Study On Angiogenesis After Hindlimb Ischemia In Sirt1-transgenic Mice

Objective: Angiogenesis is the generation of new blood vessels from existing capillaries and it provides more blood flow to body.However,abnormal angiogenesis results in a variety of pathologies.It has been reported that SIRT1 acts as a key regulator of angiogenesis.In the present study,we investigated the effect of SIRT1 on angiogenesis using a hindlimb ischemia model by ligating femoral artery in Sirt1-Tg mice.Methods:1 Breeding and genotype identification of Sirt1-Tg miceFemale Sirt1-Tg mice and the same background male(6 to 8-week-old)were crossed at the ratio of 2:1 to generate offspring.The genomic DNA was extracted from the ears tissues,and used to identify the genotypes of offspring by PCR.2 Preparation of hindlimb ischemia model of miceMale Sirt1-Tg mice and WT controls(8-week-old)were anesthetized with isoflurane under the fixed environment(temperature 24? and constant humidity).The femoral artery was ligated under microscopic vision and the hindlimb blood flow was measured before and after surgery by PeriCam PSI.The images of the hindlimb blood flow were recorded using the LASCA system.The ratios of the left/right(L/R)hindlimb blood flow were analyzed by PIMSoft.3 Measurement of hindlimb blood flow at different times after surgeryWe used the PeriCam PSI to measure the blood flow of both hindlimbs in the two groups after surgery for 3,7 and 14 days,respectively.4 Immunofluorescence staining to evaluate the expression of CD31The gastrocnemius muscles of both hindlimbs were harvested from Sirt1-Tg and WT mice at 14 days after surgery,and embedded in OCT compound for preparation of frozen sections.CD31 expression was detected by immunofluorescence staining,and the positive fluorescence intensity was detected by fluorescence microscopy.5 Western blot for the expression of VEGFWestern blot was employed to detect the expression of VEGF in gastrocnemius muscles of both hindlimbs in the two groups at 14 days after surgery.6 Statistical analysisData are expressed as mean ± SD from three independent experiments,and unpaired Student's t test was used to compare the statistical difference between the groups.Differences were considered significant at a value of P<0.05.Results:1 Identification of Sirt1-Tg miceThe PCR product of 233 bp was analyzed by agarose gel electrophoresis,and showed clearly visible bands.2 Establishment of the hindlimb ischemia model by ligating left femoral arteryLASCA showed that the blood flow signals in hindlimbs of the WT mice and Sirt1-Tg mice were symmetrical before surgery and the ratios of blood flow in hindlimbs(L/R)were 0.91±0.07 and 0.92±0.10(P>0.05).After surgery the ratios were 0.29±0.05 and 0.31±0.05(P>0.05).Compared with before surgery,the signals of left hindlimb were significantly reduced after surgery,indicating that the hindlimb ischemia model was successful.3 LASCALASCA showed that the weak blood flow signals were found in left ligated hindlimbs of the WT mice and Sirt1-Tg mice at 3 days,indicating that the angiogenesis was induced in ischemic tissues.The ratios were 0.48±0.05 and 0.47±0.08(P>0.05).The blood flow in left ligated hindlimbs gradually recovered at 7 days.And the ratios were 0.68±0.05 and 0.55±0.04(P>0.05).In WT mice,the blood flow signals of the left hindlimb were consistent with the right control at 14 days after surgery.However,it was significantly weaker in Sirt1-Tg mice than that in WT mice.And the ratios of WT mice and Sirt1-Tg mice were 0.82±0.10 and 0.66±0.07(P<0.01).4 Immunofluorescence staining for CD31Immunofluorescence staining showed that the expression of CD31 was increased in left in the two groups at 14 days after surgery compared with the right hindlimbs.However,the expression level of CD31 was lower in Sirt1-Tg mice than that in WT mice.The ratios(L/R)of relative expression in WT mice and Sirt1-Tg mice were 1.99±0.17 and 1.39±0.04(P<0.05).5 Expression of VEGFWestern blot analysis showed that compared with the right,the level of VEGF in the left gastrocnemius muscles was increase in the two groups.The increased level of VEGF was lower in Sirt1-Tg mice than that in WT mice,similar to CD31.The ratios(L/R)of relative expression in WT mice and Sirt1-Tg mice were 2.13±0.52 and 1.24±0.17(P<0.05).Conclusions1 Sirt1-Tg mice are easy to breed and reproduce.2 Angiogenesis is inhibited in ischemic areas of Sirt1-Tg mice.3 SIRT1 may be an inhibitor of the endothelial cell proliferation.