We previously reported increased expression of integrin-4 in EC treated with simvastatin (5 M, 24 h) detected by microarray analysis with increased protein levels confirmed by Western blotting (13, 29). (5 M, 16 h). Increased expression of EC inflammatory cytokines [IL-6, IL-8, monocyte chemoattractant protein (MCP)-1, regulated on activation normal T cell expressed PLX647 and secreted (RANTES)] by LPS (500 ng/ml, 4 h) was also significantly attenuated by simvastatin pretreatment (5 M, 16 h), but this effect was reversed by cotreatment with an integrin-4-blocking antibody. Finally, although simvastatin (20 mg/kg) conferred significant protection in murine ALI as evidenced by decreased bronchoalveolar lavage fluid cell counts, protein, inflammatory cytokines (IL-6, IL-1, MCP-1, RANTES), decreased Evans blue dye albumin extravasation in lung tissue, and PLX647 changes on lung histology, these effects were reversed by the integrin-4-blocking antibody (IV, 1 mg/kg, 2 h before LPS). These findings support integrin-4 as an important mediator of ALI protection by simvastatin and implicate signaling by integrin-4 as a novel therapeutic target in patients with ALI. 20 min). The optical density of the supernatant was then decided spectrophotometrically at 620 nm. The extravasated EBA concentration was calculated against a standard curve and expressed as g EBA/ml. Statistical analysis. Shapiro-Wilk testing was used to confirm data were normally distributed. Rabbit Polyclonal to GUSBL1 Student’s 0.05) among multiple-group comparisons were confirmed by one-way ANOVA and post hoc multiple-comparisons testing. Results are expressed as means SE. RESULTS Regulation of integrin-4 expression by simvastatin. We previously reported increased expression of integrin-4 in EC treated with simvastatin (5 M, 24 h) PLX647 detected by microarray analysis with increased protein levels confirmed by Western blotting (13, 29). To extend and further validate our earlier studies, we performed real-time PCR and characterized integrin-4 mRNA changes over time in human pulmonary artery EC treated with simvastatin (5 M). These studies confirmed time-dependent changes in integrin-4 mRNA levels with a significant increase evident at 6 h, whereas levels were increased even further at 16 h (Fig. 1 0.05 compared with 2 h). = 3/experimental condition, * 0.05). Role of integrin-4 in the attenuation of LPS-induced EC inflammatory responses by simvastatin. Because we have previously reported an anti-inflammatory role for integrin-4 in EC responses to LPS (3), we investigated these effects in the context of simvastatin treatment. In our initial experiments, human pulmonary artery EC were treated with simvastatin (5 M, 16 h) or vehicle either alone or with subsequent treatment with either an integrin-4-blocking antibody (20 g/ml, 2 h) or a control antibody (IgG) before LPS stimulation (500 ng/ml, 4 h). Media were then collected and IL-6 and IL-8 levels measured (Fig. 3). These studies confirmed a significant attenuation of LPS-induced IL-6 and IL-8 expression by simvastatin alone that was abrogated by treatment with the integrin-4-blocking antibody. Additional studies to assess other inflammatory cytokines revealed a significant attenuation of both monocyte chemoattractant protein (MCP)-1 and regulated on activation normal T cell expressed and secreted (RANTES) expression in LPS-stimulated EC pretreated with simvastatin (5 M, 16 h) that was significantly reversed by treatment with the integrin-4-blocking antibody (Fig. 4). Interestingly, the integrin-4-blocking antibody also affected an increase in MCP-1 expression in these studies when administered by itself. Of note, individual studies were conducted using siRNA specific for integrin-4, but these experiments confirmed an overriding effect of simvastatin resulting in increased-4 expression despite the use of siRNA that effectively inhibited integrin-4 expression when used alone. Open in a separate windows Fig. 3. The attenuation of LPS-induced EC IL-6 and IL-8 expression by simvastatin is usually mediated by PLX647 integrin-4. EC were produced to confluence and treated with simvastatin (5 M, 16 h) or vehicle before LPS (500 ng/ml, 4 h), and levels of IL-6 (= 3/condition, * 0.05). Open in a separate windows Fig. 4. The.
