The pantetheinase vanin-1 generates cysteamine, which inhibits reduced glutathione (GSH) synthesis. Vanin-1 promotes inflammation and tissue injury partly by inducing oxidative stress, and partly by peroxisome proliferator-activated receptor gamma (PPARγ) expression. Vascular smooth muscle cells (SMCs) contribute to neointimal hyperplasia in response to injury, by multiple mechanisms including modulation of oxidative stress and PPARγ. Therefore, we tested the hypothesis that vanin-1 drives SMC activation and neointimal hyperplasia. We studied reactive oxygen species (ROS) generation and functional responses to platelet-derived growth factor (PDGF) and the pro-oxidant diamide in cultured mouse aortic SMCs, and also assessed neointima formation after carotid artery ligation in vanin-1 deficiency. Vnn1 −/− SMCs demonstrated decreased oxidative stress, proliferation, migration, and matrix metalloproteinase 9 (MMP-9) activity in response to PDGF and/or diamide, with the effects on proliferation linked, in these studies, to both increased GSH levels and PPARγ expression. Vnn1−/− mice displayed markedly decreased neointima formation in response to carotid artery ligation, including decreased intima:media ratio and cross-sectional area of the neointima. We conclude that vanin-1, via dual modulation of GSH and PPARγ, critically regulates the activation of cultured SMCs and development of neointimal hyperplasia in response to carotid artery ligation. Vanin-1 is a novel potential therapeutic target for neointimal hyperplasia following revascularization.
Objective Intima formation involves smooth muscle cell (SMC) proliferation and migration that ultimately drives arterial stenosis, thrombosis, and ischemia in atherosclerosis, hypertension, and arterial revascularization. Receptor for advanced glycation endproducts (RAGE) is a transmembrane signaling receptor implicated in diabetic renal and vascular complications, and post-injury intima formation, partly via Signal transducer and activator of transcription 3 (STAT3) activation. The metabolic super-regulator Adenosine monophosphate kinase (AMPK) inhibits SMC proliferation and intima formation. AMPK activation is promoted by liver kinase B1 (LKB1), and LKB1 inhibits STAT3 activation. Here, we tested the hypothesis that RAGE promotes arterial intima formation by modulating both LKB1 and AMPK. Methods and Results RAGE ligands (the calgranulin S100A11, and glycated albumin) suppressed AMPK activation in conjunction with increased proliferation and migration of cultured SMCs. These effects were inhibited both by RAGE deficiency and by prior AMPK activation. In SMCs, RAGE ligands decreased LKB1 activity. Moreover, knockdown of both LKB1 and AMPK were associated with increased STAT3 phosphorylation levels. In response to murine carotid artery ligation, expression of RAGE and S100A11 increased, whereas AMPK and LKB1 activity decreased in situ. Conversely, LKB1 and AMPK activity increased in situ, and neointima formation was attenuated in Rage−/− mice. Conclusion The linkage of decreased LKB1 and AMPK activity with increased STAT3 in SMCs mediates the capacity of RAGE ligand-induced signaling to promote neointima formation in response to arterial injury.
Disk diffusion susceptibility tests for enterococci are frequently modified by adding 5% sheep blood (SB) to Mueller-Hinton agar; the performance standards from the National Committee for Clinical Laboratory Standards sanction this addition. Susceptibility testing of aminoglycoside antibiotics is not recommended for enterococci; in actual practice, however, some laboratories do include aminoglycoside antibiotics routinely, and others may test upon request or in selected situations. In examining 50 clinical isolates of enterococci, SB-enriched Mueller-Hinton agar frequently gave enlarged zone sizes that falsely indicated susceptibility (72% for gentamicin and tobramycin), with the average increase in zone size being 6.3 and 7.6 mm, respectively. Comparison agar dilution MICs demonstrated uniform resistance, with or without added SB. The effect was shown to be caused by heme in concentrations as low as 0.03 ,ug/ml, which, when combined with aminoglycoside antibiotics, caused a synergistic growth inhibition of the enterococci, resulting in larger aminoglycoside antibiotic zones. We postulate that the heme effect is related to a catalytic cleavage of intracellular H202 and resultant lipid peroxidation. No other organism or antimicrobial agent tested demonstrated a similar effect, although other investigators have shown a similar phenomenon with the broad-spectrum cephalosporins. Because enterococci grow well and give accurate susceptibility results on Mueller-Hinton agar without SB supplementation and because of the spectrum of definable problems with a number of antimicrobial agents, we recommend that enterococci routinely be tested without SB.
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