| BackgroundsAcute cardiovascular events due to rupture or erosion of atherosclerotic plaques are the leading cause of morbidity and mortality in atherosclerotic patients.As a result,there is a growing need for new therapies to stabilize atherosclerotic plaques.Increasing evidence indicates that immature neovessel in plaques is an important factor leading to increased vulnerability and rupture of plaques due to impaired vascular wall integrity and leakage.Therefore,stabilizing new blood vessels within the plaque may become a new target for the treatment of vulnerable plaques.Due to the imbalance between pro-angiogenic factors and pro-mature factors,the newly formed microvascular wall consists of only one layer of endothelial cells,which will cause the leaking of red blood cells,lipids and inflammatory factors into the plaques.The main regulator of angiogenesis is vascular endothelial growth factor(VEGF)-A.Preclinical studies have shown that interfering with this factor can be a potential method for treating tumors and peripheral arterial diseases.However,large-scale clinical trials using anti-VEGF-A drugs in patients with tumors and peripheral arterial disease have proven disappointing.There is still no reliable evidence on whether the inhibition of neovascularization in plaques can stabilized the vulnerability of plaques,which is caused by the lack of suitable plaque-neovascularization animal models.The lack of animal models makes huge differences between sevral research results.Herrmann et al proposed that the development of atherosclerotic lesions and cancerous lesions involve three different pathoanatomic stages:initiation,progression and complication.Research of initiation stage using a rabbit model fed an atherogenic high-fat diet for 3 weeks.The result of this study indicated that local delivery of bevacizumab(monoclonal antibody against VEGF-A)inhibited plaque angiogenesis and atherosclerosis progress.Although the progression and complication stages are highly related to the clinical situation,there are few studies on anti-angiogenesis treatment in these two stages,and inappropriate anti-angiogenesis treatment in these two stages may lead to hypoxia and endothelial cells apoptosis,in the end resulting in loss of vascular endothelium integrity(bleeding).This finding raises concerns about the importance of normalization of neovascularization within plaques in atherosclerotic plaques.Vascular normalization is a strategy.Vascular maturation growth factors can improve the coverage and maturity of pericytes.Therefore,it is important to elucidate the mechanisms and signaling pathways of vascular normalization pathways.Fibroblast growth factor(FGF)-2 is an effective growth factor that stimulates endothelial cell migration and proliferation and promotes mitosis of smooth muscle cells(SMCs).The platelet-derived growth factor-BB(PDGF-BB)/PDGF receptor-?(PDGFR-?)axis is the main signaling system for perivascular cells recruitment.Previous studies have shown that the coexistence of two angiogenic factors may have a greater functional impact than a single high level of one growth factor,even at low levels.Therefore,FGF-2+PDGF-BB may have far-reaching significance for plaque neovascularization.Here,we propose a new strategy for local delivery of FGF-2+PDGF-BB lentivirus into plaques to induce stable and persistent expression of growth factors and to"normalize" the structure of plaque neovessels,and thereby improve plaque stability.Objectives:1.To investigate the effect of intraplaque injection of FGF-2+PDGF-BB lentivirus on plaque stability2.To investigate the effect of intraplaque injection of FGF-2+PDGF-BB lentivirus on neovascularization in plaque3.Explore the effect of FGF-2+PDGF-BB on pericyte migration;4.Explore the specific mechanism of FGF-2+PDGF-BB regulating pericyte migration.Methods:1.Construction of VEGF-A,FGF-2 and PDGF-BB lentiviral vectorsThe shRNA plasmids targeting rabbit VEGF-A,FGF-2 and PDGF-BB genes were designed,and the interference efficiency of the plasmids was analyzed by Western blotting.The plasmids with the highest efficiency were selected to construct a lentiviral vector containing a green fluorescent