. When human catalase was expressed in Nox1-expressing cells, H 2O2 concentration decreased, and the cells reverted to a normal appearance, the growth rate normalized, and cells no longer produced tumors in athymic mice. A large number of genes, including many related to cell cycle, growth, and cancer (but unrelated to oxidative stress), were expressed in Nox1-expressing cells, and more than 60% of these returned to normal levels on coexpression of catalase. Thus, H2O2 in low concentrations functions as an intracellular signal that triggers a genetic program related to cell growth.

Objectives To examine selective macrophage differentiation occurring in areas of intraplaque hemorrhage in human atherosclerosis. Background Macrophage subsets are recognized in atherosclerosis but the stimulus for and importance of differentiation programs remains unknown. Methods We used freshly isolated human monocytes, a rabbit model, and human atherosclerotic plaques to analyze macrophage differentiation in response to hemorrhage. Results Macrophages characterized by high expression of both mannose and CD163 receptors preferentially exist in atherosclerotic lesions at sites of intraplaque hemorrhage. These hemoglobin (Hb)-stimulated macrophages, M(Hb), are devoid of neutral lipids typical of foam cells. In vivo modeling of hemorrhage in the rabbit model demonstrated that sponges exposed to red cells showed an increase in mannose receptor positive macrophages only when these cells contained hemoglobin (Hb). Cultured human monocytes exposed to hemoglobin:haptoglobin complexes (Hb:Hp), but not IL-4, expressed the M(Hb) phenotype and were characterized by their resistance to cholesterol loading and upregulation of ABC transporters. M(Hb) demonstrated increased ferroportin (FPN) expression, reduced intracellular iron, and reactive oxygen species (ROS). Degradation of FPN using hepcidin increased ROS, inhibited ABCA1 expression, and cholesterol efflux to ApoAI, suggesting reduced ROS triggers these effects. Knockdown of liver x receptor alpha (LXRα) inhibited ABC transporter expression in M(Hb) and macrophages differentiated in the anti-oxidant superoxide dismutase. Lastly, liver X receptor α (LXR) luciferase reporter activity was increased in M(Hb) and significantly reduced by overnight treatment with hepcidin. Collectively, these data suggest reduced ROS triggers LXRα activation and macrophage reverse cholesterol transport (RCT). Conclusions Hb is a stimulus for macrophage differentiation in human atherosclerotic plaques. A reduction of macrophage intracellular iron plays an important role in this non- foam cell phenotype by reducing ROS, which drives transcription of ABC transporters through activation of LXRα. Reduction of macrophage intracellular iron may be a promising avenue to increase macrophage RCT.

Intake of hemoglobin by the hemoglobin-haptoglobin receptor CD163 leads to a distinct alternative non-foam cell antiinflammatory macrophage phenotype that was previously considered atheroprotective. Here, we reveal an unexpected but important pathogenic role for these macrophages in atherosclerosis. Using human atherosclerotic samples, cultured cells, and a mouse model of advanced atherosclerosis, we investigated the role of intraplaque hemorrhage on macrophage function with respect to angiogenesis, vascular permeability, inflammation, and plaque progression. In human atherosclerotic lesions, CD163+ macrophages were associated with plaque progression, microvascularity, and a high level of HIF1α and VEGF-A expression. We observed irregular vascular endothelial cadherin in intraplaque microvessels surrounded by CD163+ macrophages. Within these cells, activation of HIF1α via inhibition of prolyl hydroxylases promoted VEGF-mediated increases in intraplaque angiogenesis, vascular permeability, and inflammatory cell recruitment. CD163+ macrophages increased intraplaque endothelial VCAM expression and plaque inflammation. Subjects with homozygous minor alleles of the SNP rs7136716 had elevated microvessel density, increased expression of CD163 in ruptured coronary plaques, and a higher risk of myocardial infarction and coronary heart disease in population cohorts. Thus, our findings highlight a nonlipid-driven mechanism by which alternative macrophages promote plaque angiogenesis, leakiness, inflammation, and progression via the CD163/HIF1α/VEGF-A pathway.

