Inhaled Nitric Oxide Induces Endothelial Progenitor Cells Homing To Prevent Bronchopulmonary Dysplasia

IntroductionBronchopulmonary dysplasia (BPD) is a chronic lung disease associated with high morbidity and mortality in preterm infants. The presence of a few, enlarged distal airspaces and a dysmorphic pulmonary microvascular network are the histological hallmarks of BPD. The strategy of restoring pulmonary vascular has become a novel and promising therapy for BPD. Endothelial progenitor cells (EPCs) are precursors of endothelial cells, which have the capacity of self-renewal and high proliferation. The bone marrow is a reservoir of stem/progenitor cells. EPC homing means EPCs are recruited to the site of ischemia tissue in response to endogenous or exogenous signals and substantially contribute to neovascularization. EPCs are becoming a new medicine for diseases associated with vasculogenesis. Recent evidences suggest that EPCs are involved in the pathogenesis of BPD. The reduction in the EPCs in bone marrow, circulation and lungs has emerged in animal model of BPD. The data suggests that EPCs have a protective role against the development of BPD. Studies have demonstrated that endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) play important roles in mobilization and homing of EPCs. eNOS transcription enhancer and NO donor supplementation could increase EPCs number and improve vasculogenesis in ischemic diseases. Inhaled NO was first used as a vasodilator to selectively dilate pulmonary vessels, and was then shown to have anti-inflammatory effects in the lungs. Recent data showed that inhaled NO can reduce incidence of BPD in very preterm infants, but its efficacy is controversial. Inhaled NO was reported to restore vessel density and improve lung structure in neonatal rat model of BPD. Animal studies showed that inhaled NO can promote EPCs mobilize from bone marrow, recruit to lungs, and contribute to repair of acute lung injury. We hypothesized that inhaled NO may induce transplanted EPCs homing to the lungs, and therefore prevent the development of BPD. The first part of this study is to investigate the impact of eNOS/NO signaling pathway on EPCs homing in vitro. In the second part of this study, we established the neonatal rat BPD model, studied whether inhaled NO can induce transplanted EPCs homing to prevent BPD synergistically, and evaluated its mechanism. We hope to provide some evidence for the use of EPCs transplantation combined with inhaled NO to prevent BPD.Part Ⅰ eNOS/NO Signaling Pathway Regulates EPCs Homing in vitroBackground:EPCs are precursors of endothelial cells, which have the capacity of self-renewal and high proliferation. EPCs can mobilize and home to the site of vascular injury. eNOS expression is an important feature of EPCs. eNOS can use L-arginine, oxygen and cofactors to produce NO. eNOS/NO signaling pathway is an important signaling pathway for the regulation of EPCs mobilization from bone marrow. However, the role of eNOS/NO signaling pathway in the EPCs homing is not fully understood. L-NAME is a nonspecific inhibitor of eNOS, which can competitively inhibit eNOS activity and reduce the NO production.Objectives:In this part of study, we tried to isolate, purify, and enrich rat bone marrow-derived EPCs. We also determined to investigate EPCs homing activities including migration, proliferation, tube formation in vitro, and tried to explore the role of eNOS/NO signaling pathway in these activities.Methods:Mononuclear cells were obtained from femoral and tibia bone marrow of rats, and cultured for7-10days. The abilities of binding FITC-UEA-1and intaking Dil-ac-LDL were detected by fluorescence microscopy. The level of circulating CD34+cells was detected by flow cytometry. The activities of migration, proliferation and tube formation were detected by cell culture chamber migration system, matrigel system and flow cytometry in the presence or absence of L-NAME. The mRNA and protein expression levels of CXCR4, CXCR7, VEGFR2, and eNOS were detected by real-time quantitative PCR and western blot. NO production was detected by nitrate reductase methods.Results:Colony-like cells appeared on the5th day after mononuclear cells were cultured. Typical spindle-shaped, double needle-like cells appeared on the7th-10th day. The confluence of adherent cells at this time reached about80%, and it’s the time to harvest cells and use them for in vitro studies. The percentage of FITC-UEA-1and Dil-ac-LDL double-stained cells reached to about85%. The percentage of CD34+ cells was68.2-72.4%. Cells migration, proliferation, and tube formation were significantly weakened after eNOS activity was inhibited (P<0.05, respectively). The mRNA and protein expression of CXCR4and eNOS were significantly reduced after