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Obtaining nesting material presents an optimal foraging problem, collection of materials incurs a cost in terms of risk of predation and energy spent and individuals must balance these costs with the benefits of using that material in the nest. The hazel dormouse, Muscardinus avellanarius is an endangered British mammal in which both sexes build nests. However, whether material used in their construction follows the predictions of optimal foraging theory is unknown. Here, we analyze the use of nesting materials in forty two breeding nests from six locations in Southwest England. Nests were characterized in terms of which plants were used, the relative amount of each plant, and how far away the nearest source was. We found that dormice exhibit a preference for plants closer to the nest, but that the distance they are prepared to travel depends on the plant species. Dormice traveled further to collect honeysuckle Lonicera periclymenum, oak Quercus robur, and beech Fagus sylvatica than any other plants. Distance did not affect the relative amount used, although the proportion of honeysuckle in nests was highest, and more effort was expended collecting honeysuckle, beech, bramble Rubus fruticosus and oak compared to other plants. Our results suggest that not all aspects of optimal foraging theory apply to nest material collection. However, optimal foraging theory is a useful model to examine nest material collection, providing testable predictions. As found previously honeysuckle is important as a nesting material and its presence should be taken account when assessing suitability of sites for dormice.

Objective— Liver X receptors (LXRs) are oxysterol-activated nuclear receptors that are highly expressed in macrophages and regulate lipid homeostasis and inflammation. Among putative LXR target genes, lysophosphatidylcholine acyltransferase 3 (LPCAT3) involved in the Lands cycle controls the fatty acid composition at the sn-2 position of glycerophospholipids and, therefore, the availability of fatty acids, such as arachidonic acid (AA), used for eicosanoid synthesis. The aim of our study was to determine whether LXRs could regulate the Lands cycle in human macrophages, to assess the consequences in terms of lipid composition and inflammatory response, and to work out the relative contribution of LPCAT3 to the observed changes. Approach and Results— Transcriptomic analysis revealed that LPCAT3 was upregulated by LXR agonists in human macrophages. Accordingly, LXR stimulation significantly increased lysophospholipid acyltransferase activity catalyzed by LPCAT3. Lipidomic analysis demonstrated that LXR activation increased the AA content in the polar lipid fraction, specifically in phosphatidylcholines. The LXR-mediated effects on AA distribution were abolished by LPCAT3 silencing, and a redistribution of AA toward the neutral lipid fraction was observed in this context. Finally, we observed that preconditioning of human macrophages by LXR agonist treatment increased the release of arachidonate-derived eicosanoids, such as prostaglandin E 2 and thromboxane after lipopolysaccharide stimulation, with a significant attenuation by LPCAT3 silencing. Conclusions— Altogether, our data demonstrate that the LXR-mediated induction of LPCAT3 primes human macrophages for subsequent eicosanoid secretion by increasing the pool of AA, which can be mobilized from phospholipids.

Niemann-Pick type C1 (NPC1) promotes the transport of LDL receptor (LDL-R)-derived cholesterol from late endosomes/lysosomes to other cellular compartments. NPC1-deficient cells showed impaired regulation of liver_X receptor (LXR) and sterol regulatory element-binding protein (SREBP) target genes. We observed that Apoe(-/-)Npc1(-/-) mice displayed a marked increase in total plasma cholesterol mainly due to increased VLDL, reflecting decreased clearance. Although nuclear SREBP-2 and Ldlr mRNA levels were increased in Apoe(-/-)Npc1(-/-) liver, LDL-R protein levels were decreased in association with marked induction of proprotein convertase subtilisin/kexin type 9 (Pcsk9) and inducible degrader of the LDL-R (Idol), both known to promote proteolytic degradation of LDL-R. While Pcsk9 is known to be an SREBP-2 target, marked upregulation of IDOL in Apoe(-/-)Npc1(-/-) liver was unexpected. However, several other LXR target genes also increased in Apoe(-/-)Npc1(-/-) liver, suggesting increased synthesis of endogenous LXR ligands secondary to activation of sterol biosynthesis. In conclusion, we demonstrate that NPC1 deficiency has a major impact on VLDL metabolism in Apoe(-/-) mice through modulation of hepatic LDL-R protein levels. In contrast to modest induction of hepatic IDOL with synthetic LXR ligands, a striking upregulation of IDOL in Apoe(-/-)Npc1(-/-) mice could indicate a role of endogenous LXR ligands in regulation of hepatic IDOL.

Objectives— Increasing HDL levels is a potential strategy for the treatment of atherosclerosis. Methods and Results— ITX5061, a molecule initially characterized as a p38 MAPK inhibitor, increased HDL-C levels by 20% in a human population of hypertriglyceridemic subjects with low HDL levels. ITX5061 also moderately increased apoA-I but did not affect VLDL/LDL cholesterol or plasma triglyceride concentrations. ITX5061 increased HDL-C in WT and human apoA-I transgenic mice, and kinetic experiments showed that ITX5061 decreased the fractional catabolic rate of HDL-CE and reduced its hepatic uptake. In transfected cells, ITX5061 inhibited SR-BI–dependent uptake of HDL-CE. Moreover, ITX5061 failed to increase HDL-C levels in SR-BI −/− mice. To assess effects on atherosclerosis, ITX5061 was given to atherogenic diet–fed Ldlr +/− mice with or without CETP expression for 18 weeks. In both the control and CETP-expressing groups, ITX5061-treated mice displayed reductions of early atherosclerotic lesions in the aortic arch −40%, P Conclusions— Our data indicate that ITX5061 increases HDL-C levels by inhibition of SR-BI activity. This suggests that pharmacological inhibition of SR-BI has the potential to raise HDL-C and apoA-I levels without adverse effects on VLDL/LDL cholesterol levels in humans.

