RATIONALE:The function of Nox4, a source of vascular H2O2, is unknown. Other Nox proteins were identified as mediators of endothelial dysfunction.
OBJECTIVE:We determined the function of Nox4 in ...situations of increased stress induced by ischemia or angiotensin II with global and tamoxifen-inducible Nox4 mice.
METHODS AND RESULTS:Nox4 was highly expressed in the endothelium and contributed to H2O2 formation. Nox4 mice exhibited attenuated angiogenesis (femoral artery ligation) and PEG-catalase treatment in control mice had a similar effect. Tube formation in cultured Nox4 lung endothelial cells (LECs) was attenuated and restored by low concentrations of H2O2, whereas PEG-catalase attenuated tube formation in control LECs. Angiotensin II infusion was used as a model of oxidative stress. Compared to wild-type, aortas from inducible Nox4-deficient animals had development of increased inflammation, media hypertrophy, and endothelial dysfunction. Mechanistically, loss of Nox4 resulted in reduction of endothelial nitric oxide synthase expression, nitric oxide production, and heme oxygenase-1 (HO-1) expression, which was associated with apoptosis and inflammatory activation. HO-1 expression is controlled by Nrf-2. Accordingly, Nox4-deficient LECs exhibited reduced Nrf-2 protein level and deletion of Nox4 reduced Nrf-2 reporter gene activity. In vivo treatment with hemin, an inducer of HO-1, blocked the vascular hypertrophy induced by Nox4 deletion in the angiotensin II infusion model and carbon monoxide, the product of HO-1, blocked the Nox4-deletion-induced apoptosis in LECs.
CONCLUSION:Endogenous Nox4 protects the vasculature during ischemic or inflammatory stress. Different from Nox1 and Nox2, this particular NADPH oxidase therefore may have a protective vascular function.
Formation of abdominal aortic aneurysms is a progressive inflammatory process that involves infiltration and differentiation of monocytes in the vessel wall, proliferation and migration of smooth ...muscle cells, and eventually the degradation of the internal elastic lamina, which leads to outward vascular remodeling and distension of the vessel. Because calcium channel blockers exert multiple beneficial effects on the vascular system, we investigated the effect of the benzothiazepine-type calcium channel blocker diltiazem on aneurysm formation in a mouse model. Angiotensin II infusion induced massive suprarenal aortic aneurysm formation in male apolipoprotein E-deficient mice that was blocked by cotreatment with diltiazem even if the blood pressure was controlled by coinfusion of phenylephrine. Diltiazem prevented the angiotensin II-mediated induction of proinflammatory cytokines after 7 days of angiotensin II treatment in the aortic arch attributable to a reduction in the amount of locally infiltrating macrophages. To identify the underlying mechanism, vascular segments and cultured vascular cells as well as monocytes were studied. Diltiazem failed to reduce the angiotensin II-induced expression of proinflammatory chemokines and cytokines in isolated mouse thoracic aortic segments in organ culture. Furthermore, diltiazem did not affect the recruitment of proinflammatory Ly6C(+) monocytes in vivo pointing toward an effect of the compound on gene expression in monocytes/macrophages. Indeed, diltiazem prevented the interleukin-6-induced mRNA expression of interleukin-1β and the monocyte chemoattractant protein CCL12 in peritoneal macrophages and RAW264.7 cells independent of the intracellular calcium concentration. Thus, diltiazem limits aortic aneurysm formation in mice by a blood pressure-independent anti-inflammatory effect on monocytic cells.
OBJECTIVE—Epoxyeicosatrienoic acids (EETs) have antiinflammatory effects and are required for normal endothelial function. The soluble epoxide hydrolase (sEH) metabolizes EETs to their less active ...diols. We hypothesized that knockout and inhibition of sEH prevents neointima formation in hyperlipidemic ApoE mice.
METHODS AND RESULTS—Inhibition of sEH by 12-(3-adamantan-1-yl-ureido) dodecanoic acid or knockout of the enzyme significantly increased plasma EET levels. sEH activity was detectable in femoral and carotid arteries. sEH knockout or inhibition resulted in a significant reduction of neointima formation in the femoral artery cuff model but not following carotid artery ligation. Although macrophage infiltration occurred abundantly at the site of cuff placement in both sEH and sEH, the expression of proinflammatory genes was significantly reduced in femoral arteries from sEH mice. Moreover, an in vivo 5-bromo-2′-deoxyuridine assay revealed that smooth muscle cell proliferation at the site of cuff placement was attenuated in sEH knockout and sEH inhibitor–treated animals.
CONCLUSION—These observations suggest that inhibition of sEH prevents vascular remodeling in an inflammatory model but not in a blood flow–dependent model of neointima formation.
Epoxyeicotrienoic acids (EETs) are cytochrome P450-dependent anti-hypertensive and anti-inflammatory derivatives of arachidonic acid, which are highly abundant in the kidney and considered ...reno-protective. EETs are degraded by the enzyme soluble epoxide hydrolase (sEH) and sEH inhibitors are considered treatment for chronic renal failure (CRF). We determined whether sEH inhibition attenuates the progression of CRF in the 5/6-nephrectomy model (5/6-Nx) in mice. 5/6-Nx mice were treated with a placebo, an ACE-inhibitor (Ramipril, 40 mg/kg), the sEH-inhibitor cAUCB or the CYP-inhibitor fenbendazole for 8 weeks. 5/6-Nx induced hypertension, albuminuria, glomerulosclerosis and tubulo-interstitial damage and these effects were attenuated by Ramipril. In contrast, cAUCB failed to lower the blood pressure and albuminuria was more severe as compared to placebo. Plasma EET-levels were doubled in 5/6 Nx-mice as compared to sham mice receiving placebo. Renal sEH expression was attenuated in 5/6-Nx mice but cAUCB in these animals still further increased the EET-level. cAUCB also increased 5-HETE and 15-HETE, which derive from peroxidation or lipoxygenases. Similar to cAUCB, CYP450 inhibition increased HETEs and promoted albuminuria. Thus, sEH-inhibition failed to elicit protective effects in the 5/6-Nx model and showed a tendency to aggravate the disease. These effects might be consequence of a shift of arachidonic acid metabolism into the lipoxygenase pathway.
