Relationship Of Growth Factor Expression And Inflammatory Response In Developmental Porcine Lungs

Growth factors(GFs)are diffusible proteins that act within a short distance of where they are produced to induce a variety of cellular activities through activation of diverse signaling pathways.While their function has been traditionally associated with promotion of cell proliferation,GFs are increasingly recognized as important mediators of tissue interactions in the developing lung.There is evidence suggesting that the spatial and temporal distributions of GFs in the lung are critical for normal lung development and homeostasis.Abnormal lung development was observed in genetically altered mice in whom GF expression has been disrupted,and aberrant GF signaling in human lung abnormalities,such as bronchopulmonary dysplasia(BPD), has been evidenced through clinical investigations.Besides their involvement in lung pattem formation,growth and cell differentiation during development,GFs have been also implicated in modulating injury-repair response of the lung.Altered expression of GFs has been found in a number of pathological lung conditions.Conventional mechanical ventilation(CMV)with variable fraction of oxygen is commonly used as respiratory therapy for the neonates with hypoxemic respiratory failure.However,the neonatal lungs in.early postnatal life are vulnerable to CMV and oxygen therapy because potential lung gas volumes are small,surfactant may be deficient,lung matrix is not fully developed,antioxidant capacities are low,and airspaces contain residual lung fluid.The adverse effects of CMV and oxygen therapy on immature lungs involve inflammatory damage due to alveolar overstretching or hyperoxia,and are related to an imbalance between injury severity and reparation potential of the lung tissue.Furthermore,inflammatory mediators may interfere with alveolarization in neonatal lung development,and its pathological consequence is BPD,or chronic lung disease of prematurity.Recent investigations suggest that alteration of de novo synthesis of several GFs in the lungs is vital in injury-repair response and remodeling.Among them,platelet-derived GF(PDGF)-B,insulin-like GF(IGF)-I,keratinocyte GF(KGF),hepatocyte GF(HGF),vascular endothelial GF (VEGF),and transforming GF(TGF)-β1 mediate tissue interactions and regulate a variety of cellular functions that are critical for fetal lung development,especially in septation and alveolarization.However,little is known about their postnatal response to CMV.We hypothesized that CMV in early postnatal life would affect the expression of GFs in immature lungs,which contributes to the pathogenesis of ventilator-induced lung injury(VILI).Applying adequate ventilation strategy should minimize VILI.It has been demonstrated that the use of positive end-expiratory pressure(PEEP)at 4 cmH₂O minimized alveolar protein leakage and neutrophil infiltration in surfactant-treated preterm lambs compare to that of PEEP at 7 cmH₂O.However,there are few studies reporting effects of PEEP on expression of proinflammatory cytokines and GFs in the immature lungs and the mechanisms underlying their interactions.Furthermore, surfactant and inhaled nitric oxide(iNO)are two alternative therapies for neonates suffering from severe hypoxemic respiratory failure,and reciprocal action among these combined therapies accounts for virtual effects on overall respiratory support. Recently we established a ventilated premature piglet model for testing of surfactant. Our aim in the present study was to use the same model and test a hypothesis that two levels of PEEP,in the presence of iNO and/or surfactant,should affect expression of proinflammatory cytokines and GFs differently,and delineated correlations of these factors in response to these therapies.In our study CMV resulted in a decreased expression of IGF-I,but increased expression of PDGF-B and proinflammatory cytokines in the immature lungs,which likely contribute to the pathogenesis of VILI.To further investigate effects of CMV on transcription of lung cytokines in different stages of pulmonary development,we compared a newborn and infant piglet model with low tidal volume ventilation in vivo. To our knowledge,no previous study has examined the expression of GFs and the occurrence of local inflammation during CMV with low tidal volume in healthy lungs at different developmental stages.In addition,expression patterns of GFs need to be explored during pre- and post-natal development of the porcine lung,which may be critical to understand the pathogenesis of pulmonary