RGD Microbubbles Mediated Intravascular Ultrasound Imaging To Evaluate Plaque Neovascularization

Background and Objective:Acute coronary events usually lead to death or disability in the absence of any warning.As vulnerable plaque is an important pathogenesis factor leading to acute coronary events,it is very important to identify vulnerable plaque early and accurately.Previous studies have shown that arterial and plaque neovascularization is closely related to plaque stability and cardiovascular events.However,there are two limitations on vasa vasorum imaging:on the one hand,the molecules imaging of neovascularization in nourish blood vessels and plaques requires high image resolution,limiting its application in large animals and humans;on the other hand the existing imaging technology can not distinguish between mature vasa vasorum or new formed vasa vasorum.Intravascular ultrasound(Intravascular ultrasound,IVUS)has good spatial resolution,real-time imaging and other characteristics,while integrin?v?3 is considered a new specific marker of vasa vasorum,and is closely linked with plaque stability.In this study,IVUS ultrasound enhanced imaging was performed using cRGD(cyclo(Arg-Gly-Asp))microbubbles which has a high affinity for ?v?3 integrin to verifie that whether cRGD-targeted microbubbles mediated intravascular ultrasound(IVUS)could image the plaque vasa vasorum density through enhanced gray-scale signal.Methods:1.RGD microbubbles preparation:Biotin microbubbles were prepared by lipid materials including DSPE-PEG2000-Biotin?DPPC?PEG2000 in a determinate ratio,then beening washed 3 times to remove exeess free unincorporated lipid.Streptavidin were added into Biotin microbubbles in a determinate ratio and incubated at room temperature for 15 mins,then beening washed 2 times to remove exeess free unincorporated streptavidi;At last,cRGD-biotin were added in a determinate ratio to complete targeted microbubbles preparation and incubated at 4 ? overnight.The same method was used for the preparation of cRAD-biotin conjugated isotype control microbubbles.The size and morphology of two kinds of microbubbles were observed under microscope.The average particle size and concentration of cRGD and cRAD microbubbles were measured by coulter counter.2.Animal models and groupingA total of 12 male New Zealand rabbits were purchased in Guangdong Province Experimental Animal Center,weight 2.5-3.0kg,and then randomly divided into experimental group(n = 6)and control group(n = 6).After 2 weeks of high cholesterol diet(1%cholesterol,5%lard,94%chew diet)feeding,the experimental group rabbits were anesthetized by 3%pentobarbital sodium in a concentration of 30mg/kg through ear vein injection.Punctured the rabbit's right femoral artery,then sent a 4F artery sheath into the artery.After the sheath beening fixed,a 3.5mm PTCA balloon catheter was sent into the thoracic aorta,giving 8-10kPa pressure expansion,then pulling the balloon from the thoracic aorta to the end of the abdominal aorta,repeated 3 times.Afer those procedures,800U penicillin were intraperitoneal injected for 3 days.In control group,6 rabbits were fed with a chew diet and received no intervention.2 groups were free drinking,single cage feeding for 12 weeks.3.IVUS imaging in vitroThe agarose model was formed by cooling down 500 ml 3%agarose solution into a hollow pipe.The two ends of the hollow pipe are connected with a connecting device,and the inlet end is connected with a peristaltic pump.The 40 MHz IVUS catheter probe(Boston scientific,USA)was fixed on the outflow tube of the hollow pipe.The RGD targeted microbubbles and PBS were flowed through the hollow pipe respectivly.Then observe the real time imaging of in vitro high frequency fundamental imaging characteristics.4.IVUS imaging in vivoThe experimental animals were anesthesia by injection 3%pentobarbital sodium through ear vein.Then the rabbits were fixed on the operating table.After hair removal,disinfection,left femoral artery was exposed,and then a 5F radial artery sheath was sent into the artery.IVUS catheter was located 1 cm beyond left renal artery proximal.2ml RGD and RAD microbubbles were injected into the body through ear vein in a completely randomized manner,and then washed with 5ml saline.The catheter was connected to an automated pull-back device(iLab IVUS,Boston scientific).5 minutes after injection,the catheter was pulled back at a constant speed of 0.5mm/s to the bifurcation of the common iliac artery.The image acquisition speed was 30 frames/s.After one kind of microbubbles injected,a high mechanical index ultrasound was used to completely destroy the microbubbles,and then another kind of microbubbles were performed.IVUS images were offline analysised using MCE software for color editing respectively.The MEIR value were IPP software to analyze after microbubble injection.5.HE staining and immunohistochemistryAfter IVUS imaging,the animals were euthanasia by overdose 3%sodium pentobarbital injection through the ear vein,and the abdominal aorta was fixed by 4%paraformaldehyde,and then HE staining and ?v?3 immunohistochemical staining were performed.The micro-vessel density(MVD)value were counted.6.Statistical analysisSPSS20.0 statistical software was used to analyze the data.All the data were expressed by mean ± standard deviation,and the data were analyzed by two independent samples t test or one-way ANOVA,and Bonferroni was used for multiple comparisons.P<0.05 was considered statistically significant.Result:1.Characterization of RGD targeted microbubblesCoulter counter shows that the particle size of RGD is about 1.76?8.62?m,the concentration is about 3.65×1 O8/ml;the particle size of RAD is about 1.84?8.35 m,the concentration is about 3.37×108/ml.Both RGD and RAD microbubbles were found to be uniform in size and shape with good dispersion and no obvious aggregation under the light microscope.2.IVUS in vitro imagingPBS showed no acoustic reflection characteristics while the RGD microbubbles showed good acoustic performance in the 40 MHz fundamental imaging mode.3.IVUS in vivo imagingAfter MCE color editing,in control group,there were no signal difference between baseline,RAD and RGD microbubbles contrast in the mean enhancement in the region of interest(MEIR)(P>0.05).However,in the experimental group,RGD microbubbles contrast signal was significantly higher than baseline and RAD microbubbles contrast in the MEIR(P<0.01),and the enhanced part mainly concentrated in the adventitia and atherosclerotic plaque shoulder.IPP analysis of MERI showed no difference in baseline levels(10.37±4.32)and RAD(9.99±2.81)and RGD(11.22±4.71)in the control group(P>0.05).In the experimental group,the MERI value of RGD(76.44±31.48)was significantly higher than that of baseline(76.44±31.48)and RAD(5.69±4.25)microbubbles(P<0.01).4.HE staining and immunohistochemistryHE staining showed that in the control group,there was no thickening of the intima.The smooth muscle cells arranged in neat.While the experimental group was significantly thicker intima,a large number of lipid deposition of atherosclerotic plaques.The ?v?3 immunohistochemical staining showed that there was no neovascularization in the control group,while a large number of vasa vasorum were found in the experimental group.The microvessel density(MVD)of the control group was 0.38±0.26/mm2,while in the experimental group it was 3.96 ±0.84/mm2(P<0.01).Conclusion:RGD microbubbles mediated intravascular ultrasound(IVUS)imaging can image the plaque vasa vasorum density through enhanced gray-scale signal which could be complied in the diagnosis and evaluation of the stability of atherosclerosis plaque.