Prevention of bronchopulmonary dysplasia: Finally, something that works
http://www.100md.com
《美国医学杂志》
David Geffen School of Medicine at UCLA, Torrance, USA
Abstract
Due to a lack of understanding of the molecular mechanisms involved in its pathogenesis, bronchopulmonary dysplasia (BPD) still remains a major cause of morbidity and mortality in the premature infant, and there is no effective preventive and/or therapeutic intervention. We have taken a basic biologic approach to elucidate the pathophysiology of BPD and have discovered that disruption of the alveolar Parathyroid Hormone-related Protein (PTHrP) signaling is centrally involved in this process. Further, stabilization of this signaling pathway by using exogenous PTHrP agonists can prevent and/or rescue the molecular injuries caused by insults that lead to BPD. Based upon years of work in this field, here I provide a novel and innovative molecular approach, i.e., exogenous treatment with PTHrP pathway agonists to prevent and/or treat BPD. However, to avoid any later surprises, it is important to emphasize that before translating it into human trials, this approach needs further testing and refinement in animal models.
Keywords: Chronic lung disease; Parathyroid hormone-related protein; Prematurity
Bronchopulmonary dysplasia (BPD) is a chronic pulmonary disorder that is the consequence of abnormally repaired lung damage which primarily affects premature infants. Despite tremendous advances in neonatal care in general and neonatal respiratory care in particular, BPD still remains a major cause of morbidity and mortality in the premature infant.[1] These advances may have decreased the severity of BPD, but its incidence has not decreased, and may in fact, have actually increased.[2],[3] The affected infants have both short- and long-term clinical issues, including, but not limited to a very complicated initial neonatal intensive care course, poor growth, neurodevelopmental delays, and repeated hospitalizations during the first few years of life.[4],[5],[6] Therefore, any intervention that would reduce the risk of BPD or improve its outcome is likely to have huge clinical and financial benefits.
Why Prevention Has Not Worked So Far
The primary prevention of BPD through elimination of the all important risk factor, prematurity, has not been achieved yet, and is unlikely to be achievable in the near future because of the lack of understanding of the pathophysiology of preterm labor. Up until now, the available preventive/treatment strategies for BPD have also suffered the same limitations, i.e., lack of understanding of the molecular processes involved in both normal and abnormal lung development. The preventive interventions such as antenatal and postnatal steroids, "kinder and gentler" ventilation, early closure of patent ductus arteriosus, treatment of prenatal and postnatal infections, fluid restriction, various nutrient supplements (vitamin A, polyunsaturated fatty acids, etc.), and most recently nitric oxide administration are either not effective or are associated with unacceptable side effects.[7],[8],[9],[10] Most of the these interventions are of an empiric nature, lacking a targeting of the underlying molecular processes that lead to BPD, which may precisely be the reason that none of these interventions have been effective in preventing BPD. Therefore, the available preventive strategies for BPD are not effective, not safe, and none of these is based on thorough understanding of pathogenesis of BPD. Alternate interventions that are based on a sound understanding of the pathogenesis of BPD are needed. With recent advances in understanding the molecular processes involved in both normal and abnormal lung development, in particular the mechanism of alveolar development and its failure in BPD, there is now new hope for not only preventing and treating BPD, but also possibly reversing established disease.[11]
'Old' versus 'New' BPD
Around 40 years back, when Northway described BPD, he described it as a chronic lung disease of relatively larger preterm infants who required ventilation with high pressures and oxygen for prolonged periods of time.[12] It was characterized by four well-defined clinical, radiological, and histopathological findings. The hallmarks of the now so called "Old BPD" were severe large airway injury, interstitial and alveolar edema, and extensive small airway disease with alternating areas of overinflation and fibrosis. With the introduction of antenatal steroids, routine surfactant replacement for respiratory distress syndrome, and modern respiratory care, in contrast, now the "new" BPD occurs primarily in extremely premature infants, occurs after only modest ventilatory and oxygen needs, and is not accompanied by the classic clinical and radiological stages of the "Old BPD". The most dramatic histopathologic finding is "arrested alveolarization" with minimal large or small airway disease, and relatively less inflammation and fibrosis.[2],[13]
Molecular Basis for BPD
The initiation and continued use of mechanical ventilation in infants with birth weights of < 1250 g is the strongest risk factor for the development of BPD. The surfactant deficient alveoli of such premature infants undergo breath-by-breath collapse (de-recruitment) and re-recruitment, resulting in atelectrauma, which sets in-motion the molecular cascade resulting in the structural and morphological changes characteristic of BPD. Experimental studies in preterm animals have clearly demonstrated that even a few large tidal volume breaths can result in acute lung injury that initiates the cascade of molecular events leading to BPD.[14] Given the central role of alveolar stretch in initiating molecular injury leading to BPD, it is logical to pursue a stretch sensitive gene (s) that may be expressed in the pulmonary alveolus and that is (are) essential for its structure and function. Parathyroid Hormone-related Protein (PTHrP) is one such gene. It is expressed in the developing endoderm and its receptor is present on the adepithelial mesoderm, and most importantly, PTHrP knock-out causes a specific inhibition of fetal lung development.[15] The lungs of these mice fail to transition from the pseudoglandular to the canalicular stage of lung development, i.e., failure of alveolarization, a hallmark of the "new" BPD.