protein reporter gene(GFP).2.Construction of rabbit abdominal atherosclerosis model and intraplaque injection of lentiviralNinety-six male New Zealand white rabbits(1.8-2.0kg)were fed in a clean-grade environment and underwent balloon-induced endothelial injury.After 12 weeks of high-fat feeding,IVUS-assisted intraplaque injection was performed.Six rabbits were selected for pre-experiment to verify the transfection efficiency of virus.Rabbits were divided into 6 groups:Sham group(n=15),Vector group(n?15,GFP=50?L),VEGF-A group(n=15,VEGF-A?50?L),FGF-2 group(n=15,FGF-2=50?L),PDGF-BB group(n=15,PDGF-BB=50?L),and FGF-2+PDGF-BB group(n=15,FGF-2/PDGF-BB=25?L).3.Blood lipid analysisThe experimental rabbits received intraplaque injection for 8 weeks.Before euthanasia,arterial blood was drawn from the rabbit's middle ear artery for lipid determination.The serum was centrifuged to detect the levels of low-density lipoprotein,total cholesterol,high-density lipoprotein and triglycerides in rabbits.4.Intravascular ultrasound(IVUS)assayEight weeks after the rabbits received intraplaque injection surgery,the plaque parameters of rabbits from each group were obtained by IVUS,and the plaque morphology was evaluated.5.Histopathological examinationAfter the tissue samples were collected from each group,paraffin sections were prepared for H&E staining to observe the morphology of the plaques.Sirius red staining was used to detect the collagen content in the plaques.Frozen sections were prepared,and oil red O staining was used to detect lipid content in the plaques.6.Inmunohistochemical stainingAfter taking tissue samples from each group,paraffin sections were prepared for immunohistochemical staining.The expression levels of macrophages(RAM-11),smooth muscle cells(a-SMA),CD31 and hypoxia-inducible factors(HIF-la)in plaque were detected.7.Inmunofluorescent chemical stainingAfter the rabbits were collected from each group,paraffin sections were prepared for immunofluorescence double-label staining to detect the neovascular structure(CD31/NG2),and expression level of VEGF-A,FGF-2,PDGF-BB and their related receptors FGFR-1,FGFR-2 and PDGFR-? within the plaques.8.RT-PCRTotal RNA was extracted from abdominal aortic plaques of experimental rabbits from each group,and the gene expression levels of VEGF-A,FGF-2,and their PDGF-BB in the plaque tissue were detected.9.Cell cultureThe experiments used b.END3 cells and 10T1/2 cells as the subjects of endothelial cells and pericyte cells,respectively.Pericyte cells were stimulated by FGF-2(50ng/mL),PDGF-BB(10ng/mL),and FGF-2?PDGF-BB for 5min,15min,and 60min,respectively.10.Cell scratch experimentCell scratch experiments were used to observe the effects of FGF-2(50ng/mL),PDGF-BB(10ng/mL),and FGF-2+PDGF-BB-induced pericyte migration.11.Cell transwell experimentTranswell experiments were used to observe the effects of FGF-2(50ng/mL),PDGF-BB(lOng/mL)and FGF-2+ PDGF-BB-induced pericyte migration.12.Cell migration assayEndothelial cells were labeled with PKH26,pericytes were labeled with carboxyfluorescein succinimidyl ester,and the migration rate of pericytes to endothelial cells induced by FGF-2(50ng/mL),PDGF-BB(lOng/mL),and FGF-2+PDGF-BB was observed.13.Cell InterventionTransfection of siRNA-epn 2 with small interference siRNA technology inhibited the gene expression of Epsin 2 in pericyte cells,and siRNA-N.C was used as a negative control group.14.Western-blot analysisTissue total protein was extracted from abdominal aortic plaques of experimental rabbits in each group,and the expression levels of VEGF-A,FGF--2,PDGF-BB and their related receptors FGFR-1,FGFR-2 and PDGFR-? in plaque tissue were detected.Cells of each group were collected,total protein was extracted,and protein expression levels of VEGFR-2,p-VEGFR-2,Epsinl/2,PDGFR-?,p-PDGFR-?,and GAPDH were measured.15.Statistical analysisThe data were statistically analyzed using SPSS 17.0 software and expressed as mean±standard error(Mean±SEM).P<0.05 considered the difference to be±statistically significant.Results:1.General conditions of experimental animals in each groupThe experimental animals in each group recovered fully after intraplaque injection.Two rabbits from