Objective. Hedgehog signaling not only plays crucial roles during human development but also has been implicated in the pathogenesis of several diseases in adults. The aim of the present study was to investigate the role of the hedgehog pathway in fibroblast activation in systemic sclerosis (SSc).Methods. Activation of the hedgehog pathway was analyzed by immunohistochemistry and real-time polymerase chain reaction (PCR). The effects of sonic hedgehog (SHH) on collagen synthesis were analyzed by reporter assays, real-time PCR, and Sircol assays. Myofibroblast differentiation was assessed by quantification of ␣-smooth muscle actin and stress fiber staining. The role of hedgehog signaling in vivo was analyzed by adenoviral overexpression of SHH and using mice lacking 1 allele of the gene for inhibitory receptor Patched homolog 1 (Ptch ؉/؊ mice).Results. SHH was overexpressed and resulted in activation of hedgehog signaling in patients with SSc, with accumulation of the transcription factors Gli-1 and Gli-2 and increased transcription of hedgehog target genes. Activation of hedgehog signaling induced an activated phenotype in cultured fibroblasts, with differentiation of resting fibroblasts into myofibroblasts and increased release of collagen. Adenoviral overexpression of SHH in the skin of mice was sufficient to induce skin fibrosis. Moreover, Ptch ؉/؊ mice with increased hedgehog signaling were more sensitive to bleomycin-induced dermal fibrosis.Conclusion. We demonstrated that the hedgehog pathway is activated in patients with SSc. Hedgehog signaling potently stimulates the release of collagen and myofibroblast differentiation in vitro and is sufficient to induce fibrosis in vivo. These findings identify the hedgehog cascade as a profibrotic pathway in SSc.

Microparticles (MP) are phospholipid vesicles shed by cells upon activation or apoptosis. Monocyte-derived MP upregulate the synthesis of proinflammatory mediators by lung epithelial cells; the molecular bases of such activity are unknown. Peroxisome proliferatoractivated receptors (PPAR) have been demonstrated to be involved in the modulation of nuclear factor (NF)-kB transcriptional activity and inflammation.We investigated whether the upregulation of the synthesis of proinflammatory cytokines by human lung epithelial cells induced by monocyte/macrophage-derived MP involves NF-kB activation and is modulated by PPAR-c.MP were generated by stimulation of human monocytes/macrophages with the calcium ionophore, A23187. MP were incubated with human lung epithelial cells. NF-kB translocation was assessed by electrophoretic mobility shift assay. Interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 synthesis was assessed by ELISA and RT-PCR.Stimulation of A549 alveolar cells with monocyte/macrophage-derived MP caused an increase in NF-kB activation and IL-8 and MCP-1 synthesis that was inhibited by pre-incubation with the PPAR-c agonists, rosiglitazone and 15-deoxy-D 12,14 -prostaglandin-J 2 . Parallel experiments with normal human bronchial epithelial cells largely confirmed the results. The effects of PPAR-c agonists were reversed by the specific antagonist, GW9662.Upregulation of the synthesis of proinflammatory mediators by human lung epithelial cells induced by monocyte/macrophage-derived MP is mediated by NF-kB activation through a PPAR-c dependent pathway.

Objectives Microparticles are membrane vesicles shed by cells upon activation and apoptosis. Agonists capable of inducing microparticle generation include cytokines, bacterial products, P-selectin, histamine. Cigarette smoke extract has also been recognized as an agonist involved in microparticle generation with an apoptosis-dependent mechanism. We investigated the possibility that cigarette smoke extract induces the rapid generation of proinflammatory microparticles by human mononuclear cells with a calcium-dependent mechanism. Materials and methods Human mononuclear cells were exposed to cigarette smoke extract. [Ca 2? ] i mobilization was assessed with the fluorescent probe Fluo-4 NW. Microparticles were quantified with a prothrombinase assay and by flow cytometry. Normal human bronchial epithelial cells and A549 alveolar cells were incubated with cigarette smoke extract-induced microparticles and the generation of ICAM-1, IL-8, and MCP-1 was assessed by ELISA.Results Exposure to cigarette smoke extract induced a rapid increase in [Ca 2? ] i mobilization. Microparticle generation was also increased. EGTA, verapamil and the calmodulin inhibitor, W-7, inhibited microparticle generation. Incubation of lung epithelial cells with cigarette smoke extract-induced microparticles increased the expression of proinflammatory mediators. Conclusions Exposure of mononuclear cells to cigarette smoke extract causes a rapid shedding of microparticles with a proinflammatory potential that might add to the mechanisms of disease from tobacco use.

