Motohiro Takeya

Multifunctional roles of macrophages in the development and progression of atherosclerosis in humans and experimental animals

 In atherosclerosis, macrophages are important for intracellular lipid accumulation and foam cell formation. Monocytes respond to chemotactic factors, cytokines, and macrophage growth factors produced by vascular endothelial cells, smooth muscle cells, and infiltrated cells, by migrating from peripheral blood into the arterial intima and differentiating into macrophages in atherosclerotic lesions. Although various chemotactic factors are known to induce monocyte migration, monocyte chemoattractant protein-1 is the most important and powerful inducer of migration into atherosclerotic lesions. Macrophage colony-stimulating factor is crucial for monocyte/macrophage differentiation and proliferation, and for the survival of macrophages in these lesions. A minor population of macrophages can proliferate in the atherosclerotic lesions themselves, particularly in the early stage. The macrophages express a variety of receptors, particularly scavenger receptors, and take up modified lipoproteins, including oxidized low-density lipoprotein, β-very-low-density lipoprotein, and/or enzymatically degraded low-density lipoprotein. These cells accumulate cholesterol esters in the cytoplasm, which leads to foam cell formation in lesion development. Among various scavenger receptors, class A type I and type II macrophage scavenger receptors (MSR-A I,II) play the most important role in the uptake of oxidized low-density lipoprotein by macrophages. In addition, macrophages and macrophage-derived foam cells produce ceroid and advanced glycation end-products (AGEs) and accumulate these substances in their cytoplasm. Extracellularly generated AGEs are taken up by macrophages via receptors for AGEs, including MSR-AI,II. Most foam cells die in loco because of apoptosis, and some foam cells escape from the lesions into peripheral blood. Macrophages also play multifaceted roles in inducing plaque rupture, blood coagulation, and fibrinolysis via the production of various enzymes, activators, inhibitors, and bioactive mediators. During the development of atherosclerosis, macrophages interact with vascular endothelial cells, medial smooth muscle cells, and infiltrated inflammatory cells, particularly T cells and dendritic cells. This review, based on data accumulated in studies of atherosclerosis in humans and experimental animals, focuses on the multifunctional roles of macrophages in the pathogenesis and progression of atherosclerosis.

Important Role of Local Angiotensin II Activity Mediated via Type 1 Receptor in the Pathogenesis of Cardiovascular Inflammatory Changes Induced by Chronic Blockade of Nitric Oxide Synthesis in Rats

BACKGROUND The chronic inhibition of NO synthesis by N(omega)-nitro-L-arginine methyl ester (L-NAME) upregulates the cardiovascular tissue angiotensin II (Ang II)-generating system and induces cardiovascular inflammatory changes in rats. METHODS AND RESULTS We used a rat model to investigate the role of local Ang II activity in the pathogenesis of such inflammatory changes. Marked increases in monocyte infiltration into coronary vessels and myocardial interstitial areas, monocyte chemoattractant protein-1 (MCP-1) expression, and nuclear factor-kappaB (NF-kappaB, an important redox-sensitive transcriptional factor that induces MCP-1) activity were observed on day 3 of L-NAME administration. Along with these changes, vascular superoxide anion production was also increased. Treatment with an Ang II type 1 receptor antagonist or with a thiol-containing antioxidant, N-acetylcysteine, prevented all of these changes. CONCLUSIONS Increased Ang II activity mediated via the type 1 receptor may thus be important in the pathogenesis of early cardiovascular inflammatory changes in this model. Endothelium-derived NO may decrease MCP-1 production and oxidative stress-sensitive signals by suppressing localized activity of Ang II.

