| Objective:Cathepsin S(CatS)regulates the biological activity of endothelial cells(ECs)by modifying or degrading various types of active substrates.At present,most of the studies are focused on the expression and role of CatS in atherosclerotic cardiovascular disease,but the expression and role of it in ischemic angiogenesis are rarely reported.Recently,the expression and function of peroxisome proliferator-activated receptor-gamma(PPAR-y)in the cardiovascular system and inflammatory-immune cells have received increasing attention.However,the function of CatS inhibitor(CatS-I)on ischemia-induced angiogenesis are largely unknown.Therefore,in this study,we aim to investigate the potential mechanism of CatS-I in ischemia-induced angiogenesis by a mouse limb ischemic model.Methods:The 8-week wild-type mice(21-25 g,C57/BL6,supplied by the Nagoya University Animal Laboratory,Japan)were divided into two groups according to the random number table method:control group and CatS-I group,20 for each group.In the control group,except the standard diet administration,the mice also received 5%carboxymcthyl cellulose sodium(CMC)intraperitoneally once a day for 17 days;CatS-I group was given intraperitoneal injection of CatS-I[1 mg/(kg·d)],once a day,for a total of 17 days.Three days later,mice in the two groups were anesthetized with by intraperitoneal injection of pentobarbital(60 mg/kg)and the left lower abdomen skin was incised.Then,the femoral nerve was ligated and the femoral artery roots were ligated,and thereafter the three main branches of the femoral artery were isolated and ligated.The femoral artery trunk and its branches were then resected to establish a model of lower limb ischemia.Laser flow Doppler flow imaging system was used to measure the blood flow before and immediately after the operation,and on the 4th,7th,and 14th days after operation.The ratio of blood flow area ratio between the ischemic limb and the non-ischemic limb was calculated;at the same time The skeletal muscle tissue of the same part of the lower extremities was taken at day 4 to detect the protein expression levels of the molecules involved in angiogenesis,such as peroxisome proliferator-activated receptor-y(PPAR-y)and phosphorylated protein kinase B(p-Akt),phosphorylated endothelial nitric oxide synthase(p-eNOS)and vascular endothelial growth factor(VEGF)protein levels by Western blotting;on the 14th day after surgery,skeletal l)muscle tissue from the same site of both lower extremities was used for cryosectioning.The vascular endothelial cells were labeled with CD31 monoclonal antibody immunohistochemical staining to determine the number and density of capillaries.Measured data were expressed as mean ± standard deviation.Comparisons between groups were compared by Tukey post hoc test.Count data were compared using the ?2 test.P<0.05 was considered statistically significant.Results:1.CatS-I significantly inhibited the recovery of blood flow.The ratio of ischemic to non-ischemic LDBF showed that the blood flow recovery in CatS-I group was significantly slower than that in the control group(P<0.05).2.CatS-I significantly inhibited the formation of capillary.Observed with CD31 immunohistochemical staining,the capillary formation of CatS-I group was slightly decreased in the non-ischemic muscles compared with that of controls on day 14 after surgery,but furthermore there was no statistical significance(P>0.05);However,in the ischemic muscles,the capillary density in CatS-I group was significantly reduced compare to the control group(P<0.01).3.Representative western blots show that compared with the control group,the levels of PPAR-y?p-Akt?p-eNOS and VEGF proteins were significantly decreased in CatS-I mice.Conclusion:CatS may regulate ischemia-induced neovascularization by PPAR-y activation and Akt/eNOS signaling pathway.Thus,CatS represents a new therapeutic target for ischemic cardiovascular disease. |
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