Background— Therapeutic angiogenesis by delivery of vascular endothelial growth factor (VEGF) has attracted attention. However, the role and function of VEGF in experimental restenosis (neointimal formation) after vascular intraluminal injury have not been addressed. Methods and Results— We report herein that blockade of VEGF by soluble VEGF receptor 1 ( sFlt-1 ) gene transfer attenuated neointimal formation after intraluminal injury in rabbits, rats, and mice. sFlt-1 gene transfer markedly attenuated the early vascular inflammation and proliferation and later neointimal formation. sFlt-1 gene transfer also inhibited increased expression of inflammatory factors such as monocyte chemoattractant protein-1 and VEGF. Intravascular VEGF gene transfer enhanced angiogenesis in the adventitia but did not reduce neointimal formation. Conclusions— Increased expression and activity of VEGF are essential in the development of experimental restenosis after intraluminal injury by recruiting monocyte-lineage cells.

Angiotensin II (Ang II) upregulates vascular endothelial growth factor (VEGF) and activates vascular inflammation. However, the decisive role of VEGF in Ang II–induced vascular inflammation and remodeling has not been addressed. Ang II infusion to wild-type mice increased local expression of VEGF and its receptors in cells of aortic wall and plasma VEGF, and caused aortic inflammation (monocyte infiltration) and remodeling (wall thickening and fibrosis). Hypoxia-inducible factor-1α colocalized with VEGF-positive cell types. Blockade of VEGF by the soluble VEGF receptor 1 (sFlt-1) gene transfer attenuated the Ang II–induced inflammation and remodeling. The sFlt-1 gene transfer also inhibited the increased expression of VEGF and inflammatory factors such as monocyte chemoattractant protein-1. In contrast, sFlt-1 gene transfer did not affect Ang II–induced arterial hypertension and cardiac hypertrophy. VEGF is an essential mediator in Ang II–induced vascular inflammation and structural changes through its proinflammatory actions.

Background— Stromal cell–derived factor-1α (SDF-1α) is implicated as a chemokine for endothelial progenitor cells (EPCs). We therefore hypothesized that SDF-1α gene transfer would induce therapeutic neovascularization in vivo by functioning as a chemokine of EPC. Methods and Results— To examine SDF-1α–induced mobilization of EPC, we used bone marrow–transplanted mice whose blood cells ubiquitously express β-galactosidase (LacZ). We produced unilateral hindlimb ischemia in the mice and transfected them with plasmid DNA encoding SDF-1α or empty plasmids into the ischemic muscles. SDF-1α gene transfer mobilized EPCs into the peripheral blood, augmented recovery of blood perfusion to the ischemic limb, and increased capillary density associated with partial incorporation of LacZ-positive cells into the capillaries of the ischemic limb, suggesting that SDF-1α induced vasculogenesis and angiogenesis. SDF-1α gene transfer did not affect ischemia-induced expression of vascular endothelial growth factor (VEGF) but did enhance Akt and endothelial nitric oxide synthase (eNOS) activity. Blockade of VEGF or NOS prevented all such SDF-1α–induced effects. Conclusions— SDF-1α gene transfer enhanced ischemia-induced vasculogenesis and angiogenesis in vivo through a VEGF/eNOS-related pathway. This strategy might become a novel chemokine therapy for next generation therapeutic neovascularization.

No prior study has examined the effect of intravenous injection of bone marrow mononuclear cells (MNCs) on myocardial infarction size (IS). We tested the hypothesis that transplantation of MNCs decreases IS through the release of vascular endothelial growth factor (VEGF). Immediately after ligation of the left coronary artery of immunodeficient mice, PBS or MNCs were intravenously administered. Myocardial IS was significantly less in MNCs-treated mice than in PBS-treated mice. Trace experiments showed accumulation of exogenously administered MNCs into the vicinity of infarcted myocardium. Injection of MNCs did not affect capillary density after infarction, but did reduced myocardial cell apoptosis. Blockade of VEGF by a neutralizing antibody or by gene transfer of a soluble form of Flt-1 VEGF receptor diminished the IS-limiting effects of MNCs. In conclusion, injection of MNCs can reduce myocardial IS through the release of VEGF. The MNC therapy for acute myocardial infarction might improve prognosis of patients with myocardial infarction.

