C-reactive protein (CRP) is an acute-phase protein that binds specifically to phosphorylcholine (PC) as a component of microbial capsular polysaccharide and participates in the innate immune response against microorganisms. CRP elevation also is a major risk factor for cardiovascular disease. We previously demonstrated that EO6, an antioxidized LDL autoantibody, was a T15 clono-specific anti-PC antibody and specifically binds to PC on oxidized phosphatidylcholine (PtC) but not on native PtC. Similarly, EO6 binds apoptotic cells but not viable cells. In addition, such oxidized phospholipids are recognized by macrophage scavenger receptors, implying that these innate immune responses participate in the clearance because of their proinflammatory properties. We now report that CRP binds to oxidized LDL (OxLDL) and oxidized PtC (OxPtC), but does not bind to native, nonoxidized LDL nor to nonoxidized PtC, and its binding is mediated through the recognition of a PC moiety. Reciprocally, CRP binds to PC, which can be competed for by OxLDL and OxPtC but not by native LDL, nonoxidized PtC, or by oxidized phospholipids without the PC headgroup. CRP also binds to apoptotic cells, and this binding is competed for by OxLDL, OxPtC, and PC. These data suggest that CRP binds OxLDL and apoptotic cells by recognition of a PC moiety that becomes accessible as a result of oxidation of PtC molecule. We propose that, analogous to EO6 and scavenger receptors, CRP is a part of the innate immune response to oxidized PC-bearing phospholipids within OxLDL and on the plasma membranes of apoptotic cells.atherosclerosis ͉ innate immunity ͉ scavenger receptors ͉ autoantibody EO6

Abstract-There is increasing evidence that complement activation may play a role in atherogenesis. Complement proteins have been demonstrated to be present in early atherosclerotic lesions of animals and humans, and cholesterol-induced atherosclerotic lesion formation is reduced in complement-deficient animals. Potential complement activators in atherosclerotic lesions are now a subject matter of debate. C-reactive protein (CRP) is an acute-phase protein that is involved in inflammatory processes in numerous ways. It binds to lipoproteins and activates the complement system via the classic pathway. In this study we have investigated early atherosclerotic lesions of human coronary arteries by means of immunohistochemical staining. We demonstrate here that CRP deposits in the arterial wall in early atherosclerotic lesions with 2 predominant manifestations. First, there is a diffuse rather than a focal deposition in the deep fibroelastic layer and in the fibromuscular layer of the intima adjacent to the media. In this location, CRP frequently colocalizes with the terminal complement complex. Second, the majority of foam cells below the endothelium show positive staining for CRP. In this location, no colocalization with the terminal complement proteins can be observed. Our data suggest that CRP may promote atherosclerotic lesion formation by activating the complement system and being involved in foam cell formation. (Arterioscler Thromb Vasc Biol. 1998;18:1386-1392.)

In patients with coronary artery disease, a low level of activity of red-cell glutathione peroxidase 1 is independently associated with an increased risk of cardiovascular events. Glutathione peroxidase 1 activity may have prognostic value in addition to that of traditional risk factors. Furthermore, increasing glutathione peroxidase 1 activity might lower the risk of cardiovascular events.

Abstract-Treatment of low density lipoprotein (LDL) with degrading enzymes transforms the molecule to a moiety that is micromorphologically indistinguishable from lipoproteinaceous particles that are present in atherosclerotic plaques, and enzymatically modified LDL (E-LDL), but not oxidized LDL (ox-LDL), spontaneously activates the alternative complement pathway, as do lesion lipoprotein derivatives. Furthermore, because E-LDL is a potent inducer of macrophage foam cell formation, we propose that enzymatic degradation may be the key process that renders LDL atherogenic. In this article, we report the production of two murine monoclonal antibodies recognizing cryptic epitopes in human apolipoprotein B that become exposed after enzymatic attack on LDL. One antibody reacted with LDL after single treatment with trypsin, whereas recognition by the second antibody required combined treatment of LDL with trypsin and cholesterol esterase. In ELISAs, both antibodies reacted with E-LDL produced in vitro and with lesion complement activator derived from human atherosclerotic plaques, but they were unreactive with native LDL or ox-LDL. The antibodies stained E-LDL, but not native LDL or ox-LDL, that had been artificially injected into arterial vessel walls. With the use of these antibodies, we have demonstrated that early human atherosclerotic coronary lesions obtained at autopsy as well as lesions examined in freshly explanted hearts always contain extensive extracellular deposits of E-LDL. Terminal complement complexes, detected with a monoclonal antibody specific for a C5b-9 neoepitope, colocalized with E-LDL within the intima, which is compatible with the proposal that subendothelially deposited LDL is enzymatically transformed to a complement activator at the earliest stages in lesion development. (Arterioscler Thromb Vasc Biol. 1998;18:369-378.)