•Migration testing of food packaging materials.•GC-MS analysis of caprolactam (regulated additive) and 2,4-di-tert-butylphenol (NIAS).•Method application to real food packaging material.•Salinity ...level of food simulants may influence migration of analytes from packaging.
Caprolactam and 2,4-di-tert-butyl phenol (2,4-DTBP) are substances typically found in some food contact materials (FCMs). They are known to often migrate into food, and are difficult to analyse in liquid food simulants using GC. In this work a simple salting-out Liquid-Liquid Extraction (SALLE) for the analysis of both substances in water and the official food simulant A (10 % v/v ethanol, Regulation (EU) No. 10/2011) is presented. The method, which included analytical determination by GC-MS, was optimized and validated to ensure sufficient analytical quality.
The method’s LOQs allowed the proper quantification of caprolactam at its EU legislative limit (15 mg kg−1). For 2,4-DTBP the method also revealed good sensitivity, although no official limits have been established yet. Linear regression coefficients (R2) were in all cases higher than 0.999, and recoveries ranged from 87 % and 95% for caprolactam and 2,4-DTBP, respectively. Precision was also acceptable, with the RSDs (%) below 12 %. The method proved to be adequate to be used for routine analysis.
The presence of salt during migration of caprolactam and 2,4-DTBP was also investigated in this work. Polyamide/polyethylene FCM multilayer films have been tested with water and simulant A, containing different amounts of NaCl (up to 15 % m/v), and applying different migration conditions (temperature and time). The results indicated that salinity plays an important effect on the migration of caprolactam, with the presence of salt reducing its migration in case of water and increasing it in case of simulant A. These preliminary results seem to indicate that migration testing should consider not only the well-known fatty content of a food, but also its salinity content, as it may end up affecting drastically the migration of polar substances.
Protein disulfide isomerase (PDI) and its homologs are oxidoreductases facilitating protein folding in the ER. Endo-PDI (also termed ERp46) is highly expressed in endothelial cells. It belongs to the ...PDI family but its physiological function is largely unknown. We studied the role of Endo-PDI in endothelial angiogenic responses. Stimulation of human umbilical vein endothelial cells (with TNFα (10ng/ml) increased ERK1/2 phosphorylation. This effect was largely attenuated by Endo-PDI siRNA, whereas JNK and p38 MAP kinase phosphorylation was Endo-PDI independent. Similarly, TNFα-stimulated NF-κB signaling determined by IκBα degradation as well as TNFα-induced ICAM expression was unaffected by Endo-PDI siRNA. The action of Endo-PDI was not mediated by extracellular thiol exchange or cell surface PDI as demonstrated by nonpermeative inhibitors and PDI-neutralizing antibody. Moreover, exogenously added PDI failed to restore ERK1/2 activation after Endo-PDI knockdown. This suggests that Endo-PDI acts intracellularly potentially by maintaining the Ras/Raf/MEK/ERK pathway. Indeed, knockdown of Endo-PDI attenuated Ras activation measured by G-LISA and Raf phosphorylation. ERK activation influences gene expression by the transcriptional factor AP-1, which controls MMP-9 and cathepsin B, two proteases required for angiogenesis. TNFα-stimulated MMP-9 and cathepsin B induction was reduced by silencing of Endo-PDI. Accordingly, inhibition of cathepsin B or Endo-PDI siRNA blocked the TNFα-stimulated angiogenic response in the spheroid outgrowth assays. Moreover ex vivo tube formation and in vivo Matrigel angiogenesis in response to TNFα were attenuated by Endo-PDI siRNA. In conclusion, our study establishes Endo-PDI as a novel, important mediator of AP-1-driven gene expression and endothelial angiogenic function.
Different silicone baking moulds (37 samples) were characterized with respect to potential migrating substances using
1
H-NMR, RP-HPLC-UV/ELSD and GC techniques. In all cases cyclic organosiloxane ...oligomers with the formula Si(CH
3
)
2
-O
n
were identified (n = 6 ... 50). Additionally, linear, partly hydroxyl-terminated organosiloxanes HO-Si(CH
3
)
2
-O
n
-H (n = 7 ... 20) were found in 13 samples. No substances other than siloxanes could be detected, meaning the migrants mainly consist of organopolysiloxanes. Based on this knowledge, a
1
H-NMR quantification method for siloxanes was established for the analysis of both simulants and foodstuffs. Validation of the
1
H-NMR method gave suitable performance characteristics: limit of detection 8.7 mg kg
-1
oil, coefficient of variation 7.8% (at a level of 1.0 mg kg
-1
food). Migration studies were carried out with simulants (olive oil, isooctane, ethanol (95%), Tenax) as well as preparation of different cakes. From the 1st to 10th experiment, siloxane migration into cakes only slightly decreased, with a significant dependence on fat content. Migration never exceeded a level of 21 mg kg
-1
(3 mg dm
-2
) and was, therefore, well below the overall migration limit of 60 mg kg
-1
(10 mg dm
-2
). However, migration behaviour into simulants differed completely from these results.
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