the FGF-2 group died of wound infection 5 days after the surgery.Two rabbits from the Vector group died of diarrhea 12 days after surgery.The weight of the rabbits was measured before the end of the experiment.There was no statistical difference of the weight of the rabbits from each group.2.Basic indicators of experimental animals in each groupBefore the end of the experiment,the serum lipids of the rabbits from each group were measured,and there was no difference in the serum lipid levels of low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C),triglycerides(TG),and total cholesterol(TC)from each group.By RT-PCR detection,the lentiviral vectors within the plaques from each group were effectively expressed.There was no difference in the expression levels of VEGF-A,FGF-2 and PDGF-BB in the serum of the rabbits from each group.According to the immunofluorescence double-label assay,VEGF-A,FGF-2,and PDGF-BB are widely expressed within the plaques.According to the location of cell spatial distribution within the plaques,it can be seen that compared with the control groups,in the VEGF-A group and the FGF-2 group,VEGF-A and FGF-2 were mainly expressed in macrophages.While in the PDGF-BB group and the FGF-2+PDGF-BB group,PDGF-BB was mainly expressed in smooth muscle cells.All rabbits were examined by IVUS before euthanasia.There was no difference in lumen area and outer elastic membrane area between groups.But compared with the Sham group and Vector group,the plaque area in the VEGF-A group and FGF-2 group significantly increased.3.FGF-2+PDGF-BB overexpression can reduce the area of plaque necrosis nucleiImmunofluorescent double-labeling staining of endothelial cells and smooth muscle cells showed the area of necrotic nuclei within the plaque.It was found that compared with the VEGF-A group and FGF-2 group,overexpression of FGF-2+PDGF-BB significantly reduced plaque necrotic nuclei area.4.FGF-2? PDGF-BB overexpression enhances plaque stabilityImmunohistochemical staining showed that the content of macrophages in the FGF-2+PDGF-BB group was significantly decreased compared with other groups,and the contents of smooth muscle cells and collagen were significantly increased in the PDGF-BB group and the FGF-2+ PDGF-BB group.Vulnerability index analysis showed that overexpression of FGF-2+PDGF-BB significantly increased the stability of vulnerable plaques.5.FGF-2+PDGF-BB overexpression induced fewer plaque neovessels than FGF-2 or PDGF-BB used aloneAccording to immunohistochemical staining of endothelial cell marker(CD31),compared with FGF-2 group or PDGF-BB group,the number of plaque neovessels in FGF-2 H+PDGF-BB group was significantly decreased.Pimonidazole staining showed that hypoxia levels were significantly lower in the FGF-2+PDGF-BB group than in the FGF-2 or PDGF-BB group.At the same time,western blot analysis showed that the protein level of HIF1-? in the FGF-2+PDGF-BB group was significantly lower than that in the FGF-2 or PDGF-BB group.6.FGF-2?PDGF-BB overexpression reduces intraplaque haemorrhageEvans Blue staining,H&E staining and Glycophorin A staining showed that there were almost no free red blood cells wandering within the plaques of the FGF-2+PDGF-BB group,and a large number of red blood cells were seen with the plaques in the VEGF-A group and the FGF-2 group.7.FGF-2+PDGF-BB overexpression enhances pericyte coverage of plaque neovesselsIn order to evaluate the effect of FGF-2+PDGF-BB overexpression on plaque neovessels maturity,CD31(endothelial cell marker)and NG2(peripheral cell marker)immunofluorescence double-labeling staining was used to calculate coverage of pericyte on plaque neovessels.The results indicated that compared with the control groups,FGF-2+PDGF-BB significantly increased the recruitment of pericyte to endothelial cells.Inter-endothelial connections and pericyte coverage were detected by TEM.Isolation of neovessel endothelial cells was observed in the VEGF-A and FGF-2 groups.In contrast,neovessels in the FGF-2+PDGF-BB group showed endothelial junction integrity and pericyte coverage.8.FGF-2+PDGF-BB overexpression increases the expression of FGFR-2 and PDGFR-? in plaquesFGF-FGFR and PDGF-PDGFR systems