation in apoE-deficient mice.127 Further, MCP-1–/– mice, when crossed with LDL receptor-deficient mice, had decreases in lesion size and macrophage infiltration.128 Our laboratory has recently demonstrated that very low levels of endotoxin (<1 ng/mL) cause significant increases in MCP-1 release by HCAEC, HCASMC, and human saphenous vein explants.28,29 HCASMC were particularly sensitive, showing an 4-fold increase over baseline in response to only 30 pg/mL LPS.29
IL-8 is another key chemokine implicated in atherogenesis.114 IL-8 is known to be chemotactic for neutrophils and to activate NADPH oxidase in these cells, resulting in a local increase in production of ROS.129 IL-8 is also chemotactic for T lymphocytes, which are prevalent in the fibrous cap of atherosclerotic lesions, where they may be involved in the pathogenesis of acute coronary syndromes.115,130 Importantly, IL-8 was recently shown to induce chemotaxis of freshly isolated peripheral blood monocytes and to convert monocyte rolling to firm adhesion on endothelial monolayers.131 A recent study of 2355 apparently healthy individuals revealed that elevated basal levels of IL-8 were associated with increased risk of future coronary artery disease.132 Furthermore, LDL receptor-deficient mice that were irradiated and repopulated with bone marrow cells lacking the murine homologue of the IL-8 receptor (CXCR-2) had less extensive atherosclerotic lesions and fewer infiltrating macrophages as compared with those mice receiving bone marrow cells expressing CXCR-2.133 Thus, vascular induction of IL-8 by endotoxin could contribute to atherogenesis through a variety of mechanisms.
IL-8 release in response to endotoxin has been demonstrated in a wide variety of experimental models. As with MCP-1, our laboratory has shown that very low levels of endotoxin (<1 ng/mL) cause significant increases in IL-8 release by human coronary artery cells and blood vessel explants,28,29 with HCASMC and explants responding to concentrations as low as 30 pg/mL LPS.
Adhesion Molecules and Monocyte Transmigration
The release of chemotactic cytokines plays a vital role in attracting monocytes, neutrophils, and T lymphocytes to the vessel wall. However, other molecules expressed on leukocytes and endothelium also play critical roles in the attachment and eventual transmigration of leukocytes across the endothelium. Migration begins with "rolling" of the leukocytes along the endothelial surface, a process mediated by selectins.134,135 The next step, "firm adhesion," is mediated by binding of ß2-integrins expressed on leukocytes to cellular adhesion molecules (CAMs; eg, intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule [VCAM-1]) that are expressed on the apical surface of the endothelium.134,136 Finally, leukocytes cross the endothelium via interactions between PECAM-1 molecules that are expressed by both cell types.137 Endotoxin modulates this process at multiple steps, including activation of ß2-integrins, upregulation of selectins and CAMs, and increased phosphorylation of PECAM-1.134,136,138,139 Endotoxin also increases release of PAF and expression of the PAF receptor by endothelial cells.134 Antibodies against PAF receptor partially inhibit transmigration, suggesting a role for PAF signaling in the process. As noted, many of these studies used endotoxin concentrations from 25 to >100 ng/mL; however, Rice et al recently showed that pretreatment of human saphenous vein endothelial cells with as little as 1 ng/mL of LPS caused a 3.5-fold increase in U-937 monocytic cell binding to the endothelial surface, when compared with explants treated with vehicle alone.28 In similar experiments with HCAEC, pretreatment of endothelial cells with 10 ng/mL LPS produced a 75-fold increase in U-937 binding compared with vehicle control (Stoll et al, unpublished data). Thus, low levels of endotoxin cause not only the production of chemotactic cytokines that attract monocytes and other leukocytes to the endothelium but also the increased expression of the adhesion molecules that facilitate the initial binding and eventual migration of these cells into the vessel wall.
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Statins and Endotoxin |
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Statins are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting step in cholesterol biosynthesis. Statins are widely used clinically for their efficacy in producing significant reductions in plasma cholesterol and LDL. However, several large clinical trials have suggested that the lipid-lowering effects may not completely account for the reduced incidence of cardiovascular disease seen in patients receiving statin therapy.140–142 Specifically, a number of these studies have shown that statins may also possess anti-inflammatory effects.143–145 Importantly, several recent reports suggest that statins can attenuate the effects of endotoxin in astrocytes, microglia, macrophages, and endothelial cells,146,147 and in mice in vivo.148,149 Our laboratory has demonstrated that lovastatin and atorvastatin block endotoxin-induced proinflammatory cytokine release, monocyte adhesion, and ROS production in human saphenous vein explants, as well as cultured HCAEC and HCASMC.28 These findings are consistent with recent clinical reports that statin therapy may be beneficial in sepsis.150,151
A potential mechanism for inhibition of proinflammatory endotoxin signaling by statins may be found in reduced synthesis of mevalonate, the immediate product of HMG-CoA reductase, rather than in cholesterol itself. In addition to its role as a cholesterol precursor, mevalonate is required for the prenylation of a number of proteins, which in turn determines the orientation of these proteins in the plasma membrane and/or their interactions with other signaling molecules.152 For example, isoprenylation of the small G protein p21 rac is required for its function in the assembly of NADPH oxidase, and this isoprenylation is prevented by statins.153 In addition, farnesylation of Ras has been shown to be required for its interaction with PI3K, which may be involved in endotoxin signaling.154 In cells expressing the GPI-linked mCD14, PI3 kinase may act directly on the GPI anchor of CD14, perhaps playing a role in clustering of CD14 molecules or in transducing the signal from the LPS:CD14 complex to the endotoxin receptor. Prenylation of one of the molecules in this signaling complex, which would be inhibited by statins, might be required for full activity.
