1.Effects Of VEGF On Fatty Acid Metabolism During Cardiac Hypertrophy 2.Effects Of VEGF-B On Inducing Cardiac Hypertrophy

PARTⅠEffects of VEGF on fatty acid metabolism during cardiac hypertrophyBackgroundCardiac hypertrophy is a response of the heart to a wide range of extrinsic stimuli,such as arterial hypertension,valvular heart disease,myocardial infarction,and cardiomyopathy. But the response is not infinite and the heart function will be affected without removal of the etiopathogenisis.Sometimes,it could cause sudden death.Usually cardiac hypertrophy is associated with an increase in glucose utilization and a decrease in fatty acid oxidation.That the changes in intracellular substrate and metabolite levels in cardiomyocytes are a consequence or the reason for cardiac hypertrophy is still unknown. However,not some single factor plays the whole role in the development of cardiac hypertrophy.Defects in mitochondrial fatty acid oxidation enzymes cause childhood hypertrophic cardiomyopathy,and perturbation of fatty acid oxidation in animal models causes cardiac hypertrophy,demonstrating that substrate utilization is important in the pathogenesis of hypertrophy.Vascular endothelial growth factor(VEGF) was first found by Ferrara and Gospodarowicz in 1989.It is a sub-family of growth factors,more specifically of platelet-derived growth factor family of cystine-knot growth factors.They are important signaling proteins involved in both vasculogenesis(the de novo formation of the embryonic circulatory system) and angiogenesis(the growth of blood vessels from pre-existing vasculature).It was reported that VEGF was involved in the process of myocardial hypertrophy.Peroxisome proliferator-activated receptors(PPARs) are ligand-activated transcription factors that regulate the expression of genes involved in fatty acid uptake and oxidation, and inflammation.The PPAR subfamily consists of three subtypes,PPARα,PPARβ,and PPARγ.And it has been certified that PPARβis the most important subtype in cardiac cells and plays a prominent role in the regulation of cardiac lipid metabolism. Till now,there is no report about the effects of VEGF on fatty acid oxidation during cardiac hypertrophy.Our research observed the effects of VEGF on PPARβand fatty acid oxidation during cardiac hypertrophy,exploring a new way for cardiac hypertrophy prevention and cure.Objective1.To establish a cardiac hypertrophy animal model,and culture Neonatal rat ventricular myocytes and H9C2 cells.2.To observe the changes of protein expression and fatty acid oxidation in cardiac hypertrophy model.3.To observe the influence of VEGF on PPARβand fatty acid oxidation.Methods1.Establish a cardiac hypertrophy animal modelThirty male Wistar rats were randomly divided into 3 groups:control group(n=8), operation group(n=8) and medicine group(n=8).They were fed a standard diet for 7 days before the studies began.Pressure overload was induced by constriction of the abdominal aorta at the suprarenal level with 7-0 nylon strings.For the other two groups, sham operations were done.And the rats in the medicine group were given VEGF inhibitor,pegaptanib.Hearts were collected 15 days later.2.Neonatal rat ventricular myocytes cultureNeonatal rat ventricular myocytes from 1 to 2 day old Wistar rats were prepared and cultured in Dulbecco's modified Eagle's medium containing 10%fetal bovine serum. Cells were randomly divided into 3 groups:control group(n=10),PE group(n=10) and medicine group(n=10).Cells of PE group were cultured with PE(phenylephrine) and cells of medicine group were culture with PE and pegaptanib.Then the cells were treated like this:1.Cells of PE group were cultured with PE and[~3H]leuCine and cells of medicine group were culture with PE,pegaptanib and[~3H]leuCine for 24h.Then the protein was isolated and tested.2.Cells of PE group were cultured with PE and 9,10-[~3H]palmitate and cells of medicine group were culture with PE,pegaptanib and 9,10-[~3H]palmitate for 24h.Then the results were got.3.H9C2 cells culture H9C2 cells culture were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum.Cells were randomly divided into 3 groups:control group(n=10), LPS group(n=10) and medicine group(n=10).Cells of LPS group were cultured with LPS and cells of medicine group were cultured with LPS and pegaptanib.4.RT-PCRThe RNA was extracted from ventricles of different group rats.The mRNA