Objectives-Basic fibroblast growth factor (bFGF) stimulates vascular smooth muscle cell (SMC) migration. We determined whether bFGF increases SMC reactive oxygen-species (ROS) and studied the role of ROS for SMC migration. Methods and Results-bFGF rapidly increased rat SMC ROS formation and migration through pathways sensitive to inhibition of NADPH oxidases, PI3-kinase, protein kinase C, and Rac-1. SiRNA directed against the NADPH oxidase Nox4 impaired basal but not bFGF-induced ROS formation and did not affect migration. In contrast, siRNA against Nox1 blocked the agonist-induced ROS generation as well as the bFGF-induced migration. Agonist-induced migration was also attenuated in SMC derived from Nox1 y/Ϫ mice and transduction of Nox1 restored normal migration. Likewise, SMC outgrowth in response to bFGF was attenuated in aortic segments from Nox1 y/Ϫ mice as compared with Nox1 y/ϩ mice. bFGF activated JNK but not Src in a Nox1-dependent manner. Consequently, phosphorylation of the adaptor protein paxillin, which is central for migration and secretion of matrix-metalloproteinases, were dependent on Nox1 as well as JNK but not Src. Conclusions-These

Aglaia (family Meliaceae) plants are used in traditional medicine (e.g., in Vietnam) for the treatment of inflammatory skin diseases and allergic inflammatory disorders such as asthma. Inflammatory diseases arise from inappropriate activation of the immune system, leading to abnormal expression of genes encoding inflammatory cytokines and tissue-destructive enzymes. The active compounds isolated from these plants are derivatives of rocaglamide. In this study we show that rocaglamides are potent immunosuppressive phytochemicals that suppress IFN-γ, TNF-α, IL-2, and IL-4 production in peripheral blood T cells at nanomolar concentrations. We demonstrate that rocaglamides inhibit cytokine gene expression at the transcriptional level. At the doses that inhibit cytokine production, they selectively block NF-AT activity without impairing NF-κB and AP-1. We also show that inhibition of NF-AT activation by rocaglamide is mediated by strong activation of JNK and p38 kinases. Our study suggests that rocaglamide derivatives may serve as a new source of NF-AT-specific inhibitors for the treatment of certain inflammatory diseases.

The family of NADPH oxidases represents an important source of reactive oxygen species (ROS) within the cell. Nox4 is a special member of this family as it constitutively produces H2O2 and its loss promotes inflammation. A major cellular component of inflammation is the macrophage population, which can be divided into several subpopulations depending on their phenotype, with proinflammatory M(LPS+IFNγ) and wound-healing M(IL4+IL13) macrophages being extremes of the functional spectrum. Whether Nox4 is expressed in macrophages is discussed controversially. Here, we show that macrophages besides a high level of Nox2 indeed express Nox4. As Nox4 contributes to differentiation of many cells, we hypothesize that Nox4 plays a role in determining the polarization and the phenotype of macrophages. In bone marrow-derived monocytes, ex vivo treatment with LPS/IFNγ or IL4/IL13 results in polarization of the cells into M(LPS+IFNγ) or M(IL4+IL13) macrophages, respectively. In this ex vivo setting, Nox4 deficiency reduces M(IL4+IL13) polarization and forces M(LPS+IFNγ). Nox4-/- M(LPS+IFNγ)-polarized macrophages express more Nox2 and produce more superoxide anions than wild type M(LPS+IFNγ)-polarized macrophages. Mechanistically, Nox4 deficiency reduces STAT6 activation and promotes NFκB activity, with the latter being responsible for the higher level of Nox2 in Nox4-deficient M(LPS+IFNγ)-polarized macrophages. According to those findings, in vivo, in a murine inflammation-driven fibrosarcoma model, Nox4 deficiency forces the expression of proinflammatory genes and cytokines, accompanied by an increase in the number of proinflammatory Ly6C+ macrophages in the tumors. Collectively, the data obtained in this study suggest an anti-inflammatory role for Nox4 in macrophages. Nox4 deficiency results in less M(IL4+IL13) polarization and suppression of NFκB activity in monocytes.

Rationale: Classic histology is the gold standard for vascular network imaging and analysis. The method however is laborious and prone to artefacts. Here, the suitability of ultramicroscopy (UM) and micro-computed tomography (CT) was studied to establish potential alternatives to histology.Methods: The vasculature of murine organs (kidney, heart and atherosclerotic carotid arteries) was visualized using conventional 2D microscopy, 3D light sheet ultramicroscopy (UM) and micro-CT. Moreover, spheroid-based human endothelial cell vessel formation in mice was quantified. Fluorescently labeled Isolectin GS-IB4 A647 was used for in vivo labeling of vasculature for UM analysis, and analyses were performed ex vivo after sample preparation. For CT imaging, animals were perfused postmortem with radiopaque contrast agent.Results: Using UM imaging, 3D vascular network information could be obtained in samples of animals receiving in vivo injection of the fluorescently labeled Isolectin GS-IB4. Resolution was sufficient to measure single endothelial cell integration into capillaries in the spheroid-based matrigel plug assay. Because of the selective staining of the endothelium, imaging of larger vessels yielded less favorable results. Using micro-CT or even nano-CT, imaging of capillaries was impossible due to insufficient X-ray absorption and thus insufficient signal-to-noise ratio. Identification of lumen in murine arteries using micro-CT was in contrast superior to UM.Conclusion: UM and micro-CT are two complementary techniques. Whereas UM is ideal for imaging and especially quantifying capillary networks and arterioles, larger vascular structures are easier and faster to quantify and visualize using micro-CT. 3D information of both techniques is superior to 2D histology. UM and micro-CT together may open a new field of clinical pathology diagnosis.

IL‐4 plays a pivotal role in the development of the Th2 cell mediated humoral immune response and causes IgE‐dependent allergic inflammatory diseases. Expression of IL‐4 in differentiated Th2 cells is regulated by transcription factors such as NF‐AT, AP‐1 and NF‐IL6. Recently, increasing evidence indicates that the pro‐inflammatory transcription factor NF‐κB may also participate inIL‐4 expression. In this study, we show that the IL‐4 promoter is synergistically activated by NF‐κB, NF‐AT and NF‐IL6 at the NF‐κB/NF‐AT/NF‐IL6 composite sites. In addition, we performed the chromatin immunoprecipitation technique to determine the functional relevance of NF‐κB in the activation of the IL‐4 gene in vivo. We demonstrate that NF‐κB binds to the IL‐4 promoter in vivo upon T cell activation. Inhibition of NF‐κB nuclear translocation in living cells blocked binding of NF‐κB to the IL‐4 promoter. The data provide first evidence that NF‐κB is directly involved in IL‐4 transcription.