In Vivo Angiogenesis Imaging And Multi-Modality Image Fusion Post Myocardial Infarction And Intervention In Minipig Heart

BackgroundCoronary artery disease and subsequent myocardial infarction(MI) are two leading causes of morbidity and mortality in the world. Although advances in MI therapy have improved acute survival rates, survivors are prone to development of chronic degenerative changes as a result of irreversible cardiomyocytes loss, extracellular matrix fibrosis and pathological gene programming. The active seek of an effective treatment, especially biological treatment brings hope to the recovery of myocardial infarction and heart failure induced by left ventricular remodeling. A new treatment must go through the preclinical studies on large animal models before it is used clinically. Large animal model of chronic MI with long–term postoperative survival is very important to study the molecular and cellular changes following MI and to test novel therapies for prevention of chronic negative myocardial remodeling. Traditional MI animal modeling is established via interventional balloon occlusion techniques or open chest surgery to block the blood flow of left anterior descending coronary artery(LAD). However, some issues and potential pitfalls should be recognized such as high incidence rate of malignant ventricular arrhythmias and less induced infarct size. In recent years, numerous large animals’ MI modeling have been proposed and developed. But to our knowledge, these methods still need further optimization.Angiogenesis imaging and multi-modality image fusion have been great progress for medical imaging and molecular imaging technology recently. It provides in vivo observation and comprehensive evaluation for myocardial infarction and the angiogenic process after MI from the structure, function and molecular level, which is a powerful tool for preclinical studies. Among a variety of targets for angiogenesis imaging, αvβ3-integrin is most studied. Many arginine-glycine-aspartic acid(RGD)-containing peptides probes with high affinity and selectivity using as specific ligand for αvβ3-integrin have been developed. Among them, NOTA-PRGD2 have shown optimal combinations of receptor binding, blood clearance kinetics and biodistribution. In our previous study, the tracer NOTA-PRGD2 could visualize and quantify angiogenesis in myocardial ischemia/reperfusion rats longitudinally, which showed the same trend with autoradiographic imaging and immuno-histochemistry staining ex vivo. Another pilot clinical study indicated that 68Ga-NOTA-PRGD2 pachy uptake was found at or around the ischemic region in patients post-MI and the uptake level was correlated with the phase and severity of MI. However, repeated scan can not be performed in patients with MI due to the limit of medical ethic. The MI in big animals can be kept equal infarcted size, monitored in long term and compared itself for multiple times, which can reflect the real and actual condition of angiogenesis post-MI.In most studies, the early stage post-MI is suggested for heart intervention, which is not completely consistent with clinical needs that heart failure tends to occur in the middle or late stage after MI. In terms of the route of administration, accumulating data have shown that transcatheter endocardial injection is more efficient and clinically feasible. Vascular endothelial growth factor(VEGF) is a cytokine promoting angiogenesis, which has been studied widely and deeply. Collagen binding domain(CBD) VEGF, developed by Professor Dai Jianwu’s team from Genetics and Development Institute of Chinese Academy of Sciences, can bind with type-I collagen surrounding the infarction area, so as to reduce the dilution and dissipation and improve local plasma concentration of drugs. CBD-VEGF can promote angiogenesis and improve cardiac function than ordinary VEGF, which has been validated on rat MI model and rabbit MI model, but still lacks evidence of large animal studies and in vivo imaging results. Aims1. To initiated a consistent and sustained coronary embolism model with larger infarct size, high operation success rate and short operative time by the sequential balloons-sponge embolization in minipigs so as to lay a good foundation for