Under the influence of Sonic Hedgehog, the developing endoderm expresses PTHrP and its receptor on the adjoining mesenchyme. PTHrP binding to its receptor on the mesenchyme activates the Protein Kinase A pathway, which actively down-regulates the default Wingless/Int pathway and up-regulates the adipogenic pathway through a key nuclear transcription factor, Peroxisome Proliferator Activated Receptor g (PPARg) and its down-stream regulatory genes Adipocyte Differentiation Related Protein (ADRP).[16] ADRP is necessary for the transit of neutral lipid from the lipofibroblast to the ATII cell for surfactant phospholipid synthesis. Lipofibroblasts in turn secrete leptin, which acts on its receptor on AT II cells, stimulating both surfactant phospholipid and protein synthesis. Since PTHrP stimulates leptin production by lung fibroblasts, it provides a complete growth factor-mediated paracrine loop for the synthesis of pulmonary surfactant.[17] Overall, PTHrP signaling, by inhibiting the Wnt signaling, inhibits the default myogenic phenotype, and by stimulating the PPARg signaling, induces the lipogenic phenotype, which is necessary for maintaining alveolar homeostasis through its autocrine effect on interstitial fibroblasts and its paracrine effect on ATII cells.[11] Specifically, the interstitial lipofibroblast phenotype provides protection against oxygen free radicals (i.e., protection against oxotrauma), trafficks neutral lipid substrate to ATII cells for surfactant phospholipid synthesis (i.e., protection against atelectrauma), causes ATII cell proliferation (i.e., protection against any insult causing epithelial injury), thereby promoting alveolar growth, development, and injury/repair.[11],[18] Physiologically, this would stabilize the alveolus, preventing its collapse, maintaining adequate gas-exchange and reducing the energy expenditure by decreasing the work of breathing. On the other hand, although myofibroblasts may also be important for normal lung development, these cells are the hallmark of chronic lung diseases in both the neonate and adult.[19] In the developing lung, myofibroblasts are fewer in number and localize to the periphery of the alveolar septa, where they very likely participate in the formation of new septa.[20] However, in chronic lung diseases, myofibroblasts not only increase in number but are also abnormally located in the center of the alveolar septum in great abundance. In summary, PTHrP signaling is critical in maintaining alveolar homeostasis. Under the influence of cyclic stretch, PTHrP is secreted by the AT II cell, which acts on its receptor on the adjacent lipofibroblast, which in turn secretes leptin that facilitates surfactant synthesis by the ATII cells, underlining the importance of PTHrP signaling in maintaining alveolar homeostasis. If normal PTHrP signaling is interrupted, e.g., by conditions such as prematurity, volutrauma, exposure to hyperoxia or inflammatory cytokines, all conditions that are known to be associated with BPD, normal alveolar homeostasis is altered and interstitial fibroblasts undergo molecular and phenotypic changes consistent with development of BPD. With this rationale, below I review data from various in vitro and in vivo models in which we have examined the role of the modulators of PTHrP signaling in ablating the molecular injury that leads to BPD.
Effect of Barotrauma/Volutrauma/Atelectrauma on PTHRP Signaling
The physiologic stretching of the alveolar type II cell in isolation increases the expression and production of PTHrP, potentially explaining why increased lung tidal volume stimulates pulmonary surfactant synthesis.[21] In contrast to the increased synthesis of surfactant in response to mild stretch (3-5%), overdistension of the type II cell results in down-regulation of PTHrP expression, simulating the consequences of barotrauma/volutrauma/atelectrauma.
Prevention of Oxotrauma by PTHRP/PPARγ Signaling Pathway Agonists
Since lipofibroblasts play an important role in injury-repair mechanisms in the lung, we studied the effects of hyperoxia on the fibroblast phenotype in immature and relatively mature alveolar interstitial fibroblasts, and found that exposure to hyperoxia down-regulated PTHrP signaling, augmenting the transdifferentiation of pulmonary lipofibroblasts to myofibroblasts, the central event in the pathogenesis of BPD.[22] Fetal rat lung fibroblasts (FRLF) from e (embryonic, term = e22) 18 and e21 gestation were studied. After initial culture, cell were maintained either in normoxia (21% O 2 ) or subjected to hyperoxia for 24h (95% O 2 ) at passage (P) 1 and P5. Passage 1 and 5 cells were analyzed for the expression of well-characterized lipogenic and myogenic markers [PTHrP receptor, ADRP, and a smooth muscle actin (SMA)] based on semi-quantitative competitive RT-PCR, triglyceride uptake, and leptin assay. Serial passaging and maintenance of cells in normoxia resulted in a significant decrease in the expression of the lipogenic markers from P1 to P5, spontaneously. This decrease was greater for immature (e18) than for (near-mature) e21 FRLF. However, exposing cells to hyperoxia augmented the loss of the lipogenic markers and gain of the myogenic marker from P1 to P5 in comparison to cells maintained in normoxia. This augmentation was also greater for e18 versus e21 lipofibroblasts. The changes in mRNA expression were accompanied by decreased triglyceride uptake and leptin secretion on exposure to hyperoxia. These data suggest that exposure to hyperoxia augments the transdifferentiation of pulmonary lipofibroblasts to myofibroblasts. Importantly, pretreatment with a PTHrP/PPARg signaling pathway agonist, prostaglandin J 2 (PGJ 2 ), at least partially attenuated the hyperoxia-augmented lipo-to-myofibroblast transdifferentiation.
