Mycoplasmas and their membranes are potent activators of macrophages, the active principle being lipoproteins and lipopeptides. Two stereoisomers of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 (MALP-2) differing in the configuration of the lipid moiety were synthesized and compared in their macrophage-activating potential, the R-MALP being >100 times more active than the S-MALP in stimulating the release of cytokines, chemokines, and NO. To assess the role of the Toll-like receptor (TLR) family in mycoplasmal lipopeptide signaling, the MALP-2-mediated responses were analyzed using macrophages from wild-type, TLR2-, TLR4-, and MyD88-deficient mice. TLR2- and MyD88-deficient cells showed severely impaired cytokine productions in response to R- and S-MALP. The MALP-induced activation of intracellular signaling molecules was fully dependent on both TLR2 and MyD88. There was a strong preference for the R-MALP in the recognition by its functional receptor, TLR2.
Mechanical stretch is a hallmark of arterial hypertension and leads to vessel wall remodeling, which involves matrix metalloproteinases (MMPs). Because mechanical stretch is further capable of inducing reactive oxygen species (ROS) formation via the NAD(P)H oxidase, we assessed whether mechanical stretch enhances MMP expression and activity in a NAD(P)H oxidase-dependent manner. Therefore, vascular smooth muscle cells (VSMCs) isolated from C57BL/6 mice were exposed to cyclic mechanical stretch. The impact of ROS was assessed using VSMCs isolated from p47phox-/- mice, deficient for a NAD(P)H oxidase subunit responsible for ROS formation. Transcript levels were investigated by cDNA array and confirmed by RT-PCR. ROS formation was determined by DCF fluoroscopy and MMP-2 activity by zymography. Mechanical stretch of wild-type VSMCs resulted in a rapid ROS formation and p47phox membrane translocation that is followed by an increase in Nox-1 transcripts. ROS formation was completely abrogated in p47phox-/- VSMCs. cDNA array further revealed an increase of MMP-2 mRNA in response to mechanical stretch, which was validated by RT-PCR. Using p47phox-/- VSMCs, this increase in MMP-2 mRNA was completely blunted. mRNA expression of tissue inhibitor of MMP-2 TIMP-1 and TIMP-2 and membrane-type 1 MMP was unaffected by mechanical stretch. Gelatinolytic activity of pro-MMP-2 has been increased rapidly in wild-type VSMCs and was completely abolished in p47phox-/- VSMCs. These results indicate that mechanical stretch induces ROS formation via the NAD(P)H oxidase and thereby enhances MMP-2 mRNA expression and pro-MMP-2 release. These results are consistent with the notion that in arterial hypertension, reactive oxygen species are involved in vascular remodeling via MMP activation. The full text of this article is available online at http://www.circresaha.org.
Background— Vascular lipid accumulation and inflammation are hallmarks of atherosclerosis and perpetuate atherosclerotic plaque development. Mediators of inflammation, ie, interleukin (IL)-6, are elevated in patients with acute coronary syndromes and may contribute to the exacerbation of atherosclerosis. Methods and Results— To assess the role of IL-6 in atherosclerosis, ApoE −/− – IL-6 −/− double-knockout mice were generated, fed a normal chow diet, and housed for 53±4 weeks. Mortality and blood pressure were unaltered. However, serum cholesterol levels and subsequent atherosclerotic lesion formation (oil red O stain) were significantly increased in ApoE −/− – IL-6 −/− mice compared with ApoE −/− , wild-type (WT), and IL-6 −/− mice. Plaques of ApoE −/− – IL-6 −/− mice showed significantly reduced transcript and protein levels of matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, collagen I and V, and lysyl oxidase (by reverse transcriptase-polymerase chain reaction and immunohistochemistry). Recruitment of macrophages and leukocytes (Mac3- and CD45-positive staining) into the atherosclerotic lesion was significantly reduced in ApoE −/− – IL-6 −/− mice. The transcript and serum protein (ELISA) levels of IL-10 were significantly reduced. Conclusions— Thus, a lifetime IL-6 deficiency enhances atherosclerotic plaque formation in ApoE K −/− – IL-6 −/− mice and leads to maladaptive vascular developmental processes. These observations are consistent with the notion that baseline levels of IL-6 are required to modulate lipid homeostasis, vascular remodeling, and plaque inflammation in atherosclerosis.
SummaryThe plant plasma membrane intrinsic protein, PIP1b, facilitates water transport. These features were characterized in Xenopus oocytes and it has asked whether aquaporins are relevant for water transport in plants. In order to elucidate this uncertainty Arabidopsis thaliana was transformed with an anti-sense construct targeted to the PIP1b gene. Molecular analysis revealed that the anti-sense lines have reduced steady-state levels of PIP1b and the highly homologous PIP1a mRNA. The cell membrane water permeability was analyzed by swelling of protoplasts, which had been transferred into hypotonic conditions. The results indicate that the reduced expression of the specific aquaporins decreases the cellular osmotic water permeability coefficient approximately three times. The morphology and development of the anti-sense lines resembles that of control plants, with the exception of the root system, which is five times as abundant as that of control plants. Xylem pressure measurement suggests that the increase of root mass compensates the reduced cellular water permeability in order to ensure a sufficient water supply to the plant. The results obtained by this study, therefore, clearly demonstrate that aquaporins are important for plant water transport.
