Visually Assessment Of Matrigel Angiogenesis With Ultrasound Molecular Imaging Using Microbubbles Targeted To Endothelial αv-integrins

Background and Objective:Angiogenesis is the formation of new capillaries from existent micro vessels by sprouting, migration, proliferation of endothelial cells and matrix remodeling when body is in the process of growth or experiencing wound healing, ischemic hypoxia and inflammation. As a basic physiological procedure, angiogenesis participates in the healing of all kinds of wounds, ulcers, myocardial infarction and chronic infection et al. But there are angiogenic disorders in many pathological states such as diabetic retinopathy, atherosclerosis, and solid tumors and other serious diseases which are hazardous for human beings. Therefore, high sensitive, non-invasive, targeted, quantitative evaluation of angiogenesis has the vital significance to early cardiovascular disease (mainly ischemic heart disease and peripheral arterial occlusive disease) and neoplastic diseases. The number and spatial distribution of new blood vessels can assess the early response of microvasculation reacted to growth factors to guide the delivery pro-angiogenic proteins or genes for cardiovascular disease. The early angiogenesis is able to develop early detection of tumor and micrometastasis conducive to early treatment of patients, and dynamic monitoring of new blood vessels can assess the effect of anti-cancer therapy.Ultrasonnd molecular imaging is a new online technique of noninvasive molecular imaging technologies. By virtue of connecting special ligands of specific molecules in pathologic tissue to the surface of ultrasound microbubbles, targeted ultrasound microbubbles can be constructed. Via intravenous injection, the targeted ultrasound microbubbles can gather at the target tissue and remain there for a long time, so active targeted ultrasound molecular imaging at molecular lever can be produced by contrast enhanced ultrasound. With extensive perspective and great clinical significance, evaluation of angiogenesis with targeted ultrasound molecular imaging technology has increasingly become a frontline focus in ultrasound research for the past few years. Ellegala built targeted microbubbles with echistatin to effectively evaluate neovascularization of the rat model of malignant glioma. And Leong-Poi constructed microbubbles targeted to av-integrin (MBα) and proofed that the adhesive capacity of the targeted microbubbles in neovascularization was higher than in normal vascular endothelial cells.Matrigel is a soluble basement membrane matrix extracted from ESH mouse. At room temperature, it can automatically gather and become bioactive materials similar to basement membranes of mammalian cell. Matrigel model has good operability and repeatability which has been widely applied to the experimental study as an acknowledged angiogenesis model. After subcutaneous injection of the Matrigel, endothelial cells migrate to Matrigel to form new blood vessels. Blood vessel formation takes about 10 days to complete and adding appropriate concentration of pro-angiogenic factors can speed up the angiogenesis.Basic fibroblast growth factor (FGF-2) is one of the most effective pro-angiogenic factors in vivo. It can up-regulate capillary basement membrane degradation, endothelial cell migration and proliferation, collagen synthesis and down-regulation the activity of matrix metalloproteinases. It has proved that FGF-2 plays an important role in vivo as well as in vitro to stimulate newborn blood vessels. FGF-2 can enhance the release of protein kinases which can degrade the basement membrane, thereby promoting angiogenesis, and the formation of small blood vessels.Leong-Poi et al performed contrast ultrasound study on Matrigel model with MBa and video intensity of MBa is high that of control microbubble(P<0.05). But the specificity of angiogenesis wasn't evaluated because of lack of closed group. Stieger found microvessel density measurements displayed a significant correlation with power Doppler enhanced area (r=0.65, P<0.05) with non-targeted microbubble. Although ultrasound molecular imaging of angiogenesis has achieved initial success, there is no research report about specificity and quantification of angiogenesis. Therefore, we established Matrigel model by adding different levels of FGF-2 and control model by blocking a-integrin in new blood vessels in order to investigate the assessment of angiogenesis mediated by different levels of FGF-2 with ultrasound molecular imaging using microbubbles targeted to endothelialαv-integrins and quantitative evaluation of angiogenesis preliminarily.Methods:1. Microbubbles preparation:lipid microbubbles with biotin were prepared by sonication of perfluorocarbon gas (C3H8) with aqueous dispersion of several lipids in determinate ratio. After being washed (4×) to remove excess free unincorporated lipid, streptavidin in determinate ratio were added to the lipid microbubbles with biotin, then washed (2×) to removed excess free unincorporated streptavidin and the biotin conjugated mouse anti-integrin av monoclone antibodies or biotin conjugated isotype control mAb in determinate ratio were added respectively to complete the preparation of MBa and MBc. At last, the MBa and MBc were washed (2×) to remove excess free unincorporated antibodies. Both MBαand MBc were storaged in refrigerator at 4℃. The mean diameter and density in both MBa and MBa were measured by coulter counter.2. Evaluation of MBa in vitro:Using green fluorescent-labeled antibody for antiαv-integrin monoclonal antibody to identify the linking antibodies on the MBa. To evaluate the combinding efficacy between MBa andαvSFc (Recombinant human av-integrin/Fc Chimera) with a parallel plate flow chamber at a shearing force under