^99mTc-3prgd2 Molecular Imaging Targeting Integrin ?v?3 In The Evaluation Of Doxorubicin-induced Myocardial Fibrosis In Rats

Objective:Anthracyclines can cause myocardial fibrosis through a variety of ways.Myofibroblast(MFB)is the main source of excessive collagen deposition in the process of myocardial fibrosis.Monitoring MFB by imaging specific cell markers is an effective method to predict myocardial fibrosis.^99mTc-3PRGD2 is a ^99mTc labeled dimer arginine-glycine-aspartate(RGD)-imaging peptide(RIP)targeting integrin?v?3 on the surface of MFB.This study consists of two parts,to explore 1)the feasibility of^99mTc-3PRGD2 in evaluating myocardial fibrosis induced by doxorubicin;2)the diagnostic value of ^99mTc-3PRGD2 in the progression of myocardial fibrosis.Methods:Part I:Twenty male Sprague Dawley(SD)rats were randomly divided into control group(n=10)and experimental group(n=10).The experimental group was given intraperitoneal doxorubicin injection of 1.25 mg/kg twice a week for 6 weeks.The control group was given the same amount of normal saline.Afterbolusinjection of^99mTc-3PRGD2(74–111MBq)via the tail vein,the planar dynamic anteroposterior images were acquired immediately.The acquisition lasted for 60 minutes and 60consecutive images were obtained.Thespecific binding of ^99mTc-3PRGD2 to integrin?v?3 was further confirmed by integrin receptor inhibition test usingc(RGDy K).The region of interest(ROI)of heart(H)and background(B)of mediastinum were drawn respectively,and the ROI was copied into the subsequent 59 frames by GE Xeleris workstation.The time radioactivity curve of cardiac ROI and half life time of heart(Heartt_1/2)were obtained and the heart counts percentage(%Heart)were calculated by dividing the heart ROI radioactivity count of each frame of the subsequent 59 images by that of the first frame,%Heart of 20 min,40 min and 60 min were recorded as%Heart_20min,%Heart_40min and%Heart_60min,respectively for subsequent analysis.Then the heart to background ratio(HBR)of each frame was calculated.HBR at 20 min,40min and 60 min were recorded as HBR_20min,HBR_40min and HBR_60min respectively for subsequent analysis.After the single photon imaging,the myocardial tissue was taken,frozen or fixed in neutral formaldehyde.HE staining was used to evaluate the degree of myocardial injury.The degree of myocardial fibrosis was evaluated by Masson staining.Immunofluorescence was used to detect the expression of integrin?v?3 in myocardium;Western blotting was used to detect the protein expression of the two groups,including?-SMA,integrin?v subunit and?3 subunit.The internal reference protein was GAPDH.Student t test or Mann-Whitney test were used to compare the image parameters between experimental group and the control group.P<0.05 was noted as statistical significance.Part II:Thirty male SD rats were randomly divided into baseline group(n=6)and model group(n=24).The model group was given intraperitoneal doxorubicin injection of 1.25mg/kg twice a week for 6 weeks.The baseline group was not treated.Echocardiography was performed within 48 hours before single photon imaging.Fraction shortening(FS)and left ventricular ejection fraction(LVEF)were used to evaluate left ventricular systolic function.Diastolic function was evaluated by the peak velocity of mitral valve in early diastole(E),peak velocity of mitral valve ring of lateral wall(e')and E/e'.Single photon imaging was performed in the baseline group(n=6)before administration(recorded as week 0).3rd,6th and 9th week after administration,at most 6 rats in the model group were randomly selected for single photon imaging at each time point and noted as 3-week group,6-week group and 9-week group,respectively.After single photon imaging,blood samples(about 0.2ml)were collected from the tail vein immediately,and then the rats were sacrificed to remove the heart and other major organs(liver,spleen,lung,intestine and kidney).The uptake of ^99mTc-3PRGD2 in venous blood and organ tissue samples was measured by gamma counter,and the percentage injected dose per gram of the tissue(ID/g)was calculated.Myocardial tissue sampling,myocardial pathological staining and image analysis were the same as Part I.Three rats in each group were randomly selected for Western blot analysis for?-SMA,integrin?v and?3 subunit,CD31 and CD68,and the internal reference protein was GAPDH.Univariate analysis of variance(ANOVA)and post hoc student Newman Keuls test/Kruskal Wallis test and post hoc Conover test were used to compare the differences of cardiac function parameters,myocardial injury score,CVF,%ID/g and imaging parameters among the groups.Pearson test was used to analyze the correlation between%ID/g and integrin?3 subunit expression.P<0.05 was noted as statistical significance.Results:Part I:Six rats in the experimental group and six rats in the control group completed all the scanning.In the two groups,the radioactivity of cardiac ROI was the highest in the first frame,and then gradually decreased,and the radioactive tracer was discharged through the urinary system.