In a further series of experiments, using [1,2-13C 2 ]-D-glucose tracer and gas chromatography/mass spectrometry, we metabolically profiled e18 and e21 FRLF with and without hyperoxia exposure at passages 1, 4, 7, and 10.[23] For this series of studies, glucose carbon redistribution between the nucleic acid ribose, lactate, and palmitate synthetic pathways and ADRP expression by RT-PCR were examined. Exposure to hyperoxia at each passage caused a decrease in ADRP mRNA expression. This passage- dependent transdifferentiation is accompanied by a moderate increase in the synthesis of nucleic acid ribose from glucose through the non-oxidative steps of the pentose cycle. E18 fibroblasts showed over an 85% decrease in the de novo synthesis of palmitate from glucose, while e21 fibroblasts showed a less pronounced 32% to 38% decrease in de novo lipid synthesis in hyperoxia-exposed cultures. From these data The authors conclude that (1) there is a maturation-dependent sensitivity to hyperoxia; (2) transdifferentiation of the fibroblast, as demonstrated by changes in ADRP expression, is accompanied by metabolic enzyme changes affecting ribose synthesis from glucose, and (3) hyperoxia specifically inhibits lipogenesis from glucose. These molecular and metabolomic data were further complemented by "genome-wide" microarray analysis of RNA extracted from P1 and P10 e19 FRLF with or without exposure to hyperoxia (95% O 2 for 24h). Rat chip RAE230 , which has 15,923 RNA transcripts, including most genes of interest, e.g., PTHrP, PPARg, Adipophilin (moderate homology with ADRP) was used. "Genome-wide" expression was analyzed using Affymetrix GeneChip Operating Software and Netaffx, Genespring 6.2 (Silicon Genetics), and Gene Microarray Pathway Profiler (Gladstone Institute). In accord with our molecular and metabolomic data, cluster analysis of the microarray data confirmed the down-regulation of cholesterol and fatty acid synthetic genes and up-regulation of fatty acid degradation and Wnt signaling pathway genes on passaging e19 FRLF from P1 to P10, and on exposure to hyperoxia at passages 1 and 10, thereby confirming lipo-to-myofibroblast transdifferenation under these conditions. Based on these data, it appears that hyperoxia induces lipo-to-myofibroblast transdifferentiation, which may be an important mechanism for hyperoxia-induced lung injury and is likely to be a key element in the pathophysiology of BPD.
Following present investigation in vitro studies, as outlined above, it is next determined whether upon exposure to hyperoxia, pulmonary alveolar lipo-to-myofibroblast transdifferentiation occurs in vivo , and whether treatment with a potent PTHrP/PPARg signaling pathway agonist, Rosiglitazone (RGZ), would prevent this process.[24] Newborn Sprague Dawley rat pups were exposed to normoxia (21% O 2 ), hyperoxia alone (95% O 2 for 24h), or hyperoxia with RGZ (95% O 2 for 24h + RGZ, 3 mg/Kg, administered intraperitoneally). Subsequently, pups were sacrificed and lung tissue was analyzed by morphometry, and by RT-PCR, Western hybridization, and immunohistochemistry for the expression of key lipogenic and myogenic markers. We observed a significant decrease in the expression of lipogenic markers and a significant increase in the expression of myogenic markers in the hyperoxia alone group. Exposure of rat pups to 24 hours of hyperoxia dramatically affected normal postnatal lung development. Histologic examination of lungs obtained from hyperoxic pups revealed a remarkable arrest of alveolarization compared with their normoxic controls. Hyperoxia-exposed lungs demonstrated relatively large air spaces, thinned interstitial, and decreased secondary septal crests compared to air-exposed controls. In lungs from hyperoxic animals, septal thickness was reduced significantly compared to control lungs. Furthermore, quantitative analysis of alveolar number demonstrated a 50% reduction in average radial alveolar counts in hyperoxic animals compared to controls. Pre-treatment with RGZ almost completely prevented the hyperoxia-induced changes in lung morphology, including the effects on septal thickness and radial alveolar counts. The hyperoxia-induced morphologic, molecular, and immunohistochemical changes were almost completely prevented by RGZ. This was the first evidence of in vivo lipo-to-myofibroblast transdifferentiation and its almost complete prevention by RGZ, suggesting that administration of PPARg agonists might be a novel, effective strategy to prevent the hyperoxia-induced lung molecular injury that has been implicated in the pathogenesis of BPD.
Prevention of Infection-Induced Lung Injury by PTHrP/PPARγ Signaling Pathway Agonists
Since lung inflammation is a key factor that predisposes preterm infants to BPD, in a series of studies,[25] we determined the effects of lipopolysaccharide (LPS) on PTHrP-driven pulmonary epithelial-mesenchymal interactions that have been shown to be essential in the maintenance of lung homeostasis. Lung explants derived from e19.5 Sprague Dawley rat pups were treated with LPS (0-50 ml) with or without a PTHrP pathway agonist, PGJ 2 , for up to 72h. There were acute (24 hour) significant increases in the expression of PTHrP, PPARg, ADRP, and Surfactant Protein-B (SP-B), without any significant effects on the expression of aSMA. This was followed (72 hours) by significant decreases in the expression of PTHrP, PPARg, ADRP, and SP-B, accompanied by a significant increase in the expression of aSMA, the key functional marker of BPD. These changes were completely prevented by concomitant treatment with PGJ 2 , providing an integrated mechanism for the acute stimulation of lung maturation accompanied paradoxically by BPD following intrauterine inflammation. Further, as in present studies in vivo hyperoxia model, treatment with a specific agonist of epithelial-mesenchymal interactions prevented the inflammation-induced molecular lung injury that is known to result in BPD.