Rho-Kinase Mediates Angiotensin II-Induced Monocyte Chemoattractant Protein-1 Expression in Rat Vascular Smooth Muscle Cells

Recently, it was shown that Rho-kinase plays an important role in blood pressure regulation. However, it is not known whether Rho-kinase is involved in atherogenesis. Monocyte chemoattractant protein-1 (MCP-1) is an important chemokine that regulates monocyte recruitment and atherogenesis. Therefore, we examined the role of Rho and Rho-kinase in the angiotensin (Ang) II-induced expression of MCP-1. Ang II dose- and time-dependently enhanced the expression of MCP-1 mRNA and the protein production in vascular smooth muscle cells. CV11974, an Ang II type 1 receptor (AT1-R) specific antagonist inhibited the enhancement of MCP-1 expression by Ang II, suggesting that the effect of Ang II is mediated by the AT1-R. Botulinum C3 exotoxin, a specific inhibitor of Rho, suppressed Ang II-induced MCP-1 production. To examine the role of Rho-kinase in Ang II-induced MCP-1 expression, we used adenovirus-mediated overexpression of the dominant negative mutant of Rho-kinase (AdDNRhoK) or Y-27632, a specific inhibitor of Rho-kinase. Both AdDNRhoK and Y-27632 strongly inhibited Ang II-induced MCP-1 expression. Although inhibition of extracellular signal-regulated protein kinase (ERK) by PD 098,059 also inhibited Ang II-induced MCP-1 expression, Y-27632 did not affect Ang II-induced activation of ERK. These results indicate that Rho-kinase plays a critical role in Ang II-induced MCP-1 production independent of ERK. The Rho-Rho-kinase pathway may be a novel target for the inhibition of Ang II signaling and the treatment of atherosclerosis.

Probucol Attenuates Left Ventricular Dysfunction and Remodeling in Tachycardia-Induced Heart Failure Roles of Oxidative Stress and Inflammation

Background—Oxidative stress and inflammation are potentially involved in the pathogenesis of heart failure (HF). We examined whether antioxidant and antiinflammatory treatment with probucol decreases myocardial oxidative stress and inflammation and attenuates the progression of left ventricular (LV) dysfunction and remodeling (dilatation) in tachycardia-induced HF. Methods and Results—We studied 3 groups of dogs: a sham-operated control group and 2 other groups that underwent ventricular pacing at 240 bpm with and without probucol treatment (100 mg/kg IP per week) for 4 weeks. Dogs that underwent ventricular pacing for 4 weeks developed signs of HF, such as a reduction in the LV ejection fraction and increases in the LV end-diastolic dimension and LV end-diastolic pressure. Myocardial oxidative stress, as measured by electron spin resonance spectroscopy with 4-hydroxy-2,2,6,6,-tetramethyl-piperidine-N-oxyl (hydroxy-TEMPO), was significantly increased. There was an increase in myocardial monocyte infiltration, monocyte chemoattractant protein-1 expression, and renin-angiotensin system and matrix metalloproteinase activity. Probucol treatment prevented increases in oxidative stress, inflammation, and matrix metalloproteinase activity and attenuated LV dysfunction and remodeling. Conclusions—Probucol attenuated LV dysfunction and remodeling, possibly through its antioxidant and/or antiinflammatory effects in ventricular pacing–induced HF. These data suggest that inflammatory disorders, which cause an abnormal interaction between failing myocardium and activated monocytes, have an important role in the progression of HF.

Identification in Human Atherosclerotic Lesions of GA-pyridine, a Novel Structure Derived from Glycolaldehyde-modified Proteins

Glycolaldehyde (GA) is formed from serine by action of myeloperoxidase and reacts with proteins to form several products. Prominent among them isN ε-(carboxymethyl)lysine (CML), which is also known as one of the advanced glycation end products. Because CML is formed from a wide range of precursors, we have attempted to identify unique structures characteristic of the reaction of GA with protein. To this end, monoclonal (GA5 and 1A12) and polyclonal (non-CML-GA) antibodies specific for GA-modified proteins were prepared. These antibodies specifically reacted with GA-modified and with hypochlorous acid-modified BSA, but not with BSA modified by other aldehydes, indicating that the epitope of these antibodies could be a specific marker for myeloperoxidase-induced protein modification. By HPLC purification from GA-modifiedN α-(carbobenzyloxy)-l-lysine, GA5-reactive compound was isolated, and its chemical structure was characterized as 3-hydroxy-4-hydroxymethyl-1-(5-amino-5-carboxypentyl) pyridinium cation. This compound named as GA-pyridine was recognized both by 1A12 and non-CML-GA, indicating that GA-pyridine is an important antigenic structure in GA-modified proteins. Immunohistochemical studies with GA5 demonstrated the accumulation of GA-pyridine in the cytoplasm of foam cells and extracellularly in the central region of atheroma in human atherosclerotic lesions. These results suggest that myeloperoxidase-mediated protein modification via GA may contribute to atherogenesis.

