Abstract 5260: Digitoxin But Not Digoxin Elicits Anti-inflammatory And Vasoprotective Properties In Endothelial Cells: Therapeutic Implications For The Treatment Of Atherosclerosis ?
Expression of proinflammatory chemokines, impaired function of eNOS and apoptosis in the endothelium are crucial initiating steps in the pathogenesis of atherosclerosis. Recent observations pointed to potential anti-inflammatory properties of digitoxin. Therefore, we investigated if digitoxin inhibits chemokine expression (MCP-1, IL-8), activates eNOS, and inhibits apoptosis in endothelial cells (EC) as well as the underlying signaling pathways affected by digitoxin. Digitoxin but not digoxin (10–30 nM) potently inhibited cytokine-induced expression of MCP-1 & IL-8 in EC and capacity of corresponding cell culture supernatants on monocyte migration (ELISA, transwell-migration, -90%, n=5, p<0.01). Furthermore, digitoxin but not digoxin prevented IL-1β-induced activation of NF-κB-signaling and p44/42-MAPK without affecting activation of JNK and p38-MAPK (westernblot (WB): Iκ-Bα-degradation, nuclear translocation NF-κB-p65, phospho-p44/42,-JNK,-p38, n=5). Inhibition of NF-κB-signaling (IKK-2-inhibitor sc-514, n=5, p<0.01) but not of MEK1 (PD98059), activator of p44/42-MAPK, mimicked the observed inhibitory effects of digitoxin on proinflammatory processes. Moreover, digitoxin inhibited NF-κB-signaling at the level of TAK-1/IKK (WB: phospho-TAK-1,-IKK,-IκB-α, Iκ-Bα, n=5). Additionally, digitoxin prevented TNF-α-induced apoptosis in EC (PI-staining/FACS, caspase-3 activity, -95%, n=5, p<0.01) accompanied by marked activation of Akt (WB: phospho-Akt, n=5). Inhibition of PI-3-kinase (LY294002), activator of Akt, prevented the anti-apoptotic properties and reduced the inhibitory action of digitoxin on NF-κB-signaling and MCP-1 expression (n=5, p<0.05). Finally, digitoxin activated eNOS (WB: eNOS/phopho-eNOS; NO-release, n=5, p<0.05), which was blocked (n=5, p< 0.05) by inhibition of PI-3-kinase (LY294002), Ca2+/Calmodulin-dependent-proteinkinase-II (CAMK-II, KN-93) and chelating [Ca 2+]i (BAPTA-AM). Digitoxin elicits anti-inflammatory and vasoprotective properties by blocking NF-κB- and activating PI-3-kinase/Akt-signaling as well as CAMK-II in EC, indicating a potential therapeutical implication for the treatment of atherosclerosis.