key: cord-0049207-51b9ld7d authors: Zhang, Zhao; Lu, Dong-shi; Zhang, Dan-qing; Wang, Xin; Ming, Yu; Wu, Zhou-yang title: Targeted Antagonism of Vascular Endothelial Growth Factor Reduces Mortality of Mice with Acute Respiratory Distress Syndrome date: 2020-08-29 journal: Curr Med Sci DOI: 10.1007/s11596-020-2236-7 sha: 9f2e4347f54fe2ee9de733cf7bb2c2a09c631777 doc_id: 49207 cord_uid: 51b9ld7d Acute respiratory distress syndrome (ARDS) is associated with a mortality of 45%. Our previous research indicated that anti-vascular endothelial growth factor (VEGF) could maintain the normal structure and function of the respiratory barrier. However, systemic application of VEGF antagonists would lead to animal death. This study attempts to study the targeted drug delivery for ARDS. In this study, we used soluble fms-like tyrosine kinase-1 (sFlt)-targeted ultrasound microbubbles to antagonize the effect of VEGF on lung tissue. Ninety male BALB/c mice were randomly assigned to 6 groups: phosphate buffer saline (PBS) group (PBS+PBS); blank group (PBS+empty microbubbles); lipopolysaccharide (LPS) group (LPS+PBS); ARDS group (LPS+empty microbubbles); control group (PBS+sFlt microbubbles); and treatment group (LPS+sFlt microbubbles). After administration of LPS or PBS in the corresponding groups, the sFlt-targeted microbubbles or empty microbubbles were injected into the blood circulation. Then the lungs were irradiated with ultrasound, which ruptured the drug-loaded microbubbles and helped release drugs to the lung tissues targeted. The lung injury score, lung wet/dry ratio (W/D), liver and kidney functions, and the mortality of the mice in all groups were investigated at the predetermined time point. The difference in mortality between groups was examined by Fisher test. Other data were analyzed by one-way analysis of variance (ANOVA). A value of P<0.05 indicates that the difference was significant. The results showed that the PaO(2) levels were normal in the PBS group, the blank group, and the control group. The LPS group and ARDS group showed significant hypoxia. PaO(2) was improved significantly in the treatment group. The lung injury score and W/D were normal in the PBS group, the blank group, and the control group. The lung injury score and W/D increased significantly in the LPS group and ARDS group and decreased in the treatment group (P<0.05). The mortality rate of the ARDS model was 60% (95% confidence interval 47.5%–72.5%), and that with sFlt-targeted microbubbles was significantly lower at only 40% (95% confidence interval 27.5%–52.5%, P<0.05). It was concluded that anti-VEGF with sFlt targeted ultrasound microbubbles attenuated the lung injury and ultimately reduced the 7-day mortality effectively. It might be a suitable therapeutic tool for the treatment of ARDS. Acute respiratory distress syndrome (ARDS) is a common critical disease in clinic and is associated with a mortality of around 45% [1] . Our previous studies indicated that anti-vascular endothelial growth factor (VEGF) with soluble fms-like tyrosine kinase-1 (sFlt) could maintain the normal structure and function of respiratory membrane in lung [2, 3] . However, it has been reported that systemic application of sFlt might reduce vascular permeability and affect the function of other organs such as liver and kidney, with clinical manifestations being anuria, malaise, or even death [4, 5] . It appears that the adverse reaction of sFlt has restricted its clinical application. The approach as how to make sFlt target the lung may play an important role in the treatment of ARDS. Recent advancements in ultrasound microbubblemediated drug delivery technology has shown that this approach can improve spatially confined delivery of drugs to target tissues and reduce the toxicity [6, 7] . In this study, sFlt was loaded into microbubble pockets and injected into animals. After the drug-loaded microbubbles entered the blood circulation, the lungs were irradiated with ultrasound, which ruptured the drug-loaded microbubbles and enabled drug release to occur in the lung tissues targeted. The lung injury score and mortality were measured to evaluate the benefits of sFlt-loaded ultrasound microbubbles. The sFlt-targeted microbubbles were prepared by incubating sFlt (soluble vascular endothelial cell growth factor receptor-1, 471-F1, R&D Systems, USA) with a mixture of 1,2-distearoyl-sn-glycero-3phosphocholine (4 mg/mL), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (2 mg/mL), and polyethylene glycol (2 mg/mL) at 37°C for 20 min [2, 7] . The mixture was filled with perfluorobutane gas and then mechanically shocked for 45 s prior to being stayed for 5 min and rinsed for 3 times with PBS. The average particle size of the microbubbles and the concentration were measured using an optical microscope. Encapsulation efficiency (EE) of sFlt-targeted microbubbles was determined with a sFlt ELISA Kit (Mouse sVEGFR1/ Flt-1 DuoSet ELISA, DY471, R&D Systems, USA). The absorbance (A) developed in proportion to the amount of specific cytokine was recorded using an enzyme mark instrument (SPECTRAMAX 190, Molecular Devices Corporation, USA) at 450 nm wavelength. A standard curve was thus generated to calculate the concentrations of sFlt. The EE is defined as follows: Drug EE=(Total dosage in the system-Free dosage in