are two signalling pathways involved in pericyte recruitment in angiogenic vessels.Immunofluorescence staining and Western Blot showed that the protein expression levels of FGFR-2 and PDGFR-? in the plaques of FGF-2+PDGF-BB group were significantly increased.Therefore,FGF-2+PDGF-BB overexpression can effectively activate the signaling pathways related to neovascular maturation.9.FGF-2+PDGF-BB promote pericytes migrationCell scratch test and transwell chamber migration experiment confirmed that compared with the control group,FGF-2+PDGF-BB treatment can significantly increase pericyte migration.10.FGF-2+PDGF-BB promote pericytes migrate to endothelial cellsPericyte-endothelial cell adhesion experiments were used to detect the migration of free pericytes to endothelial cells.Fluorescence image analysis showed that compared with the control group,FGF-2+PDGF-BB treatment increased the migration of pericytes to tube-forming endothelial cells.11.FGF-2+PDGF-BB time-dependently inhibits phosphorylation of VEGFR-2 in pericytesFGF-2,PDGF-BB,and FGF-2+PDGF-BB were used to stimulate pericytes for 5min,15min,and 60min,respectively.Western blot results showed that FGF-2+PDGF-BB significantly inhibited VEGFR-2 phosphorylation at 5min,15min,and 60min compared with NT group.12.FGF-2+PDGF-BB time-dependent induction of Epsin2 expression in pericytesFGF-2,PDGF-BB,and FGF-2+PDGF-BB stimulated pericytes for 5min,15min,and 60min,respectively.Western blot results showed that FGF-2+PDGF-BB significantly increased Epsin-2 expression at 5min,15min,and 60min compared with NT group.Western blot results showed that Epsin-2 siRNA significantly inhibit Epsin2 protein expression in pericytes in FGF-2?PDGF-BB group.And compared with NT group,inhibition of Epsin2 expression in pericytes could significantly reduce PDGFR-? phosphorylation and increase VEGFR-2 phosphorylation in FGF-2+PDGF-BB group.Pericyte-endothelial cell adhesion experiments showed that compared with NT group,inhibiting the expression of Epsin2 in pericytes could significantly reduce the migration and adhesion of pericytes to endothelial cells in FGF-2+PDGF-BB group.Conclusion:1 Immature plaques neovessels increase plaque vulnerability;2 FGF-2?PDGF-BB overexpression reduce intraplaque hemorrhage;3 FGF-2+PDGF-BB overexpression increase the maturation of neovessels in the plaque;4 FGF-2+PDGF-BB induces the expression of Epsin-2 in pericytes,thereby inhibiting the phosphorylation of VEGFR-2 in pericytes and increasing the migration of pericytes.Backg-roundsAtherosclerotic plaque rupture followed by the thrombosis is the primary cause of acute cardio-and cerebral vascular events with high mortality and morbidity.These rupture prone plaques,which are termed vulnerable plaques,are characterized by active inflammation hallmarked by macrophage infiltration,as well as typical large lipid core and thin fibrous cap.In comparison,neutrophils are scarcely detected in plaques although they are the largest population of circulating leukocytes.However,the neutrophil is evidenced trafficking to the injured endothelia caused by atherogenic factors or inflammatory chemocants.Neutrophil influx can also be imaged co-localized with macrophage within vulnerable plaques.After the short life span(1-2 days),apoptotic neutrophils are engulfed by macrophage and this rapid phagocytosis polarizes macrophages to anti-atherosclerotic phenotype to restore homeostasis.It assembles the immune system innate to defense the invading pathogens so that the activated neutrophil will "target" the atherosclerosis and subsequently transmigrate into the plaque in response to endothelial injury.This intrinsic immunological response can be used to increase the local drug concentration.Wu et al.reported doxorubicin-internalized neutrophil for inflamed brain tumor therapy on this basis and tumor homing.But different hemodynamics should be pointed out that the atherosclerotic plaques are lesions in large to medial size artery with high shear stress.This hinders the attachment of drug carriers to the endothelia and accumulation of delivered drug within the plaque.Targeted motifs or ligands,such as connect selections,integrin,and cell adhesion