expressions of ANF and PDK4 were determined by RT-PCR.5.Western-blot analysisWestern-blot analysis was used to determine the protein expression of VEGF and PPARβ.Results1.Operation group showed a higher HW/BW and more ANF expression than control group,while the data of the medicine group were lower than those of the operation group.2.PE induced the ANF expression and its effect was reversed by VEGF inhibitor, pegaptanib.3.PE decreased neonatal rat cardiomyocytes fatty acid oxidation,and its effect was reversed by VEGF inhibitor,pegaptanib.4.LPS increased the expression of VEGF and decreased the expression of PPARβ,and pegaptanib decreased the expression of VEGF and increased the expression of PPARβ.Conclusion1.A cardiac hypertrophy animal model was established by 15 days of constriction of the abdominal aorta in male Wistar rats.2.VEGF expression was increased,PPARβexpression was decreased,and fatty acid oxidation was decreased during cardiac hypertrophy.While,all the results were reversed by VEGF inhibitor,pegaptanib.3.VEGF inhibitor can promote the fatty acid oxidation of hypertrophic cardiomyocytes. PARTⅡEffects of VEGF-B on inducing cardiac hypertrophyBackgroundPathologic cardiac hypertrophy develops in response to increases in afterload and represents a common intermediary in the development of heart failure.Left ventricular hypertrophy is an independent risk factor for several adverse outcomes,including cardiac mortality,arrhythmias,and myocardial infarction.Results from numerous studies suggest that reducing heart mass in patients with pathologic cardiac hypertrophy may reduce morbidity and mortality and improves patient outcomes.However,the only treatments currently proven to reverse both structural and functional cardiac abnormalities associated with pathologic cardiac hypertrophy are anti-hypertensive therapies and aortic valve replacement(for aortic stenosis),both of which have a limited success rate. Understanding the underlying processes regulating plasticity of the heart will allow us to identify specific pathways against which to target new therapies and may improve the long-term outcomes of patients with pathologic cardiac hypertrophy.Vascular endothelial growth factor(VEGF) was first found by Ferrara and Gospodarowicz in 1989.Members of the vascular endothelial growth factor(VEGF) family,currently comprising 6 mammalian proteins,are major regulators of blood and lymphatic vessel development and growth.VEGF is essential for vasculogenesis and angiogenesis.VEGF-B has a wide tissue distribution,being most abundant in the myocardium,skeletal and vascular smooth muscle.VEGF-B has also been implicated in protecting the brain from ischemic injury.However,the ability of VEGF-B to stimulate angiogenesis directly is poor in many tissues.And role of VEGF-B was studied in various models of pathological angiogenesis using mice lacking VEGF-B or overexpressing VEGF-B,which suggested that VEGF-B having a relatively restricted angiogenic activity in the ischemic heart.VEGF-B did not stimulate vessel growth when delivered into muscle or periadventitial tissue via adenoviral vectors.On the contrary,VEGF-B overexpressed in endothelial cells of transgenic mice was able to potentiate,rather than initiate,angiogenesis.And unlike VEGF,VEGF-B did not increase vascular permeability. VEGF-B exists as 2 isoforms,VEGF-B167 and VEGF-B186,generated by alternative splicing.Both isoforms bind to VEGF receptor(VEGFR)-1 and neuropilin-1 but not to the major mitogenic endothelial cell receptors VEGFR-2 or VEGFR-3.Here we have studied the effects of VEGF-B on inducing cardiac hypertrophy with mice overexpressing VEGF-B,exploring a new way for cardiac hypertrophy prevention and cure.Objective1.To establish a VEGF-B overexpression animal model.2.To establish a cardiac hypertrophy reversal animal model.3.To establish a cardiac atrophy animal model.4.To explore the effects of VEGF-B on inducing cardiac hypertrophy by different index examination.Methods1.Establishment of the VEGF-B overexpression animal model VEGF-B overexpression mice were gift from another lab.We got the VEGF-B+/+ homozygote mice by mating and PCR genotyping.2.Establishment of the cardiac hypertrophy reversal animal model Forty VEGF-B+/+ mice were randomly divided into 2 groups:experiment group(n=20) and experiment-sham group(n=20);and Forty wild type mice were randomly divided into 2 groups:control