| IntroductionVascular injury is very common in many lung diseases in children. Serious lung diseases such as acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), neonatal respiratory distress syndrome (NRDS), meconium aspiration syndrome (MAS) and bronchopulmonary dysplasia (BPD) carry high mortality and moribidity in children. The histologic changes of lung tissues in these diseases are characterized by extensive microvascular damage. Therefore, it is crucial to restore pulmonary endothelial function and therapeutics targeted at endothelium reparation may be promising strategy for threatment of lung injury in children.Endothelial progenitor cell (EPC), mainly derived from bone marrow, is the precursor of endothelium cells and has the ability of self-renew and proliferation. EPC can be mobilizied from bone marrow, and migrate to the peripheral circulation where they contribute to the repair of injured endothelium and the formation of new blood vessels. The cardiovascular field has tremendously advanced the understanding of the relationship between EPC and damaged endothelium in cardiovascular disease. Recently, investigators in pulmonary medicine have become interested in the utility of EPC in disorders involving the pulmonary vasculature. But these studies mainly focused on adult lung diseases and the data is little about the lung injury in children. Therefore, we performed this study to investigate the level of circulating EPC in infants with RDS along with its’ mobilizating factors. Furthermore, we established an oleic acid induced ALI model in piglet and to compare the circulating EPC level in ALI with different severity, to elucidate the EPC mobilization mechanism, and to investigate the effect of iNO on EPC level and reparation of lung injury. We hope to explore the role of EPC in RDS and ALI, and propose a novel promising approach in the clinical therapy with EPC Part Ⅰ Level of Circulating CD34+Cells and its Relationship with Clinical Outcome in RDS InfantsBackground RDS remains an important cause of mortality and morbidity in preterm infants despite exogenous surfactant and lung protective ventilation strategy are widely applied. Both mechanical ventilation and hyperoxia may contribute to acute and chronic lung injury at alveolar and vascular compartments, leading to development of BPD. Many investigators have undretakn researches on the changes of bone marrow derived stem and progenitor cells in diseases of adult lung, but the role of these cells in neonatal lung disease has not been characterized. It is reported that the number of circulating EPC was decreased after hyperoxia exposure, and thereby the vascular growth was arrested in the lung, leading to the development of BPD. A recently research reported that extremely preterm infants who displayed lower numbers of EPC had an increased risk of developing BPD. However, it is unclear whether circulating CD34+cells contribute to the repair of lung injury and consequently improve the outcome of RDS infants.Objectives To investigate whether the level of circulating CD34+cells was elevated along with its mobilizing cytokines in neonatal with RDS; and to deterime whether the circulating CD34+cell level is associated with clinical outcome of RDS.Methods Forty-one RDS infants admitted to the neonatal intensive care units (NICU) were enrolled in the study from February2007to May2008. Preterm (n=20) and term infants (n=14) without diffuse lung diseases admitted to neonatal ward during the same period served as controls. Circulating CD34+cells were identified by flow cytometry. Plasma concentrations of vascular endothelial growth factor (VEGF), stromal cell-derived factor-1(SDF-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were determined by immunochemical assays. Patient information was collected at the same time, and correlation was analysed between the level of CD34+cells and the short term outcome.Results Forty-one RDS infants in comparison with20preterm and14term controls were enrolled in this study. The RDS infants had a significant higher number of CD34+cells [25(6-174) cells/μl] compared with preterm [15(1-100) cells/μl] and term controls [9(2-22) cells/μl], suggesting that bone marrow derived stem and progenitor cells were mobilizied in RDS. An inverse correlation was observed between CD34+cell level and SNAPPE-Ⅱ on admission in RDS (r=-0.473, P<0.01), but not in the controls. The median number of CD34+cells was higher in RDS survivors than in the non-survivors [26(6-174) vs.4(8-11) cells/μl; P<0.05), as reflected by a trend of lower level of CD34+cells with prolonged duration of ventilation (r=-0.396, P<0.05). Plasma concentration of SDF-1was significantly higher in RDS than in the preterm controls (12.6±1.8vs.9.6±1.8ng/ml, P<0.01). Moreover, there was a correlation between the level of CD34+cells and the plasma SDF-1concentration (r=0.305, P<0.01). This suggested that SDF-1participate in the mobilization and homing of CD34+cells in RDS. In the preterm and term control infants, there was an inverse correlation between the number of CD34+cells and GA (r=-0.373, P<0.05).Conclusions The level of circulating CD34+cells was elevated in RDS and was associated with improved outcome. The plasma