Febuxostat, a Xanthine Oxidase Inhibitor, Decreased Macrophage Matrix Metalloproteinase Expression in Hypoxia.

Macrophages in the atheroma region produce matrix metalloproteinases (MMPs) and decrease plaque stability. Tissue oxygen tension decreases in the arterial wall of the atherosclerotic region. Hypoxia inducible factor (HIF)-1α plays a critical role in the transcriptional activation of hypoxia inducible genes. However, the precise roles of HIF-1α independent pathways in hypoxic responses are largely unknown. Xanthine oxidase (XO) is an enzyme that utilizes molecular oxygen and produces reactive oxygen species (ROS). Here, we show that ROS derived from XO increases MMP-3, -10, and -13 expression in murine macrophages. We found that the transcript levels of macrophage MMP-3, -10, and -13 were increased in hypoxic conditions. Hypoxia induced MMP expression in HIF-1α deficient macrophages. N-acetylcysteine (NAC) or febuxostat, an XO inhibitor, suppressed MMP expression in murine macrophages. Febuxostat decreased the incidence of plaque rupture in apolipoprotein-E-deficient mice. Our results indicate that febuxostat stabilized atherosclerotic plaque via suppressing the activities of macrophage MMP-9 and -13. Febuxostat administration is a potential therapeutic option in the management of atherosclerotic patients.

Macrophage-derived angiopoietin-like protein 2 accelerates development of abdominal aortic aneurysm.

OBJECTIVE: Recently, we reported that angiopoietin-like protein 2 (Angptl2) functions in various chronic inflammatory diseases. In the present study, we asked whether Angptl2 and its associated chronic inflammation contribute to abdominal aortic aneurysm (AAA). METHODS AND RESULTS: Immunohistochemistry revealed that Angptl2 is abundantly expressed in infiltrating macrophages within the vessel wall of patients with AAA and in a CaCl(2)-induced AAA mouse model. When Angptl2-deficient mice were used in the mouse model, they showed decreased AAA development compared with wild-type mice, as evidenced by reduction in aneurysmal size, less severe destruction of vessel structure, and lower expression of proinflammatory cytokines and matrix metalloproteinase-9. However, no difference in the number of infiltrating macrophages within the aortic aneurysmal vessel wall was observed between genotypes. AAA development was also significantly suppressed in wild-type mice that underwent Angptl2-deficient bone marrow transplantation. Expression levels of proinflammatory cytokines and metalloproteinase-9 in Angptl2-deficient macrophages were significantly decreased, and those decreases were rescued by treatment of Angptl2 deficient macrophages with exogenous Angptl2. CONCLUSIONS: Macrophage-derived Angptl2 contributes to AAA development by inducing inflammation and degradation of extracellular matrix in the vessel wall, suggesting that targeting the Angptl2-induced inflammatory axis in macrophages could represent a new strategy for AAA therapy.

Mitochondrial dysfunction and increased reactive oxygen species impair insulin secretion in sphingomyelin synthase 1-null mice.

Sphingomyelin synthase 1 (SMS1) catalyzes the conversion of ceramide to sphingomyelin. Here, we generated and analyzed SMS1-null mice. SMS1-null mice exhibited moderate neonatal lethality, reduced body weight, and loss of fat tissues mass, suggesting that they might have metabolic abnormality. Indeed, analysis on glucose metabolism revealed that they showed severe deficiencies in insulin secretion. Isolated mutant islets exhibited severely impaired ability to release insulin, dependent on glucose stimuli. Further analysis indicated that mitochondria in mutant islet cells cannot up-regulate ATP production in response to glucose. We also observed additional mitochondrial abnormalities, such as hyperpolarized membrane potential and increased levels of reactive oxygen species (ROS) in mutant islets. Finally, when SMS1-null mice were treated with the anti-oxidant N-acetyl cysteine, we observed partial recovery of insulin secretion, indicating that ROS overproduction underlies pancreatic ß-cell dysfunction in SMS1-null mice. Altogether, our data suggest that SMS1 is important for controlling ROS generation, and that SMS1 is required for normal mitochondrial function and insulin secretion in pancreatic ß-cells.

The endoplasmic reticulum stress-C/EBP homologous protein pathway-mediated apoptosis in macrophages contributes to the instability of atherosclerotic plaques.