disorders in early life. Molecular mechanisms that regulate the expression of GFs in the setting of lung injury have not been well identified.However,there are several reasons to believe that inflammation may have effects on the expression of GFs in the developmental lung. First,inflammation is the main pathway through which the effects of insults,such as volutrauma,oxygen toxicity or infection are translated into lung injury,followed by repair or remodeling process which is controlled by GFs.Second,in the present in vivo study a close correlation was found in the mRNA expression of interleukin (IL)-1βor IL-6 with PDGF-B,and an inverse correlation with IGF-I.Thus,in vitro studies concerning effects of proinflammatory cytokines on GF expression are needed to elucidate interactions among these cytokines.We built procedure for isolation of typeâ…¡alveolar epithelial cells(AEC-â…¡)from newbom piglet,and used the primary AEC-â…¡as experimental model in the in vitro study.Objective1.To investigate effects of PEEP,in the presence of iNO and/or surfactant,on expression of proinflammatory cytokines and GFs in premature(day 99 of gestination)piglet lungs,and delineate correlations of these cytokines in response to these therapies.2.To compare effects of CMV with low tidal volume(6-8 ml/kg)on newborn(1 day after birth)and infant(4 weeks after birth)porcine lungs by examing expression of GFs and occurrence of local inflammation.3.To explore expression patterns of GFs during pre- and post-natal development of porcine lung(day 97-100 of gestination,1 day and 4-5 weeks after birth).4.To investigate effects of proinflammatory cytokines(rhIL-1βand rhIL-6)on expression of GFs(PDGF-B and IGF-I)in AEC-â…¡in vitro,and estimate effects of alteration in GFs expression on AEC-â…¡proliferation and mRNA expression of surfactant associated protein(SP)-A/B. Methods1 Effects of PEEP,iNO and surfactant on expression of GFs and proinflammatory cytokines in preterm piglet lungsPreterm piglets at GA of 97-100 d(85%gestation)were obtained by cesarean section.After initial stabilization for 15 min of ventilation,those animals with PaO₂/FiO₂≤250 mmHg were enrolled and randomly assigned to groups as either low PEEP at 5-6 cmH₂O(PEEP5)or high PEEP at 10-12 cmH₂O(PEEP10),and immediately allocated to different subgroups(n=6)receiving surfactant and/or iNO; PEEP5(C5),PEEP5+NO(NO5),PEEP5+PS(PS5),PEEP5+NO+PS(SNO5), PEEP10(C10),PEEP10+NO(NO10),PEEP10+PS(PS10),PEEP10+NO+PS (SNO10).The ventilators were initially set at a frequency of 50 breaths/min,peak inspiratory pressure(PIP)of 20 cmH₂O,PEEP of 5-6 cmH₂O or 10-12 cmH₂O, inspiration time of 0.4 s,and fraction of inspired oxygen(FiO₂)of 80%.The ventilator settings were adjusted to provide a tidal volume of 6-8 ml/kg and to maintain PaO₂ at 50-80 mmHg and PaCO₂ at 35-60 mmHg while PEEP was kept constant.A porcine lung surfactant was used for the PS and SNO at 100 mg/kg body weight instilled in bolus into the lungs via the endotracheal tube.NO gas was continuously supplied to ventilator circuit at 10 parts per million in volume(ppm) during the whole ventilation.After treating for 6 hours,the piglets were sacrificed and right lung tissue was preserved for measurement of mRNA expression of GFs (PDGF-B,IGF-I,KGF,HGF,VEGF,TGF-β1)andpro-inflammatorycytokines (IL-1β,IL-6,IL-8,TNF-α).Left lung was taken for histopathology and immunohistochemical analysis of GFs.The results were compared with those from non-ventilated preterm piglets(SB,n=6).2 Effects of CMV with low tidal volume on expression of GFs and proinflammatory cytokines in newborn and infant porcine lungsHealthy piglets of postnatal age 1 day and 4 weeks were subject to CMV for 24 hours(MV group,n=5 or 6),each with its own non-ventilated littemates as control (SB group,n=6).The ventilators were initially set at a frequency of 40(newborn piglet)or 30(infant piglet)breaths/min,PIP of 6-10 cmH₂O,PEEP of 0 cmH₂O,an inspiration-to-expiration ratio of 1;1.5-2,and FiO₂ of 30%.After 10 min of stabilization of mechanical ventilation,ventilator settings,lung mechanics and blood gas values were recorded and this time was designated as 0 hour of the ventilation during the treatment.These parameters were measured at each hour up to 24 hours. The ventilator settings were adjusted to provide a tidal volume of 6-8 ml/kg and to maintain pH at 7.25-7.45,PaO₂＞60 mmHg and PaCO₂ at 30-50 mmHg.At the end of experiment,lung tissue from the tip of right mid-lobe was used for determining wet-to-dry weight ratio(W/D).The rest of right mid-lobe was immediately frozen in liquid nitrogen for