Oxidized low density of lipoprotein (oxLDL) is the major lipid found in atherosclerotic lesion and elevated plasma oxLDL is recognized to be a risk factor of atherosclerosis. Whether plasma oxLDL could be transported across endothelial cells and initiate atherosclerotic changes remains unknown. In an established in vitro cellular transcytosis model, the present study found that oxLDL could traffic across vascular endothelial cells and further that the regulation of endogenous ceramide production by ceramide metabolizing enzyme inhibitors significantly altered the transcytosis of oxLDL across endothelial cells. It was found that acid sphingomyelinase inhibitor, desipramine (DES), and de novo ceramide synthesis inhibitor, myriocin (MYR), both decreasing the endogenous ceramide production, significantly inhibited the transcytosis of oxLDL. Ceramidase inhibitor, N-oleoylethanolamine (NOE), and sphingomyelin synthase inhibitor, O-Tricyclo[5.2.1.02,6]dec-9-yl dithiocarbonate potassium salt (D609), both increasing the endogenous ceramide production, significantly upregulated the transcytosis of oxLDL. In vivo, injection of fluorescence labeled oxLDL into mice body also predisposed to the subendothelial retention of these oxidized lipids. The observations provided in the present study demonstrate that endogenous ceramide contributes to the transcytosis of oxLDL across endothelial cells and promotes the initiating step of atherosclerosis—the subendothelial retention of lipids in vascular wall.

The first two authors contributed equally to this work Allergic rhinitis (AR) is characterized by IgE-mediated immediate hypersensitivity and usually progresses to chronic nasal inflammation, with depression as one of its comorbidities. The importance of treating the depression in AR patients has been increasingly recognized. Desipramine is a representative of tricyclic-antidepressant agents. In the present study we investigate whether desipramine has therapeutic effects on AR inflammation. BALB/C mice were sensitized by intraperitoneal injection of ovalbumin (OVA), followed by repeated challenge with OVA intranasally. Desipramine was administered orally to treat the mice. The nasal symptoms (sneezing, nasal scratching etc.) ofAR were evaluated to determine the severity ofAR. Cytokines in the nasal lavage fluid (NALF), including interferon-y (IFN-y), interleukin 4 (IL-4) and serum OVA-specific immunoglobulin E (IgE) antibody were measured by ELISA. The regulatory T cells (T reg ) and T helper cells 17 (Thl7) were quantified by flow cytometric analysis. As a result, the repeated oral administration of desipramine attenuated the nasal symptoms (sneezing and nasal rubbing) in AR mice. Desipramine also suppressed the serum OVA-specific IgE and IL-4 levels, but had no effect on IFN-y level. Moreover, desipramine treatment up regulated CD4+CD25+Foxp3+T reg cells, which were found down-regulated in established AR mice. Meanwhile, desipramine administration attenuated CD4+IL-17+T h17 cells, which were significantly increased inAR mice. These results suggest that the antidepressant drug, desipramine, also has anti-allergic action, which was possibly achieved by reducing allergen-specific IgE and Th2 cytokine production and maintaining a balance between T reg and Th17 cells. Thus, this study provide the first evidence that desipramine may be utilized to treat allergic diseases, especially for those allergic patients with depression or depression patients with allergy.

The present study addressed whether dual oxidase 1 (Duox1), a predominant isoform of NADPH oxidase in bronchial epithelial cells, is also activated through assembling of Duox1 and its partners such as p47 phox due to lipid raft (LR) clustering. By gradient ultracentrifugation to isolate LR fractions in bronchial epithelial cells, it was found that Duox1 or p47 phox was translocated into LR fractions when stimulated by tumor necrosis factor-α (TNF-α). Confocal microscopic analysis revealed that LRs were aggregated or clustered in the membrane, which were colocalized with Duox1 or p47 phox. Ceramide, a hydrolysis product of sphingomyelin, was also found colocalized with Duox1 or p47 phox upon stimulation. In the presence of the commonly used LR disruptor, methyl-β-cyclodextrin (MCD), or the acid sphingomyelinase (ASMase) inhibitor, desipramine (DES), TNF-α-stimulated aggregation, translocation, and colocalization of LR components and Duox1 or its partners was abolished. Functionally, TNF-α-stimulated H2O2 production was also blocked by MCD and DES (194.6 ± 15.4% vs. 90.6 ± 15.9% and 148.8 ± 20.4%), and the activation of the pivotal proinflammatory transcription factor, NF-κB, by TNF-α was reversed by MCD and DES as well as by small interfering RNAs of Duox1 or ASMase. Our results for the first time demonstrate that Duox1-mediated redox signaling in bronchial epithelial cells is associated with LR clustering dependent on the production of ceramide through ASMase.

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