Transplanted endothelial progenitor cell improves lung structure in neonatal rats exposed to hyperoxia

Abstract

Objective To study the effect of transplanted endothelial progenitor cell (EPC) on hyperoxia-induced lung injury in neonatal rats. Methods Rat bone marrow mononuclear cells were cultured in endothelial cell growth medium to obtain EPCs, which were identified by morphology, phagocytosis and CD34+ analyses. Sixty neonatal Sprague-Dawley rats were allowed to acclimate in room air for 24 h after birth, and were then divided into four groups (15 per group), including the air group, the hyperoxia group, the EPCs transplantation group and the Nω-nitro-L-arginine methyl ester (L-NAME) intervention group. Neoborn rats in the Air and Hyperoxia groups were fed in the room air or hyperoxia (85% oxygen) for 28 days. For rats in transplantation group were exposed continuously to hyperoxia for 28 days, and got an EPC (1×105 cells) injection on the 21st day. Rats in Intervention group were exposed continuously to hyperoxia for 28 days, got an EPC (1×105 cells) injection on the 21st day, and a daily injection of L-NAME from day 21 to day 28, with a daily dose of 20 mg/kg. Levels of circulating CD34+ cells and serum VEGF expression were detected. Specimens from lung tissues were analyzed by immunohistochemistry or immunofluorescence. The expression of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2) and eNOS were detected by real-time polymerase chain reaction and Western-blotting. NO production were detected by nitrate reductase assay. One-way ANOVA and Bonferroni test were used for statistical analysis. Results (1) The cultured cells had a typical cobblestone appearance; double positive cell binding of fluorescein Ulex Europaeus agglutinin-1 and uptake of Dil-labeled acetylated low density lipoprotein accounted for approximately 85% of the total number of cells. CD34+ cells accounted for 68.2%-72.4% of total cultured cells. (2) Circulating CD34+ cells in the air group, hyperoxia group, EPC transplantation group and L-NAME intervention group were (1.91±0.34)%, (1.06±0.10)%, (1.47±0.06)% and (0.77±0.11)% (F=32.710, P=0.000). The number of circulating CD34+ cells in the hyperoxia group was lower than the air group, in the EPC transplantation group the number of these cells was higher than the hyperoxia group, and in the L-NAME intervention group the number of these cells was lower than that in the EPC transplantation group, and the differences between these two groups were statistically significant (P<0.05, respectively). Serum VEGF in the four groups was (7.90±2.72), (6.38±0.72), (14.00±1.66) and (11.70±1.91) pg/ml, respectively. The difference between the four groups was statistically significant (F=22.809, P=0.000), and serum VEGF in the EPC transplantation group was higher than that in the hyperoxia group (P<0.05). (3) Transplanted EPCs could engraft in pulmonary vascular endothelium and alveolar interstitium, and L-NAME intervention significantly reduced the engraftment of EPCs in the lungs (10.7±0.47 / field vs 16.95±0.5 /field, t=17.820, P=0.000). (4) There were significant differences in the radial alveolar count (RAC) and number of microvessels between the four groups (F=859.580 or 211.150, P=0.000, respectively). RAC and the number of microvessels in the hyperoxia group were less than those in the air group (7.98±0.23 vs 13.12±0.20, 3.98±0.42 vs 9.50±0.22, P<0.05, respectively). The number of microvessels in the EPC transplantation group was 5.40±0.41, being higher than that in the hyperoxia group (P<0.05). (5) VEGF mRNA in lungs in the hyperoxia group was lower than that in the air group (0.23±0.16 vs 1.05±0.33, P<0.05); in the EPC transplantation group, VEGF mRNA was higher than that in the hyperoxia group (0.69±0.09 vs 0.23±0.16, P<0.05); and in the L-NAME intervention group, VEGF mRNA was lower than that in the EPC transplantation group (0.31±0.08 vs 0.69±0.09, P<0.05). VEGF protein in the lungs in the hyperoxia group was lower than the air group (0.52±0.01 vs 0.82±0.01, P<0.05), and was higher in the EPC transplantation group than the hyperoxia group (0.58±0.05 vs 0.52±2501, P<0.05). VEGFR2 mRNA in the hyperoxia group was lower than the air group (0.35±0.13 vs 1.07±0.45, P<0.05). eNOS mRNA in the hyperoxia group was lower than the air group (0.46±0.10 vs 1.05±0.36, P<0.05). eNOS protein in the hyperoxia group was lower than the air group (0.32±0.01 vs 0.51±0.03, P<0.05), and was higher in the EPC transplantation group than the hyperoxia group (0.86±0.02 vs 0.32±0.01, P<0.05). Conclusion Transplanted EPC can engraft in the lung tissue, improving alveolar and pulmonary vascular development, which may be associated with upregulation of the expression of eNOS and VEGF in lung. Key words: Endothelial progenitor cells; Stem cell transplantation; Bronchopulmonary dysplasia; Vascular endothelial growth factor A; Nitric Oxide Synthase; Rats