%Heart and HBR curves began to decrease rapidly,and then gradually slowed down.At each time point,the concentration of radionuclide of cardiac ROI in the experimental group was higher than that in the control group.After blocking the receptor with c(RGDy K),the uptake of ^99mTc-3PRGD2 in soft tissues and solid organs decreased significantly,similar to the background.At the same time,the excretion of imaging agent was accelerated.There were significant differences in Heartt_1/2 and%heart and HBR at each evaluated time point(20 min,40 min and 60min)between the control group and the experimental group.Compared with the experimental group,%heart and HBR of the experimental+blocking group were significantly decreased at the evaluated time points.HE staining showed disarranged myocardial cells with myofibrillar loss and vacuolization in the experimental group,and the median score was 0.5(1.0,2.0).In the control group,the cells were arranged orderly,without obvious above-mentioned appearance,and the median score was 0(0,0).There was significant difference between the two groups(M-W test,P<0.0001).Masson staining showed that myocardial interstitial fibrosis was obvious in the experimental group,but rare in the control group except perivascular myocardial interstitial fibrosis.CVF in the experimental group was significantly higher than that in the control group(8.00±2.18%vs 1.91±0.26%,M-W test,P<0.0001).In the control group,integrin?v?3 was mainly expressed in vascular structure and overlapped with?-SMA+/desmin+vascular smooth muscle.In addition to vascular structure,MFBs(?-SMA+/desmin-)expressed integrin?v?3 in the experimental group.Western blot showed that the expression of integrin?v,?3 subunit and?-SMA in the experimental group were significantly higher than those in the control group.Part II:All animals in the baseline group(n=6)completed the experiment successfully.There were 6,6 and 5 animals in 3-week group,6-week group and 9-week group respectively.Echocardiography showed that there was no significant difference in cardiac function parameters between the 3-week group and the baseline group,suggesting that left ventricular systolic function and diastolic function were maintained in this group.FS,LVEF,E and e'of the 6-week group were lower than those of thebaseline group,and E/e'was significantly higher than that of the baseline group.In the 9-week group,FS and LVEF were further decreased compared with the 6-week group,and E/e'was further increased compared with the 6-week group,suggesting that the systolic and diastolic functions of the 9-week group were further decreased compared with the 6-week group.At each time point,the concentration of radionuclide of cardiac ROI in each model group was higher than that in the baseline group.In the pairwise comparison of imaging parameters,the Heartt_1/2 of 9-week group was significantly longer than any other group.There was no significant difference in%heart at each evaluated time point(20 min,40min and 60 min)between the 3-week group and the baseline group;compared with the baseline group or the 3-week group model,%Heart of the 6-week and 9-week groups were higher at each evaluated time point.In addition,compared with the baseline group,the HBR at each time point in the 3-week group were significantly higher,the HBR in the 6-week group was further higher than that in the 3-week group,and the HBR in the9-week group was lower than that in the 6-week group.HE staining showed disarranged myocardial cells with myofibrillar loss and vacuolization in the 3-week group,and then the degree of myocardial injury aggravated with time.Masson staining showed that the myocardial interstitial fibrosis in the 3-week group was slightly increased than that in the baseline group,significantly aggravated in the 6-week group,and more severe in the9-week group,while CVF was significantly increased correspondingly.The expression of integrin?v,?3 subunit and?-SMA in model group were significantly higher than those in baseline group.In the model group,the expression of?v,?3 subunit and?-SMA peaked in the 6-week group and decreased in the 9-week group.The expression of CD31(endothelial marker)in each model group was lower than that in the control group.The expression of CD68(macrophage marker)increased slightly in the 3-week group but returned to the baseline level in the 6-week/9-week groups.This suggested that the up regulation of integrin?v?3 expression in the model group was mainly due to the activation of MFB.Compared with the baseline group,the myocardial%ID/g of the3-week group was significantly increased,and the myocardial%ID/g of the 6-week group was further increased.Compared with the 6-week group,the mean value of%ID/g in the 9-week group was slightly lower,but not statistically significant(P=0.42).Compared with the baseline group,liver%ID/g and blood%ID/g were higher in the6-week and 9-week groups,while there was no significant difference in spleen,lung,intestine or kidney%ID/g.Myocardial%ID/g was significantly correlated with integrin?3 expression with r=0.90(95%CI:0.68-0.97,P=0.0001).Since high blood%ID/g could affect myocardial imaging agent uptake in the 9-week group,this study further evaluated the correlation between integrin?3 expression and myocardial/blood%ID/g ratio(Pearson test,coefficient r=0.91,95%CI:0.71-0.98,P<0.0001).Conclusion:1.^99mTc-3PRGD2 single photon imaging can specifically target MFB in the process of doxorubicin induced myocardial fibrosis for molecular imaging;2.^99mTc-3PRGD2 single photon imaging has early diagnostic value for doxorubicin induced myocardial fibrosis,which can be earlier than the cardiac function parameters measured by conventional echocardiography.HBR is a better semi-quantitative parameter.