Abstract-Infiltration of monocytes into the arterial wall is an early cellular event in atherogenesis. Recent evidence shows that C-reactive protein (CRP) is deposited in the arterial intima at sites of atherogenesis. In this study, we demonstrate that CRP deposition precedes the appearance of monocytes in early atherosclerotic lesions. CRP is chemotactic for freshly isolated human blood monocytes. A specific CRP receptor is demonstrated on monocytes in vitro as well as in vivo, and blockage of the receptor by use of a monoclonal anti-receptor antibody completely abolishes CRP-induced chemotaxis. CRP may play a major role in the recruitment of monocytes during atherogenesis. (Arterioscler Thromb Vasc

A crucial cause of the decreased bioactivity of nitric oxide (NO) in cardiovascular diseases is the uncoupling of the endothelial NO synthase (eNOS) caused by the oxidative stress-mediated deficiency of the NOS cofactor tetrahydrobiopterin (BH 4 ). The reversal of eNOS uncoupling might represent a novel therapeutic approach. The treatment of apolipoprotein E knockout (ApoE-KO) mice with resveratrol resulted in the up-regulation of superoxide dismutase (SOD) isoforms (SOD1-SOD3), glutathione peroxidase 1 (GPx1), and catalase and the down-regulation of NADPH oxidases NOX2 and NOX4 in the hearts of ApoE-KO mice. This was associated with reductions in superoxide, 3-nitrotyrosine, and malondialdehyde levels. In parallel, the cardiac expression of GTP cyclohydrolase 1 (GCH1), the rate-limiting enzyme in BH 4 biosynthesis, was enhanced by resveratrol. This enhancement was accompanied by an elevation in BH 4 levels. Superoxide production from ApoE-KO mice hearts was reduced by the NOS inhibitor L-N G -nitro-arginine methyl ester, indicating eNOS uncoupling in this pathological model. Resveratrol treatment resulted in a reversal of eNOS uncoupling. Treatment of human endothelial cells with resveratrol led to an up-regulation of SOD1, SOD2, SOD3, GPx1, catalase, and GCH1. Some of these effects were preventable with sirtinol, an inhibitor of the protein deacetylase sirtuin 1. In summary, resveratrol decreased superoxide production and enhanced the inactivation of reactive oxygen species. The resulting reduction in BH 4 oxidation, together with the enhanced biosynthesis of BH 4 by GCH1, probably was responsible for the reversal of eNOS uncoupling. This novel mechanism (reversal of eNOS uncoupling) might contribute to the protective effects of resveratrol.

