| Objective:Amot protein expression in wound granulation tissues sampled from diabetic patients with foot ulcers and diabetic mice with hindlimb ischemic ulcers were determined. Vascular endothelial cells (VECs) were cultured in high-glucose culture media at anaerobic condition, and assayed for the Amot expression. The regulatory mechanisms of the Amot on angiogenesis in diabeteic ischemic ulcer-healing were investigated.Methods:1. The diabetic patients with foot ulcers were divided into four groups according to their ankle brachial index(ABI) and glycosylated haemoglobin A1c(GHbA1c):group A ABI≥0.7, GHbA1c<9%; group B ABI≥0.7, GHbA1c≥9%; group C ABI＜0.7, GHbA1c<9 and group D ABI＜0.7, GHbA1c≥9%. The patients with foot burns served as control group. The wound granulation tissues were sampled these patients. Western blot was used to determine the Amot expression. High glucose and ischemia effects on the Amot expression were evaluated and the relationships between the variance of the Amot expression and ulcer-healing were also studied.2. An animal model of ischemic ulcers at hindlimb was established in streptozotocin induced diabetic mouse. Then after, the Amot mRNA expressions in the wound granulation tissues of these mice were assayed by real-time fluorescent quantitation. The correlation between the Amot mRNA expression and the wound-healing in the hyperglycemia and ischemia conditions was analyzed.3. The VECs were cultured in a culture media with different glucose concentrations at anaerobic condition. The Amot expression of the VECs cultured in different conditions were detected by Western blot.4. The Amot siRNA was designed and applied to downregulate the Amot expression in VECs. The silencing effects were confirmed by Western blot. Wound healing experiments and angiogenesis experiments were carried out in the Amot knockdown VECs.Results:1. The Amot were expressed in the wound granulation tissues both from the diabetic patients and the diabetic ischemic mice.2. Expression of p80-Amot in the control group and the group A were higher than those of the group B and D, who had poor blood glucose control (p<0.05). There was no significant different in ulcer prognosis between groups.3. The Amot mRNA expressions in the wound granulation tissues of mouse hindlimb ulcers were different. Compared with other groups, the diabetic ischemic ulcer group had the lowest level (p<0.01). The non-diabetic non-ischemic ulcer group had higher level of expression than that of the diabetic non-ischemic ulcer group (p<0.01).4. In VECs cultured in different glucose concentrations at anaerobic condition, p130-Amot had the higher expression in the two groups of low glucose concentrations than those of other four groups (p<0.01). However, between the two groups of low glucose concentrations, the expression of p130-Amot was lower in VECs cultured at anaerobic condition (p<0.01).5. Compared to the normal VECs, the Amot expression in the VECs transfected Amot siRNA was decreased 93%. These Amot defective VECs exhibited an attenuated cell migration in the wound healing experiments and had a lesser tube formation in the angiogenesis experiments.Conclusions:1.Amot participates the process of the wound-healing in diabetic foot ulcers.2.High glucose and ischemia or anoxia decreases the Amot expression, and high glucose has a more significant negtive effect on the Amot expression.3.After the Amot expression downregulated in VECs, the cell functions of migration and angiogenesis are impaired significantly. Therefore, Amot may play a crucial role in the pathogenesis of the impaired wound-healing in diabetic foot ulcers. In future, Amot may be a new target in the treatment of poor-healing diabetic foot ulcers.
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