measurement of myeloperoxidase(MPO)activity and mRNA expression of GFs and proinflammatory cytokines.Bronchoalveolar lavage(BAL)of the right lung was performed,and total phospholipids(TPL),total proteins(TP)and disaturated phosphatidylcholine(DSPC)of BAL fluid(BALF)were measured.The left lung was perfusion fixed and taken for histopathology and immunohistochemical analysis of GFs.3 Effects of proinflammatory eytokines on primary newborn piglet AEC-â…¡AEC-â…¡s were isolated from newborn piglet lung tissue,and the cell type purity was assessed by percentage of cells alkaline phosphatase(AKP)positive.The cells were seeded in 6-well plates at a density of 4×10⁵ cells/cm² and grown in DMEM supplemented with 10%fetal bovine serum(FBS)in 5%CO₂-95%air atmosphere at 37℃.After 24 hours culture,the unattached cells were removed.At this time point, cell number was counted(cell number A).Fresh DMEM containing 10%FBS,with or without(control group)various concentrations(1 ng·ml^-1,10 ng·ml^-1,100 ng·ml^-1) of rhIL-1βor rhIL-6,was added to the plate and the cells were then incubated for up to 48 hours.At the end of experiment,cell number was counted again(cell number B) and RNA was isolated from cells using TRIzol reagent for measurement of mRNA expression of PDGF-B,IGF-I and SP-A/B.Cell proliferation was presented as cell number B/cell number A(×100%).Two independent experiments were performed.In the above experiment rhIL-1βand rhIL-6 inhibited the cell proliferation and mRNA expression of SP-A and IGF-I in AEC-â…¡.To prove that rhIL-1βand rhIL-6 exert the actions on AEC-â…¡(inhibit cell proliferation and SP-A mRNA expression) through IGF-I-dependent mechanisms by decreasing mRNA expression of IGF-I, AEC-â…¡s were co-cultured with anti-IGF-I antibody for 48 hours.Cell proliferation and mRNA expression of SP-A/B were then measured. Results1 Effects of PEEP,iNO and surfactant on expression of GFs and proinflammatory cytokines in preterm piglet lungs1.1 Effects of PEEP,iNO and surfactant on mRNA and protein expression of GFsVentilated subgroups(except for NO5 and SNO5)had enhanced PDGF-B,but decreased IGF-I,mRNA expression compared with SB(P＜0.05).There was decreased PDGF-B,in contrast to enhanced IGF-I,mRNA expression in NO5,NO10,SNO5 and SNO10 compared with C5,C10,PS5 and PS10.Significant difference was seen in NO5 and SNO5 compared to C5 or PS5(P＜0.01).The PEEP10 subgroups had enhanced expression of PDGF-B than in the corresponding PEEP5 ones,especially between NO5 and NO10(P＜0.01),SNO5 and SNO10(P＜0.01)whereas the expression of IGF-I mRNA was opposite to that of the PDGF-B(P＜0.05).For the expression of KGF,HGF,VEGF and TGF-β1,no significant changes were found in both PEEP5 and PEEP 10 subgroups.By immunohistochemistry,PDGF-B and IGF-I were expressed in mesenchyme and epithelia of premature lungs.The PEEP5 subgroups showed mild immunostainning of PDGF-B compared to the corresponding ones in the PEEP10, whereas it was more prominent in C5,PS5,C10 and PS10 than that in NO5,SNO5, NO10 and SNO10.In contrast,there was an obvious trend of decreased IGF-I expression in C10 and PS10 compared to C5 and PS5,and the immunostaining in NO5,SNO5,NO10 and SNO10 was more intense than that seen in C5,PS5,C10 and PS10.Immunostaining of KGF was located in mesenchyme and epithelial cells in airways and alveoli.No significant difference was found between the PEEP5 and PEEP 10 subgroups.1.2 Effects of PEEP,iNO and surfactant on mRNA expression of proinflammatory cytokinesVentilated subgroups had higher mRNA expression of proinflammatory cytokines (IL-1β,IL-6,IL-8 and TNF-α)compared with SB.There was a trend of decreased IL-1βand IL-6 mRNA expression in NO5,NO10,SNO5 and SNO10 compared with C5,C10,PS5 and PS10.There was also a trend of alleviated expression of IL-1βand IL-6 mRNA in the PEEP5 subgroups than in the corresponding ones in PEEP10.The expressions of IL-1βand IL-6 mRNA in both NO5 and SNO5 were lower than C5 or PS5(P＜0.05),respectively.A significant decrease of IL-1βmRNA was seen between NO10 vs.C10(P＜0.01),SNO10 vs.PS10(P＜0.01).NO10 and SNO10 had lower IL-8 mRNA expression than C10 and PS10 (P＜0.05).No significant differences of expression of TNF-αmRNA were found among the PEEP5 and PEEP10 subgroups.No significant differences for the expression of IL-8 and TNF-αmRNA were found between the PEEP5 and PEEP10 subgroups with the same treatment.1.3 Correlation analysis of the parametersExpressions of IL-1β,IL-6 mRNA were closely correlated to PDGF-B(P＜0.001), but inversely to IGF-I(P＜0.001),PaO₂/FiO₂ and Cdyn(P＜0.05)at 6 h,and that of IL-8 to KGF and