the carrier)/Total dosage in the system×100%. The clinical ultrasound system as described in the literature (Accusonic, Metron Medical Australia Pty. Ltd., Carrum Downs, Australia) was used to irradiate the chest of a mouse model [7] . The therapy ultrasound was delivered at 3 MHz, 2 w/cm 2 , duty cycle 20% for 2 min. Ultrasound irradiation of the lungs with this energy density is expected to rupture microbubbles and release drugs. The microbubble solution of 1 mL was centrifuged at 4°C for 12 000 r/min, and 500 μL supernatant was taken to determine free dosage in the carrier. The microbubbles of the precipitates were broken by irradiation with the above-mentioned ultrasonic intensity, and the drug dosage in microbubbles was measured. The two dosages were added to get the total dosage in the system, and then EE was calculated. Ethical approval for this investigation was obtained from the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology (IACUC Number: 354). All animal experiments complied with the ARRIVE guidelines. Male BALB/c mice (4-6 weeks old, 20±2 g) were obtained from the Animal Center of Tongji Medical College and the experiments were carried out in this center. The mice were kept in an animal facility for at least 3 days prior to experiments to allow adaptation to the environment and confirmation of their health. The animals were maintained on a lightdark cycle with light from 8:00 to 20:00 at 25°C. Each animal received rodent laboratory chow and water ad libitum. All experiments were approved by the animal care committee. Ninety mice were randomly divided into 6 groups with 15 mice in each group: PBS group (PBS+PBS); blank group (PBS+empty microbubbles); lipopolysaccharide (LPS) group (LPS+PBS); ARDS group (LPS+empty microbubbles); control group (PBS+sFlt microbubbles); and treatment group (LPS+sFlt microbubbles). All mice were anesthetized with pentobarbital sodium (40 mg/kg, intraperitoneal injection). The mice in the LPS group, ARDS group, and the treatment group were intraperitoneally injected with 10 mg/kg LPS (2 mg/mL, Escherichia coli 055: B5, Sigma Chemical, USA) [8] . The mice in the PBS group, blank group, and the control group were intraperitoneally injected with equal volume of PBS. Right after LPS or PBS administration, sFlt microbubbles of 50 µg/kg (5 µg/mL) were infused via the tail vein to each mouse in the control and the treatment groups [9] . The mice in the PBS group and the LPS group were given equal volume of PBS. The mice in the blank and ARDS groups were given equal volume of empty microbubbles. The animals were then irradiated with chest ultrasound at 3 MHz, 2 w/cm 2 , duty cycle 20% for 2 min to target the release of sFlt in lung tissue. The average treatment time per animal was about 4-5 min. Five mice were randomly selected from each group and the arterial blood samples were collected after 6 h. Blood gas analysis was carried out with a blood gas analyzer (ABL700, Radiometer Co., Denmark). Glutamic-pyruvic transaminase (GPT) and blood urea nitrogen (BUN) were measured with an automatic biochemistry analyzer (AU640, OLYMPUSOPTICAL Co., Ltd., Japan) to evaluate the function of liver and kidney respectively [10] . Immediately after cardiac puncture, the mouse was killed by exsanguination and the thorax was opened with a midline thoracotomy. The right upper lobes of the lungs were harvested and immersed in 10% formalin until processing in paraffin wax for histopathological examination. The lung tissues of 0.5 g were made into homegenate, and the supernatants were frozen at -20°C. The right lower lobes of the lungs were fixed with glutaraldehyde and 4% phosphate solution mixture. The left lung was weighed (wet weight) using an electronic balance in a clean test tube and then dried in a constant temperature drying box (80°C, 24 h). The dry weight was weighed and the wet weight to dry weight ratio (W/D) was calculated. The remaining 60 mice were returned to their original feeding environment to allow feeding and drinking freely. The behavior and 7-day mortality of the mice were closely observed. The right upper lobes of the lung slides were examined with a light microscope. The lung injury score was recorded as described previously. The right lower lobes of the lung slides were examined by an electron microscope (Tecnai G2 12, FEI Co., Holland). The VEGF level in the lung tissue homogenate was determined using an enzyme-linked immunosorbent assay kit for mouse VEGF according to the manufacturer's instruction (MMV00, R&D Systems Incorporation, USA). The VEGF levels in different groups were measured and calculated as reported previously [2] . Statistical analysis was conducted using SPSS 23.0 for windows (SPSS Co., Ltd., USA). Data were expressed as means±SD except for lung injury scores which were represented as median (range). The lung injury scores were analyzed using the Kruskal-Wallis rank test followed by Mann-Whitney U test. The difference in mortality between groups was examined by Fisher test. The other data were analyzed by oneway analysis of variance (ANOVA). A value of P<0.05 indicates that the difference was significant. The light microscopy showed that the sFlttargeted microbubbles were uniform and the surface was smooth (fig. 1 ). The particle size ranged from 1.8 to 5.1 μm, with an average particle