Amlodipine (a new class of calcium channel antagonist) has been shown to limit the progression of arteriosclerosis and decrease the incidence of cardiovascular events. The mechanisms underlying the beneficial effects of amlodipine, however, remain unclear. Therefore, we hypothesized that amlodipine attenuates the development of arteriosclerosis through the inhibition of inflammation in vivo. Long-term inhibition of nitric oxide (NO) by administration of a NO synthase inhibitor, Nω-nitro-l-arginine methyl ester (l-NAME), to rats induces coronary vascular inflammation [monocyte infiltration, monocyte chemoattractant protein-1 (MCP-1) expression, increased activity of angiotensin-converting enzyme (ACE)], and arteriosclerosis. Here, we used the rat model to investigate the anti-inflammatory effects of amlodipine in vivo. Treatment with amlodipine markedly inhibited the l-NAME-induced increase in vascular inflammation, oxidative stress, and local ACE and Rho activity and prevented arteriosclerosis. Interestingly, amlodipine prevented the l-NAME-induced increase in MCP-1 receptor CCR2 expression in circulating monocytes. Amlodipine markedly attenuated the high mortality rate at 8 wk of treatment. These data suggest that amlodipine attenuated arteriosclerosis through inhibiting inflammatory disorders in the rat model of long-term inhibition of NO synthesis. The anti-inflammatory effects of amlodipine seem to be mediated not only by the inhibition of local factors such as MCP-1 but also by the decrease in CCR2 in circulating monocytes. Inhibition of the MCP-1 to CCR2 pathway may represent novel anti-inflammatory actions of amlodipine beyond blood pressure lowering.

The in vitro antiproliferative and apoptosis inducing properties of the nonsteroidal anti-inflammatory drugs (NSAIDs) like acetyl salicylic acid (aspirin) and indomethacin were investigated in T98G human glioblastoma cells to explore their potential role in the chemoprevention of human glioma. The biological effects induced by aspirin and indomethacin on T98G cells, in which the expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) were confirmed by RT-PCR and immunostaining, were investigated by studying cell proliferation and apoptosis assays. The antiproliferative effects occurred in a dose- and time-dependent manner on T98G cells by the treatment with 0.1 -2 mM aspirin and 25-100 microM indomethacin. Moreover, aspirin displayed the greatest growth inhibition within 24 h. Approximately 90% growth inhibition occurred following treatment either with 2 mM aspirin or 100 microM indomethacin by 72 h and induction of apoptosis was confirmed by DNA laddering and TUNEL assay. Our in vitro findings indicate that aspirin and indomethacin have an antiproliferative effect on T98G human glioblastoma cells at toxic concentrations.

Choroidal neovascularization (CNV) is directly related to visual loss in some eye diseases, such as age-related macular degeneration. Although several human histological studies have suggested the participation of macrophages in CNV formation, the precise mechanisms are still not fully understood. In this study, we elucidated the role of ocular-infiltrating macrophages in experimental CNV using CCR2 knockout (KO) mice, wild-type mice, and C57BL/6 (B6) mice. CCR2 is the receptor of monocyte chemoattractant protein-1, and the number of infiltrating macrophage and the area of CNV were significantly reduced in CCR2 KO mice. Enriched ocular-infiltrating macrophages from B6 mice actually showed angiogenic ability in a dorsal air sac assay. Moreover, their expression of class II, CD40, B7-1 and B7-2 molecules, and the mRNA for potential angiogenic factors, such as vascular endothelial growth factor, basic fibroblast growth factor, and tumor necrosis factor α, was also observed. Collectively, we conclude that ocular-infiltrating macrophages play an important role in CNV generation.

Thyroid hormone has a broad effect on cardiovascular system. 3,3′,5-triiodo- l -thyronine (T3), a biologically active form of thyroid hormone, increases cardiac contractility. T3 causes arterial relaxation and reduction of systemic vascular resistance, resulting in an increase in cardiac output. However, the molecular mechanisms of vascular relaxation by T3 are incompletely characterized. We studied the effect of T3 on the angiotensin (Ang) II type 1 receptor (AT 1 R) expression in vascular smooth muscle cells. T3 dose-dependently decreased expression levels of AT 1 R mRNA, with a peak at 6 hours of stimulation. Binding assay using [ 125 I]Sar 1 -Ile 8 -Ang II revealed that AT 1 R number was decreased by stimulation with T3 without changing the affinity to Ang II. T3 reduced calcium response of vascular smooth muscle cells to Ang II by 26%. AT 1 R promoter activity measured by luciferase assay was reduced by 50% after 9 hours of T3 administration. mRNA stability was also decreased by T3. Real-time quantitative reverse transcription–polymerase chain reaction and Western blot analysis revealed that AT 1 R mRNA and protein were downregulated in the aorta of T3-treated rats. These results suggest that T3 downregulates AT 1 R expression both at transcriptional and posttranscriptional levels, and attenuates biological function of Ang II. Our results suggest that downregulation of AT 1 R gene expression may play an important role for T3-induced vascular relaxation.

Background—Monocyte infiltration into the arterial wall and its activation is the central event in atherogenesis. Thus, monocyte chemoattractant protein-1 (MCP-1) might be a novel therapeutic target against atherogenesis. We and others recently reported that blockade or abrogation of the MCP-1 pathway attenuates the initiation of atheroma formation in hypercholesterolemic mice. It remains unclear, however, whether blockade of MCP-1 can limit progression or destabilization of established lesions.Methods and Results—We report here that blockade of MCP-1 by transfecting anN-terminal deletion mutant of the MCP-1 gene limited progression of preexisting atherosclerotic lesions in the aortic root in hypercholesterolemic mice. In addition, blockade of MCP-1 changed the lesion composition into a more stable phenotype, ie, containing fewer macrophages and lymphocytes, less lipid, and more smooth muscle cells and collagen. This strategy decreased expression of CD40 and the CD40 ligand in the atherosclerotic plaque and normalized the increased chemokine (RANTES and MCP-1) and cytokine (tumor necrosis factor α, interleukin-6, interleukin-1β, and transforming growth factor β1) gene expression. These data suggest that MCP-1 is a central mediator in the progression and destabilization of established atheroma.Conclusions—The results of the present study suggest that the inflammatory responses mediated by MCP-1 are important in atherosclerosis and its complications.

