The Effect Of VEGF Mediated Skeletal Muscle Fiber Switch On Angiogenesis

Diabetic foot has become a serious global public health problem,peripheral arterial disease(PAD)is the most important factor leading to diabetic foot ulcer.Effectively mitigating the damage of high glucose on endothelial cells,promoting angiogenesis is the main way to solve the PAD.Current researches mostly concentrate on endothelial cells,but few studies focus on the relationship between skeletal muscle and angiogenesis.As we know,there are mainly two phenotypes of striated skeletal muscle fibers:oxidative and glycolytic fiber,the former owns higher oxidative metabolism and oxygen delivery capacity.Studies have shown that oxidative muscle fibers have higher ability of glucose uptake.We found that,VEGF significantly increased capillary density in mice,and skeletal muscle shifted from a glycolytic to an oxidative phenotype.The project aims to explore the possibility of increasing the content of oxidative muscle fibers,promoting the ability of glucose uptake of skeletal muscle,improving microenvironment surrounding the vascular endothelial cells,enhancing the function of endothelial cells,promoting angiogenesis in the lower limb,and finally improving diabetic lower limb ischemia,through the regulation of skeletal muscle fiber types.We will also explore the signaling pathways of this process,lay the theoretical and experimental foundation for future clinical application of gene therapy,and solve the problem of glucose metabolism disorder and lower limb ischemia in diabetic patients who can not exercise.Part one The effect of endurance training on fiber type composition and capillary densityObjective: The purpose of this study was to determine the changes in fiber type composition and capillary density in a mouse model of endurance training。Methods: The C57BL/6 mice were divided into two groups,Separate groups of mice that either were sedentary or voluntarily ran for 4 weeks were used for quantification of VEGF protein.All mice were killed at 1w,2w,4w timepoints and the gastrocnemius muscles were harvested for analysis.VEGF protein concentration were detected by Enzyme-linked immunosorbent assay(ELISA).The fiber type composition and capillary density in both exercise and non-exercise mouse model were observed by immunofluorescence with an antibody against the endothelial cell marker CD31 and MHC IIa antibody.Citrate synthase activity were tested to explore the level of oxidative metabolism.Results:1 VEGF protein expression after voluntary running.VEGF protein concentration in gastrocnemius muscle was 449.164 ±15.280,556.818±22.659,549.366±14.410 pg/ml in the 1w,2w and 4w trained mice,respectively,higher than 205.962±9.712pg/ml in the sedentary mice.(P< 0.05).2 The fiber type composition and capillary density in both exercise and non-exercise mouse modelWe performed indirect immunofluorescence using antibodies specific for CD31 and MHC IIa.The percentage of type IIa myofibers and capillary density in gastrocnemius muscle increased significantly after voluntary running.3 Citrate synthase activity changes in skeletal muscleCompared to sedentary group(0.023±0.002μmol/min/ml),Citrate synthase activity at 1w,2w,4w(0.029±0.002,0.036±0.001,0.034±0.001μmol/min/ml)in skeletal muscle was significantly increased(P<0.05).Part two Determine whether increased VEGF is required for skeletal muscle adaptability in a mouse model of diabetic lower limb ischemiaObjective: To determine the effect of VEGF on fiber type composition and angiogenesis in a diabetic lower limb ischemia mouse model.Methods: The C57BL/6 mice were fed with high-fat diet for at least 6weeks,intraperitoneal injection of STZ induced diabetes model(100mg/kg),under femoral artery ligation,cause the left lower limb ischemia model.The mice underwent i.m.injection with adenovirus of Ad-GFP or Ad-VEGF-GFP.Fluorescent microscope was used to observe the transfection efficiency in ischemia limb muscles.VEGF protein concentration were detected by Enzyme-linked immunosorbent assay(ELISA).The oxidative myofiber were observed by immunofluorescence with MHC IIa antibody.The blood perfusion in ischemic skeletal muscle was evaluated by Laser doppler perfusion imaging(LDPI).Citrate synthase activity were tested to explore the level of oxidative metabolism.Results:1 Observing VEGF adenovirus transfection efficiency by fluorescence microscopy of GFP-tag.The transfection efficiency of Ad-VEGF-GFP and Ad-GFP is high and continued