Role of Monocyte Chemoattractant Protein-1 in Cardiovascular Remodeling Induced by Chronic Blockade of Nitric Oxide Synthesis

BackgroundChronic inhibition of endothelial nitric oxide (NO) synthesis by the administration of N&ohgr;-nitro-l-arginine methyl ester (L-NAME) to rats induces early vascular inflammatory changes (monocyte infiltration into coronary vessels and monocyte chemoattractant protein-1 [MCP-1] expression) as well as subsequent arteriosclerosis (medial thickening and perivascular fibrosis) and cardiac fibrosis. However, the role of MCP-1 in this process is not known. Methods and ResultsWe investigated the effect of a specific monoclonal anti–MCP-1 neutralizing antibody in rats treated with L-NAME to determine the role of monocytes in the regulation of cardiovascular remodeling. We found increased expression of MCP-1 mRNA in vascular endothelial cells and monocytes in inflammatory lesions. Cotreatment with an anti–MCP-1 antibody, but not with control IgG, prevented the L-NAME–induced early inflammation and reduced late coronary vascular medial thickening. In contrast, the anti–MCP-1 antibody did not decrease the development of perivascular fibrosis, the expression of transforming growth factor (TGF)-&bgr;1 mRNA, or systolic pressure overload induced by L-NAME administration. ConclusionsThese results suggest that MCP-1 is necessary for the development of medial thickening as well as monocyte recruitment. In contrast, the pathogenesis of fibrosis may involve other factors, such as TGF-&bgr;1.

Localization of Human Acyl-Coenzyme A:Cholesterol Acyltransferase-1 (ACAT-1) in Macrophages and in Various Tissues

To investigate the distribution of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) in various human tissues, we examined tissues of autopsy cases immunohistochemically. ACAT-1 was demonstrated in macrophages, antigen-presenting cells, steroid hormone-producing cells, neurons, cardiomyocytes, smooth muscle cells, mesothelial cells, epithelial cells of the urinary tracts, thyroid follicles, renal tubules, pituitary, prostatic, and bronchial glands, alveolar and intestinal epithelial cells, pancreatic acinar cells, and hepatocytes. These findings showed that ACAT-1 is present in a variety of human tissues examined. The immunoreactivities are particularly prominent in the macrophages, steroid hormone-producing cells, followed by hepatocytes, and intestinal epithelia. In cultured human macrophages, immunoelectron microscopy revealed that ACAT-1 was located mainly in the tubular rough endoplasmic reticulum; immunoblot analysis showed that the ACAT-1 protein content did not change with or without cholesterol loading; however, on cholesterol loading, about 30 to 40% of the total immunoreactivity appeared in small-sized vesicles. These vesicles were also enriched in 78-kd glucose-regulated protein (GRP 78), a specific marker for the endoplasmic reticulum. Immunofluorescent microscopy demonstrated extensive colocalization of ACAT-1 and GRP 78 signals in both the tubular and vesicular endoplasmic reticulum before and after cholesterol loading. These results raise the possibility that foam cell formation may activate an endoplasmic reticulum vesiculation process, producing vesicles enriched in the ACAT-1 protein.

Angiopoietin-related growth factor (AGF) promotes epidermal proliferation, remodeling, and regeneration.