Background-We have recently demonstrated that activity of red blood cell glutathione peroxidase-1 is inversely associated with the risk of cardiovascular events in patients with coronary artery disease. The present study analyzed the effect of glutathione peroxidase-1 deficiency on atherogenesis in the apolipoprotein E-deficient mouse. Methods and Results-Female apolipoprotein E-deficient mice with and without glutathione peroxidase-1 deficiency were placed on a Western-type diet for another 6, 12, or 24 weeks. After 24 weeks on Western-type diet, double-knockout mice (GPx-1 Ϫ/Ϫ ApoE Ϫ/Ϫ ) developed significantly more atherosclerosis than control apolipoprotein E-deficient mice. Moreover, glutathione peroxidase-1 deficiency led to modified atherosclerotic lesions with increased cellularity. Functional experiments revealed that glutathione peroxidase-1 deficiency leads to increased reactive oxygen species concentration in the aortic wall as well as increased overall oxidative stress. Peritoneal macrophages from double-knockout mice showed increased in vitro proliferation in response to macrophage-colony-stimulating factor. Also, we found lower levels of bioactive nitric oxide as well as increased tyrosine nitration as a marker of peroxynitrite production. Key Words: antioxidants Ⅲ atherosclerosis Ⅲ nitric oxide O xidative stress is defined as an imbalance between the production and degradation of reactive oxygen species (ROS). Enzymatic inactivation of ROS is achieved mainly by superoxide dismutases, catalase and the glutathione peroxidases. 1 Indeed, glutathione and the glutathione peroxidases constitute the principal antioxidant defense system in mammalian cells. [2][3][4] Glutathione peroxidase-1 (GPx-1), the ubiquitous intracellular form and key antioxidant enzyme within many cells, including the endothelium, consumes reduced glutathione to convert hydrogen peroxide to water and lipid peroxides to their respective alcohols. 5 It also acts as a peroxynitrite reductase. 6 Because of its major role in the prevention of oxidative stress, GPx-1 may be an important antiatherogenic enzyme. 7 In fact, we have recently shown in patients with coronary artery disease that a low activity of red blood cell GPx-1 is associated with an increased risk of cardiovascular events independently from traditional risk factors or atherosclerosis. 8 A mouse model of GPx-1 deficiency is available. These animals appear healthy and are fertile. 9 However, a recent in vitro study showed increased cell-mediated oxidation of low-density lipoprotein (LDL) in this model. 10 Furthermore, GPx-1 deficiency causes endothelial dysfunction in mice 11 that is aggravated by hyperhomocysteinemia. 12 GPx-1 deficiency is accompanied by increased periadventitial inflammation, neointima formation, and collagen deposition surrounding the coronary arteries. 13 GPx-1 activity is decreased or absent in carotid atherosclerotic plaques, and the absence of GPx-1 activity in atherosclerotic lesions has been linked to the development of more severe lesions in...

Abstract-Complement activation occurs in temporal correlation with the subendothelial deposition of LDL during early atherogenesis, and complement also plays a pathogenetic role in promoting lesion progression. Two lesion components have been identified that may be responsible for complement activation. First, enzymatic degradation of LDL generates a derivative that can spontaneously activate complement, and enzymatically degraded LDL (E-LDL) has been detected in the lesions. Second, C-reactive protein (CRP) colocalizes with complement C5b-9, as evidenced by immunohistological studies of early atherosclerotic lesions, so the possibility exists that this acute phase protein also fulfills a complement-activating function. Here, we report that addition of LDL and CRP to human serum did not result in significant C3 turnover. Addition of E-LDL provoked complement activation, which was markedly enhanced by CRP.Binding of CRP to E-LDL was demonstrated by sucrose flotation experiments. Binding was Ca 2ϩ -dependent and inhibitable by phosphorylcholine, and the complement-activating property of E-LDL was destroyed by treatment with phospholipase C. These results indicated that CRP binds to phosphorylcholine groups that become exposed in enzymatically degraded LDL particles. Immunohistological studies complemented these findings in showing that CRP colocalizes with E-LDL in early human atherosclerotic lesions. Thus enzymatic, nonoxidative modification of tissue-deposited LDL can be expected to confer CRP-binding capacity onto the molecule. The ensuing enhancement of complement activation may be relevant to the development and progression of the atherosclerotic lesion. Key Words: atherogenesis Ⅲ corrective protein Ⅲ complement Ⅲ LDL C omplement and C-reactive protein (CRP) are emerging as 2 components that may play important roles in atherogenesis. Early studies indicated that activated complement components 1,2 and CRP 3,4 are present in atherosclerotic lesions, and the demonstration followed that in situ C5b-9 generation occurred in temporal correlation with lipid deposition. 5 The search for a complement-activating entity led to the isolation of an LDL derivative, termed lesion complement activator (LCA), that had the capacity to activate the alternative complement pathway. 6 We are considering that the high content of free cholesterol in the LCA particles is important because unesterified cholesterol activates complement. 7 Similar lipidic moieties were isolated in other laboratories, 8,9 although their capacity to activate complement was not tested. Furthermore, fused LDL particles micromorphologically similar to LCA were visualized in extracellular location by deep freeze-etch electronmicroscopy in arteries of cholesterol-fed rabbits. 10 Collectively, these studies indicated that tissue-deposited LDL is modified extracellularly to yield lipid droplets with a high content of free cholesterol that have intrinsic complement-activating capacity.Subsequently, it was shown that LDL, but not HDL or VLDL, could be transformed...