8,9-Dehydrohispanolone-15,16-lactol diterpene prevents LPS-triggered inflammatory responses by inhibiting endothelial activation

Abstract

Endothelial activation contributes to lung inflammatory disorders by inducing leucocyte recruitment to pulmonary parenchyma. Consequently, vascular-targeted therapies constitute promising strategies for the treatment of inflammatory pathologies. In the present study, we evaluated the effect of 8,9-dehydrohispanolone-15,16-lactol diterpene (DT) on lung endothelium during inflammation. Lung endothelial cells pre-treated with DT and activated with lipopolysaccharide (LPS) or tumour necrosis factor-α (TNF-α) exhibited reduced expression of the pro-inflammatory cytokines Cxcl10, Ccl5 and Cxcl1, whereas the anti-inflammatory molecules IL1r2 and IL-10 were induced. Consistent with this result, DT pre-treatment inhibited nuclear factor κB (NF-κB) nuclear translocation, by interfering with IκBα phosphorylation, and consequently NF-κB transcriptional activity in endothelium activated by LPS or TNF-α. Furthermore, DT, probably through p38 signalling, induced transcriptional activation of genes containing activator protein 1 (AP-1)-binding elements. Inhibition of p38 prevented IL1r2 mRNA expression in endothelium incubated with DT alone or in combination with LPS or TNF-α. Accordingly, conditioned medium (CM) from these cells failed to stimulate leucocytes as measured by a reduction in adhesive ability of the leucocyte cell line J774 to fibronectin (FN). Additionally, DT reduced the expression of the endothelial adhesion molecules E-selectin, vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) after activation. Similarly, expression of VCAM-1 and ICAM-1 molecules on the lung endothelial layer of C57/BL6 mice pre-treated with DT and challenged with LPS were unchanged. Finally, inhibition of vascular adhesion molecule expression by DT decreased the interaction of J774 cells with lung endothelial cells in an inflammatory environment. Our findings establish DT as a novel endothelial inhibitor for the treatment of inflammatory-related diseases triggered by Gram-negative bacteria or by the associated cytokine TNF-α.

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

“…Cytokines are small proteins released by the endothelial layer in the inflammatory context and strongly regulate leukocyte behavior. Pro-inflammatory members, such as Ccl5, Cxcl10 and Cxcl1, bind to their specific receptors on the leukocyte surface and signal to induce leukocyte interaction with the newly expressed adhesion molecules on the vascular layer (E-selectin, VCAM-1 and ICAM-1), and leukocyte migration to the pathological area ( Figure 1, Figure 2, Figure 3) 8, 10,18 . Other members of the same family of proteins are involved in the resolution of the inflammation and consequently tissue repair.…”

“…Other members of the same family of proteins are involved in the resolution of the inflammation and consequently tissue repair. The expression of these anti-inflammatory cytokines is relevant for chronic inflammatory diseases because they impede leukocyte recruitment and favor immunity clearance 10,19,20 . To select possible endothelial inflammatory regulators, it is recommended to perform this cytokine expression assay and evaluate the endothelial adhesion molecules expression in the presence of the compounds of interest.…”

“…The LPS binding to its cell surface receptor triggers complex intracellular signaling cascades led by MAP kinases ERK 1/2, JNK, and p38 and the NF-kB signalosome to induce the inflammatory transcriptional program. Many of these pathways are common to other inflammatory stimuli such as TNF-α or IL-1 10,22 . The endothelial activation is determined by detecting the phosphorylated form of the MAP kinase members as shown for ERK 1/2 in Figure 4.…”

“…. The MLEC-04 cell line has been validated in the literature to be an appropriate system to study endothelial activation 9,10 . Based on research interests, these approaches can be easily extrapolated to any endothelial or leukocyte systems and inflammatory profile.…”

Suppressive Effects of GSS on Lipopolysaccharide-Induced Endothelial Cell Injury and ALI via TNF-αand IL-6

“…LPS binds and activates Toll-like receptor 4 (TLR4) of ECs, which results in massive release of various proinflammatory mediators, such as interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α). Overexpression of TNF-α and IL-6 is associated with ALI, multiple organ dysfunction syndrome (MODS), and mortality [15,16].…”