Prevention of Nicotine-Induced Lung Injury by PTHRP/PPARγ Signaling Pathway Agonists
The authors tested the hypothesis that in vitro nicotine exposure disrupts specific epithelial-mesenchymal paracrine signaling pathways and results in pulmonary lipo-to-myofibroblast transdifferentiation, resulting in altered pulmonary development and function.[26] WI38 cells, a human embryonic pulmonary fibroblast cell line, were treated with nicotine with or without specific agonists of the alveolar fibroblast lipogenic pathway, PTHrP, dibutyryl cAMP or the potent PPARg stimulant RGZ for 7 days. Subsequently, expression of the key lipogenic and myogenic markers was examined. Nicotine treatment resulted in significantly decreased expression of lipogenic and increased expression of myogenic markers in a dose-dependent manner, indicating that nicotine exposure causes lipo-to-myofibroblast transdifferentiation. The nicotine-induced lipo-to-myofibroblast transdifferen-tiation was completely prevented by concomitant treatment with PTHrP, dibutyryl cAMP, RGZ, and by transiently overexpressing PPARg, suggesting the possibility of complete prevention of nicotine-induced lung injury by PTHrP/PPARg signaling pathway agonists. Indeed in our subsequent studies, we demonstrated that by augmenting PTHrP/PPARg signaling pathway in vivo, we were able to completely block the nicotine-induced increase in alveolar type II cell proliferation, once again underpinning the critical role of PTHrP/PPARg signaling in nicotine-induced lung injury.[27]
Significance of PTHRP Signaling in Human BPD
Since PTHrP secreted by pulmonary alveolar type II cells is a key physiologic paracrine factor in maintaining alveolar homeostasis, we hypothesized that its levels in the tracheal aspirates (TA) of ventilated Very Low Birth Weight Infants (VLBWI) would correlate with the development of BPD.[28] The authors examined whether TA PTHrP content during the first week of life correlated with the later development of BPD. Forty VLBWIs (birth weight 943 ± 302 grams (mean ± SD); gestational age 27 ± 2 weeks; 21 males and 19 females), who were ventilated for Respiratory Distress Syndrome, were studied. The TAs were collected once daily until the infants were extubated and assayed for PTHrP. The levels of TA PTHrP were correlated with the later development of BPD. PTHrP in the TA during the first week of life was significantly lower in those infants who developed BPD (12/40) than among those who did not (28/40). The PTHrP levels also correlated with the duration of mechanical ventilation needed in these infants. A PTHrP level of ￡ 1.32 pg/mg protein predicted the later development of BPD maximally [84.6% correct classifications (true positives + true negatives)], with a sensitivity = 76.9% and specificity = 88.5%. Using a TA PTHrP level of 1.32 pg/mg protein as the cutoff, the authors constructed Kaplan-Meier curves to compare the duration of ventilation needed between the two groups, i.e., ￡ and > than 1.32 pg/mg protein TA PTHrP level. Infants with TA PTHrP levels greater than 1.32 pg/mg protein were off ventilatory support significantly earlier. To determine how PTHrP levels compared with the other known predictors of BPD, such as birth weight (< 1000 g), GA (< 28 wk), and sex (male gender), we performed multivariate logistic regression analysis for these 4 variables in predicting the development of BPD. Of these variables, a PTHrP level of ￡ 1.32 pg/mg protein was the strongest predictor of BPD and remained so after adjusting for the other 3 variables, i.e., birth weight of < 1000 g, GA of < 28 wk, and male gender. From these data, we concluded that lower TA PTHrP content during the first week of life in ventilated VLBWI inversely correlates with prolonged ventilation and the later development of BPD.
Prevention of Molecular Lung Injuries Leading to BPD with PTHrP/PPARγ Agonists
It is clear from the work outlined above that we have systematically demonstrated the central role of PTHrP-driven epithelial-mesenchymal signaling in maintaining alveolar homeostasis in barotrauma, oxotrauma, infection and nicotine injury. The lipofibroblast expresses both ADRP and leptin in response to PTHrP signaling from the alveolar type II cell, resulting in direct protection of the mesoderm against oxidant injury, and protection against atelectasis by augmenting surfactant phospholipid and protein synthesis. Disruption of PTHrP signaling down-regulates the adipogenic signaling and up-regulates the myogenic signaling pathways, causing myofibroblast transdifferentiation.[11],[22],[23],[24],[25],[26],[27] Unlike lipofibroblasts, myofibroblasts cannot promote AT II cell growth and differentiation,[11] leading to the failed alveolarization characteristic of BPD. A variety of factors associated with failed alveolarization- barotrauma, oxotrauma, infection and nicotine- all cause myofibroblast transdifferentiation in vitro[11],[22],[23],[25],[26] and in vivo.[24], [27] More importantly, the authors have shown that PTHrP/PPARg signaling pathway agonists such as PGJ 2 and RGZ can prevent or rescue myofibroblast transdifferentiation effectively, therefore, preventing the inhibition of alveolarization in the developing lung.
In summary, we have outlined a novel and innovative molecular approach to prevent BPD that is based upon sound understanding of the molecular processes involved in its pathogenesis. Therefore, after trying a host of different approaches that have been utilized so far and that have essentially been futile in preventing BPD, we might have discovered a rationale approach that works. However, it still needs some fine tuning before it is ready for clinical use. In the mean time, we need to exploit every little opportunity to adopt and refine NICU care practices that minimize lung damage. These include but are not limited to maximizing the administration of antenatal steroids and early postnatal surfactant, avoidance or minimization of ventilatory support, and acceptance of lower target range for pulse oximetry during oxygen supplementation.
References
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3. Jobe AH, Bancalari E. Bronchopulmonary dysplasia. Am J Respir Crit Care Med 2001; 163: 1723-1729.
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5. Short EJ, Klein NK, Lewis BA, Fulton S, Eisengart S, Kercsmar C et al . Cognitive and academic consequences of bronchopulmonary dysplasia and very low birth weight: 8-year-old outcomes. Pediatrics 2003; 112(5): e359.