Increased hepatic oxidative stress with ethanol administration is hypothesized to be caused either by enhanced pro-oxidant production or decreased levels of antioxidants or both. We used the intragastric feeding rat model to assess the relationship between hepatic antioxidant enzymes and pathological liver injury in animals fed different dietary fats. Male Wistar rats (5 per group) were fed ethanol with either medium-chain triglycerides (MCTE), palm oil (PE), corn oil (CE), or fish oil (FE). Control animals were fed isocaloric amounts of dextrose instead of ethanol with the same diets. The following were evaluated in each group: liver pathology, lipid peroxidation, manganese superoxide dismutase (MnSOD) levels, copper-zinc SOD (CuZnSOD) levels, glutathione peroxidase (GPX) levels, and catalase (CAT) levels. All enzymes were evaluated using activity assays and immunoblots. Rats fed FE showed the most severe pathology (fatty liver, necrosis, and inflammation), those fed CE showed moderate changes, those fed PE showed fatty liver only, and those fed MCTE were normal. Parameters indicative of lipid peroxidation (conjugated dienes and thiobarbituric acid-reactive substances) were also greater in rat livers from animals fed the diets high in polyunsaturated fatty acids (CE and FE). CuZnSOD, GPX, and CAT activities showed an inverse correlation (r ‫؍‬ ؊.92, P F .01) with severity of pathological injury, with the lowest levels for both enzymes found in FE-fed rats. Decreased enzyme activity in CE-and FE-fed rats was accompanied by similar decreases in immunoreactive protein. Ethanol administration did not cause significant decreases in enzyme activity in groups that showed no necroinflammatory changes (MCTE and PE). MnSOD activity showed no significant change in any ethanol-fed group. Our results show that decreases in CuZnSOD, GPX, and CAT occur in rats showing pathological liver injury and also having the highest levels of lipid peroxidation. These results suggest that feeding dietary substrates that enhance lipid peroxidation can exacerbate both ethanol-induced oxidative damage as well as necroinflammatory changes. The decrease in activity of antioxidant enzymes observed in animals fed diets high in polyunsaturated fatty acids and ethanol could possibly increase the susceptibility to oxidative damage and further contribute to ethanol-induced liver injury. (HEPATOLOGY 1998;27:1317-1323.)

The low-density lipoprotein receptorrelated protein (LRP) is a member of the LDL receptor gene family that binds several ligands, including tissue-type plasminogen activator (tPA). tPA is found in blood, where its primary function is as a thrombolytic enzyme, and in the central nervous system where it mediates events associated with cell death. Cerebral ischemia induces changes in the neurovascular unit (NVU) that result in brain edema. We investigated whether the interaction between tPA and LRP plays a role in the regulation of the permeability of the NVU during cerebral ischemia. We found that the ischemic insult induces shedding of LRP's ectodomain from perivascular astrocytes into the basement membrane. This event associates with the detachment of astrocytic end-feet processes and the formation of areas of perivascular edema. The shedding of LRP's ectodomain is significantly decreased in tPA deficient (tPA ؊/؊ ) mice, is increased by incubation with tPA, and is inhibited by the receptor-associated protein (RAP). IntroductionThe low-density lipoprotein receptor-related protein (LRP) is a member of the LDL receptor gene family composed of a 515-kDa heavy chain noncovalently bound to an 85-kDa light chain containing a transmembrane and a cytoplasmic domain. 1 LRP mediates the internalization of apoE-enriched lipoprotein particles, 2 ␣-2-macroglobulin-protease complexes, 3 and several other ligands, including plasminogen activators, proteinase-inhibitor complexes, clotting factors, and the amyloid precursor protein (APP). 1 LRP has also been implicated in cellular signal transduction pathways 4 and neurotransmission. 5 Like other signaling receptors such as Notch 6 and APP, 7 LRP undergoes a ␥-secretase-likedependent cleavage of its cytoplasmic site with release of its intramembranous domain. 8 This process is preceded by shedding of the receptor's ectodomain which may increase substrate availability for the enzymes that are required for the cleavage of the intramembranous or cytosolic sites. 8,9 Tissue-type plasminogen activator (tPA) is a ligand for LRP, 10,11 on binding to LRP induces a transient tyrosine phosphorylation of its cytoplasmic domain, and induces increased synthesis of 13 Animal studies have demonstrated that following the onset of cerebral ischemia there is an increase in endogenous tPA activity within the ischemic tissue, [14][15][16] and that either genetic deficiency of tPA 14,17 or its inhibition with neuroserpin 15,18 are associated with neuronal survival and decrease in the volume of the ischemic lesion.The neurovascular unit (NVU) is a dynamic structure consisting of endothelial cells, the basal lamina, astrocytic end-feet processes, pericytes, and neurons. 19,20 One of the functions of the NVU is to form a barrier, known as the blood-brain barrier (BBB), that regulates the entry of selected molecules from the blood into the central nervous system (CNS). 21,22 During cerebral ischemia the permeability of the NVU increases, resulting in the development of cerebral edema, 23,24 which is a ...