further heart intervention.2. To investigate the dynamic angiogenesis formation in porcine infarcted heart tracking by αvβ3 targeted positron emission tomography(PET) probe 68Ga-NOTA-PRGD2 and attempted to perform the multi-modality image fusion to present angiogenesis more distinctly and visually.3. To perform the transcatheter endocardial injection with CBD-VEGF, ordinary VEGF and PBS in minipigs with old MI, and compare the difference in three groups with assessment of multi-modality imaging and angiogenesis imaging. Methods1.Eighteen healthy minipigs(25-30 kg) were randomly divided into 3 groups for left anterior descending artery(LAD) occlusion: BO group with temporary balloon occlusion for 60 mins, HB group with half-balloon embolism, BBS group with two sequential half-balloons and one sponge as the embolism clot. The incidence of ventricular fibrillation(VF), total mortality, operating time and vascular recanalization 3 months post-MI were recorded and compared. Echocardiography, multimodality nuclear medical imaging and histology staining were applied for evaluation of cardiac function and infarct size.2.Acute myocardial infarction(AMI) in eight minipigs established by sequential embolization was confirmed by electrocardiogram, echocardiography and 18F-fluorodeoxyglucose(FDG) PET/CT imaging pre-MI and post-MI. Before PET/CT scan for angiogenesis, minipigs were intravenously injected with 68Ga-NOTA-PRGD2 solution at the dose of approximately 1.85 MBq(0.05 m Ci) per kilogram body weight. Cardiac PET/CT scan was performed to evaluate the standardized uptake value(SUV) of region of interest(ROI) with 68Ga-NOTA-PRGD2 at pre-MI, 3 days, 1 week, 3 weeks, 6 weeks, 12 weeks and 24 weeks post operation. Syngo MI software and MIM 6.4 software were used to analyze the single and series plane imaging results respectively. Contralateral myocardium was considered as non ROI. Cardiac Dual-source Computed Tomography(DSCT) and Computed Tomography Angiography(CTA) with consistent posture of minipigs was performed 1 week and 12 weeks post-MI in order to acquire the anatomical morphology of heart and coronary artery clearly, which is important to image registration. After that, image segmentation, registration and image fusion in three dimension were performed with multi-modality images.3.12 minipigs with MI for 3 months and 2 healthy minipigs of same age were involved. Old MI model were established and screened by echocardiography and follow-up with coronary angiography to guarantee the stability as baseline. The ultrasonic data and SPECT/CT data were obtained before intervention. 12 minipigs with old MI were randomly divided into 3 groups(CBD-VEGF 200μg, ordinary VEGF 200μg and PBS 2ml in each minipig), and underwent transcatheter endocardial injection respectively. At pre-operation and 1 month, 2 months and 3 months after intervention, the animals of three groups were all examined by 68Ga-NOTA-PRGD2 PET/CT. Coronary angiography were performed at 3 months after intervention. The three intervention groups and healthy control group were assessed with echocardiography and SPECT/CT at 3 months and 9 months after intervention. The infarct size were compared in three groups at 9 months after intervention. Results:1.13 out of total 18 minipigs survived after the operation, while 5 animals were died with VF(3 in BO group,1 in HB group, 1 in BBS group) with an 83.3%(5/6 minipigs) acute procedural survival rate in two embolism groups. The operating duration were(60.0±0.5)mins,(21.4±5.2) mins and(31.2±4.7) mins in three groups respectively. LAD recanalization was found in 3 animals of HB group but none in BBS group by angiography follow-up. And the total perfusion deficit(TPD) of the BBS group is larger than HB group(P<0.05, n=10). The infarct size were found more stable and larger in HB group and BBS group than that in BO group(P<0.05,n=13).2.8 out of 10 minipigs underwent the MI operation alived. The survived minipigs are all suffered from MI with anterior and anteroseptal wall in mid-level of left ventricle and the infarcted size was equivalent approximately assessed by echocardiography. The patchy or cloddy angiogenesis in 3D tracking by 68Ga-NOTA-PRGD2 tracer occured in the border zone around the septal MI region