6. Chye JK, Gray PH. Rehospitalization and growth of infants with bronchopulmonary dysplasia: a matched control study. J Paediatr Child Health 1995; 31(2): 105-111.
7. Barrington KJ, Finer NN. Treatment of Bronchopulmonary Dysplasia - A Review. Clinics in Perinatology 1998; 25(1): 177-202.
8. Ambalavanan N, Tyson JE, Kennedy KA, Hansen NI, Vohr BR, Wright LL et al . National Institute of Child Health and Human Development Neonatal Research Network. Vitamin A supplementation for extremely low birth weight infants: outcome at 18 to 22 months. Pediatrics 2005; 115: e249-54.
9. Ballard RA, Truog WE, Cnaan A, Martin RJ, Ballard PL, Merrill JD et al . NO CLD Study Group. Inhaled nitric oxide in preterm infants undergoing mechanical ventilation. N Engl J Med 2006; 355: 343-353.
10. Doyle LW, Halliday HL, Ehrenkranz RA et al . Impact of postnatal systemic corticosteroids on mortality and cerebral palsy in preterm infants: effect modification by risk for chronic lung disease. Pediatrics 2005; 115: 655-661.
11. Torday JS, Torres E, Rehan VK. The role of fibroblast transdifferentiation in lung epithelial cell proliferation, differentiation, and repair in vitro. Pediatr Pathol Mol Med 2003; 22(3): 189-207.
12. Northway WH Jr, Rosan RC, Porter DY. Pulmonary disease following respirator therapy of hyaline-membrane disease. Bronchopulmonary dysplasia. N Engl J Med 1967; 276(7): 357-368.
13. Husain AN, Siddiqui NH, Stocker JT. Pathology of arrested acinar development in postsurfactant bronchopulmonary dysplasia. Hum Pathol 1998; 29: 710-717.
14. Bjorklund LJ, Ingimarsson J, Curstedt T, John J, Robertson B, Werner O et al . Manual ventilation with a few large breaths at birth compromises the therapeutic effect of subsequent surfactant replacement in immature lambs. Pediatr Res 1997; 42(3): 348-355.
15. Rubin LP, Kovacs CS, De Paepe ME, Tsai SW, Torday JS, Kronenberg HM. Arrested pulmonary alveolar cytodifferentiation and defective surfactant synthesis in mice missing the gene for parathyroid hormone-related protein. Dev Dyn 2004; 230(2): 278-289.
16. Torday JS, Rehan VK. Deconvoluting lung evolution using functional/comparative genomics. Am J Respir Cell Mol Biol 2004; 31: 8-12.
17. Torday JS, Rehan VK. Stretch-stimulated surfactant synthesis is coordinated by the paracrine actions of PTHrP and leptin. Am J Physiol Lung Cell Mol Physiol 2002; 283: L130-135.
18. Torday JS, Torday DP, Gutnick J, Qin J, Rehan V. Biologic role of fetal lung fibroblast triglycerides as antioxidants. Pediatr Res 2001; 49(6): 843-849.
19. Powell DW, Mifflin RC, Valentich JD, Crowe SE, Saada JI, West AB. Myofibroblasts. I. Paracrine cells important in health and disease. Am J Physiol 1999; 277(1 Pt 1): C1-9.
20. Bostrom H, Willetts K, Pekny M, Leveen P, Lindahl P, Hedstrand H et al. PDGF-A signaling is a critical event in lung alveolar myofibroblast development and alveogenesis. Cell 1996; 85(6): 863-873.
21. Torday JS, Sanchez-Esteban J, Rubin LP. Paracrine mediators of mechanotransduction in lung development. Am J Med Sci 1998; 316: 205-208.
22. Rehan V, Torday J. Hyperoxia augments pulmonary lipofibroblast-to-myofibroblasttransdifferentiation. Cell Biochem Biophys 2003; 38: 239-250.
23. Boros LG, Torday JS, Paul Lee WN, Rehan VK. Oxygen-induced metabolic changes and transdifferentiation in immature fetal rat lung lipofibroblasts. Mol Genet Metab 2002; 77: 230-236.
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25. Rehan VK, Dargan-Batra SK, Wang Y, Cerny L, Sakurai R, Santos J et al . The Mechanism for the Paradoxical Decrease in Respiratory Distress Syndrome and Increase in Bronchopulmonary Dysplasia Associated With Chorioamnionitis. ( In press, American Journal of Physiology ).
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27. Rehan VK, Wang Y, Sugano S, Patel S, Santos J, Sakurai R et al . In Utero Nicotine Exposure Alters Fetal Rat Lung Alveolar Type II cell Proliferation, Differentiation, and Metabolism ( In press, American Journal of Physiology ).
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Abstract
Due to a lack of understanding of the molecular mechanisms involved in its pathogenesis, bronchopulmonary dysplasia (BPD) still remains a major cause of morbidity and mortality in the premature infant, and there is no effective preventive and/or therapeutic intervention. We have taken a basic biologic approach to elucidate the pathophysiology of BPD and have discovered that disruption of the alveolar Parathyroid Hormone-related Protein (PTHrP) signaling is centrally involved in this process. Further, stabilization of this signaling pathway by using exogenous PTHrP agonists can prevent and/or rescue the molecular injuries caused by insults that lead to BPD. Based upon years of work in this field, here I provide a novel and innovative molecular approach, i.e., exogenous treatment with PTHrP pathway agonists to prevent and/or treat BPD. However, to avoid any later surprises, it is important to emphasize that before translating it into human trials, this approach needs further testing and refinement in animal models.
