Abstract 347: S100A1 is Critical for Neoangiogenesis Following Hind-Limb Ischemia
Recent data from our lab suggests that S100A1, an EF-hand type Ca++-handling protein present in endothelial cells (ECs), is necessary for nitric oxide (NO) production. NO regulates mobilization, proliferation and migration of ECs, and is also important for VEGF action in neoangiogenesis. NO is known to be produced by endothelial-type NO synthase (eNOS) in an Akt- and Ca++-dependent manner. In the present study, we set out to investigate a potential role of S100A1 in neoangiogenesis. For this purpose, we removed the right common femoral artery in 12-week-old S100A1 knock-out (SKO) mice and age-matched wild-type (WT) mice. Blood flow (BF) was measured in the tibial posterior artery before, immediately after, and at 1 and 2 weeks after surgery using echo-doppler and found to be similar between SKOs and WT mice in the non-ischemic hindlimbs (NI HLs). In contrast, there was no BF in the ischemic HL immediately after surgery in either group. On day 15, WTs showed partial restoration of BF (59±5 % of the contralateral NI HLs, n=10), whereas SKOs had significantly less BF restoration (17±6 %, p<0.05, n=10). BF was also assessed by injecting dyed beads and measuring the release of dye in digested muscles of the ischemic and NI HLs. We found reduced microspheres dilution in SKOs as compared to WTs (0.21±0.04 vs. 0.63±0.07, p<0.05, n=4), indicating again reduced BF. Interestingly, a significant percentage of SKOs suffered from leg amputation after femoral artery resection, whereas WTs did not show any significant pathological signs in the ischemic HLs. Clinical score, which measures severity of HL impairment, was significantly higher in SKOs as compared to WTs (4.3±0.5 vs. 0.6±0.3, p<0.01 χ2). Capillary per muscle fiber density (C/F) was found to be significantly lower in SKOs compared to WTs in tibial muscle sections. Finally, western blot analysis of gastrocnemius muscle showed increased protein levels of VEGF, increased Akt activation and Akt-induced eNOS phosphorylation in SKOs compared to WTs, most probably representing a compensatory mechanism for reduced NO production due to the S100A1 absence. Taken together, these results suggest that endogenous endothelial S100A1 plays a crucial role in the regulation of the angiogenic response to peripheral artery ischemia.