| Part 1:Hirudin Promotes Angiogenesis by Modulating the Cross-talk between p38 MAPK and ERK in Rat Ischemic Skin Flap TissueBackground:Random skin flap is common for repairing wound, reconstructing the function and improving skin appearance. If a high length to width ratio random skin flap is raised, the distal tissue of the flap inevitably subject to ischemia and subsequent necrosis which often results in partial loss of the flap. However, the knowledge about natural hirudin functional role in angiogenesis is still unclear.Objective:In the present study we tested the hypothesis that natural hirudin induces angiogenesis by regulate the balance of VEGF and endostatin in random skin flap using in vivo model. We also determined whether the MAPKs signaling played a role in the natural hirudin associated angiogenesis.Methods:Two caudally based dorsal skin flaps (7.5 cm long and 1.5 cm wide) were created on the either sides of dorsal midline respectively. Forty flaps were assigned to control and natural hirudin groups. The flaps were injected with natural hirudin (natural hirudin group) or normal saline (control group) immediately after surgery and again on postoperative day 1,2 and 3. Photographs of the flaps were taken postoperatively at day 6 for evaluate the viable flap area and the necrotic flap area. Total proteins of flaps were isolated on postoperatively day 0,1,2,4 and 6 to analyze expression of proteins via western blot assay. The microvascular density of the flap tissue was analyze on postoperatively day 4 and 6.Results:At postoperatively day 6, the flap survival rate in hirudin group (90 ± 5.8%) was significantly larger than that in the control (62 ±7.1%) group, p < 0.01. Histological analysis of flaps in control group demonstrated that partial necrosis was observed in epidermal layer and dermal layer at postoperative day 4. In contrast, early neovascularization was seen in the dermal and subdermal layers in hirudin-treated flaps. At postoperative day 4, MVD in sections from hirudin group was significantly higher than that in the control (mean±SD:31.50±5.68 vs 15.90±5.44 per field, p< 0.01). At postoperative day 6, MVD in sections from hirudin group was 35.90±6.76 per field, whereas massive necrosis and no microvessel were found in sections from control group. We observed that the production of thrombin was upregulated in ischemic flap tissue from day1 to day 6, and hirudin treatment could limit the suddent increase of thrombin at day 1 and day 2(p< 0.01). We observed that the expression of VEGF in both two groups was elevated after operation, but the level of VEGF in hirudin group was higher than that in the control group (p<0.01). In hirudin group, the expression of endostatin was decrease on postoperative day 1,2 and 4 compared with control group (p<0.05). In flaps treated with hirudin there was a dramatic decrease (p<0.01) in TSP-1 production from postoperative day 1 to 6. Topical application of hirudin increased ERK1/2 phosphorylation, while attenuated the phosphorylation of p38 MAPK. Thrombin treatment could reverse the effects of hirudin on ERK1/2 and p38 MAPK activity. Inhibition of p38 MAPK activation resulted in significant decrease of TSP-1 expression, but not endostatin. After 3 h of p38 MAPK inhibition, the activation of ERK1/2 was increased significantly (p<0.01). However, inhibition of the MEK/ERK pathway by PD98059 had no significant effect on p38 MAPK phosphorylation (p>0.05), indicating that a unidirectional cross-talk between p38 MAPK and ERK might exist in rat ischemic flap tissue. Inhibition of PAR1 caused significant decreased phosphorylation of p38 MAPK (p<0.01) and increased the phosphorylation of ERK1/2 (p<0.01). These results were similar to the observations in hirudin-treated flaps. although addition of hirudin in SCH79797-treated flaps did not provide a further decrease of p38 MAPK activity (p> 0.05), the level of ERK1/2 activity in flap treats with SCH79797 together with hirudin increased (p<0.01) to the same level as that of flap treats with 2 ATU of hirudin alone, suggesting that there are another pathways that involved in the hirudin-mediated ERK1/2 activity.Conclusion:In summary, our study demonstrated that hirudin treatment not only increase VEGF expression but also decrease endostatin and TSP-1 production, which may potentially contribute to the hirudin mediated effects of improving angiogenesis in ischemic flap tissue. Our data also suggest that a cross-talk from p38 MAPK to ERK pathway appears to exist in ischemic flap tissue, and hirudin may exerts its angiogenesis effect via inhibits the thrombin-induced negative cross-talk of p38 MAPK-ERK. This novel pathway may play a crucial role in the improvement of skin flap survival.Part 2:The Effects of Hirudin on the Thrombin-induced Apoptosis of SD rat Vascular Endothelial Cells in HypoxiaBackground:We previously reported that topical application of natural hirudin can improve microcirculation and promoted neovascularization in ischemic flap tissue, and thereby improve flap survival. However, the knowledge about natural hirudin functional role in vascular endothelial cells (VECs) has not been previously described.Objective:To investigat the influences of hirudin on_the apoptosis of SD rat vascular endothelial cells induced by hypoxia.Methods:Confirmation of VECs in flap tissue was done by means of CD34 staining, and the cell apoptosis was detected by Tunel assay. In this way, the apoptosis rate of VECs of ischemic flap tissue was evaluated. VECs were isolated from SD rat aorta. Then, the cells were identified by CD31 immunohistochemical methods and flow cytometry. Cells were divided into Nor group(cultrued in normal condition), Cont group(hypoxia only), Tho group (2 U/ml of thrombin under hypoxia),hir group (2ATU/ml of hirudin under hypoxia), Low-T+H group (1ATU/ml of hirudin+2/ml U of thrombin under hypoxia) and High-H+T group (2ATU/ml of hirudin+2/ml U of thrombin under hypoxia). After 6 h of hypoxia, total proteins of each group was isolated, and the cell apoptosis was evaluated via Tunel assay.Results:Apoptosis rate of VECs in hirudin-treated flap tissue was 36.44±9.57%, it is lower than that in the control group (87.24±8.34%) (p<0.01). Under the hypoxia condition,2 U/ml of thrombin could increase the apoptosis rate of VECs(p<0.01). However, when the hirudin was added to the medium, the thrombin-induecd cell apoptosis was agonisted by hirudin(p<0.01). Hypoxia could stimulated the phosphorylation of p38 MAPK, and addition of thrombin would further increase the p38 MAPK phosphorylation(p<0.01). Hirudin could suppresses the thrombin-induced p38 MAPK phosphorylation(p<0.01).20 μM of SB203580 reatment could significantly inhibit the p38 MAPK phosphorylation and suppresses apoptosis of VECs. Interestingly, although addition of 2 U/ml of thrombin in VECs did not provide a further increase of cell apoptosis. Our immunoblot analysis revealed that the phosphorylation of ERK1/2 was significantly increased (p<0.01) by SB203580 treatment in ischemic flap tissue, suggesting that a cross-talk of p38 MAPK-ERK appears to exist in VECs under hypoxia. As shown in the Low-T+H group, hirudin treatment was result in an increase of ERK1/2 phosphorylation and decrease of Bim phosphorylation(p<0.01).Conclusion:Under the hypoxia, thrombin could the increases p38 MAPK phosphorylation and attenuate the ERK1/2 phosphorylation, and promote VECs apoptosis. Hirudin may exerts its anti-apoptotic effect via inhibits the thrombin-induced negative cross-talk of p38 MAPK-ERK under hypoxia condition. |
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