The Function And Mechanism Study Of Aerobic Exercise-induced FSTL1 In Promoting Cardiac Angiogenesis In LDLR-KO Mice

BackgroundCoronary microvascular disease is a clinical syndrome in which the structure and function of the anterior and small arteries of the coronary artery are abnormal,leading to angina pectoris or myocardial ischemia.Endothelial dysfunction is the main pathogenesis of coronary microvascular disease.In atherosclerosis and coronary microvascular disease caused by high levels of low-density lipoprotein(LDL),high concentrations of LDL in the blood could be internalized by endothelial cells and oxidized into oxidized LDL(ox-LDL).Ox-LDL can cause the expression of inflammatory factors increased,endothelial dysfunction,and functional abnormalities of the anterior and small arteries.Promoting angiogenesis is undoubtedly an effective way to treat coronary microvascular disease.Multiple clinical studies have confirmed that aerobic exercise not only alters the metabolism of blood lipids and glucose in the host but also regulates the expression and distribution of angiogenesis-related cytokines,thereby inducing angiogenesis.Previous studies have shown that myokines secreted by skeletal muscles may be the main molecular mechanism.Follistatin-like 1(FSTL1)is one of the most important myokines,which has been shown to promote angiogenesis in several diseases.However,it is not clear whether FSTL1 can promote angiogenesis in coronary microvascular disease.Therefore,the study of the mechanism of FSTL1 on ox-LDLinduced endothelial cell dysfunction and its effects on the process of angiogenesis is beneficial for understanding clinical aerobic exercise rehabilitation therapy,providing new treatment strategies and potential targets for the clinical treatment of coronary microvascular disease.Objectives1.To explore the pathology of coronary microcirculation in low density lipoprotein receptor-knockout(LDLR-KO)mice and establish an animal model of coronary microvascular disease.2.To explore the effect of aerobic exercise on coronary microcirculation of LDLRKO mice,and explore the role of aerobic exercise-induced FSTL1 in it.3.To explore the effect of FSTL1 on the function of vascular endothelial cells using in vivo and in vitro experiments.4.To explore the role and possible mechanism of FSTL1 in ox-LDL-induced dysfunction of vascular endothelial cells.Methods1.LDLR-KO mice were fed a high-fat diet to establish a coronary microvascular disease model.Observe the degree of coronary microvascular lesion,cardiac vascular density,and cardiac function changes of model mice after aerobic exercise training,and evaluate the effect of aerobic exercise on coronary microcirculation of LDLR-KO mice fed with the high-fat diet.2.The weights,lipid,and glucose concentrations of LDLR-KO mice fed with the high-fat diet after aerobic exercise were measured to determine the protective factors of aerobic exercise in improving coronary microvascular disease.3.FSTL1 concentrations in serum,skeletal muscle,and heart tissue of LDLR-KO mice fed with the high-fat diet after aerobic exercise were detected.After inhibiting the expression of FSTL1 in mice with adeno-associated virus(AAV),FSTL1 concentrations in heart tissue were also detected to verify the main source of FSTL1 in heart tissue during aerobic exercise.4.After inhibiting the expression of FSTL1 in LDLR-KO mice fed with the highfat diet,aerobic exercise training was conducted to evaluate the effect of aerobic exercise-induced FSTL1 on coronary microcirculation by observing the changes in cardiac vascular pathology,cardiac vascular density,and cardiac function.At the same time,angiogenesis-related proteins in heart tissue were detected to evaluate the relationship between aerobic exercise-induced FSTL1 and angiogenesis-related proteins.5.Using Ox-LDL to stimulate human umbilical vein endothelial cells(HUVEC)to mimic the dysfunction of vascular endothelial cells under high blood lipid conditions.the effect of FSTL1 on the angiogenesis and the expression of the angiogenesis-related protein of HUVEC were observed.Results1.After feeding with the high-fat diet,LDLR-KO mice can form obvious atherosclerotic plaque in the aorta,the coronary microvascular wall was infiltrated with foam cells,besides,inflammatory cells can be seen around coronary microvascular,and lipid vacuoles can be seen in the myocardium.2.After feeding with the high-fat diet,the microvessel density in the heart tissue of LDLR-KO mice was lower than that of C57BL mice.3.After aerobic exercise training,the blood LDL concentration and blood glucose level of LDLR-KO mice were not significantly different compared to control mice.4.After aerobic exercise training,the left ventricular ejection fraction of LDLRKO mice was significantly higher than that of control mice,and the density of blood vessels between myocardium was also increased significantly.5.After aerobic exercise training,the concentration of FSTL1 in blood,myocardial and coronary microvascular wall of LDLR-KO mice was significantly higher than that of control mice.However,after inhibiting the expression of FSTL1,aerobic exercise could not increase the concentration of blood and myocardial FSTL1 in LDLR-KO mice,while the concentration of FSTL1 in coronary microvascular wall was decreased.6.After aerobic exercise training,no obvious foam cells were found in the coronary microvascular wall of LDLR-KO mice compared with control mice.However,after inhibiting the expression of FSTL1,foam cells still existed in the coronary microvascular wall of LDLR-KO mice after aerobic exercise.In addition,after inhibiting the expression of FSTL1,LDLR-KO mice also had lower cardiac ejection fraction and lower cardiac vascular density compared to control mice after aerobic exercise training.7.After aerobic exercise training,the expression of vascular endothelial growth factor A(VEGFA)and phospho-endothelial nitric oxide synthase(p-eNOS)in the heart tissue of LDLR-KO mice was increased compared to that in control mice,however,inhibiting the expression of FSTL1 decreased the expression of vascular VEGFA and eNOS in the heart tissue after aerobic exercise training.8.FSTL1 treatment for 24 hours and 48 hours can promote the proliferation of HUVEC cells in a concentration-dependent manner.9.FSTL1 can significantly increase the ability of HUVEC to form tubules,while ox-LDL significantly decreased the ability of HUVEC to form tubules and even leads to cell death.FSTL1 pretreatment can reduce the damage of ox-LDL to HUVEC.10.FSTL1 treatment for 48 hours increased the expression of VEGFA and peNOS in HUVEC,and this effect could be blocked by inhibiting Akt signalingConclusions1.LDLR-KO mice could develop coronary microvascular lesions after high-fat feeding.2.Aerobic exercise can improve coronary microvascular lesion and promote cardiac angiogenesis in LDLR-KO mice.3.FSTL1 induced by aerobic exercise has cardiovascular protective effect on LDLR-KO mice.4.FSTL1 promotes angiogenesis by improving endothelial cell function via Akt pathway.Innovation and significanceThis study was the first to explore the protective effect of FSTL1 induced by aerobic exercise on coronary microvessels of LDLR-KO mice fed with the high-fat diet.FSTL1 has been shown to promote angiogenesis in vivo and in vitro,and may become a new strategy and molecular target for the treatment of coronary microvascular disease by promoting angiogenesis.This study provides an experimental basis for the use of FSTL1 in the treatment of coronary microvascular diseases.