TGF-β1(P＜0.001,P＜0.05,respectively).Expression of IGF-I mRNA was correlated to PaO₂/FiO₂(P＜0.05),while PDGF-B was inversely correlated to PaO₂/FiO₂(P＜0.001),and that of IGF-I and KGF to Cdyn(P＜0.05).2 Effects of CMV with low tidal volume on expression of GFs and inflammatory response in newborn and infant porcine lungs2.1 Effects of CMV with low tidal volume on expression of GFs in newborn and infant porcine lungsNo significant differences for the expression of PDGF-B,IGF-I,KGF,HGF, VEGF and TGF-β1 mRNA were found between the ventilated and nonventilated newborn or infant porcine lungs.2.2 Inflammatory responses in newborn and infant porcine lungs during CMV with low tidal volumeNo significant differences for MPO activity and the expression of IL-1β,IL-6, IL-8 and TNF-αmRNA were found between the ventilated and nonventilated newborn or infant piglet lungs.In newborn piglets ventilated for 24 hours the W/D was significantly higher than nonventilated controls(P＜0.05).In contrast,ventilation in infant piglets was not associated with a significant increase in W/D versus nonventilated infant piglets.2.3 Effects of CMV with low tidal volume on phospholipid content in BALF of newborn and infant pigletsSignificant decreases in DSPC and DSPC/TP were found in ventilated newborn piglets compared to nonventilated controls(P＜0.05;P＜0.01).Significant decreases in TPL and DSPC were found in ventilated infant piglets compared to nonventilated controls(P＜0.05). 2.4 Expression patterns of GFs during pre- and post-natal development of porcine lungExpression of PDGF-B and IGF-I mRNA was higher in preterm piglet lungs than newborn and infant piglet lungs(P＜0.01).Expression of HGF mRNA was higher in preterm and newborn piglet lungs than infant piglet lungs(P＜0.05).No significant differences for the expression of KGF,VEGF and TGF-β1 mRNA were found among different stages of lung development.3 Effects of proinflammatory cytoldnes on primary newborn piglet AEC-â…¡3.1 Effects of proinflammatory cytokines at different concentrations on mRNA expression of GFs in AEC-â…¡Effects of rhIL-1βand rhIL-6 on expression of IGF-I mRNA in AEC-â…¡were dose-dependent.Expression of IGF-I mRNA decreased with the increase of concentrations of rhIL-1βand rhIL-6.In contrast,rhIL-1βand rhIL-6 had no effects on expression of PDGF-B mRNA in AEC-â…¡.Expression of IGF-I mRNA was lower in rhIL-1β(100 ng·ml^-1)group than control group and rhIL-1β(1 ng·ml^-1)group (P＜0.01;P＜0.05).RhIL-6(10 ng·ml^-1)group and rhIL-6(100 ng·ml^-1)group had decreased expression of IGF-I mRNA compared to control group(P＜0.05).3.2 Effects of proinflammatory eytokines at different concentrations on AEC-â…¡proliferation and mRNA expression of SP-A/BCell numbers were lower in rhIL-1β(100 ng·ml^-1)group than control group and rhIL-1β(1 ng·ml^-1)group(P＜0.05).Expression of SP-A mRNA decreased with the increase of concentration of rhIL-1β.RhIL-1β(100 ng·ml^-1)group had decreased expression of SP-A mRNA compared to control group(P＜0.05).There was a trend of decreased cell numbers and expression of SP-A mRNA in rhIL-6-treated groups compared to control group.Expression of SP-A mRNA was lower in rhIL-6(100 ng·ml^-1)group than rhIL-6(1 ng·ml^-1)group(P＜0.05).No significant difference for expression of SP-B mRNA was found among groups.3.3 Effects of IGF-I pathway on AEC-â…¡proliferation and mRNA expression of SP-A/BCell numbers and expression of SP-A/B mRNA were lower in anti-IGF-I group than control group(P＜0.05). Conclusions1.Mechanical ventilation in early postnatal life caused inflammatory injury in immature lungs by increasing expression of proinflammatory cytokine(IL-1-β, IL-6,IL-8 and TNF-α)mRNA,and increased PDGF-B,but decreased IGF-I, mRNA expression.2.Adequate PEEP with iNO mitigated the lung inflammation by decreasing expression of proinflammatory cytokine mRNA,and restored expression of PDGF-B and IGF-I in immature lungs.3.Expressions of IL-1β,IL-6 mRNA were closely correlated to PDGF-B,but inversely to IGF-I,indicating that PDGF-B and IGF-I expression may be affected by IL-1βand IL-6 during inflammatory lung injury.4.CMV with low tidal volume for 24 hours did not cause inflammatory response and GF expression change in newborn and infant porcine lungs.5.Expressions of PDGF-B and IGF-I were higher in preterm piglet lungs than newborn and infant piglet lungs,indicating that the two cytokines may play a critical role in late fetal lung development.6.In vitro study rhIL-1βand rhIL-6 inhibited AEC-â…¡proliferation and SP-A mRNA expression through IGF-I-dependent mechanisms by decreasing mRNA expression of IGF-I.