until 2 weeks.2 VEGF protein expression after intramuscular delivery of AdVEGF-GFP.Two weeks after injection,VEGF protein concentration in Ad-VEGF-GFP(653.373 ± 55.348 pg/ml)was significantly higher than the control group Ad-GFP(180.339±15.000 pg/ml)(P <0.05).3 The change in fiber type compositionThe percentage of type IIa myofibers in gastrocnemius muscle increased significantly following Ad-VEGF-GFP injection,observed by immunofluorescence.4 Citrate synthase activity changes in skeletal muscleCompared to Ad-GFP group(0.018 ± 0.002μmol/min/ml),Citrate synthase activity at Ad-VEGF-GFP(0.030±0.003μmol/min/ml))in gastrocnemius muscle was significantly increased(P< 0.05).5 Blood Perfusion of lower limbs evaluated by Laser DopplerThe blood perfusion in ischemic skeletal muscle of Ad-VEGF-GFP wassignificantly higher than Ad-GFP group(P<0.05),suggested that VEGF induces angiogenesis.Part three Observe the effect and mechanism of oxidative myofibers on vascular endothelial cell activity by upregulating oxidative fiber by VEGF in vitro.Objective: To determine the effect of VEGF on fiber type composition,and the effect and mechanism of oxidative myofibers on vascular endothelial cell activity.Methods: C2C12 myoblasts were cultured and differentiated into myotubes.Expression of VEGF receptor 1 and 2 mRNA in myotubes was detected by semiquantitative RT-PCR.The effect of recombinant protein(rhVEGF)of different concentrations(5,10,20ng/ml)on C2C12 myotubes was studied at various time points(6h,12 h,24h).PBS+BSA-treated cells served as control.Increased mRNA expression for the oxidative myofiber characteristic genes Myhc 2a was detected by real-time quantitative PCR.Oxidative myofibers and HUVEC were co-cultured.The capacity for tubular formation and migration of HUVEC was observed by microscope.Glucose concentrations in medium were determined by the glucose oxidase method.The expression levels of PGC-1α,COXIV and GLUT4 were determined by western blot.Results:1 Expression of VEGF receptor 1 and 2 mRNA in myotubes was confirmed by semiquantitative RT-PCR.2 The expression of oxidative myofiber(Myhc 2a)gradually increased after treatment with rhVEGF.There was a significant increased expression of mRNA for oxidative myofiber 12 hours after treatment with 20ng/ml rhVEGF(P<0.0001).The oxidative myofibers were detected by immunofluorescence.3 The change in the capacity for tubular formation and migration of HUVECGroup A(control group)HUVEC surved as a control,group B(normalC2C12 myotubes control group)C2C12 myotubes and HUVEC were co-cultured,the capacity for tubular formation and migration of HUVEC did not change significantly,showing that normal myotubes have no effect on HUVEC.Group D C2C12 myotubes was treated with 20ng/ml VEGF 12 h,then oxidative myofibers and HUVEC were co-cultured,the capacity for tubular formation and migration of HUVEC is significantly increased,compared with group C(VEGF control group),which HUVEC was treated with20ng/mlVEGF 12 h,suggest that oxidative myofibers promotes HUVEC migration and tube formation ability.There were most obvious differences between the groups at 24 h.4 Glucose consumptionCompared with the control group(0.829 ± 0.150mmol/L),myotubes glucose consumption of VEGF group(2.126 ± 0.220 mmol/L)increased significantly(P<0.05),but less than the positive control insulin group(2.918±0.182 mmol/L).5 The expression of PGC-1α,GLUT4 and COXIV myotubesWith the treatment with rhVEGF,the expression of PGC-1α,GLUT4 and COXIV in myotubes was significantly increased.Conclusions:1 Endurance training induces increased VEGF expression,angiogenesis,oxidative myofiber transformation,and higer oxidative metabolic activity.2 VEGF induces fiber type switching,increases blood perfusion,meantime oxidative metabolic activity is enhanced.3 VEGF increases oxidative fiber and mitochondrial biogenesis by upregulating PGC-1α,and promotes the ability of glucose uptake in skeletal muscle,improves microenvironment surrounding the vascular endothelial cells,thus enhancing the function of endothelial cells.4 Through the regulation of skeletal muscle fiber types mediated by VEGF,we can enhance the function of endothelial cells,promote angiogenesis in the lower limb,and finally improve diabetic lower limb ischemia.Thisresearch lays a theoretical and experimental foundation for future clinical application of gene therapy,and will solve the problem of glucose metabolism disorder and lower limb ischemia in diabetic patients who can not exercise.