molecules(CAMs),lend support through binding capacity,yet it might not be sufficient.In the other hand,lots of cytokines are involved in the inflammation within a vulnerable plaque,for which cannot be targeted enough through one single molecular marker.In comparison,neutrophil holds the ability to roll at high shear stress by cell flattening,catch bonds,long tethers and so-called '"slings" around rolling cells.They can travel through blood flow and migrate into atherosclerotic lesions.Thus,neutrophil makes it an attractive cell-based drug delivery system to targeted intervention for vulnerable plaques.Microbubbles have been well accepted as one of the multifunctional carriers since they are capable of not only the drug delivery but also the contrast enhancement and targeting augment under ultrasound.The underline mechanism involves micro-hemodynamics,sonoporation,acoustic radiation force,etc.which can benefit the enrichment of microbubbles at the target site.Anne Rix et al.reported that drugs could be delivered into atherosclerotic plaques via microbubbles.In this study,we constructed a neutrophil-microbubble complex,coincided as neutrophil-Microbubble,to mimic the neutrophil's response to inflammation at vulnerable plaques.Meanwhile,microbubbles serve as the carrier,acoustic tracer and enrichment amplifier.Neutrophils were used as "Trojan horses" to convoy this bionic multifunctional balloon to the vulnerable plaques rapidly and firmly.Objectives:1.The Neutrophil-Microbubblecomplex was prepared and characterized;2.The ability of complex adhesion to endothelial cells was confirmed under static and flow cavity conditions in vitro;3.To explore the binding ability of complex to aortic plaques in Apo E-/-mice;Methods:1.Isolation of Peripheral Neutrophils.Percoll gradient method described by Wu et al.was used for the isolation of neutrophils from the whole blood of male Apo E-/-mice.Blood samples were collected in heparin tubes,purified by centrifugation(400 g,10 min,4?),and then diluted in PBS containing heparin.The cell pellets were then carefully added into the top of a three-layer Percoll gradient of 78%,69%,and 52%diluted in PBS and then centrifuged at 1500xg for 30 min at room temperature.The neutrophils were withdrawn from the 69%/78%interface and the upper part of the 78%layer.After the lysis of residual erythrocytes at 4?,neutrophils with high purity were obtained.2.Bionic multifunctional Neutrophil-Microbubble assemblyBiotinylated microbubbles with a perfluoro propane(C3F8)gas core were made by sonification.The shell was composed of 1,2-distearoyl-sn-glycerol-3-phosphocholine(DSPC),1,2-distearoyl-sn-glycerol-3-phosphoethanolamine(DSPE)-PEG(2000)and DSPE-PEG(2000)-biotin.Microbubbles had an average diameter of 1.8 ?m and were stored in sealed penicillin bottle with a C3F8 gas headspace to prevent deflation.To make conjugated microbubbles,100?l biotinylated microbubbles were washed twice with PBS/C3F8 by centrifugation(400g,3 min,4?)to remove superfluous biotin and resolved in PBS/C3F8.Next,1 mg/ml streptavidinwas added and the mixture were incubated at 24? for 25 min.Microbubbles was again washed to remove superfluous streptavidin by centrifugation.Next,1?g biotinylated rat-anti-mice-CD11b was added and the mixture was incubated at 24? for 25 min.These targeted microbubbles were again washed and resolved in DMEM medium.Final concentration of the microbubbles was determined using a Multisizer 3 Coulter Counter.Neutrophils were then incubated with anti-CD11b antibody conjugated microbubbles in a 100:1 ratio under continuous rotation at room temperature for 25 min.And then Neutrophil-balloon was analyzed for the number of microbubbles per Neutrophil-balloon.3.Cell viability assayCell viability assay involved use of Cell Counting Kit-8(CCK-8).Neutrophils(5 X 103 per well)or Neutrophil-balloon(5×103 per well)were seeded in a 96-well plate in 5%CO2 at 3 7?.After removing the media,cells were incubated with CCK-8 reagent for 2 h.Optical density(OD)was measured at 450 nm.Cell viability was calculated as[(ODNeutrophil+ODblank)/(ODcontrrol+ODblank)]×100%.4.Indocyanine green(ICG)loaded neutrophilICG was dissolved in 100?L DMSO and