We report here the identification of an angiopoietin-related growth factor (AGF). To examine the biological function of AGF in vivo, we created transgenic mice expressing AGF in epidermal keratinocytes (K14-AGF). K14-AGF mice exhibited swollen and reddish ears, nose and eyelids. Histological analyses of K14-AGF mice revealed significantly thickened epidermis and a marked increase in proliferating epidermal cells as well as vascular cells in the skin compared with nontransgenic controls. In addition, we found rapid wound closure in the healing process and an unusual closure of holes punched in the ears of K14-AGF mice. Furthermore, we observed that AGF is expressed in platelets and mast cells, and detected at wounded skin, whereas there was no expression of AGF detected in normal skin tissues, suggesting that AGF derived from these infiltrated cells affects epidermal proliferation and thereby plays a role in the wound healing process. These findings demonstrate that biological functions of AGF in epidermal keratinocytes could lead to novel therapeutic strategies for wound care and epidermal regenerative medicine.

Molecular Mechanism and Role of Endothelial Monocyte Chemoattractant Protein-1 Induction by Vascular Endothelial Growth Factor

Objective—We investigated the role of monocyte chemoattractant protein-1 (MCP-1) in vascular endothelial growth factor (VEGF)–induced angiogenesis and vascular permeability and the underlying molecular mechanism of VEGF-induced endothelial MCP-1 expression in vitro and in vivo. Methods and Results—We used an anti–MCP-1 neutralizing antibody for specific inhibition of MCP-1. VEGF increased tubule formation in the angiogenesis assay and vascular permeability in the Miles assay, and these effects were markedly inhibited by anti–MCP-1 antibody. Using a luciferase MCP-1 promoter-gene assay, we found that the activator protein-1 (AP-1) binding site of the MCP-1 promoter region contributes to the increase in MCP-1 promoter activity by VEGF. To specifically inhibit AP-1, we used recombinant adenovirus containing a dominant-negative c-Jun (Ad-DN-c-Jun). Ad-DN-c-Jun inhibited VEGF-induced endothelial MCP-1 mRNA expression and promoter activity in vitro. In vivo gene transfer of DN-c-Jun into rat carotid artery, with the hemagglutinating virus of the Japan liposome method, significantly blocked VEGF-induced MCP-1 and macrophage/monocyte (ED1) expression in endothelium. Conclusions—These results reveal that endothelial MCP-1 induced by VEGF seems to participate in angiogenesis, vascular leakage, or arteriosclerosis. AP-1 plays a critical role in the molecular mechanism underlying induction of MCP-1 by VEGF.

Production, characterization, and interspecies reactivities of monoclonal antibodies against human class A macrophage scavenger receptors

Class A macrophage scavenger receptor (SR-A) is one of the major receptors of macrophages and plays important roles in atherogenesis and host defense mechanisms. To assess the role of SR-A, monoclonal antibodies were generated by immunizing SR-A-deficient mice with a recombinant protein of human type I SR-A as immunogen. Four antibodies (SRA-C6, SRA-D10, SRA-E5, and SRA-F8) were confirmed to be specific for SR-A by Western blot analysis. In early atherosclerotic lesions, these antibodies recognized scattered macrophages in intima and foamy macrophages in the periphery of atheromatous cores. Interestingly, foamy macrophages in the core lesion were only weakly stained. In other organs, the antibodies recognized tissue macrophages such as alveolar macrophages, Kupffer cells in the liver, red pulp macrophages in the spleen, sinus macrophages in lymph nodes, and interstitial macrophages in various organs. Perivascular macrophages in the brain (Mato cells) were also positive for these antibodies. Freshly isolated blood monocytes were negative; however, they became positive for these antibodies after 1 day in culture. At 3-5 days in culture, the reaction intensity became stronger along their differentiation towards macrophages. Dendritic cells such as interdigitating cells of lymphoid tissues and epidermal Langerhans cells were invariably negative. In the reaction with animal tissues, each antibody showed a unique reaction pattern. Among four antibodies, SRA-E5 recognized SR-A molecules in all animal species examined, including rats and mice. These antibodies will be useful tools for the study of SR-A in atherogenesis and various other pathological conditions in humans and animal species.