Keywords: Chronic lung disease; Parathyroid hormone-related protein; Prematurity
Bronchopulmonary dysplasia (BPD) is a chronic pulmonary disorder that is the consequence of abnormally repaired lung damage which primarily affects premature infants. Despite tremendous advances in neonatal care in general and neonatal respiratory care in particular, BPD still remains a major cause of morbidity and mortality in the premature infant.[1] These advances may have decreased the severity of BPD, but its incidence has not decreased, and may in fact, have actually increased.[2],[3] The affected infants have both short- and long-term clinical issues, including, but not limited to a very complicated initial neonatal intensive care course, poor growth, neurodevelopmental delays, and repeated hospitalizations during the first few years of life.[4],[5],[6] Therefore, any intervention that would reduce the risk of BPD or improve its outcome is likely to have huge clinical and financial benefits.
Why Prevention Has Not Worked So Far
The primary prevention of BPD through elimination of the all important risk factor, prematurity, has not been achieved yet, and is unlikely to be achievable in the near future because of the lack of understanding of the pathophysiology of preterm labor. Up until now, the available preventive/treatment strategies for BPD have also suffered the same limitations, i.e., lack of understanding of the molecular processes involved in both normal and abnormal lung development. The preventive interventions such as antenatal and postnatal steroids, "kinder and gentler" ventilation, early closure of patent ductus arteriosus, treatment of prenatal and postnatal infections, fluid restriction, various nutrient supplements (vitamin A, polyunsaturated fatty acids, etc.), and most recently nitric oxide administration are either not effective or are associated with unacceptable side effects.[7],[8],[9],[10] Most of the these interventions are of an empiric nature, lacking a targeting of the underlying molecular processes that lead to BPD, which may precisely be the reason that none of these interventions have been effective in preventing BPD. Therefore, the available preventive strategies for BPD are not effective, not safe, and none of these is based on thorough understanding of pathogenesis of BPD. Alternate interventions that are based on a sound understanding of the pathogenesis of BPD are needed. With recent advances in understanding the molecular processes involved in both normal and abnormal lung development, in particular the mechanism of alveolar development and its failure in BPD, there is now new hope for not only preventing and treating BPD, but also possibly reversing established disease.[11]
'Old' versus 'New' BPD
Around 40 years back, when Northway described BPD, he described it as a chronic lung disease of relatively larger preterm infants who required ventilation with high pressures and oxygen for prolonged periods of time.[12] It was characterized by four well-defined clinical, radiological, and histopathological findings. The hallmarks of the now so called "Old BPD" were severe large airway injury, interstitial and alveolar edema, and extensive small airway disease with alternating areas of overinflation and fibrosis. With the introduction of antenatal steroids, routine surfactant replacement for respiratory distress syndrome, and modern respiratory care, in contrast, now the "new" BPD occurs primarily in extremely premature infants, occurs after only modest ventilatory and oxygen needs, and is not accompanied by the classic clinical and radiological stages of the "Old BPD". The most dramatic histopathologic finding is "arrested alveolarization" with minimal large or small airway disease, and relatively less inflammation and fibrosis.[2],[13]
Molecular Basis for BPD
The initiation and continued use of mechanical ventilation in infants with birth weights of < 1250 g is the strongest risk factor for the development of BPD. The surfactant deficient alveoli of such premature infants undergo breath-by-breath collapse (de-recruitment) and re-recruitment, resulting in atelectrauma, which sets in-motion the molecular cascade resulting in the structural and morphological changes characteristic of BPD. Experimental studies in preterm animals have clearly demonstrated that even a few large tidal volume breaths can result in acute lung injury that initiates the cascade of molecular events leading to BPD.[14] Given the central role of alveolar stretch in initiating molecular injury leading to BPD, it is logical to pursue a stretch sensitive gene (s) that may be expressed in the pulmonary alveolus and that is (are) essential for its structure and function. Parathyroid Hormone-related Protein (PTHrP) is one such gene. It is expressed in the developing endoderm and its receptor is present on the adepithelial mesoderm, and most importantly, PTHrP knock-out causes a specific inhibition of fetal lung development.[15] The lungs of these mice fail to transition from the pseudoglandular to the canalicular stage of lung development, i.e., failure of alveolarization, a hallmark of the "new" BPD.