SDF-1concentration was correlated with the level of CD34+cells, suggesting that the CD34+cells might be involved in the reparation of neonatal lung injury. Part Ⅱ Effects of Inhaled Nitric Oxide on Circulating Endothelial Progenitor Cells Level and Vascular Reparation in Piglets with Oleic Acid Induced Acute Lung InjuryBackground Acute lung injury (ALI), and its severe form, acute respiratory distress syndrome (ARDS) are major causes of death in intensive care units in children. Despite the advances in supportive care and pharmacologic treatment, recent data indicate that the mortality of acute respiratory failure from ALI is still high at about40%. In our country, the mortality of ARDS in a clinical trial of25children hospitals is about61%in2004, and the value is about44.8%in recently reported data in2005-2006. Efforts to find ideal therapies have been unrevealing. The histologic changes of lung tissue in ARDS are characterized by destruction of pulmonary endothelium, and the injured alveolar-capillary barrier leads to increased pulmonary vascular permeability. The therapies targeted at repairing or limiting of endothelial damage might be a key point of lung injury reparation. Recently, Burnham et al reported that endothelial progenitor cells (EPC) numbers were twofold higher in patients with ALI compared with healthy volunteers, and higher EPC colony counts was associated with improved survival. Experiment studies indicated that infusion of EPC was benefit to ALI. However, these studies also raised some issues. It would be impossible to collect cells from a patient before disease onset to use later on illness. A more feasible possibility might be to use mobilization agents to enhance endogenous EPC release. NO is an essential molecular in bone marrow EPC mobilization. Inhaled NO can diffuse into blood fastly and its effects can be limited in the local lung issue. At the same time, NO can interact with Hb and its product SNO has a systemic effect. It is unclear whether inhaled NO can promote the recruitment of bone marrow EPC. And the investigation on this issue will provide novel therapy for ALI.Objectives To compare the level of EPC in peripheral blood and bone marrow in piglets with different severity of ALI; to investigate the effects of inhaled NO on level of EPC in circulating and bone marrow; and to explore the mechanism of EPC recruitment.Methods Twenty-six healthy,7-10kg piglets were anesthetized and orally intubated, followed by mechanical ventilation with air using pressure controlled ventilation.They were then randomly treated in5groups (n=5each):control (C), moderate ALI with P/F<300mmHg (M), severe ALI with P/F<200mmHg (SM), the severe ALI with subcutaneous injection of G-CSF at a dose of10μg/kg daily for7days (GM), the severe ALI with inhaled NO at10ppm (NM). ALI was induced by intravenous infusion of oleic acid. Moderate ALI was defined as PaO2/FiO2≤300mmHg, dynamic lung compliance (Cdyn) decreased by more than30%compared to the baseline level. Severe ALI was defined as PaO2/FiO2≤200mmHg, dynamic lung compliance (Cdyn) decreased by more than50%compared to the baseline level. Animals were ventilated for24-72h. After treatment they were weaned and feeded for recovery until day7. At baseline,0h,24h,72h and168h, blood RT was performed, blood and bone marrow EPC were measured by flow cytometry analysis, plasma concentration of SDF-1、VEGF、SCF and IL-6、IL-8、IL-10were determined by immunochemical assays. At7d, animals were sacrificed by overdose of10%potassium chloride, lung tissues and bronchoalveolar lavage fluid (BALF) were collected. Lung tissue from the tip of right mid-lobe was used for determining wet-to-dry weight ratio (W/D). The expression of CD34、KDR、CD133、VEGF、 SDF-1、CXCR4mRNA in lung tissues were measured by real-time polymerase chain reaction (real-time PCR) at the end of the experiment. Left lung was taken for histopathology and immunohistochemical analysis of VEGFR2、endothelial NO synthase (eNOS、pulmonary vascular density and lung tissue apoptosis. Bone marrow sections were taken for histopathology and bone marrow plasma concentrations of NO and MMP-9were also determined.Results There were no intergroup differences in body weight, sex and baseline condition of blood gas values. We divided the subjects into two parts for better analysis (different injury severity group and different intervention group).1. Oxygenation and lung mechanics:a) Oxygenation:Bseline values of OI, PaO2/FiO2and A-aDO2had no significant difference across all groups. At different time points (0h,2h,6h), the OI was significantly higher in the SM group than C and M group (P<0.05). The OI was higher in SM than C group at12h, but no difference was seen between SM and M group. The changes of PaO2/TiO2were similar with OI. There were significant differences in A-aD02between SM and C group at2and12h. No difference was found at other time points. In different intervention groups, a trend of improvement for OI was seen in NM compared with SM group. Moderate improvement was seen in NM compared with SM group for A-aDO2at24h (P<.05).b) Lung mechanics:At baseline, no difference was seen for the Cdyn and Vd/Vt across all groups. Among groups with different severity of lung