Peroxisome proliferator-activated receptor-γ (PPARγ) ligands are widely used in patients with insulin resistance and diabetes. Because coronary artery disease is a major complication for such patients, it is important to determine the effects of PPARγ activation on arteriosclerosis. Long-term inhibition of endothelial NO synthesis by administration of N ω -nitro- l -arginine methyl ester (L-NAME) to rats induces coronary vascular inflammation (monocyte infiltration, monocyte chemoattractant protein-1 [MCP-1] expression) and subsequent arteriosclerosis. We examined the effects of pioglitazone (a PPARγ ligand) in this rat model to determine whether PPARγ activation with pioglitazone inhibits arteriosclerosis by its indirect effects on metabolic conditions or by direct effects on the cells participating to the pathogenesis of arteriosclerosis. We found that pioglitazone did not affect metabolic states, systolic blood pressure, or serum NO levels, but did prevent the L-NAME–induced coronary inflammation and arteriosclerosis. Pioglitazone did not reduce local expression of MCP-1 but markedly attenuated increased expression of the MCP-1 receptor C-C chemokine receptor 2 (CCR2) in lesional and circulating monocytes. PPARγ activation with pioglitazone prevented coronary arteriosclerosis, possibly by its antiinflammatory effects (downregulation of CCR2 in circulating monocytes). Inhibition of the CCR2-mediated inflammation may represent novel antiinflammatory actions of pioglitazone beyond improvement of metabolic state.

Background — It remains unclear whether vascular endothelial growth factor (VEGF) is a proarteriosclerotic or an antiarteriosclerotic factor. We recently reported that long-term inhibition of nitric oxide by administering Nω -nitro- l -arginine methyl ester (L-NAME) induces coronary vascular inflammation and arteriosclerosis. Methods and Results — We used this animal model to investigate the role of VEGF in arteriosclerosis. We blocked VEGF activity in vivo by transfecting with plasmid DNA encoding the murine soluble FLT-1 ( sFLT-1 ) gene into thigh muscle. Soluble FLT-1 can suppress VEGF activity both by sequestering VEGF and by functioning as a dominant-negative inhibitor of VEGF receptors. We observed vascular inflammation associated with increased VEGF expression within 3 days of L-NAME administration, which was prevented by pretreatment with ACE inhibitor, angiotensin II receptor antagonist, or neutralizing monocyte chemoattractant protein-1 antibody. The sFLT-1 gene transfer attenuated the early vascular inflammation and prevented late arteriosclerosis. The sFLT-1 gene transfer also inhibited increased expression of monocyte chemoattractant protein-1 and transforming growth factor-β, indicating creation of a positive feedback loop to cause arteriosclerosis. Conclusions — VEGF is necessary in the development of arteriosclerosis by mediating monocyte recruitment and activation in this model.

We report a case of sarcomatous transformation of a prolactin (PRL)-producing pituitary adenoma in a 27-year-old man. He originally presented with bitemporal visual disturbance, headache, and hyperprolactinemia 8 years earlier. Tumor shrinkage was confirmed by magnetic resonance imaging (MRI) during treatment with dopamine-receptor agonist. However, 3 years later transsphenoidal surgery had to be performed because of tumor re-growth. Histopathological examination revealed a PRL-producing adenoma with fibrotic changes. One year later, he presented with right-sided visual disturbance, and tumor re-growth was confirmed using MRI. He underwent transcranial surgery, followed by radiation therapy (50 Gy in 25 fractions). The histological and immunostaining features were similar in both specimens obtained from the two operations. Four years later, he presented with left-sided visual disturbance, and tumor re-growth was confirmed using MRI. The mass lesion dramatically increased in size within 2 months, and partial removal of the tumor by craniotomy was performed. The specimen was histologically diagnosed as malignant fibrous histiocytoma (MFH). Regardless of aggressive chemotherapy, his clinical symptoms and imaging findings worsened rapidly. He died 7 months after the diagnosis of MFH. Because patients with pituitary tumor undergoing radiotherapy face the possibility of developing such neoplasm, long-term follow-up is required.

Liver X Receptors (LXRs) α and β are oxysterol-activated nuclear receptors involved in the control of lipid metabolism and inflammation. Pharmacological activation of LXR is promising in the treatment of atherosclerosis since it can promote cholesterol efflux from macrophages and prevent foam cell formation. However, the development of LXR agonists has been limited by undesirable side-effects such as hepatic steatosis mediated by LXRα activation. Therefore, it has been proposed that targeting LXRα activators to extrahepatic tissues or using LXRβ-specific activators could be used as alternative strategies. It is not clear whether these molecules will retain the full atheroprotective potential of non-selective agonists. Our aim was therefore to determine the contribution of LXRα and LXRβ to the control of cholesterol efflux in human macrophages. LXRα and/or LXRβ expression was suppressed by small interfering RNAs in human primary macrophages treated or not with synthetic LXRα/β dual agonists T0901317 and GW3965. We observed that LXRβ silencing had no detectable impact on the expression of LXR-target genes such as ABCA1 and ABCG1. Moreover it did not affect cholesterol efflux. In contrast, LXRα silencing reduced the response of these LXR-target genes to LXR agonist and inhibited cholesterol efflux to ApoA-I, HDL2 or to endogenous ApoE. Importantly, no differences were observed between LXRα and LXRα/β knockdown conditions. Altogether, our data demonstrate that LXRβ activation is unable to maintain maximal cholesterol efflux capacities in human primary macrophages when LXRα expression is impaired. In contrast to earlier mouse studies, LXRα levels appear as a limiting factor for macrophage cholesterol efflux in humans.

