IntroductionTo Invest. 1995. 96:60-68.)

Toll-like receptors (TLRs) and the downstream adaptor molecule myeloid differentiation factor 88 (MyD88) play an essential role in the innate immune responses. Here, we demonstrate that genetic deficiency of TLR4 or MyD88 is associated with a significant reduction of aortic plaque areas in atherosclerosis-prone apolipoprotein E-deficient mice, despite persistent hypercholesterolemia, implying an important role for the innate immune system in atherogenesis. Apolipoprotein E-deficient mice that also lacked TLR4 or MyD88 demonstrated reduced aortic atherosclerosis that was associated with reductions in circulating levels of proinflammatory cytokines IL-12 or monocyte chemoattractant protein 1, plaque lipid content, numbers of macrophage, and cyclooxygenase 2 immunoreactivity in their plaques. Endothelial-leukocyte adhesion in response to minimally modified low-density lipoprotein was reduced in aortic endothelial cells derived from MyD88-deficient mice. Taken together, our results suggest an important role for TLR4 and MyD88 signaling in atherosclerosis in a hypercholesterolemic mouse model, providing a pathophysiologic link between innate immunity, inflammation, and atherogenesis.

Background-Inflammation is implicated in atherogenesis and plaque disruption. Toll-like receptor 2 (TLR-2) and TLR-4, a human homologue of drosophila Toll, play an important role in the innate and inflammatory signaling responses to microbial agents. To investigate a potential role of these receptors in atherosclerosis, we assessed the expression of TLR-2 and TLR-4 in murine and human atherosclerotic plaques. Methods and Results-Aortic root lesions of high-fat diet-fed apoE-deficient mice (nϭ5) and human coronary atherosclerotic plaques (nϭ9) obtained at autopsy were examined for TLR-4 and TLR-2 expression by immunohistochemistry. Aortic atherosclerotic lesions in all apoE-deficient mice expressed TLR-4, whereas aortic tissue obtained from control C57BL/6J mice showed no TLR-4 expression. All 5 lipid-rich human plaques expressed TRL-4, whereas the 4 fibrous plaques and 4 normal human arteries showed no or minimal expression. Serial sections and double immunostaining showed TLR-4 colocalizing with macrophages both in murine atherosclerotic lesions and at the shoulder region of human coronary artery plaques. In contrast to TLR-4, none of the plaques expressed TLR-2. Furthermore, basal TLR-4 mRNA expression by human monocyte-derived macrophages was upregulated by ox-LDL in vitro. A potential role for infection in the development of atherosclerosis has been considered for several decades, but interest in this topic has recently reemerged because of several recent observations. Accumulating evidence has implicated specific infectious agents, including Chlamydia pneumoniae, in the progression and/or destabilization of atherosclerosis. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] Recent studies suggest that chlamydia lipopolysaccharide (LPS) induces foam-cell formation, whereas its heat-shock protein (chlamydia HSP60) induces oxidative modification of LDL. 5,18 Chlamydia HSP60 has been implicated in the induction of deleterious immune responses in human chlamydial infection and has been found to colocalize with infiltrating macrophages in the atheroma lesions. 19 Collectively, these data support a potential role for C pneumoniae in the development and progression of atherosclerosis and suggest that this organism may indeed play an active role in atheroma development. Available data, however, also underscore the current lack of a complete understanding of the molecular mechanisms that link C pneumoniae infection to innate immunity and trigger the signals for enhanced inflammation and atherogenesis. Conclusions-OurLPS, a major component of the outer surface of Gramnegative bacteria, activates the proinflammatory transcription factor nuclear factor (NF)-B in endothelial cells and macrophages. 20,21 Recently, human Toll-like receptor-4 (TLR-4), a human homologue of drosophila Toll, has been identified as Currently, more than 10 human TLRs have been identified, and at least 10 human homologues of drosophila Toll have been sequenced. Whereas TLR-4 is used by enteric Gramnegative bacteria and LPS, TLR-2 is use...

Dystrophic or ectopic mineral deposition occurs in many pathologic conditions, including atherosclerosis. Calcium mineral deposits that frequently accompany atherosclerosis are readily quantifiable radiographically, serve as a surrogate marker for the disease, and predict a higher risk of myocardial infarction and death. Accelerating research interest has been propelled by a clear need to understand how plaque structure, composition, and stability lead to devastating cardiovascular events. In atherosclerotic plaque, accumulating evidence is consistent with the notion that calcification involves the participation of arterial osteoblasts and osteoclasts. Here we summarize current models of intimal arterial plaque calcification and highlight intriguing questions that require further investigation. Because atherosclerosis is a chronic vascular inflammation, we propose that arterial plaque calcification is best conceptualized as a convergence of bone biology with vascular inflammatory pathobiology.P laque structure and composition importantly impact clinical expression of atherosclerosis. Molecular medicine in the 21st century has turned toward a comprehensive understanding of the dynamic processes that influence the composition and stability of atheroma and of how structural plaque components impact clinical outcomes. Recently, increasing interest has focused on understanding how atherosclerotic pathology is related to a common plaque constituent: calcium mineral deposits. Pathologists have long known that calcified atherosclerotic arteries can contain tissue that is histomorphologically indistinguishable from bone (1, 2). Important studies in the last decade have now spawned a model wherein calcification in atherosclerotic plaque is viewed as an active, complex, and therefore presumably regulated process that exhibits intriguing similarities to new bone formation, or remodeling. Ectopic and dystrophic mineral deposition and extracellular matrix calcification can occur in numerous pathologic conditions by passive precipitation. Here we focus on one specific type of mineral deposition with high relevance to cardiac pathology: intimal arterial calcification in the context of atherosclerotic plaque. The emerging view is that plaque calcification represents a meeting of bone biology with chronic plaque inflammation. Remarkable cellular ontogenetic versatility in atherosclerosis appears to effect profound structural alterations, with significant ramifications for plaque stability and clinical outcomes. Clinical SignificanceAtherosclerotic lesions frequently become calcified. The process can begin early and accelerates as the disease progresses and more complex lesions develop. Calcium deposits in coronary arteries indicate the presence of plaque, but the converse statement that an absence of coronary calcium indicates an absence of atheromatous plaque is not true (1). Because calcification is a surrogate measure of coronary atherosclerosis, clinical interest has focused on the usefulness of noninvasive detection of calci...