mixed with 400 DMEM(plus 10%fetal bovine serum)to a final concentration of 2 mg/ml.Mix 300?L ICG and 300 ?L serum-free DMEM with 5?L protamine sulfate solution to a final concentration of 10 mg/ml.The neutrophils were dissociating evenly with pipette three times.Add the prepared protamine/ICG solution and incubate for 1 h at 37 ?.Then centrifuge at 400 rpm for 5 minutes.The ligand conjugation efficiency of targeted MBs was detected according to the fluorescence intensity of ICG by using Synergy 4 microplate reader.5.Inflammation responsive adhesion of Neutrophil-Microbubble in vitroThe MBIgG,MBCD11b or Neutrophil-balloon(5×106/ml)was added into a 24-well plate pre-coated with bEnd.3 cells stimulated by lOng/ml TNF-? and 60mg/L ox-LDL.The plate was sealed,inverted and rotated for 5 min.PBS was used to remove the free Neutrophil-balloon and MBs,and then the number of attached Neutrophil-balloon and MBs were counted under an optical microscope at five random bright fields of view.6.Resistance to high shear stress in vitro by use of flow chamberThe parallel-plate flow chamber setup was reported previously.The flow devices were maintained in 37? incubator with 5%CO2.The Neutrophil-balloon,MBCD11b and MBIgG were drawn into bEnd.3 cells coated ?-Slide chamber at a shear stress of 4 dynes/cm2 for 1,2,4 and 8 min.Then,these three kinds of microbubbles were exposed to a range of lower shear stress of 4,8,12 and 16 dynes/cm2 for 4 min.7.Western blot analysisTotal protein of bEnd.3 cells was extracted,and protein concentrations were examined by a BCA assay kit.Polyvinylidene fluoride(PVDF)membranes were incubated with specific primary antibodies for intercellular adhesion molecule-1(ICAM-1),and ?-actin at 4? for 12 h,and then incubated with horseradish peroxidase conjugated secondary antibodies.Protein bands were visualized by enhanced chemiluminescence kit.The individual bands' densitometric intensity(area×density)on Western blots was measured by the Image J software.Sample loadings were normalized to ?-actin expressions.8.Atherosclerotic animal modelAll male ApoE-/-mice and C57 mice were purchased from the Southern Medical University.The study protocol was approved by the Medical Ethics Committee on Animal Research of the Shenzhen institutes of Advanced Technology,Chinese Academy of Sciences(Ethics No.KY2016-090).All procedures followed the principles of laboratory animal care and guidelines from the National Institutes of Health.Fifteen ApoE-/-mice were assigned evenly into three groups and fed with atherogenic diet(0.25%cholesterol,15%cocoa butter)for 8,16 or 24 weeks respectively.Age-matched C57 mice(n=15)served as the control group were also involved following the same grouping and feeding protocol.9.Acoustic tracing of the bionic behavior of Neutrophil-balloon in vivoUltrasonography was performed vivo using Vevo 2100 with a MS250 nonlinear transducer(center frequency,18 MHz;lateral resolution,165 ?m;axial resolution,75?m;transmit power,10%;dynamic range,40 dB).After anaesthetized by isoflurane in oxygen(2 L/min),50 ?L MBIgG,MBcD11b,or Neutrophil-Microbubble(1×107)was injected through the tail vein,followed by 0.1 mL normal saline injection.Four minutes later,the long axis view of the aortic arch was acquired from the right parasternal window.A high-power ultrasound destruction pulse sequenced was used to acquire a series of 500 ultrasonographic frames as described before.10.ICG transportation in vivoIn anesthetized mice,ICG loaded neutrophil and ICG loaded Neutrophil-Microbubble were injected through tail vein access.After euthanasia,aortas of mice in each group were dissected for fluorescence assay.The dissected aortas were embedded in ultrasonic coupling agent and imaged at emission wavelength 780 nm by homemade photoacoustic microscopy(spatial resolution,100 um).11.Histology and immunohistochemistryThe arteries were swiftly removed.Each specimen was fixed with 4%paraformaldehyde fixative and embedded in paraffin for haematoxylin and eosin(H&E)staining.For immunostaining,serial cross-sections with a thickness of ?m were stained with an anti-SMC antibody(1:200 dilution)and an anti-CD68 antibody(1:200 dilution)for characterization of