Improvement Effects And Mechanism Of Diagnostic Ultrasound Combined With Microbubbles On Myocardial Angiogenesis In Rat Model Of Diabetic Cardiomyopathy

Part Ⅰ Effect of Diagnostic Ultrasound Combined with Microbubbles on the Activity of High Glucose Human Umbilical Vein Endothelial Cells Cultured with High Glucose and its MechanismBackgroundDiabetes mellitus(DM)is an endocrine and metabolic disease caused by high blood glucose levels,which can lead to endothelial dysfunction,myocardial fibrosis and oxidative stress,accelerate the process of atherosclerosis and endogenous myocardial cell damage.It eventually causes a set of changes in the myocardial structure and function,resulting diabetic cardiomyopathy(DCM).DCM is a common cardiovascular complication independent of coronary artery disease and hypertension.Vascular lesions are important for the pathological basis of DCM.Vascular endothelial cells attaching to the inner wall of blood vessels should keep the integrity of the vascular wall and homeostasis,which directly affect the procession of DCM.Studies have shown that high glucose in vitro can lead to apoptosis of vascular endothelial cells and destroy the integrity of endothelial cells.PI3K-Akt-eNOS pathway may be involved in diabetic endothelial cell dysfunction.Therefore,it is important to do some research to improve vascular endothelial function and inhibit the apoptosis of vascular endothelial cells,in order to prevent the development of DCM.Diagnostic ultrasound(DUS)is a kind of low-intensity ultrasound(LIUS),which has long been used in clinic safely.Ultrasound targeted microbubble destruction(UTMD)technology,which uses ultrasound exposure to release of drug and gene,has been developed as a noninvasive and target specific method in angiogenesis therapy of cardiovascular disease.LIUS in combination with microbubbles has been shown to increase vascular endothelial growth factor(VEGF),inducing angiogenesis,used in ischemic diseases.This study is detected low intensity diagnostic ultrasound combined microbubble contrast agent can protect the injury by high glucose of human umbilical vein endothelial cells(HUVECs),and investigate the mechanisms whether is related to the activation of PI3K-Akt signal pathways.ObjectiveTo observe the effects of diagnostic ultrasound combined with microbubbles on HUVECs in migration ability and the expression of PI3K,Akt and eNOS under high glucose condition,and investigate the possible molecular mechanism of the effects of diagnostic ultrasound combined with microbubbles on HUVECs activity.MethodHUVECs were cultured with HG(35mmol/L glucose)for the indicated time(24 h)to establish the injury model.The cells were divided into four groups:normal group,MB group,Glucose group and VFLISH group.Cell migration was evaluated by cell scratch test,and the expression proteins of PI3K,Akt and eNOS were detected by immunofluorescence method.The parameters of ultrasonic irradiation were 3.Ocycle,50%sound power,0.82MI,and 1 min,which determined determined by the previous cell-volume effect-aging experiment.ResultsThe migration rate of endothelial cells was time-dependent.There was no significant difference in cell migration rate between the MB group and the Normal group at the same observation time point(all P>0.05).At the observation points at 3h,6h,9h and 12h,the migration rate of endothelial cells in the Glucose group showed statistical differences compared with the Normal group(P=0.045;P=0.003;P=0.004;P<0.001).The migration rate of endothelial cells in the VF1ASH group increased significantly at 6h,9h and 12h,which was significantly different from that in the Glucose group(P=0.04;P=0.033;P=0.047).There was no significant difference in the expression of PI3K,AKT and eNOS between the Normal group and the MB group by immunofluorescence using fluorescence labeling of cell marker proteins.(all P=1.000).The expression of PI3K,Akt and eNOS were significantly decreased in Glucose group compared with the Normal group and the MB group(all P<0.001).The expression of PI3K,Akt and eNOS were significantly increased in VF1ASH group compared with Glucose group(P<0.001;P=0.003;P<0.001).Compared with the Normal group,there were no statistical differences in the level of PI3K and eNOS in the VF1ASH group(P=0.149;P=0.229),while the expression of Akt was significantly decreased(P<0.001).Conclusion1.High glucose can decrease the HUVECs cell activity and inhibit the cell migration ability,which were significantly lower than that of normal group.The microbubble contrast agent did not inhibit the cell proliferation and it was safe for endothelial cells.2.Diagnostic ultrasound combined with microbubbles can increase the activity of human umbilical vein endothelial cells in high glucose state,and the mechanism may be related to the activation of PI3K-Akt-eNOS signaling pathway,to provide some theoretical support for the clinical transformation of the technology.Part Ⅱ:Diagnostic Ultrasound Combined with Microbubbles Promotes Myocardial