physiologic flow conditions.3. Animal preparation:Matrigel angiogenesis was created by subcutaneous injection of matrigel in Kunming mice. Two different levels of FGF-2 was adding to Matrigel to form matrigel at the FGF-2 concentrations of 1μg/mL and 0.5μg/mL.20 mice were randomly divided into 2 groups injected with two concentrations of Matrigel. Mice were routinely anesthetized and 0.6 ml of enriched Matrigel (4℃) was injected subcutaneously in the left ventral region. Matrigel hardened to form discrete ellipsoid plugs.4. CEU imaging:CEU of matrigel plugs was performed in 10 anesthetized mice 10 days after matrigel injection. All experimental mice were performed with CEU respectively by using MBa and MBc, the intravenous injection of 4×106 microbubbles were made in random order with 30 minutes interval. After five minutes of intravenous injection, microbubbles in the circulation were eliminated, the ultrasound signal (video intensity, VI) from MBa and MBc were measured by second harmonic CEU imaging with pulsing interval time (PI) of ten seconds and a mechanical index (MI) of 0.18, transmission frequency of 7.0 MHz and receiving frequency of 14.0 MHz. After the first picture of CEU imaging being taken, the microbubbles were destroyed by two to three seconds of continuous imaging with a high MI of 1.9 and the background- subtracted VI of tumor was measured. Analysis of VI was performed by MCE software and color-coding for visual analysis was performed.5. Examination of pathology and immunofluorescence:After CEU imaging, all Matrigel plugs of experimental mice were harvested for the examination of pathology and immunofluorescence.6. Determination of median fluorescence:intensity and micro vessel count:50 regions with the same area were selected in the fluorescence photograph and median fluorescence:intensity was measured by imagepro software6.0 in these fifty regions.①Grayscale images acquisition;②Color reverse;③Optical density correction;④Selecting intensity threshold 10-200 in count/size to filter out impurities.⑤set filter value to30 pixels;⑥IOD/area representing the median fluorescence intensity and then calculating the average of the 50 regions.Micro vessels count:Average of the 50 regions was calculated subject to a lumen structure.Results:1. Results for microbubble preparation:The density of MBαand MBc is about 3.07～4.75×108/ml and 2.80～4.56×108/ml, the mean sizes for MBa and MBc were about 2.08±0.65μm and 2.12±1.13μm respectively.2. Results for evaluation of MBa in vitro:The mouse anti-mouse av-integrin monoclonal antibodies linked well to the surface of microbubble, which was observed with fluorescence microscopy. MBa andαvSFc enjoyed a good targeted combination with a shearing force (<1.0dyn/cm2) under physiologic flow conditions, which was observed in the parallel plate flow chamber.3. Results for CEU imaging:Significant ultrasound imaging was observed in the peripheral zone and the surrounding tissue of the Matrigel with MBa. While there was no CEU imaging in the center of the Matrigel. Six min later no significant CEU imaging was observed in the surrounding tissue, while the peripheral zone was still clearly visible. The CEU imaging with MBc was the same as that with MBa except no obvious CEU imaging was observed in the Matrigel. After blocking with antibody against av-integrin, significant CEU imaging could be observed in the Matrigel with MBa and MBc, but six min later there was no significant CEU imaging.4. Results for CEU-derived IV:In high lever group, VI of the matrigel was significantly higher for MBa as compared with MBc(P<0.05). After blocking with antibody against av-integrin, a great decrease was observed in the MBa group (P<0.05) while no significant difference was noted for MBc (P>0.05). The trend in low dosage group was similar to that in high lever group.5. Results for examination of pathology and immunofluorescence:HE showed abundant new vessels in which there were red blood cells in the peripheral zone of the matrigel plugs with a large amount of white blood cells (mononuclear cells) infiltration. Neovessels in matrigel was positive for av-integrin which was observed with fluorescence microscopy. There were more newborn blood vessels in high lever group than that in low lever group and high micro vessel density which is more closed to the central zone could be seen in high lever group. Immunofluorescence showed abundant av-integrin expressed within matrigel in which the amount of av-integrin in high lever group is much than that in low lever group. After blocking with antibody against av-integrin, there was hardlyαv-integrin in endothelial cells.6. VI of the matrigel was 1.4 times for MBa in high level compared with that in low level. After blocked with antibody againstαv-integrin, VI was 1.37 times for MBa in high level compared with that in low level. The median fluorescence intensity in high level was 0.32 times higher than that in low level. After blocked with antibody, the median fluorescence intensity in high level was 0.32 times higher than that in low level. Micro vessels count per unit was 1.2 times as much as that in low level. After blocked with antibody, micro vessels count per unit was 1.16 times as much as that in low level.Conclusions:1. Microbubbles targeted toαv-integrin can be successfully constructed by combining anti av-integrin monoclonal antibodies to lipid microbubbles via "avidin-biotin" bridging chemistry.2. Ultrasound molecular imaging with microbubbles targeted to av-integrin can be effective and specific in evaluating the angiogenesis in a murine model of subcutaneous matrigel plugs.3.αv-integrin can be an ideal target for molecular imaging of angiogenesis and assessment of angiogenesis in matrigel has good specificity and a great application prospect in quantify study.