Inflammatory cell activation plays a key role in atherosclerotic plaque growth and acute complications. While secretion of proteases and inflammatory cytokines are likely involved in the development of plaque instability, the precise mechanistic pathways are not well understood.Based on our previous study, we crossed Toll-like receptor 3 (Tlr3)(-/-) mice with a unique BALB-Apoe(-/-)Npc1(-/-) plaque complication-susceptible mouse model, as well as the widely-used B6-Ldlr(-/-) atherosclerosis model, to test the role of TLR3 signaling in the development of plaque instability. TLR3-deficient mice showed no change in aortic root lesion area, but displayed a marked increase in collagen and smooth muscle cell (SMC) content of lesions. Notably, Apoe(-/-)Npc1(-/-)Tlr3(-/-) mice exhibited a 50% reduction in the incidence of medial destruction, a precursor to aortic aneurysm formation. MMP-2 activity was markedly reduced in aortic extracts from Apoe(-/-)Npc1(-/-)Tlr3(-/-) compared to controls, while both MMP-2 and -9 activities were reduced in Ldlr(-/-)Tlr3(-/-) extracts. Consistent with the in vivo data, TLR3 deficiency suppressed MMP-2 activity induced by TNF-α or polyinosine-polycytidylic acid in macrophages from Apoe(-/-)Npc1(-/-) mice.TLR3 plays a critical role in regulating the degradation of extracellular matrix in lesions, in part by modulation of macrophage MMP-2 and -9 activities.

The molecular events linking lipid accumulation in atherosclerotic plaques to complications such as aneurysm formation and plaque disruption are poorly understood. BALB/c- Apoe −/− mice bearing a null mutation in the Npc1 gene display prominent medial erosion and atherothrombosis, whereas their macrophages accumulate free cholesterol in late endosomes and show increased cathepsin K ( Ctsk ) expression. We now show increased cathepsin K immunostaining and increased cysteinyl proteinase activity using near infrared fluorescence imaging over proximal aortas of Apoe −/− , Npc1 −/− mice. In mechanistic studies, cholesterol loading of macrophage plasma membranes (cyclodextrin–cholesterol) or endosomal system (AcLDL+U18666A or Npc1 null mutation) activated Toll-like receptor (TLR) signaling, leading to sustained phosphorylation of p38 mitogen-activated protein kinase and induction of p38 targets, including Ctsk , S100a8 , Mmp8 , and Mmp14 . Studies in macrophages from knockout mice showed major roles for TLR4, following plasma membrane cholesterol loading, and for TLR3, after late endosomal loading. TLR signaling via p38 led to phosphorylation and activation of the transcription factor Microphthalmia transcription factor, acting at E-box elements in the Ctsk promoter. These studies suggest that free cholesterol enrichment of either plasma or endosomal membranes in macrophages leads to activation of signaling via various TLRs, prolonged p38 mitogen-activated protein kinase activation, and induction of Mmps , Ctsk , and S100a8 , potentially contributing to plaque complications.

Background— Two macrophage ABC transporters, ABCA1 and ABCG1, have a major role in promoting cholesterol efflux from macrophages. Peritoneal macrophages deficient in ABCA1, ABCG1, or both show enhanced expression of inflammatory and chemokine genes. This study was undertaken to elucidate the mechanisms and consequences of enhanced inflammatory gene expression in ABC transporter–deficient macrophages. Methods and Results— Basal and lipopolysaccharide-stimulated thioglycollate-elicited peritoneal macrophages showed increased inflammatory gene expression in the order Abca1 −/− Abcg1 −/− > Abcg1 −/− > Abca1 −/− >wild-type. The increased inflammatory gene expression was abolished in macrophages deficient in Toll-like receptor 4 (TLR4) or MyD88/TRIF. TLR4 cell surface concentration was increased in Abca1 −/− Abcg1 −/− > Abcg1 −/− > Abca1 −/− > wild-type macrophages. Treatment of transporter-deficient cells with cyclodextrin reduced and cholesterol-cyclodextrin loading increased inflammatory gene expression. Abca1 −/− Abcg1 − bone marrow–derived macrophages showed enhanced inflammatory gene responses to TLR2, TLR3, and TLR4 ligands. To assess in vivo relevance, we injected intraperitoneally thioglycollate in Abcg1 −/− bone marrow–transplanted, Western diet–fed, Ldlr -deficient mice. This resulted in a profound inflammatory infiltrate in the adventitia and necrotic core region of atherosclerotic lesions, consisting primarily of neutrophils. Conclusions— The results suggest that high-density lipoprotein and apolipoprotein A-1 exert anti-inflammatory effects by promoting cholesterol efflux via ABCG1 and ABCA1 with consequent attenuation of signaling via Toll-like receptors. In response to a peripheral inflammatory stimulus, atherosclerotic lesions containing Abcg1 −/− macrophages experience an inflammatory “echo,” suggesting a possible mechanism of plaque destabilization in subjects with low high-density lipoprotein levels.