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor superfamily. TWEAK acts on responsive cells via binding to a small cell-surface receptor named fibroblast growth factor-inducible-14 (Fn14). TWEAK can stimulate numerous cellular responses including cell proliferation, migration, and proinflammatory molecule production. The present study investigated whether TWEAK plays a role in the regulation of the permeability of the neurovascular unit (NVU). We found that intracerebral injection of TWEAK in wild-type mice induces activation of the nuclear factor-B (NF-B) pathway and matrix metalloproteinase-9 (MMP-9) expression in the brain with resultant disruption in the structure of the NVU and increase in the permeability of the blood-brain barrier (BBB). TWEAK did not increase MMP-9 activity or BBB permeability when injected into mice genetically deficient in the NF-B family member p50. Furthermore, we report that inhibition of TWEAK activity during cerebral ischemia with an Fn14-Fc decoy receptor results in significant preservation of the integrity of the NVU with attenuation of cerebral ischemia-induced increase in the permeability of the BBB. We conclude that the cytokine TWEAK plays a role in the disruption of the structure and permeability of the NVU during physiological and pathological conditions.

Objectives We recently reported that lowering of macrophage free intracellular iron increases expression of cholesterol efflux transporters ABCA1 and ABCG1 by reducing generation of reactive oxygen species. In this study, we explore whether reducing macrophage intracellular iron levels via pharmacologic suppression of hepcidin can increase macrophage-specific expression of cholesterol efflux transporters and reduce atherosclerosis. Methods and Results To suppress hepcidin, increase expression of the iron exporter ferroportin (FPN), and reduce macrophage intracellular iron, we used a small molecule inhibitor of BMP signaling, LDN 193189 (LDN). LDN (10 mg/kg i.p. bid) was administered to mice and its effects on atherosclerosis, intracellular iron, oxidative stress, lipid efflux, and foam cell formation were measured in plaques and peritoneal macrophages. Long-term LDN administration to Apo E (-/-) mice increased ABCA1 immunoreactivity within intraplaque macrophages by 3.7-fold (n=8; p=0.03), reduced oil-red-o positive lipid area by 50% (n=8; p=0.02) and decreased total plaque area by 43% (n=8; p=0.001). LDN suppressed liver hepcidin transcription and increased macrophage FPN, lowering intracellular iron and hydrogen peroxide production. LDN treatment increased macrophage ABCA1 and ABCG1 expression, significantly raised cholesterol efflux to ApoA-1 and decreased foam cell formation. All preceding LDN-induced effects on cholesterol efflux were reversed by exogenous hepcidin administration, suggesting that modulation of intracellular iron levels within macrophages as the mechanism by which LDN triggers these effects. Conclusion These data suggest that pharmacologic manipulation of iron homeostasis may be a promising target to increase macrophage reverse cholesterol transport and limit atherosclerosis.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor superfamily. TWEAK acts via binding to a cell surface receptor named Fn14. To study the role of this cytokine in the regulation of the permeability of the neurovascular unit (NVU) during cerebral ischemia, TWEAK activity was inhibited in wild-type mice with a soluble Fn14-Fc decoy receptor administered either immediately or 1 h after middle cerebral artery occlusion (MCAO). Administration of Fn14-Fc decoy resulted in faster recovery of motor function and a 66.4%610% decrease in Evans blue dye extravasation when treatment was administered immediately after MCAO and a 46.1%613.1% decrease when animals were treated 1 h later (n = 4, P < 0.05). Genetic deficiency of Fn14 resulted in a 60%612.8% decrease in the volume of the ischemic lesion (n = 6, P < 0.05), and a 87%622% inhibition in Evans blue dye extravasation 48 h after the onset of the ischemic insult (n = 6, P < 0.005). Compared with control animals, treatment with Fn14-Fc decoy or genetic deficiency of Fn14 also resulted in a significant inhibition of nuclear factor-jB pathway activation, matrix metalloproteinase-9 activation and basement membrane laminin degradation after MCAO. These findings show that the cytokine TWEAK plays a role in the disruption of the structure of the NVU during cerebral ischemia and that TWEAK antagonism is a potential therapeutic strategy for acute cerebral ischemia.