group(n=20) and control-sham group(n=20).Mice of experiment group and control groups were anesthetized,and a longitudinal incision was made at the level of the suprasternal notch,allowing for visualization of the aortic arch,left carotid, and innominate artery.An 8-0 suture was used to encircle the aorta between the origin of the left carotid and the innominate arteries.One end of the suture was cut short while the other was left longer,lying on top of the pretracheal muscle and underneath the skin for easy future removal according to the schedule.For the other two groups,sham operations were done.3.Establishment of the cardiac atrophy animal model VEGF-B+/+ mice were randomly divided into 2 groups:experiment group(n=12) and experiment-sham group(n=12);and wild type mice were randomly divided into 2 groups: control group(n=12) and control-sham group(n=12).Mice of experiment group and control groups were given dexamethasone subcutaneous injection daily for 2 weeks;and the mice of the other two groups were given saline vehicle subcutaneous injection daily for 2 weeks.4.Echocardiogram examinationAccording to the schedule,transthoracic echocardiogram was performed.Mice were placed supine and the anterior chest wall was shaved.Echocardiograms were performed. Conventional images included 2-dimensional,M-mode,and continuous wave and pulsed Doppler images.5.Hemodynamic assessment of aorta flowTo assess the aortic constriction after TAC and TAC reversal,right and left carotid artery flow was assessed using a 20 MHz probe driven by a high frequency pulsed Doppler signal processing workstation.6.Real-time RT-PCRThe RNA was extracted from ventricles of different group rats.The mRNA expressions of BNP andβMHC,etc.were determined by real-time RT-PCR.7.Histology and stainingHearts were perfused and processed for histology and stained with H&E or lectin.Cell area was determined.Results1.The HW/BW,LVM/BW and thickness of ventricular walls in the cardiac hypertrophy reversal animal model:no change happened on control-sham group;values of experiment group were higher than those of control group and reached their peak at 4W;the values of experiment-sham group reached their peak at 1WR.2.Hemodynamic assessment of aorta flow:the VR/VL of control group and experiment group increased after TAC and decreased after TAC reveal;the VR/VL of control-sham group and experiment-sham group did not change.3.Histology showing the thickness of the ventricles in the cardiac hypertrophy reversal animal model:no change happened on control-sham group;the value of experiment group was higher than that of control group and reached its peak at 4W;the value of experiment-sham group reached its peak at 1WR.4.Histology showing cross section of muscle bundles:at 4W,experiment group＞experiment-sham group＞control-sham group＞control group;at 4WR,experiment group=experiment-sham group＞control-sham group=control group.5.Cardiomyocyte area in the cardiac hypertrophy reversal animal model:no change happened on control-sham group;the value of experiment group was higher than that of control group and reached its peak at 4W;the value of experiment-sham group reached its peak at 2WR.6.βMHC mRNA,BNP mRNA,SMA mRNA,TIMP1 mRNA,TIMP2 mRNA and MMP2 mRNA expression:the values of experiment group and control group reached their peak at 4W,and the values of experiment-sham group reached their peak at 2WR.7.AWDA,PWD,AWS,PWS,LVM/BW,LVW/TL and HW/BW in the atrophy model: the values of control group were smaller than the others,and the values of experiment-sham group were larger than the others.8.Histology showing the ventricle thickness in the atrophy model:experiment group and control-sham group had no change;control group got thinner ones,and experiment-sham group got thicker ones.9.Cardiomyocyte area in the atrophy model:experiment group and control-sham group had no change;control group got smaller ones,and experiment-sham group got larger ones.Conclusion1.The models of VEGF-B overexpression,cardiac hypertrophy reversal,and cardiac atrophy were successfully established.2.In the cardiac hypertrophy reversal animal model,VEGF-B overexpression induced cardiac hypertrophy and its effect was attenuated little after cardiac hypertrophy reversal.3.In the cardiac atrophy animal model,VEGF-B overexpression neutralized the atrophy inducing effect of dexamethasone.4.VEGF-B can induce cardiac hypertrophy.