Background— The formation of an occluding thrombus on a ruptured or eroded atherosclerotic plaque is the hallmark event leading to acute coronary syndromes, myocardial infarction, and sudden death in humans. However, other species are highly resistant to plaque complications, and the specific processes predisposing to plaque destabilization and thrombosis are poorly understood. Methods and Results— Mice carrying a null mutation of a gene regulating intracellular cholesterol transport (the Niemann-Pick C1 [ Npc1 ] gene) were crossed with apolipoprotein E ( Apoe ) knockout mice to examine the effect of Npc1 on atherosclerotic lesion formation. Double-mutant mice showed greater lesion area compared with Apoe −/− littermates. Remarkably, the double mutants also developed large, protruding thrombi associated with the plaques and prominent medial degradation with inflammatory cell infiltration into the adventitia. Genetic studies suggested that the BALB background was permissive for plaque complications compared with C57BL/6J, and a BALB susceptibility locus was mapped by linkage analysis to chromosome 6. Examination of clotting parameters in double-knockout mice revealed that native clotting times were shortened and thrombin-antithrombin complex and soluble CD40 ligand levels were elevated compared with wild-type controls. In addition, cathepsin K was induced in Npc1 −/− macrophages, and cathepsin K immunostaining and elastase activity were increased in proximal aortas of double-mutant mice compared with controls. Conclusions— A defect in intracellular cholesterol trafficking caused by the Npc1 null mutation predisposes to increased lesion formation, atherothrombosis, and medial degradation. Plaque complications may require a procoagulant state and an increased protease activity, leading to plaque destabilization.

We report a case of intracranial germ cell tumor that showed pathological changes from neurohypophyseal germinoma to mixed germ cell tumors consisting exclusively of undifferentiated sarcomatous component after radiochemotherapy. Three surgical specimens and autopsied brain from the patient were histologically examined. An initial specimen from the neurohypophyseal tumor was diagnosed as germinoma with a two-cell pattern. Five years later, after repeated radiochemotherapy, the second specimen resected from the right temporal lobe showed mixed germ cell tumors consisting of the three components of germinoma, choriocarcinoma, and immature teratoma. Six months later after intensive radiotherapy, the right temporal tumor recurred and was surgically removed. The histological diagnosis was mixed germ cell tumors with abundant immature teratoma component. The patient died of uncontrollable tumor growth with repeated intratumoral hemorrhages. The autopsied brain showed sarcoma with angionecrosis. This pathological alteration indicated an increase in the sarcomatous component after undergoing various treatments. We discuss the histological changes of intracranial germ cell tumor modified by treatment.

We previously showed that nonsteroidal anti-inflammatory drugs (NSAID) such as sulindac sulfide, which has chemopreventive activity, modulate the expression of several genes detected by microarray analysis. Activating transcription factor 3 (ATF3) was selected for further study because it is a transcription factor involved in cell proliferation, apoptosis, and invasion, and its expression is repressed in human colorectal tumors as compared with normal adjacent tissue. In this report, we show that ATF3 mRNA and protein expression are up-regulated in HCT-116 human colorectal cancer cells following treatment with NSAIDs, troglitazone, diallyl disulfide, and resveratrol. To ascertain the biological significance of ATF3, we overexpressed full-length ATF3 protein in the sense and antisense orientations. Overexpression of ATF3 in the sense orientation decreased focus formation in vitro and reduced the size of mouse tumor xenografts by 54% in vivo. Conversely, overexpression of antisense ATF3 was protumorigenic in vitro, however, not in vivo. ATF3 in the sense orientation did not modulate apoptosis, indicating another mechanism is involved. With microarray analysis, several genes relating to invasion and metastasis were identified by ATF3 overexpression and were confirmed by real-time reverse transcription-PCR, and several of these genes were modulated by sulindac sulfide, which inhibited invasion in these cells. Furthermore, overexpression of ATF3 inhibited invasion to a similar degree as sulindac sulfide treatment, whereas antisense ATF3 increased invasion. In conclusion, ATF3 represents a novel mechanism in which NSAIDs exert their anti-invasive activity, thereby linking ATF3 and its gene regulatory activity to the biological activity of these compounds.

Angiotensin II (Ang II) is implicated in atherogenesis by activating inflammatory responses in arterial wall cells. Ang II accelerates the atherosclerotic process in hyperlipidemic apoE−/− mice by recruiting and activating monocytes. Monocyte chemoattractant protein-1 (MCP-1) controls monocyte-mediated inflammation through its receptor, CCR2. The roles of leukocyte-derived CCR2 in the Ang II-induced acceleration of the atherosclerotic process, however, are not known. We hypothesized that deficiency of leukocyte-derived CCR2 suppresses Ang II-induced atherosclerosis.Methods and Results—A bone marrow transplantation technique (BMT) was used to develop apoE−/− mice with and without deficiency of CCR2 in leukocytes (BMT-apoE−/−CCR2+/+ and BMT-apoE−/−CCR2−/− mice). Compared with BMT-apoE−/−CCR2+/+ mice, Ang II-induced increases in atherosclerosis plaque size and abdominal aortic aneurysm formation were suppressed in BMT-apoE−/−CCR2−/− mice. This suppression was associated with a marked decrease in monocyte-mediated inflammation and inflammatory cytokine expression.Conclusion—Leukocyte-derived CCR2 is critical in Ang II-induced atherosclerosis and abdominal aneurysm formation. The present data suggest that vascular inflammation mediated by CCR2 in leukocytes is a reasonable target of therapy for treatment of atherosclerosis.