size of 2.9 μm. Fig. 2 showed the standard curve of sFlt concentration and (A) values. The EE of sFlt-targeted microbubbles was 89.73%±6.85% . Fig. 3 showed that the experimenter injected drugloaded microbubbles into the tail vein of mice and monitored whether the microbubbles reached target organs. There were no microbubbles in lung at 0 s 4B ). Through the next step of ultrasound irradiation, the drug-loaded microbubbles were released to lung tissue. Mice in ARDS group and treatment group showed obvious erect hair, chills, malaise, shortness of breath, and then sleepiness clumps. They did not eat, their eyes were covered with secretions, and some mice The mice in the PBS group, blank group, and the control group showed normal lung injury score and W/D ( fig. 5C and 5D ) and those in the LPS and ARDS groups exhibited significantly increased lung injury score and W/D. As compared with those in the ARDS group, mice in the treatment group presented decreased lung injury score and W/D (P<0.05). The PaO 2 levels were normal in the PBS group, the blank group, and the control group (fig. 5E ). The LPS group and ARDS group showed significant hypoxia (P<0.05). PaO 2 was improved significantly in the treatment group (P<0.05). As shown in fig. 5F , the VEGF levels in the LPS group, the ARDS group, and the treatment group were significantly higher than those in the PBS group, the blank group, and the control group (P<0.05). As shown in fig 5G, the mortality rate of the ARDS model was 60% (95% confidence interval 47.5%-72.5%), and that with sFlt-targeted microbubbles was significantly lower at only 40% (95% confidence interval 27.5%-52.5%, P<0.05). Fig. 6 showed the ultrastructure of lung tissue in different groups. The tight junction of lung tissue in the PBS group ( fig. 6A), blank group (fig. 6B) , and control group ( fig. 6E ) was closely connected and the respiratory membrane was thin. The tight junction in LPS group ( fig. 6C ) and ARDS group ( fig. 6D ) was widely loosened, the respiratory membrane was thickened, and leukocytes infiltrated into the lung tissue. In the treatment group ( fig. 6F ), the tight connection was basically normal, and the shape of Our research indicated that LPS intraperitoneal injection brought about shortness of breath, cyanosis, and increased W/D and lung injury score. The ARDS model was characteristic of extensive morphological lung damage, such as edema, incrassation of the alveolar wall, and infiltration of inflammatory cells into alveolar and interstitial spaces [11] . This model was fully consistent with the etiology, pathophysiology and clinical characteristics of ARDS as reported previously. In this study, the mortality rate of ARDS group was 60%, which was slightly higher than the clinical mortality as reported by other researchers [12] . This will make it easier to study the effects of protective drugs. In this study, sFlt-targeted microbubbles were stable and had high encapsulation efficiency [13] . After the drug-loaded microbubbles entered the blood circulation, the lungs were irradiated by ultrasound, which ruptured the drug-loaded microbubbles and enabled drug release to occur in the lung tissues targeted. The encapsulation and targeted release of microbubbles retarded liver and kidney damage resulting from direct systemic administration of sFlt [14, 15] . Moreover, sFlt-targeted microbubbles decreased pulmonary edema and maintained the normal structure and function of respiratory membrane in lung tissue. These pieces of evidence suggested that sFlt-targeted microbubbles reached the lung tissue accurately and blocked the effect of VEGF in the target organ. This work indicated that the increased VEGF participated in the damage of three layer structure of respiratory barrier through tight junction proteins. In line with our previous studies, anti-VEGF targeted with sFlt-targeted microbubbles could decrease the loosening of tight connection. And sFlt-targeted microbubbles could decrease the lung injury score and pulmonary edema (W/D), repair the alveolar epithelial barrier, and improve the gas exchange function of ARDS model [16] [17] [18] . As a result, the sFlt-targeted microbubbles could effectively improve the gas exchange function of lung. Our previous study have shown that VEGF played an important role in the pathogenesis of inflammation [2] . Anti-VEGF with sFlt could maintain the normal structure and function of respiratory membrane in lung tissue via tight junction proteins. However, sFlt systemic application has been shown to reduce vascular permeability and affect the function of other organs, such as liver and kidney with clinical manifestations being anuria, malaise, and even death [14, 15] . Therefore, in our previous studies, systemic application of sFlt did not reduce the mortality of animal models. In this study, we targeted the release of sFlt in lung tissue, which can effectively improve the mortality of ARDS animal model. This is the most important result of this study. This study makes sFlt closer to clinical application. To sum up, our research indicates that anti-VEGF targeted with sFlt-targeted microbubbles works to maintain the normal function of the liver and kidneys and could be a suitable therapeutic tool for the treatment of ARDS. 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