injury, from0h to6h, the Cdyn was significantly decreased in SM group compared with C and M group, the difference was diminished at subsequent assessment time. The Vd/Vt was higher in SM group than in the C group. No difference was observed at other timepoint. Among groups with different intervention, a trend of improvement was seen for Cdyn in NM and GM group compared to SM group. A significant improvement was observed for Vd/Vt in NM compared with SM at12h and24h (P<0.05).2. Hemodynamics and lung water variables:a) CO and blood pressure:At the point of model established, the CO underwent a temporally reduce in all groups, but no difference was seen across groups. There was no difference in the alteration of blood pressure.b) EVLW、ELWI and PVPI:Baseline values of EVLW、ELWI and PVPI had no significant difference across all groups. Among groups with different severity of injury, the EVLW and ELWI were increased in SM in contrast to C group at2h and12h. The PVPI was significantly increased in SM compared to M group at2h,6h,12h and24h. Among groups with different intervention, an improvement of ELWI and PVPI was seen in the NM group in comparison with SM group (P<0.05).3. Level of EPC in different severity of lung injury:a) Level of EPC in peripheral blood:There was no significant difference among groups in the percentage of EPC to MNC at baseline. The percentages of CD34+and CD34+KDR+cells were significantly elevated in M group compared to C group at0h. The M group had significantly higher percentage of all subpopulation of EPC in contrast to C group and had significantly higher percentage of CD34+KDR+and KDR+CD133+cells in contrast to SM group at24h. No significant difference was found among groups in the number of EPC, and there was a trend of similar changes with EPC percentage.b) Level of EPC in bone marrow:There was no difference among groups in the percentage of EPC. At24h, the level of EPC was increased in SM compared to C group, and the KDR+CD133+cells and CD133+cells were increased in SM in contrast to M group (P<0.05).c) The EPC level in lung:The CD34mRNA was significantly higher in M group compared with SM and C group. A similar trend of increase was found for the expression of KDR and CD133mRNA, but no statistical significance was observed.4. Effect of inhaled NO on level of EPC and the reparation of lung injury:a) Level of EPC in peripheral blood:There was no difference among groups in the percentage of EPC to MNC. There were trend of increase in the percentage of CD34+cells, CD34+KDR+cells, KDR+CD133+cells and CD133+cells in NM group in contrast to SM group, but not statistically significant. The number of CD34+cells was higher in NM group than the SM group at0h. At24h, the numbers of CD34+cells, CD34+KDR+cells and KDR+CD133+cells were higher in NM group compared with SM group (P<0.05). The NM group had a higher number of KDR+CD133+cells than SM group at168h.b) Level of EPC in bone marrow:There was no difference among groups. The NM group had a tendency of decreasing for EPC at24h and72h compared with SM group. c) The EPC level in lung:The expression of CD34and KDR mRNA were significant increased in NM compared with SM group.d) Plasma mobilizating factors:The M group had a significant higher plasma concentration of SDF-1than SM and C group at24h. The plasma concentration of SDF-1and VEGF were higher in NM than SM and C group at72h.e) Plasma inflammation cytokines:Plasma IL-8level was increased at24h compared to the baseline level in GM group, and come down rapidly at the subsequent time points. There was no difference for IL-6and IL-10level at different time points.f) Factors associated with mobilization in bone marrow:The NO in bone marrow was decreased in SM and GM group at24h, and the NM group had an elevated NO at72h compared with SM and GM. The MMP-9in SM group was lower than C group at24h and72h. And the NM group had an increased MMP-9at72h compared with SM group.g) Cytokines and receptors in lung tissue:The expression of VEGF and SDF-1mRNA was significantly higher in NM group compared to SM group and C group (P<0.05). A trend of increase was found in the expression of CXCR4mRNA. Moreover, the VEGFR2and eNOS expression was significant higher in NM group than the C and SM groups.h) Variables of lung injury repair:The lung tissue had a mild neutrophil infiltration in NM group while prominent pathological changes were found in SM group. There was modest neutrophil infiltration in GM group. Moreover, the pulmonary vascular density in NM was significantly higher compared with SM. And the comparision of LPI showed that the NM had an improved LPI compared with SM. The number of apoptosis cells was decreased in NM group compared with SM and GM group.Conclusions In a piglet model with different severity of ALI, impaired mobilization and homing of EPC from bone marrow were observed. Inhaled NO had preventive role in this pathological process by promoting mobilization and homing of EPC to the injured lungs through systemic effects or enhanced expression of SDF-1and VEGF, resulting in improved pulmonary vascular permeability, lung injury and apoptosis alleviation, and enhanced lung tissue reparation. |
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