SummaryThe importance of inflammation as a driver of pathology is no longer confined to autoimmune and infectious diseases. In line with convincing experimental data as well as abundant clinical findings the current view of atherosclerosis points to inflammation as a critical regulator of atherosclerotic plaque formation and progression leading to the fatal clinical endpoints myocardial infarction, stroke or sudden cardiac death. The underlying mechanisms have been a matter of intense research during the last decades. In this regard, the interleukin-6 (IL-6) cytokines and their signalling events have been shown to contribute to both, atherosclerotic plaque development and plaque destabilisation via a variety of mechanisms. These involve the release of other pro-inflammatory cytokines, oxidation of lipoproKeywords Cytokines, atherosclerosis, inflammation, signal transduction teins by phospholipases, stimulation of acute phase protein secretion, the release of prothrombotic mediators, and the activation of matrix metalloproteinases. Moreover, the formation of reactive oxygen species generated by vascular enzyme systems may play a critical role in the regulation of IL-6 indicating a cross talk between vasoactive substances i.e. angiotensin II or adrenalin and pro-inflammatory cytokines such as IL-6. In this review we will summarise and discuss the underlying molecular and cellular mechanisms how IL-6 as an early and central regulator of inflammation contributes to atherosclerosis and how this knowledge can be integrated into the clinical context.
Accumulating evidence suggests a critical role of increased reactive oxygen species production for left ventricular (LV) remodeling and dysfunction after myocardial infarction (MI). An increased myocardial activity of the NAD(P)H oxidase, a major oxidant enzyme system, has been observed in human heart failure; however, the role of the NAD(P)H oxidase for LV remodeling and dysfunction after MI remains to be determined. MI was induced in wild-type (WT) mice (n=46) and mice lacking the cytosolic NAD(P)H oxidase component p47(phox) (p47(phox)-/- mice) (n=32). Infarct size was similar among the groups. NAD(P)H oxidase activity was markedly increased in remote LV myocardium of WT mice after MI as compared with sham-operated mice (83+/-8 versus 16.7+/-3.5 nmol of O(2)(-) x microg(-1) x min(-1); P<0.01) but not in p47(phox)-/- mice after MI (13.5+/-3.6 versus 15.5+/-3.5 nmol of O(2)(-) x microg(-1) x min(-1)), as assessed by electron-spin resonance spectroscopy using the spin probe CP-H. Furthermore, increased myocardial xanthine oxidase activity was observed in WT, but not in p47(phox)-/- mice after MI, suggesting NAD(P)H oxidase-dependent xanthine oxidase activation. Myocardial reactive oxygen species production was increased in WT mice, but not in p47(phox)-/- mice, after MI. LV cavity dilatation and dysfunction 4 weeks after MI were markedly attenuated in p47(phox)-/- mice as compared with WT mice, as assessed by echocardiography (LV end-diastolic diameter: 4.5+/-0.2 versus 6.3+/-0.3 mm, P<0.01; LV ejection fraction, 35.8+/-2.5 versus 22.6+/-4.4%, P<0.05). Furthermore, cardiomyocyte hypertrophy, apoptosis, and interstitial fibrosis were substantially reduced in p47(phox)-/- mice as compared with WT mice. Importantly, the survival rate was markedly higher in p47(phox)-/- mice as compared with WT mice after MI (72% versus 48%; P<0.05). These results suggest a pivotal role of NAD(P)H oxidase activation and its subunit p47(phox) for LV remodeling/dysfunction and survival after MI. The NAD(P)H oxidase system represents therefore a potential novel therapeutic target to prevent cardiac failure after MI.
These data clarify, for the first time, the critical involvement of, in particular, the transsignaling of IL-6 in CAD and warrant further investigation of sgp130Fc as a novel therapeutic for the treatment of CAD and related diseases.
Summary A new aquaporin from Nicotiana tabacum (cv. Samsun) was characterized. It shares sequence homology to the Arabidopsis thaliana PIP1 protein family. By two‐phase partitioning and immunoblot analysis, plasma membrane localization could be demonstrated. The corresponding mRNA is highly abundant in roots and flowers, while it is rarely expressed in leaves and stems. Functional expression in Xenopus oocytes revealed that NtAQP1 can mediate glycerol transport in addition to water flow. However, NtAQP1 is impermeable for Na + , K + and Cl – ions. The water permeability and selectivity could not be modulated by addition of mercurials or the activity of cAMP‐dependent protein kinases.
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