WKYMVm hexapeptide, a strong formyl peptide receptor 2 agonist, attenuates hyperoxia-induced lung injuries in newborn mice

www.nature.com/scientificreports OPEN Received: 8 August 2018 Accepted: 13 February 2019 Published: xx xx xxxx WKYMVm hexapeptide, a strong formyl peptide receptor 2 agonist, attenuates hyperoxia-induced lung injuries in newborn mice Young Eun Kim1, Won Soon Park2,3, So Yoon Ahn2, Dong Kyung Sung3, Se In Sung2, Jae Ho Kim4 & Yun Sil Chang1,2,3 The hexapeptide WKYMVm, which is a strong formyl peptide receptor (FPR) 2 agonist, exhibits pro-angiogenic, anti-inflammatory and anti-apoptotic properties. However, its therapeutic efficacy in bronchopulmonary dysplasia (BPD) has not been tested to date. Here, we investigated whether WKYMVm attenuates hyperoxia-induced lung inflammation and ensuing injuries by upregulating FPR2. The proliferation and tube formation ability of human umbilical vein endothelial cells (HUVECs), along with the level of extracellular signal regulated kinase (ERK) phosphorylation, were evaluated in vitro. Newborn mice were randomly exposed to 80% oxygen or room air for 14 days starting at birth. WKYMVm (2.5 mg/kg) was intraperitoneally administrated daily from postnatal day (P) 5 to P8. At P14, mice were sacrificed for histopathological and morphometric analyses. Along with upregulation of FPR2 and p-ERK, WKYMVm promoted HUVEC cell proliferation and tube formation in vitro. Additionally, WKYMVm promoted proliferation of human pulmonary microvascular endothelial cells (HULEC-5a) and murine pulmonary endothelial and epithelial cells in vitro. WKYMVm significantly attenuated hyperoxia-induced lung inflammation, as evidenced by increased inflammatory cytokines, neutrophils, and alveolar macrophages, and resultant lung injuries, which included impaired alveolarization and angiogenesis, an increased number of apoptotic cells, and reduced levels of growth factors in vivo, such as vascular endothelial growth factor and hepatocyte growth factor. WKYMVm attenuates hyperoxiainduced lung injuries and lung inflammation by upregulating FPR2 and p-ERK. Despite recent advances in neonatal intensive care medicine, bronchopulmonary dysplasia (BPD), a chronic lung disease that occurs in premature infants receiving prolonged mechanical ventilation and oxygen supplementation, still remains a major cause of mortality and morbidity in survivors with few effective treatments1,2. Although BPD has a multifactorial aetiology, inflammation has been known to play a key role in the pathogenesis of BPD lung injuries including impaired alveolarization and angiogenesis3,4. Therefore, there is an urgent need to develop safe and effective anti-inflammatory agents as potential novel therapeutic candidates for BPD. Recent studies have shown that the WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met) hexapeptide, a strong formyl receptor (FPR) 2 agonist, has pleiotropic anti-inflammatory, pro-angiogenic, anti-apoptotic and immunomodulatory effects5 in various animal models of sepsis6, ulcerative colitis7, myocardial infarction8, ischemic hindlimb9 and diabetic cutaneous wound healing10. These data support the development of WKYMVm as a novel and effective anti-inflammatory therapeutic agent. However, the precise role of FPR2 in the pathogenesis of BPD and the functional significance of the FPR2 agonist WKYMVm in attenuating hyperoxia-induced neonatal lung injuries remain to be clarified. 1 Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University, Seoul, South Korea. 2Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea. 3Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, South Korea. 4Department of Physiology, School of Medicine, Pusan National University, Yangsan, South Korea. Young Eun Kim and Won Soon Park contributed equally. Correspondence and requests for materials should be addressed to Y.S.C. (email: ) Scientific Reports | (2019) 9:6815 | https://doi.org/10.1038/s41598-019-43321-4 1 www.nature.com/scientificreports www.nature.com/scientificreports/ Thus, in this study, we investigated the therapeutic efficacy of the FPR2 agonist WKYMVm in attenuating hyperoxia-induced lung inflammation and ensuing lung injuries, including impaired alveolarization and angiogenesis in newborn mice. Materials and Methods Isolation and culture of mouse lung endothelial cells. After 1- to 2-week-old mice (BALB/c) were anesthetized with ketamine/xylazine (140/14 mg/kg), ice-cold DMEM was injected via the right ventricle to flush the lungs of blood. One millilitre of collagenase type II (10 mg/ml) (GIBCO, Grand Island, NY) and DNase I (20 µg/ml) (Sigma-Aldrich, St. Louis, MO, USA) were quickly instilled through the trachea into the lungs, and then, the lungs were chopped as fine as possible. Chopped lungs were subsequently removed and incubated with 5 ml of collagenase II in a 50 ml tube for 30 min in a 37 °C shaking incubator. After the 40 min incubation, 25 ml of 1 × PBS was added to the tube. The tube was then vigorously shaken for 30 sec to dissolve the lung, and the resulting tissue/cell suspension was filtered through a 100 µm and a 40 µm strainer. Fetal bovine serum (FBS) was added to quench collagenase activity. The cells were centrifuged at 300 g for 10 min. The cells were washed once with 10 ml of HBSS/0.75% BSA and centrifuged again. After resuspension with 1 ml of sterile MACS buffer (PBS/0.75% BSA/2 mM EDTA), the cells were transferred to a new tube and centrifuged again at 400 g for 10 min. The cells were resuspended with 90 µl of MACS buffer and 10 µl of CD31-conjugated microbeads (Miltenyi Biotech, Bergish Gladbach, Germany). One millitre of MACS buffer was added to the cells, and the entire volume was applied to the column. The column was washed three times, and the cells were eluted. The cells were centrifuged at 400 g for 5 min and resuspended in 0.1% gelatin-coated plates. The purity of endothelial cells was determined with CD31 FACS analysis (Supplementary Fig. S2A). Isolation and culture of rat lung epithelial cells. After 4- to 8-week-old Sprague-Dawley rats were anesthetized with ketamine/xylazine (140/14 mg/kg), ice-cold DMEM was injected via the right ventricle to flush the lung of blood. A tracheal cannula was carefully inserted into the lung. We attached the barrel of a 1 ml syringe to the opening of the tracheal cannula and then slowly injected 1 ml of DMEM into the lung. We detached the syringe from the tracheal cannula and poured the lavage fluid from the lung. We repeated this procedure at least 6 times to remove as many macrophages as possible. The lungs were digested by instilling 2 ml of elastase (10 U/ml) at 37 °C and incubating for 20 min. The digested lungs were transferred to a Petri dish. After trimming away the trachea and major bronchi, the lung parenchyma was chopped using curved scissors into small, 1 to 2 cm2 pieces. Five millilitres of FBS was added to stop the digestion. Then, 1 (...truncated)