smooth muscle cells and macrophage.Secondary antibodies were used according to protocols of horseradish peroxidase and diaminobezidin(DAB)chromogenic technique.12.Statistical analysisAll data analyses were performed by SPSS 18.0.P<0.05 was considered statistically significant.Continuous variables are presented as the mean±standard deviation.Differences in multiple groups were analyzed using one-way ANOVA.The significance of the differences between the two groups was tested using Dunnett's T3 test.The relationships between acoustic parameters and histological data were analyzed by Pearson correlation analysis.Results:1.Characterization of composites and detection of acoustic propertiesEach neutrophil was conjugated with 3.4±1.2 microvesicles with an average diameter of 8.4±1.2?m.Although neutrophils have a short life span,the preparation process and ultrasound(500 Hz,5%,0.35 Mpa,255 W/cm2)did not affect their activity.We also prepared MBIgG and MBCD11b as controls for study.2.Targeted adhesion of the complex to b.End.3 cells in vitro inflammation modelIn order to verify the in vitro cell-targeting function of Neutrophil-Microbubble,static adhesion experiments and parallel plate flow chamber systems were used.The results confirmed that the complex could bind to bEnd.3 cells with significantly higher affinity than MBIgG or MBCD11b.In addition,E-selectin and ICAM-1 specific inhibitor 2,4-disubstituted thieno[2,3-c]pyridine(A-205804)are able to inhibit the binding ability of the Neutrophil-Microbubble or MBCD11b to b.END3 cells.3.In vivo ultrasound imaging detection of the complexThe in vivo verification of the Neutrophil-Microbubble high-affinity biomimetic behavior was performed on an Apo E-/-mouse model.The results indicated that the imaging intensity of Neutrophil-balloon was significantly higher than that of MBigG and MBCD11b(P<0.05),after 10 weeks of high fat diet.Neutrophil-Microbubble and MBCD11b ultrasound molecular imaging tests were performed after 8 weeks,16 weeks,and 24 weeks of high-fat diet.Results indicated that with the increase of high-fat time,the imaging effect of the Neutrophil-Microbubble complex was significantly higher than that of MBCD11b(P<0.05).At each time point,the vascular tissue of the mouse aorta-imaging site was selected for histological staining of Oil O-Red and Picric-Sirius red and immunohistological staining of anti-a-actin and anti-CD68.The results showed that the content of macrophages and lipids increased with the time of high-fat feeding,while the contents of collagen and smooth muscle cells decreased,eventually leading to an increase in plaque vulnerability(P<0.05).According to Pearson correlation analysis,the macrophage content and vulnerability index were significantly positively correlated with the imaging intensity of the complex(both P<0.01).In summary,under high aortic shear forces,the targeting of the complex to the plaque will increase with the degree of plaque inflammation.4.Compound Indocyanine Green(ICG)releaseThe fluorescence intensity of the plaques was detected after 0.5h,1h,3h,6h,12h and 24h after the mice were injected with the ICG-complex through the tail vein.Results showed that the fluorescence intensity reached its peak at 1 hour after injection and gradually decayed at about 24 hours.In addition,when the concentration of the ICG-complex gradually increased from 5 x 107 cells/ml to 1 x 109 cells/ml,the fluorescence intensity of the plaques gradually increased.The fluorescence intensity of the plaque is also significantly related to the area within the plaque.The area of plaque at each time point was statistically analyzed at 8,16 and 24 weeks by oil red-O staining.Pearson correlation analysis indicated that the fluorescence intensity of the plaque was positively correlated with the area of the plaque(P<0.01).Conclusion:1.The Neutrophil-Microbubble was successfully assembled;2.The adhesion ability of the Neutrophil-Microbubble complex under static and high shear conditions was verified in vitro;3.The complex can bind with plaques,and the imaging intensity of complex was significantly correlated with vulnerable index. |
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