Oxidized lipoproteins have been identified in atherosclerotic plaques and in early lesions in humans as well as in animals. There is accumulating evidence that such oxidized lipoproteins have an important role in atherosclerosis. Treatment of endothelial cells with altered lipoproteins stimulates monocyte binding as well as the production of chemotactic factors for monocytes. Both these findings could be relevant to the accumulation of monocytes-macrophages in the arterial wall during the early stages of lesion development. We now report that treatment of endothelial cells (EC) with modified low-density lipoproteins obtained by mild iron oxidation or by prolonged storage, results in a rapid and large induction of the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage CSF (M-CSF) and granulocyte CSF (G-CSF). These growth factors affect the differentiation, survival, proliferation, migration and metabolism of macrophages/granulocytes, and G-CSF and GM-CSF also affect the migration and proliferation of EC. Because EC and macrophages are important in the development of atherosclerosis, the expression of the CSFs by these cells could contribute to the disease.

Pathologists have recognized arterial calcification for over a century. Recent years have witnessed a strong resurgence of interest in atherosclerotic plaque calcification because it: 1) can be easily detected noninvasively; 2) closely correlates with the amount of atherosclerotic plaque; 3) serves as a surrogate measure for atherosclerosis, allowing preclinical detection of the disease; and 4) is associated with heightened risk of adverse cardiovascular events. There are two major types of calcification in arteries: calcification of the media tunica layer (sometimes called Mönckeberg's sclerosis), and calcification within subdomains of atherosclerotic plaque within the intimal layer of the artery. There are important similarities and differences between these two entities. Of particular interest are increasing parallels between cellular and molecular features of arterial calcification and bone biology, and this has led to accelerating interest in understanding how and why bone-like mineral deposits may form in arteries. Here, we review the two major pathological types of arterial calcification, the proposed models of calcification, and endocrine and genetic determinants that affect arterial calcification. In addition, we highlight areas requiring further investigation.

Background-Matrix metalloproteinases (MMPs) are expressed in atherosclerotic plaques, where in their active form, they may contribute to vascular remodeling and plaque disruption. In this study, we tested the hypothesis that membrane type 1 MMP (MT1-MMP), a novel transmembrane MMP that activates pro-MMP-2 (gelatinase A), is expressed in human atherosclerotic plaques and that its expression is regulated by proinflammatory molecules. Cultured SMCs constitutively expressed MT1-MMP mRNA and protein, which increased 2-to 4-fold over control in a time-dependent manner within 4 to 8 hours of exposure to IL-1␣, TNF-␣, and ox-LDL (thiobarbituric acid-reactive substances, 13.4 nmol/mg LDL protein), whereas native LDL had no effect. Flow cytometry revealed MT1-MMP expression by human monocyte-derived M, which increased 3.8-fold over baseline within 6 hours after exposure to 10 ng/mL TNF-␣. Conclusions-This

Background-Macrophages in human atherosclerotic plaques produce a family of matrix metalloproteinases (MMPs), which may influence vascular remodeling and plaque disruption. Because oxidized LDL (ox-LDL) is implicated in many proatherogenic events, we hypothesized that ox-LDL would regulate expression of MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in monocyte-derived macrophages. Methods and Results-Mononuclear cells were isolated from normal human subjects with Ficoll-Paque density gradient centrifugation, and adherent cells were allowed to differentiate into macrophages during 7 days of culture in plastic dishes. On day 7, by use of serum-free medium, the macrophages were incubated with various concentrations of native LDL (n-LDL) and copper-oxidized LDL. Exposure to ox-LDL (10 to 50 g/mL) increased MMP-9 mRNA expression as analyzed by Northern blot, protein expression as measured by ELISA and Western blot, and gelatinolytic activity as determined by zymography. The increase in MMP-9 expression was associated with increased nuclear binding of transcription factor NF-B and AP-1 complex on electromobility shift assay. In contrast, ox-LDL (10 to 50 g/mL) decreased TIMP-1 expression. Ox-LDL-induced increase in MMP-9 expression was abrogated by HDL (100 g/mL). n-LDL had no significant effect on MMP-9 or TIMP-1 expression. Conclusions-These data demonstrate that unlike n-LDL, ox-LDL upregulates MMP-9 expression while reducing TIMP-1 expression in monocyte-derived macrophages. Furthermore, HDL abrogates ox-LDL-induced MMP-9 expression. Thus, ox-LDL may contribute to macrophage-mediated matrix breakdown in the atherosclerotic plaques, thereby predisposing them to plaque disruption and/or vascular remodeling. (Circulation. 1999;99:993-998.)