Under the influence of Sonic Hedgehog, the developing endoderm expresses PTHrP and its receptor on the adjoining mesenchyme. PTHrP binding to its receptor on the mesenchyme activates the Protein Kinase A pathway, which actively down-regulates the default Wingless/Int pathway and up-regulates the adipogenic pathway through a key nuclear transcription factor, Peroxisome Proliferator Activated Receptor g (PPARg) and its down-stream regulatory genes Adipocyte Differentiation Related Protein (ADRP).[16] ADRP is necessary for the transit of neutral lipid from the lipofibroblast to the ATII cell for surfactant phospholipid synthesis. Lipofibroblasts in turn secrete leptin, which acts on its receptor on AT II cells, stimulating both surfactant phospholipid and protein synthesis. Since PTHrP stimulates leptin production by lung fibroblasts, it provides a complete growth factor-mediated paracrine loop for the synthesis of pulmonary surfactant.[17] Overall, PTHrP signaling, by inhibiting the Wnt signaling, inhibits the default myogenic phenotype, and by stimulating the PPARg signaling, induces the lipogenic phenotype, which is necessary for maintaining alveolar homeostasis through its autocrine effect on interstitial fibroblasts and its paracrine effect on ATII cells.[11] Specifically, the interstitial lipofibroblast phenotype provides protection against oxygen free radicals (i.e., protection against oxotrauma), trafficks neutral lipid substrate to ATII cells for surfactant phospholipid synthesis (i.e., protection against atelectrauma), causes ATII cell proliferation (i.e., protection against any insult causing epithelial injury), thereby promoting alveolar growth, development, and injury/repair.[11],[18] Physiologically, this would stabilize the alveolus, preventing its collapse, maintaining adequate gas-exchange and reducing the energy expenditure by decreasing the work of breathing. On the other hand, although myofibroblasts may also be important for normal lung development, these cells are the hallmark of chronic lung diseases in both the neonate and adult.[19] In the developing lung, myofibroblasts are fewer in number and localize to the periphery of the alveolar septa, where they very likely participate in the formation of new septa.[20] However, in chronic lung diseases, myofibroblasts not only increase in number but are also abnormally located in the center of the alveolar septum in great abundance. In summary, PTHrP signaling is critical in maintaining alveolar homeostasis. Under the influence of cyclic stretch, PTHrP is secreted by the AT II cell, which acts on its receptor on the adjacent lipofibroblast, which in turn secretes leptin that facilitates surfactant synthesis by the ATII cells, underlining the importance of PTHrP signaling in maintaining alveolar homeostasis. If normal PTHrP signaling is interrupted, e.g., by conditions such as prematurity, volutrauma, exposure to hyperoxia or inflammatory cytokines, all conditions that are known to be associated with BPD, normal alveolar homeostasis is altered and interstitial fibroblasts undergo molecular and phenotypic changes consistent with development of BPD. With this rationale, below I review data from various in vitro and in vivo models in which we have examined the role of the modulators of PTHrP signaling in ablating the molecular injury that leads to BPD.
Effect of Barotrauma/Volutrauma/Atelectrauma on PTHRP Signaling
The physiologic stretching of the alveolar type II cell in isolation increases the expression and production of PTHrP, potentially explaining why increased lung tidal volume stimulates pulmonary surfactant synthesis.[21] In contrast to the increased synthesis of surfactant in response to mild stretch (3-5%), overdistension of the type II cell results in down-regulation of PTHrP expression, simulating the consequences of barotrauma/volutrauma/atelectrauma.
Prevention of Oxotrauma by PTHRP/PPARγ Signaling Pathway Agonists
Since lipofibroblasts play an important role in injury-repair mechanisms in the lung, we studied the effects of hyperoxia on the fibroblast phenotype in immature and relatively mature alveolar interstitial fibroblasts, and found that exposure to hyperoxia down-regulated PTHrP signaling, augmenting the transdifferentiation of pulmonary lipofibroblasts to myofibroblasts, the central event in the pathogenesis of BPD.[22] Fetal rat lung fibroblasts (FRLF) from e (embryonic, term = e22) 18 and e21 gestation were studied. After initial culture, cell were maintained either in normoxia (21% O 2 ) or subjected to hyperoxia for 24h (95% O 2 ) at passage (P) 1 and P5. Passage 1 and 5 cells were analyzed for the expression of well-characterized lipogenic and myogenic markers [PTHrP receptor, ADRP, and a smooth muscle actin (SMA)] based on semi-quantitative competitive RT-PCR, triglyceride uptake, and leptin assay. Serial passaging and maintenance of cells in normoxia resulted in a significant decrease in the expression of the lipogenic markers from P1 to P5, spontaneously. This decrease was greater for immature (e18) than for (near-mature) e21 FRLF. However, exposing cells to hyperoxia augmented the loss of the lipogenic markers and gain of the myogenic marker from P1 to P5 in comparison to cells maintained in normoxia. This augmentation was also greater for e18 versus e21 lipofibroblasts. The changes in mRNA expression were accompanied by decreased triglyceride uptake and leptin secretion on exposure to hyperoxia. These data suggest that exposure to hyperoxia augments the transdifferentiation of pulmonary lipofibroblasts to myofibroblasts. Importantly, pretreatment with a PTHrP/PPARg signaling pathway agonist, prostaglandin J 2 (PGJ 2 ), at least partially attenuated the hyperoxia-augmented lipo-to-myofibroblast transdifferentiation.
In a further series of experiments, using [1,2-13C 2 ]-D-glucose tracer and gas chromatography/mass spectrometry, we metabolically profiled e18 and e21 FRLF with and without hyperoxia exposure at passages 1, 4, 7, and 10.[23] For this series of studies, glucose carbon redistribution between the nucleic acid ribose, lactate, and palmitate synthetic pathways and ADRP expression by RT-PCR were examined. Exposure to hyperoxia at each passage caused a decrease in ADRP mRNA expression. This passage- dependent transdifferentiation is accompanied by a moderate increase in the synthesis of nucleic acid ribose from glucose through the non-oxidative steps of the pentose cycle. E18 fibroblasts showed over an 85% decrease in the de novo synthesis of palmitate from glucose, while e21 fibroblasts showed a less pronounced 32% to 38% decrease in de novo lipid synthesis in hyperoxia-exposed cultures. From these data The authors conclude that (1) there is a maturation-dependent sensitivity to hyperoxia; (2) transdifferentiation of the fibroblast, as demonstrated by changes in ADRP expression, is accompanied by metabolic enzyme changes affecting ribose synthesis from glucose, and (3) hyperoxia specifically inhibits lipogenesis from glucose. These molecular and metabolomic data were further complemented by "genome-wide" microarray analysis of RNA extracted from P1 and P10 e19 FRLF with or without exposure to hyperoxia (95% O 2 for 24h). Rat chip RAE230 , which has 15,923 RNA transcripts, including most genes of interest, e.g., PTHrP, PPARg, Adipophilin (moderate homology with ADRP) was used. "Genome-wide" expression was analyzed using Affymetrix GeneChip Operating Software and Netaffx, Genespring 6.2 (Silicon Genetics), and Gene Microarray Pathway Profiler (Gladstone Institute). In accord with our molecular and metabolomic data, cluster analysis of the microarray data confirmed the down-regulation of cholesterol and fatty acid synthetic genes and up-regulation of fatty acid degradation and Wnt signaling pathway genes on passaging e19 FRLF from P1 to P10, and on exposure to hyperoxia at passages 1 and 10, thereby confirming lipo-to-myofibroblast transdifferenation under these conditions. Based on these data, it appears that hyperoxia induces lipo-to-myofibroblast transdifferentiation, which may be an important mechanism for hyperoxia-induced lung injury and is likely to be a key element in the pathophysiology of BPD.