ObjectivesTissue fi brosis is a leading cause of death in patients with systemic sclerosis (SSc). Effective antifi brotic treatments are not available. Here, the authors investigated inhibition of hedgehog signalling by targeting Smoothened (Smo) as a novel antifi brotic approach. Methods The activation status of the hedgehog pathway was assessed by immunohistochemistry for Gli transcription factors and by quantifi cation of hedgehog target genes. Hedgehog signalling was inhibited by the selective inhibitor LDE223 and by small interfering RNA against Smo in the models of bleomycin-induced dermal fi brosis and in tight-skin-1 mice. Results Hedgehog signalling is activated in SSc and in murine models of SSc. Inhibition of Smo either by LDE223 or by small interfering RNA prevented dermal thickening, myofi broblast differentiation and accumulation of collagen upon challenge with bleomycin. Targeting Smo also exerted potent antifi brotic effects in tight-skin-1 mice and did prevent progression of fi brosis and induced regression of pre-established fi brosis. Conclusions Inhibition of hedgehog signalling exerted potent antifi brotic effects in preclinical models of SSc in both preventive and therapeutic settings. These fi ndings might have direct translational implications because inhibitors of Smo are already available and yielded promising results in initial clinical trials.

Our data demonstrate that hedgehog pathways and TGF-β signalling both converge to GLI2 and that GLI2 integrates those signalling to promote tissue fibrosis. These findings may have translational implications as non-selective inhibitors of GLI2 are in clinical use and selective molecules are currently in development.

The reversal of multidrug resistance by 22 molecules [8-aryl-8-hydroxy-5-R'-8H-[1,4]thiazino[3,4-c][1,2,4]oxadiazol-3-ones (1a-i) and 8-aryl-8-alkoxy-5-methyl-8H-[1,4]thiazino[3,4-c][1,2,4]oxadiazol-3-ones (2a-m)] related to myocardial-calcium-channel-modulator diltiazem was studied in multidrug resistant A2780/DX3 and their sensitive counterpart A2780 cells. MTT, cytofluorimetry assays, and fluorescence microscopy analyses were used to define activity and accumulation of doxorubicin with or without the diltiazem-like modulators. Of the 22 molecules, 1a, 2f, 2g, and 2m were able to overcome the established criteria for the selection in A2780/DX3 cells (IC(50) reduction > or = 25%), but only 2f, 2g, and 2m caused a significant increase of intracellular accumulation of doxorubicin. In conclusion, experiments lead to the identification of three diltiazem-like molecules able to increase the intracellular accumulation of doxorubicin by inhibiting the MDR1 function, thus potentiating its antiproliferative activity in multidrug resistant A2780/DX3 cells.

We report herein the reversal of multidrug resistance-1 (MDR1) in A2780/DX3 cells by the two nifedipine-like compounds 1 and 2 that are part of a library of 1,4-dihydropyridines (1,4-DHPs) calcium-channel modulators bearing in C-4 a different substituted imidazo[2,1-b]thiazole system. By methylthiazol tetrazolium (MTT) assay, cytofluorimetry, and fluorescence microscopy we evaluated their ability to reverse MDR in our cell system. Moreover, together with compound 3 (the diltiazem-like 8-(4-chlorophenyl)-5-methyl-8-[(2Z)-pent-2-en-1-yloxy]-8H-[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one) we analyzed their ability to potentiate the triggering of apoptosis after exposure to doxorubicin, through the nuclear morphological analysis after 4',6-diamidino-2-phenylindole (DAPI), the fluorescein isothiocyanate (FITC)-Annexin-V/propidium iodide (PI) staining and the caspase activity determination. Our results demonstrate that compounds 1 and 2, at concentrations showing a very low (5%) or absent inhibition of cell proliferation, in combination with doxorubicin enhance its antiproliferative activity (from 30% to 54% IC(50) reduction) in A2780/DX3 cells through an increase of doxorubicin intracellular accumulation. These compounds together with compound 3, which has already been demonstrated to act as a potent inhibitor of MDR1 function, were also able to significantly potentiate the activation of the apoptosis machinery triggered by the exposure to doxorubicin. In conclusion, our results identify two new molecules structurally related to the calcium-channel blocker nifedipine, but characterized by a very low LTCC blockers activity, able to potentiate the antiproliferative and apoptotic activities of doxorubicin through an increase of its intracellular concentration likely caused by the inhibition of MDR1 function.

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