Osteopontin is an important mediator of alcoholic liver disease via hepatic stellate cell activation

Figure 6 Alcohol-induced plasmin activation is mediated via over-expression of Osteopontin. A: In vivo: Plasmin activity (pNA mol/L per minute) was significantly inhibited with alcohol (6 g/kg) administration compared to saline control both in WT and Osteopontin (OPN)^-/- mice. Conversely, basal plasmin activity was significantly higher in untreated OPN^-/- compared to WT mice. ìPA and buffer alone were used as positive and negative controls, respectively. ^aP < 0.05 vs saline in WT; ^cP < 0.05 vs saline in OPN^-/-; ^eP < 0.05 vs alcohol in WT; In vitro: B: Alcohol (10 mmol/L) significantly increased plasmin activity > 5-fold in LX2 (lane 2) compared to untreated cells (lane 1), which was significantly inhibited with 4-MP (lane 3), anti-OPN antibody (lane 4), OPN-R3 aptamer (lane 5) and OPN siRNA (siOPN) (lane 6). ^aP < 0.05 vs untreated cells; ^cP < 0.05 vs LX2; C: Compared to untreated cells (lane 1), LX2 cell migration (% migrated/non-migrated) significantly increased by 1.7-fold with alcohol (10 mmol/L) (lane 2) and was inhibited with 4-MP (lane 3). Blocking OPN with anti-OPN antibody (lane 4) and OPN-R3 aptamer (lane 5) and OPN binding with anti-ITGAV antibody (lane 7) significantly reduced LX2 cell migration. Cell migration was moderately reduced with anti-CD44v6 and did not reach significance. Confocal (panel) shows typical images of migrated cells for each treatment. Migration was quantitated by counting Hoechst stained cells (blue nuclei) using confocal images in at least six fields per treatment. ^aP < 0.05 vs untreated cells; ^dP < 0.01 vs alcohol.