Angiogenesis and Functional Improvements in Rat Model of Diabetic CardiomyopathyBackgroundMicrovascular insufficiency plays an important role in the development of DCM,which is mainly manifested by abnormal microcirculation anatomical function,resulting in reduced myocardial perfusion and reduced cardiac function.Therapeutic angiogenesis has been mainly used for the treatment of ischemic diseases,but there is still a lack of safe and efficient delivery system in the future clinical application.The process of angiogenesis starts with the activation of endothelial cells,then proliferates and migrates,and promotes angiogenesis in existing small vessels.In the first part of the study,we found that DUS combined with MB can significantly improve the viability and migration of endothelial cells,protect endothelial cells and regulate angiogenesis.This study sought to verify the preclinical performance of SonoVue microbubbles(MB)combined ultrasound(US)treatment on myocardial angiogenesis in the rat model of DCM to improve the myocardial microcirculation,so as to achieve the purpose of effective treatment of diabetic cardiomyopathy.Maybe this combined strategy might be a promising option for early intervention of DCM in diabetic patients and provide a new,non-invasive,effective,safe method for the treatment of diabetic cardiomyopathy.ObjectiveThis study sought to observe the intervention effect of SonoVue microbubbles(MB)combined ultrasound(US)on cardiac function in the rat model of DCM,observe the effects of different cavitation parameters on myocardial angiogenesis,and investigate the optimal ultrasonic parameters to improve the myocardial microcirculation of DCM.MethodsThe male Sprague-Dawley(SD)rats were induced DCM by streptozotocin through intraperitoneal injecting and fed with high-fat diet.We detected the body weight and blood sugar of the rats,evaluate d the pathological changes of the pancreas and myocardium to determine whether the DCM model was successfully constructed.After the DCM model was established,the rats were divided into the normal group,DCM model group,and US+MB group,while the US+MB group was divided into four subsets according to different pulse lengths(PL)(8 cycles;18 cycle;26 cycle;36 cycle).After all interventions,all rats underwent conventional echocardiography to examine the cardiac function.The rats were sacrificed and myocardial tissue was examined by histology and morphometry evaluations to detect the myocardial protective effect of SonoVue MBs using US techniques.The ultrastructural changes of the cardiac microvascular were examined under electron microscope.CD31 immunohistochemistry was used to identify the myocardial capillary density(MCD)and evaluated the improvement of DCM myocardial microvascular generation using US+MB.ResultsWe successfully made the DCM rats model,found that the myocardial cells in the DCM Model group showed hypertrophy,fibrosis,and focal necrosis using stained from the pathologic result.Endothelial cells of small arteries were hyperplasia and degeneration in myocardium,as well as inflammatory cell infiltration,myocardial capillary lumen narrowed,thickening of basement membrane.The collagen volume fraction(CVF)of myocardium increased significantly compared with normal group(P<0.001).After two weeks of intervention treatment,myocardial hypertrophy,fibrosis and cardiomyocytosis were significantly reduced in the US+MB group.Collagen fiber was significantly reduced in the 26cycle group and the 36cycle group,and it showed that there were no statistically significant difference in CVF compared with that in normal group(P=0.450;P=0.534).The ultrastructural changes of the cardiac microvascular were examined under electron microscope.We found that the surface of cardiac microvascular in the DCM model group was more burred and continuality was missing,the endothelial cell membrane was incomplete,and the heterochromatin of the inner nucleus increased and agglomerated,which indicated the pathological state of these microvessels.The damage of myocardium arterioles in DCM rats was improved after US+MB intervention.The traditional echocardiographic measurements showed that there was no significant difference in LVIDd and IVS among all the groups(F=0.567,P=0.725;F=1.053,P=0.394),and there was statistically significant difference in LVEF and LVFS in the DCM model group and normal control group(P=0.002;P<0.001).A distinct improvement was observed in DCM model rats in the levels of the ultrasonic measurements after UTMD intervention.The 26 cycle group showed the highest LVEF and LVFS after treatment,which had no statistically significance when compared to the normal group(P=0.557;P=0.463).The DCM model group demonstrated a gradual decrease in the MCD of myocardial tissue when compared with that in the control group(P<0.001).Moreover,each US-MBs intervention group showed significant increase in MCD than that in the DCM control group,the level of MCD in the 26 cycle group showed highest level among the US-MB groups