OBJECTIVE: To elucidate whether and how the endoplasmic reticulum (ER) stress-C/EBP homologous protein (CHOP) pathway in macrophages is involved in the rupture of atherosclerotic plaques. METHODS AND RESULTS: Increases in macrophage-derived foam cell death in coronary atherosclerotic plaques cause the plaque to become vulnerable, thus resulting in acute coronary syndrome. The ER stress-CHOP/growth arrest and DNA damage-inducible gene-153 (GADD153) pathway is induced in the macrophage-derived cells in atherosclerotic lesions and is involved in plaque formation. However, the role of CHOP in the final stage of atherosclerosis has not been fully elucidated. Many CHOP-expressing macrophages showed apoptosis in advanced ruptured atherosclerotic lesions in wild-type mice, whereas few apoptotic cells were observed in Chop(-/-) mice. The rupture of atherosclerotic plaques was significantly reduced in high cholesterol-fed Chop(-/-)/Apoe(-/-) mice compared with Chop(+/+)/Apoe(-/-) mice. Furthermore, using mice that underwent bone marrow transplantation, we showed that expression of CHOP in macrophages significantly contributes to the formation of ruptures. By using primary cultured macrophages, we further showed that unesterified free cholesterol derived from incorporated denatured low-density lipoprotein was accumulated in the ER and induced ER stress-mediated apoptosis in a CHOP-Bcl2-associated X protein (Bax) pathway-dependent manner. CONCLUSIONS: The ER stress-CHOP-Bax-mediated apoptosis in macrophages contributes to the instability of atherosclerotic plaques.

Synoviocyte-derived angiopoietin-like protein 2 contributes to synovial chronic inflammation in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by symmetrical polyarticular synovitis of the diarthrodial joints. Several proinflammatory cytokines derived from both infiltrating inflammatory cells and activated resident cells within the RA joint play a fundamental role in the processes that cause inflammation. However, anticytokine treatment is beneficial but not curative, the effects are only partial, and nonresponses are common. Therefore, an effort has been made to identify other key regulators of inflammation in articular structures to develop new therapies to suppress synovial inflammation and joint destruction in RA. Adipose tissue-derived angiopoietin-like protein 2 (Angptl2) activates an inflammatory cascade in endothelial cells and induces chemotaxis of monocytes/macrophages in obesity, resulting in initiation and propagation of inflammation within adipose tissues and obesity-related metabolic diseases. Angptl2 mRNA and protein are abundantly expressed in hyperplastic rheumatoid synovium of RA patients, especially in fibroblast-like and macrophage-like synoviocytes, but not in B and T lymphocytes. Angptl2 concentration in joints of RA patients was also significantly increased in comparison with patients with osteoarthritis, which in comparison with RA represents a significantly lower inflammatory grade form of arthritis. Notably, Angptl2 promoted increased chemotactic activities of CD14+CD16- monocytes from synovial fluid of RA patients. Therefore, Angptl2 acts as an important rheumatoid synovium-derived inflammatory mediator in RA pathogenesis.

Molecular mechanisms of the LPS-induced non-apoptotic ER stress-CHOP pathway.

The expression of C/EBP homologous protein (CHOP), which is an endoplasmic reticulum (ER) stress-induced transcription factor, induces apoptosis. Our previous study demonstrated that lipopolysaccharide (LPS)-induced CHOP expression does not induce apoptosis, but activates a pro-IL-1beta activation process. However, the mechanism by which CHOP activates different pathways, depending on the difference in the inducing stimuli, remains to be clarified. The present study shows that LPS rapidly activates the ER function-protective pathway, but not the PERK pathway in macrophages. PERK plays a major role in CHOP induction, and other ER stress sensors-mediated pathways play minor roles. The induction of CHOP by LPS was delayed and weak, in comparison with CHOP induction by ER stress-inducer thapsigargin. In addition, LPS-pre-treatment or overexpression of ER chaperone, IgH chain binding protein (BiP), prevented ER stress-mediated apoptosis. LPS plus IFN-gamma-treated macrophages produce a larger amount of nitric oxide (NO) in comparison with LPS-treated cells. Treatment with the NO donor, SNAP (S-nitro-N-acetyl-dl-penicillamine), induces CHOP at an earlier period than LPS treatment. The depletion of NO retards CHOP induction and prevents apoptosis in LPS plus IFN-gamma-treated cells. We concluded that apoptosis is prevented in LPS-treated macrophages, because the ER function-protective mechanisms are induced before CHOP expression, and induction level of CHOP is low.

Angiopoietin-like protein 2 promotes chronic adipose tissue inflammation and obesity-related systemic insulin resistance.

Recent studies of obesity have provided new insights into the mechanisms underlying insulin resistance and metabolic dysregulation. Numerous efforts have been made to identify key regulators of obesity-linked adipose tissue inflammation and insulin resistance. We found that angiopoietin-like protein 2 (Angptl2) was secreted by adipose tissue and that its circulating level was closely related to adiposity, systemic insulin resistance, and inflammation in both mice and humans. Angptl2 activated an inflammatory cascade in endothelial cells via integrin signaling and induced chemotaxis of monocytes/macrophages. Constitutive Angptl2 activation in vivo induced inflammation of the vasculature characterized by abundant attachment of leukocytes to the vessel walls and increased permeability. Angptl2 deletion ameliorated adipose tissue inflammation and systemic insulin resistance in diet-induced obese mice. Conversely, Angptl2 overexpression in adipose tissue caused local inflammation and systemic insulin resistance in nonobese mice. Thus, Angptl2 is a key adipocyte-derived inflammatory mediator that links obesity to systemic insulin resistance.