eNOS activity was inhibited (P<0.05, respectively), while CXCR7and VEGFR2were influenced little. NO production was dramatically decreased after eNOS activity was inhibited (P<0.05).Conclusion:Rat bone marrow-derived EPCs were successful isolated and purified. L-NAME significantly reduced the eNOS expression and NO production of EPCs. The activities of EPCs migration, proliferation, and tube formation were weakened when eNOS was inhibited by L-NAME, which suggested that the eNOS/NO signaling pathway play an important role in EPCs’activities. Part Ⅱ Inhaled NO Induces Transplanted EPCs Homing to Prevent Bronchopulmonary Dysplasia in Neonatal RatsBackground:Many risk factors are involved in the cause of BPD, and the treatment of BPD is also diverse. Inhaled NO and stem cell therapy for BPD is still in the experimental stage. The indications of inhaled NO in neonates are persistent pulmonary hypertension and progressive hypoxemic respiratory failure. Recent data showed that inhaled NO can reduce incidence of BPD in very preterm infants, but its efficacy is controversial. Animal experiments showed that bone marrow-derived angiogenic cells injected into the pulmonary circulation could restore lung alveolar and vascular growth of BPD. Some studies showed that inhaled NO can mobilize EPCs from bone marrow of preterm piglets, increase the engraftment of EPCs in the lungs, reduce lung vascular injury, and promote lung repair. We speculated that the combination of inhaled NO with EPCs transplantation could achieve a synergy therapy for BPD.Objectives:In this part of study, we tried to evaluate the efficacy of combining transplanted EPCs and inhaled NO to prevent neonatal rat’s BPD, and we also tried to explore whether inhaled NO could induce EPCs homing to the lung and its mechanism.Methods:BPD model was established by exposing newborn rats continuously to hyperoxia (oxygen concentration was maintained at0.85) for28days. EPCs labeled with BrdU in vitro were injected into the circulation of newborn rats through tail veins. The experiment was designed in6groups,15newborn rats in each group. Air group (C group):Newborn rats were fed in the room air for28days. High oxygen group (O group):Newborn rats were exposed continuously to hyperoxia for28days. High oxygen+EPCs transplantation group (E group):Newborn rats were exposed continuously to hyperoxia for28days; EPCs (1×105cells) were injected through tail veins on the22th day. High oxygen+EPCs+L-NAME group (L group):Newborn rats were exposed continuously to hyperoxia for28days; EPCs (1×105cells) were injected through tail veins on the22th day; L-NAME was injected intraperitoneally daily in the last7days (20mg/kg/day). High oxygen+EPCs+inhaled NO group (N group):Newborn rats were exposed continuously to hyperoxia for28days; EPCs (1×105cells) were injected through tail veins on the22th day; Since the22th day of hyperoxia, newborn rats was inhaling NO (NO concentration was maintained at5ppm) continuously for7days. High oxygen+EPCs+inhaled NO+L-NAME group (S group):Newborn rats were exposed continuously to hyperoxia for28days; EPCs (1×105cells) were injected through tail veins on the22th day; Since the22th day of hyperoxia, newborn rats was inhaling NO (NO concentration was maintained at5ppm) continuously for7days; L-NAME was injected intraperitoneally daily in the last7days (20mg/kg/day). Regular replacements of feed, bedding, exchang of maternal rats were carried out daily. The growth and respiration of newborn rats were observed and recorded, and the oxygen concentration, humidity and temperature were monitored. The concentration of inhaled NO was monitored on real-time. The level of circulating CD34+cells at the end of the experiment was detected by flow cytometry, and serum VEGF expression was detected by ELISA. Specimens from lung tissues were collected after pulmonary circulation was flushed through the right ventricle. The right upper and middle lobes were used for detecting VEGF, VEGFR2, eNOS and SDF-1mRNA expression by real-time fluorescence quantitative PCR. The right lower lobe was used for detecting protein levels of VEGF, VEGFR2and eNOS by western blot. The right vice lobe was used for detecting NO production by nitrate reductase assay. The left lung tissue was fixed in4%paraformaldehyde, and embedded in paraffin. Morphological changes and radial alveolar count (RAC) were analyzed by immunohistochemistry. The expression of factor VIII in the lungs was examined by the immunohistochemistry. The engraftment of transplanted EPCs in lungs was evaluated by immunofluorescence. Results:1、BPD model establishmentAfter exposure to hyperoxia for two weeks, newborn rats lost hair luster, gained weight slowly, were significantly short-of-breath. At the end of the experiment, the body weight of litters