and close to the blocked coronary artery demonstrated by multi-modality image fusion. The regional accumulation of 68Ga-NOTA-PRGD2 can be detected at 3 days(0.78 ± 0.13 vs. 0.47 ± 0.15, P > 0.05) presented as SUVavg in single plane and peaked between 1 and 3 weeks(1.01 ± 0.12 vs. 0.59 ± 0.14, P < 0.05 and 1.55 ± 0.20 vs. 0.81 ± 0.17, P< 0.05, respectively). The focal accumulation decreased but still kept at a higher level at 6 weeks and 12 weeks post operation(0.86 ± 0.12 vs. 0.50 ± 0.10, P < 0.05 and 0.80 ± 0.15 vs. 0.40 ± 0.07, P < 0.05). Until 24 weeks post operation, the SUVavg of ROI in single plane were still higher than that of non ROI, but statistical difference were not found(0.59±0.10 vs 0.41±0.09, P>0.05). Meanwhile, SUVmax in single plane, SUVavg and SUVmax in series plane remained the same trend. In addition, the volume of ROI presented dynamic changes and peaked at 3 weeks post-MI, with the value of 7.00 ± 1.05 cubic centimeters. Multi-modality image fusion in 3D further present the location and volume of angiogenesis distinctly.3.All of the 12 old MI minipigs were successfully conducted with transcatheter endocardial injection at 3 months post-MI. During the operation, frequent premature ventricualr contraction and nonsustained ventricular tachycardia occured, while there was no ventricular fibrillation. All of the minipigs involved in the experiment were successfully conducted with a series of image examinations. Total perfusion deficit(TPD) in CBD-VEGF gradually decreased at 3 months and 9 months after intervention, and appeared significant difference compared with ordinary VEGF group and PBS group(P <0.05), which was respectively(25.0±1.6)%,(34.0±1.2)% and(35.5±1.5)% at 9 months after intervention. It can be seen from the horizontal long axis of heart that the septal myocardial perfusion in CBD-VEGF group was improved 9 months after intervention. The SUVavg of 68Ga-NOTA-PRGD2 increased at 1 month, 2 months and 3 months after the intervention, while the PBS group and ordinary VEGF group tended to decrease over time and appeared significant difference(P<0.05) among the three groups at 3 months after the intervention, which were(0.96±0.08),(0.62±0.10) and(0.59±0.07) respectively. The SUVmax of 68Ga-NOTA-PRGD2 appeared same trend at different time points after the intervention. However, cardiac function and regional wall motion in CBD-VEGF showed no significant improvement evaluated by echocardiography and SPECT/CT. The infarct size also showed no significant difference in three groups. Conclusions1.A new modeling method of myocardial infarction in minipig was established and validated. Compared with the classical modeling induced by balloon transient blocking, the method of sequential balloons-sponge embolization could induce consistent myocardial infarction with larger infarct size, total and sustained embolization, higher operation success rate, shorter time and better repeatability, which was a promising improvement of traditional modeling of myocardial infarction.2.PET imaging of 68Ga-NOTA-PRGD2 could be applied to noninvasive long-term observation of the angiogenic process and the dynamic change of angiogenesis post-MI in large animals was revealed. A software platform of cardiac multimodality image fusion was established by cooperation and the fused image could provide more comprehensive and objective evaluation on myocardial infarction itself and angiogenesis post-MI, and also it could present the position and volume of angiogenic signals more accurately and visually. It clearly and definitely confirmed that lump-like or cloddy angiogenesis mainly occured in the border area of the anteroseptal infarction and normal myocardium in anterior wall myocardial infarction, which is located in the severe ischemic area near the distal of embolus in anterior descending artery..3. CBD-VEGF was superior to ordinary VEGF and PBS by promoting angiogenesis and improving myocardial perfusion after percutaneous endocardial delivery in old MI model, meanwhile improvement of cardiac function and reduced infarct size weren’t be discovered. Multimodality cardiac imaging and 68Ga-NOTA-PRGD2 angiogenesis molecular image with target of integrin-αvβ3 contributed to the evaluation of intervention effect.