and no statistically significant difference compared to the normal group(P=0.625).Conclusion1、From morphologic observation and echocardiography,the DCM rats had a series of structural abnormalities of cardiac myocardium compared to the normal rats,which further verified the damage of hyperglycemia to the myocardium of rats.2、The US-MB groups exerted cardioprotective effect in DCM rats,improve the structure and function of myocardium,while the 26 cycle group showed significant therapeutic effects on the cardiac functions in DCM rats,and which may be related to the increase of myocardial microcirculation perfusion.3、This strategy using SonoVue MB and US can improve the efficacy of angiogenesis,improved reparative neovascularization and increased cardiac perfusion,even reverse the progress of cardiac dysfunction and pathological abnormalities,especially using the 26 cycle parameters.Under further study,this combined strategy might provide a novel approach for early intervention of DCM in diabetic patients.Part Ⅲ The Possible Mechanism of Impro ving Myocardial Angiogenesis by Diagnostic Ultrasound Combined with Microbubbles in Rat Model of Diabetic CardiomyopathyBackgroundDiabetic cardiomyopathy(DCM)is caused by metabolic disorders caused by high glucose state on myocardial microvascular damage,which can lead to impaired microvascular function.Therefore,promoting myocardial angiogenesis is important to improve myocardial function of DCM.Low intensity ultrasound can induce angiogenesis combined with microbubbles,which has been used in the treatment of ischemic cardiovascular disease.In the first study,we observed that the diagnostic low intensity ultrasound combined with SonoVue microbubbles contrast agent increased myocardial microangiogenesis and improved myocardial systolic function in DCM rats.The exact mechanism of this technique promoting angiogenesis is still not fully clear.In the first part of this study,the results show that low-intensity therapeutic ultrasound can promote the angiogenesis of human umbilical vein endothelial cells by activating the PI3K-Akt-eNOS signaling pathway.Therefore,we hypothesized that DUS combined with microbubbles may antagonize diabetic endothelial cell injury by activating PI3K-Akt-eNOS signaling pathway,promote myocardial angiogenesis,improve myocardial microcirculation,and ultimately improve cardiac function.ObjectiveIn this study,we detect the protein expressions and phosphorylation levels of PI3K,Akt and eNOS using immunohistochemistry,Western blotting and other methods,observe the possible mechanism of DUS combined with microbubbles on myocardial microcirculation in rats with DCM,so as to provide new ideas and experimental basis for the treatment of DCM.MethodsThe male Sprague-Dawley(SD)rats were induced DCM by streptozotocin through intraperitoneal injecting and fed with high fat diet.The rats were divided into three groups:normal group(normal),DCM model group(DCM model),and the SonoVue microbubble+US group(US+MB),and the US+MB group was used the pulse lengths of 26 cycle according to the research part I.After 2 weeks of intervention using diagnostic ultrasound combined with VFLASH,all rats were sacrificed and serum oxidation indexes were detected.and the expression of PI3K-Akt-eNOS pathway protein in rat myocardial tissue was detected by Western blot and immunohistochemistry.ResultsThe protein level of P13K、Akt and PI3K phosphorylation in myocardial tissue of rats in DCM model group were significantly decreased compared with normal control group,as well as the US+MB group(all P<0.001).The protein level of PI3K、Akt and PI3K phosphorylation in myocardial tissue of rats increased in US+MB group compared with DCM model group(P=0.034;P=0.040;P=0.036),while the phosphorylation level of Akt was not significantly different(P=1.000).the protein level of eNOS and p-eNOS were slightly increased compared with US+MB group and DCM model group,but the difference was not statistically significant(P=0.344;P=1.000).Immunohistochemical results generally showed that the protein expressions of PI3K,Akt and eNOS in the myocardium of rats in the DCM model group and the US+MB group were significantly decreased compared with the normal group(all P<0.001).The levels of PI3K and eNOS in myocardium of rats in US+MB group were higher than those in DCM model group,which showed significant difference with P value of 0.04 and 0.027.The level of Akt was higher than those in DCM model group,but was not significantly different(P=1.000).Conclusions1.High glucose can affect the expression of PI3K-Akt-eNOS signaling pathway protein in the myocardium of DCM rats,which affect the myocardial microvascular perfusion,leading to myocardial function impairment.2.DUS combined with MB may promote the synthesis and release of downstream factor eNOS through activation of PI3K-Akt pathway,increase myocardial angiogenesis,improve myocardial microvessel density,enhance myocardial microcirculation to some extent,and thus protect myocardial function.