exposed to hyperoxia was significantly lower than that of rats exposed to room air. Lung histology of neonatal rats exposed to hyperoxia demonstrated enlargement of distal air spaces with a simplification of lung structure. However, the lung structure of neonatal rats exposed to room air maintained normal.2、Inhaled NO combined with transplanted EPCs improved alveolar numbers and pulmonary vascular abnormality in neonatal BPD modelThere was significant difference of RAC among6groups. RAC of neonatal rats exposed to hyperoxia (the O, E, L, N and S groups) decreased significantly compared with that of rats exposed to room air. RAC in the N group was significantly higher than that in other hyperoxia groups (the O, E, L and S groups). Vessel density of rats exposed to hyperoxia was strikingly decreased compared with that of rats exposed to room air reflected by factor Ⅷ staining. Vessel density in the N group was significantly improved compared with that in other hyperoxia groups (the O, E, L and S groups).3、Inhaled NO combined with transplanted EPCs increased the level of circulating CD34+cellsThere was significant difference of circulating CD34+cells among6groups. The level of CD34+cells in hyperoxia groups (the O, E L and S groups) reduced significantly compared with that in the air group. The levels of CD34+cells were improved significantly in the E and N groups compared with that in groups treated with eNOS inhibitor (the L and S groups). The level of CD34+cell increased dramatically in the N group compared with that in other4hyperoxia groups (the O, E, L and S groups). There was no significant difference in the level of CD34+cells between the O and L group, while inhaled NO can significantly increase the level of CD34+cells.4、Engraftment of transplanted EPCs in the lungsThe transplanted EPCs could engraft in the lungs. The engraftment in the N group increased significantly compared with that in other groups (the E, L and S groups). The engraftment in the E group was more robust than that in the L group.5、The impact of inhaled NO combined with transplanted EPCs on the expression of homing molecules (VEGF/VEGFR2, eNOS, SDF-1and NO)(1) Serum VEGF levelThere was significant difference of serum VEGF level among6groups. The serum VEGF level in the O group decreased significantly compared with that in the room air group. The serum VEGF level increased significantly in EPCs intervention groups (the E, L, N and S groups) compared with that in the EPCs non-intervention groups (the C and O groups).(2) VEGF/VEGFR2, eNOS and SDF-1mRNA expression in lungsThere was significant difference in the VEGF mRNA expression among6groups. The VEGF mRNA expression reduced significantly in hyperoxia intervention groups (the O, E, L, N and S groups) compared with that in the room air group. The VEGF mRNA expression increased significantly in the E group compared with that in the L and S group; however, there was little change compared with that in the N group.There was significant difference in the VEGFR2mRNA expression among6groups. VEGFR2mRNA expression reduced significantly in hyperoxia intervention groups (the O, E, L, N and S groups) compared with that in the air group. There was no significant difference in VEGFR2mRNA expression among the O, E, L, N and S groups.There was significant difference of the eNOS mRNA expression among6groups. The eNOS mRNA expression decreased significantly in the O, L, and S groups compared with that in the air group. There was no significant difference between the E and N groups. The eNOS mRNA expression significantly improved in the N group compared with that in the O group.No difference was found in SDF-1mRNA expression among all6groups.(3) The VEGF/VEGFR2and eNOS protein expressionThere was significant difference of VEGF protein expression among6groups. The VEGF protein expression reduced significantly in the O, E, L and S groups compared with that in the C and N groups. There was no difference between the C and N groups. There was no significant difference among the O, E, L and S groups.No significant difference was found in VEGFR2protein expression among all6groups.There was significant difference in eNOS protein expression among6groups. eNOS expression increased significantly in the EPCs intervention groups (the E, L, N and S groups) compared with that in EPCs non-intervention groups (the C and O groups). There was no difference among EPCs intervention groups (the E, L, N and S groups).(4) NO productionNo difference was found in production of NO among all6groups.Conclusion:NO inhalation combined with EPCs transplantation can improve abnormalities of alveoli and vessels in BPD. Inhaled NO can induce transplanted EPCs homing to the lung, which may be associated with the upregulation of eNOS expression in lungs.