We have shown that the inhibitory effect of statins on endotoxin signaling in human saphenous vein explants and coronary artery endothelial cells is caused by inhibiting a geranylgeranylated protein, probably a member of the Rho GTPase family.28,147 Alternatively or additionally, because expression of the critical immunomodulatory molecule CD14 is believed to be regulated by oxidative stress,155 and endotoxin itself is known to increase free radical production,28,156 the antioxidant effects of statins may act to blunt this aspect of endotoxin signaling.157
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Species Specificity of the Response to Endotoxin |
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Species-dependent differences in response to endotoxin have been recognized for many years. In general, sheep, pigs, goats, and humans are extremely responsive, whereas rats and mice are relatively insensitive, requiring high doses of endotoxin to elicit a response.158–163 Significant functional differences exist even between different rodent species. LPS increases survival of rats, but not mice, during hyperoxia; this difference is caused by a greater LPS-induced induction of MnSOD mRNA and protein in rats.161 Similar differences in LPS responsiveness have been reported between rats and gerbils or hamsters.162,163 Among primates, chimpanzees share the generally high endotoxin sensitivity of humans, although baboons are insensitive, with a responsiveness comparable to that of rodents.164
Individual tissues isolated from different species also vary widely in their responses to endotoxin. Further, the functional diversity of endothelial cells from different vascular beds is well recognized.165,166 For example, endothelial cells from bovine and sheep pulmonary artery, bovine brain microvasculature and aorta, and human kidney display extensive cell lysis in response to endotoxin, whereas endothelial cells from human umbilical vein and pulmonary artery and vein do not.167 Although bovine aorta, pulmonary artery, and mesenteric artery endothelial cells are responsive to LPS, bovine aortic smooth muscle cells fail to respond even in the presence of 100-fold higher endotoxin levels (up to 100 µg/mL).168 In contrast, our laboratory’s results with human coronary artery cells show that the human coronary artery smooth muscle cells are 2 orders of magnitude more sensitive to LPS than the coronary artery endothelial cells.29 Finally, whereas the molecular interactions of endotoxin with mononuclear cells have been studied extensively, it is not clear whether endotoxin engages and activates vascular cells by similar mechanisms.
It has been known for many years that tetra-acylated LPS or lipid A (lipid IVA) and Rhodobacter sphaeroides lipid A are LPS antagonists in human cells but display LPS mimetic activity in murine169 and hamster cells.170 Further, humans, but not mice, can distinguish between the wild-type penta-acylated LPS of Pseudomonas aeruginosa and the far more bioactive hexa-acylated P. aeruginosa LPS found in the lungs of patients with cystic fibrosis.171 Recently, these differences have been attributed to structural differences in both TLR-4 and MD-2 in the two species. Lien et al cloned TLR-4 from hamster macrophages and found that human THP-1 cells expressing the hamster TLR-4 responded to lipid IVa as an LPS mimetic, as if they were hamster in origin.172 Thus, cells heterologously overexpressing TLR-4 from different species acquired a pharmacological phenotype with respect to recognition of lipid A substructures that corresponded to the species of origin of the TLR-4. Similar studies with MD-2 have also delineated species-specific differences between human and murine MD-2.173 For example, taxol activates the MD-2/TLR-4 signaling pathway in mice, but not in humans;174 the difference has been traced to a single amino acid, Gln22 in murine MD-2.174,175
Collectively, these studies indicate that important differences in innate immune system recognition and function between different species and vascular beds preclude generalization of findings with endotoxin across a wide range of experimental models.
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Summary |
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In summary, accumulating experimental and epidemiological evidence suggests that chronic inflammation associated with low-level endotoxemia, such as that resulting from chronic subclinical infections, may be an important risk factor for cardiovascular disease. Endotoxin may trigger or accelerate atherosclerosis through multiple mechanisms, including increases in ROS, chemotactic and proinflammatory cytokines and other acute phase reactants, and adhesion molecules. All of these latter events may contribute importantly to invasion of the vascular wall by monocytes/macrophages, neutrophils, and T lymphocytes. There is evidence that statins, in addition to their lipid-lowering properties, may play a protective role by blocking at least some of these proinflammatory pathways. Overall, the studies discussed here may provide a paradigm for the role of other microbial agents in cardiovascular disease. Finally, in evaluating the existing literature on endotoxin and endotoxin signaling, the reader should bear in mind that reported findings in nonhuman mammals and, in particular, in studies using very high, nonphysiological concentrations of endotoxin, may not be relevant to an understanding of the role of endotoxin in human cardiovascular disease.
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Footnotes |
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Consulting Editor for this article was Alan M. Fogelman, MD, Professor of Medicine and Executive Chair, Departments of Medicine and Cardiology, UCLA School of Medicine, Los Angeles, Calif.
Received July 12, 2004; accepted September 23, 2004.
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