Objective— Vascular endothelial growth factor (VEGF) is upregulated after arterial injury. Its role in the pathogenesis of neointimal formation after periadventitial injury, however, has not been addressed. Methods and Results— Expression of VEGF and its receptors but not that of placental growth factor markedly increased with the development of neointimal formation in hypercholesterolemic mice after cuff-induced periarterial injury. Transfection with the murine soluble Flt-1 (sFlt-1) gene to block VEGF in vivo in mice inhibited early inflammation and later neointimal formation. The sFlt-1 gene transfer did not affect plasma lipid levels but attenuated increased expression of VEGF, Flt-1, Flk-1, monocyte chemoattractant protein-1, and other inflammation-promoting factors. Mice with Flt-1 kinase deficiency also displayed reduced neointimal formation. Conclusions— Inflammatory changes mediated by VEGF and Flt-1 signals play an important role in the pathogenesis of neointimal formation after cuff-induced periadventitial injury. VEGF might promote neointimal formation by acting as a proinflammatory cytokine.

The induction of cyclooxygenase-2 (COX-2) expression is associated with more aggressive gliomas and poor survival. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit COX activity and have antitumorigenic properties. In this report, our initial aim was to determine if indomethacin would alter gene expression as measured by suppression subtractive hybridization (SSH). Three up-regulated and four down-regulated genes by indomethacin treatment were identified. Laminin gamma1, an extracellular matrix molecule, was the most significantly repressed gene. The repression of laminin gamma1 by indomethacin was confirmed by Northern and Western blot analyses and occurred in a concentration- and time-dependent manner at the protein level. Among several NSAIDs tested, only sulindac sulfide and indomethacin suppressed laminin gamma1 protein expression, and this repression was observed in both COX-expressing and -deficient cell lines, suggesting that laminin gamma1 repression by COX inhibitors was independent of COX. Indomethacin, at a concentration that represses laminin gamma1, inhibited glioblastoma cell invasion that was partially restored with additional human laminin protein containing gamma1 chain. The repression of laminin gamma1 by NSAIDs may be related to attenuation of invasion of brain tumors. These findings are important in understanding the chemopreventive activity of some NSAIDs and could be relevant for designing therapeutic strategies against glioblastoma.

Activated monocytes are present in the arterial walls of hypertensive patients and animals. Monocyte chemoattractant protein-1 (MCP-1), which controls monocyte function through its receptor (CCR2), is implicated in hypertensive inflammatory changes in the arterial wall. The role of CCR2 expression on monocytes in hypertension-induced vascular remodeling, however, has not been addressed. We hypothesized that CCR2 on monocytes is critical in hypertension-induced vascular inflammation and remodeling. Hypertension was induced by infusion of angiotensin II (Ang II) into wild-type mice, CCR2-deficient (CCR2−/−) mice, and bone marrow-transferred mice with a leukocyte-selective CCR2 deficiency (BMT-CCR2−/−). In wild-type mice, Ang II increased CCR2 intensity in circulating monocytes, which was prevented by an Ang II type-1 (AT1) receptor blocker or blunted in AT1receptor-deficient mice. Enhanced CCR2 intensity on monocytes was observed in hypertensive patients and rats, and was reduced by treatment with the Ang II receptor blocker, supporting the clinical relevance of the observation in mice. In CCR2−/−and BMT-CCR2−/−mice, Ang II-induced vascular inflammation and vascular remodeling (aortic wall thickening and fibrosis) were blunted as compared with control mice. In contrast, Ang II-induced left ventricular hypertrophy developed in CCR2−/−and BMT-CCR2−/−mice. The present study suggests that CCR2 expression in monocytes has a critical role in vascular inflammation and remodeling in Ang II-induced hypertension, and possibly in other forms of hypertension.

Objective—Chronic inflammatory processes might be involved in the progression and destabilization of atherosclerotic plaques. Therefore, identification of the mechanism underlying arterial inflammatory function might lead to the development of novel therapeutic strategies. Angiotensin II (AngII) is implicated in atherogenesis by activating the vascular inflammation system, mainly through monocyte chemotaxis. Therefore, we hypothesized that AngII increases plaque size and promotes destabilization of established atheromas by activating the monocyte chemoattractant protein-1 (MCP-1) pathway.Methods and Results—We report here that 4-week infusion of AngII not only increased plaque size but also induced a destabilization phenotype (ie, increased macrophages and lipids and decreased collagen and smooth muscle cells) of pre-existing atherosclerotic lesions of hypercholesterolemic mice. AngII also enhanced the gene expression of inflammatory cytokines (TNFα, IL-6, etc.) and chemokines (MCP-1, CCR2, etc). Blockade of MCP-1, by transfecting the deletion mutant of the human MCP-1 gene into the skeletal muscles, limited AngII-induced progression and destabilization of established atherosclerotic lesions and suppressed the induction of proinflammatory genes.Conclusions—These data suggest that MCP-1 functions as a central inflammatory mediator in the AngII-induced progression and changes in plaque composition of established atheroma.

