12: Inhibition of phosphorylation of HSP27 decreases vasospasm in human saphenous vein

Abstract

Saphenous vein is used as a conduit for peripheral vascular and coronary reconstruction. However, surgical manipulation during harvest of the vessel often leads to vasospasm. Impaired relaxation is one of the main reasons for vein graft spasm. The small heat shock proteins, HSP20 and HSP27, coordinately regulate vascular smooth muscle (VSM) tone. We hypothesized that inhibiting the phosphorylation of HSP27 may increase sodium nitroprusside (SNP) induced phosphorylation of HSP20 and relaxation of saphenous vein. Human saphenous vein (HSV) rings were equilibrated in a muscle bath, treated with contractile and relaxing agents and the force generated was recorded. Muscle rings were frozen, and extracted proteins were analyzed for HSP20 and HSP27 phosphorylation by gel electrophoresis and western blotting. HSV contracted dose-dependently to norepinephrine (NE) and relaxed dose-dependently to sodium nitroprusside (SNP), a nitric oxide donor. Pre-incubation of HSV with SB203580 (50 μM), an inhibitor of p38 MAP Kinase, which is upstream of MAPKAP kinase II (the kinase that directly phosphorylates HSP27), increased SNP (1 μM) relaxation of HSV from 20% +/- 8% to 62% +/- 12% (p< 0.05, n=5, +/-SEM) . SB203580 pre-treatment also reduced the magnitude of NE induced contraction by (50% +/1 9%). SB treatment decreased the basal phosphorylation of HSP27 (Ser 82 and Ser 15) by 68% and increases SNP stimulated phosphorylation of HSP20 (Ser 16) by 20%. These results suggest that SB203580 treatment can reduce the magnitude of agonist induced contraction. This was associated with a decrease in the basal level of HSP27 phosphorylation. In addition, decreases in HSP27 phosphorylation were associated with increased SNP induced phosphorylation of HSP20 and relaxation. These results suggest that the two small heat shock proteins, HSP20 and HSP27 may have opposing effects in mediating vascular tone and that inhibiting the phosphorylation of HSP27 may be a therapeutic approach for preventing vasospasm.