Early growth response factor-1 (Egr-1) is a transcription factor that is rapidly activated after vascular injury and thus might contribute to vascular proliferation and inflammation. We hypothesized that Egr-1 might therefore be a therapeutic target against restenosis. Hypercholesterolemic rabbits were intraluminally administered synthetic DNA as a 'decoy' against Egr-1 immediately after carotid artery balloon injury. Efficient transfection was confirmed by the delivery of a fluorescence-labeled decoy. Gel mobility-shift assay showed increased Egr-1 activity after balloon injury and its prevention by Egr-1 decoy transfection in vivo. Egr-1 decoy transfection attenuated early inflammation and proliferation and later neointimal hyperplasia. In addition, Egr-1 decoy transfection reduced gene expression and protein production of Egr-1-dependent genes such as platelet-derived growth factor-B, transforming growth factor-beta1, and monocyte chemoattractant protein-1. The Egr-1 pathway has an essential role in the pathogenesis of neointimal hyperplasia after balloon injury in hypercholesterolemic rabbits. This decoy strategy is a potential practical form of therapy for human restenosis.

Background— Peroxisome proliferator–activated receptor-γ activators have recently been implicated as regulators of cellular proliferation and inflammatory response such as cytokine expression. Because proinflammatory cytokines play a critical role in left ventricular (LV) remodeling after myocardial infarction (MI), we examined the effects of pioglitazone treatment in an experimental model of chronic heart failure. Methods and Results— Mice with extensive anterior MI were treated with placebo or pioglitazone (3 mg · kg −1 · d −1 ) as a dietary supplement for 4 weeks starting 6 hours after surgery. Infarct size and glucose levels were similar among all groups. LV cavity dilatation and dysfunction by echocardiography were significantly attenuated in MI mice given pioglitazone. LV end-diastolic pressure was increased in MI mice and was significantly reduced by pioglitazone treatment. Pioglitazone partially normalized LV dP/dt max and dP/dt min , indices of LV contractile function, which were significantly reduced in MI mice. Improvement of LV function by pioglitazone was accompanied by a decrease in myocyte hypertrophy and interstitial fibrosis and a reduced expression of tumor necrosis factor-α, transforming growth factor-β, and monocyte chemoattractant protein-1 genes in the noninfarcted LV from MI mice. LV inducible nitric oxide synthase and gelatinase B protein levels were increased in MI and were not altered by pioglitazone treatment. Conclusions— Pioglitazone improved LV remodeling and function in mice with post-MI heart failure. This effect was associated with an attenuated LV expression of inflammatory cytokines and chemokines. Peroxisome proliferator–activated receptor-γ ligands have promise as preventive and therapeutic agents against heart failure.

Deep-seated gliomas, including thalamic gliomas, have a poor prognosis because of difficulty of accessibility for surgery. In addition, an infiltrative pattern of the tumor is related to a poor prognosis. In this study, the infiltrative/invasive profile of the proliferating tumor cells of a right thalamic glioma was evaluated in an autopsied brain. A 71-year-old man died from extensive infiltration of a right thalamic glioma. The distribution of the proliferating tumor cells at the right thalamic tumor level was represented by the topographic map of MIB-1 labeling indices (LI) on the whole-brain coronal slice, and this map was analyzed with pathological findings and postmortem T2-weighted magnetic resonance imaging (MRI). The highest MIB-1 LI was 24% for the whole autopsy brain at the thalamic tumor level, whereas the MIB-1 LI was 21% for the biopsy sample of the right thalamic glioma. Because this patient survived only 9 months after diagnosis of the tumor as anaplastic astrocytoma, it was confirmed that 21% MIB-1 LI of the biopsy sample was relevant to his prognosis. The topographic map of MIB-1 LI showed that the proliferating tumor cells of the right thalamic glioma invaded the ventricular walls and the contralateral thalamus by the periventricular route, but there was no exophytic extension to the cortex. In conclusion, topographic analysis of the proliferative potential detected by MIB-1 immunostaining provides information on the growth pattern of human glioma.

Prevention of restenosis after coronary intervention is a major clinical challenge, which highlights the need of new therapeutic options. Vascular injury may involve inflammatory responses that accelerate the recruitment and activation of monocytes through the activation of chemotactic factors, including monocyte chemoattractant protein-1 (MCP-1). However, there is no definitive evidence supporting the role of MCP-1 in restenosis. We recently devised a new strategy for anti-MCP-1 gene therapy by transfecting an N-terminal deletion mutant of the MCP-1 gene into skeletal muscles. We demonstrate here that this strategy suppressed monocyte infiltration/activation in the injured site and markedly inhibited restenotic changes (neointimal hyperplasia) after balloon injury of the carotid artery in rats and monkeys. This strategy also suppressed the local production of MCP-1 and inflammatory cytokines. Therefore, monocyte infiltration and activation mediated by MCP-1 are essential in the development of restenotic changes after balloon injury. This strategy may be a useful form of gene therapy against human restenosis.