Following present investigation in vitro studies, as outlined above, it is next determined whether upon exposure to hyperoxia, pulmonary alveolar lipo-to-myofibroblast transdifferentiation occurs in vivo , and whether treatment with a potent PTHrP/PPARg signaling pathway agonist, Rosiglitazone (RGZ), would prevent this process.[24] Newborn Sprague Dawley rat pups were exposed to normoxia (21% O 2 ), hyperoxia alone (95% O 2 for 24h), or hyperoxia with RGZ (95% O 2 for 24h + RGZ, 3 mg/Kg, administered intraperitoneally). Subsequently, pups were sacrificed and lung tissue was analyzed by morphometry, and by RT-PCR, Western hybridization, and immunohistochemistry for the expression of key lipogenic and myogenic markers. We observed a significant decrease in the expression of lipogenic markers and a significant increase in the expression of myogenic markers in the hyperoxia alone group. Exposure of rat pups to 24 hours of hyperoxia dramatically affected normal postnatal lung development. Histologic examination of lungs obtained from hyperoxic pups revealed a remarkable arrest of alveolarization compared with their normoxic controls. Hyperoxia-exposed lungs demonstrated relatively large air spaces, thinned interstitial, and decreased secondary septal crests compared to air-exposed controls. In lungs from hyperoxic animals, septal thickness was reduced significantly compared to control lungs. Furthermore, quantitative analysis of alveolar number demonstrated a 50% reduction in average radial alveolar counts in hyperoxic animals compared to controls. Pre-treatment with RGZ almost completely prevented the hyperoxia-induced changes in lung morphology, including the effects on septal thickness and radial alveolar counts. The hyperoxia-induced morphologic, molecular, and immunohistochemical changes were almost completely prevented by RGZ. This was the first evidence of in vivo lipo-to-myofibroblast transdifferentiation and its almost complete prevention by RGZ, suggesting that administration of PPARg agonists might be a novel, effective strategy to prevent the hyperoxia-induced lung molecular injury that has been implicated in the pathogenesis of BPD.
Prevention of Infection-Induced Lung Injury by PTHrP/PPARγ Signaling Pathway Agonists
Since lung inflammation is a key factor that predisposes preterm infants to BPD, in a series of studies,[25] we determined the effects of lipopolysaccharide (LPS) on PTHrP-driven pulmonary epithelial-mesenchymal interactions that have been shown to be essential in the maintenance of lung homeostasis. Lung explants derived from e19.5 Sprague Dawley rat pups were treated with LPS (0-50 ml) with or without a PTHrP pathway agonist, PGJ 2 , for up to 72h. There were acute (24 hour) significant increases in the expression of PTHrP, PPARg, ADRP, and Surfactant Protein-B (SP-B), without any significant effects on the expression of aSMA. This was followed (72 hours) by significant decreases in the expression of PTHrP, PPARg, ADRP, and SP-B, accompanied by a significant increase in the expression of aSMA, the key functional marker of BPD. These changes were completely prevented by concomitant treatment with PGJ 2 , providing an integrated mechanism for the acute stimulation of lung maturation accompanied paradoxically by BPD following intrauterine inflammation. Further, as in present studies in vivo hyperoxia model, treatment with a specific agonist of epithelial-mesenchymal interactions prevented the inflammation-induced molecular lung injury that is known to result in BPD.
Prevention of Nicotine-Induced Lung Injury by PTHRP/PPARγ Signaling Pathway Agonists
The authors tested the hypothesis that in vitro nicotine exposure disrupts specific epithelial-mesenchymal paracrine signaling pathways and results in pulmonary lipo-to-myofibroblast transdifferentiation, resulting in altered pulmonary development and function.[26] WI38 cells, a human embryonic pulmonary fibroblast cell line, were treated with nicotine with or without specific agonists of the alveolar fibroblast lipogenic pathway, PTHrP, dibutyryl cAMP or the potent PPARg stimulant RGZ for 7 days. Subsequently, expression of the key lipogenic and myogenic markers was examined. Nicotine treatment resulted in significantly decreased expression of lipogenic and increased expression of myogenic markers in a dose-dependent manner, indicating that nicotine exposure causes lipo-to-myofibroblast transdifferentiation. The nicotine-induced lipo-to-myofibroblast transdifferen-tiation was completely prevented by concomitant treatment with PTHrP, dibutyryl cAMP, RGZ, and by transiently overexpressing PPARg, suggesting the possibility of complete prevention of nicotine-induced lung injury by PTHrP/PPARg signaling pathway agonists. Indeed in our subsequent studies, we demonstrated that by augmenting PTHrP/PPARg signaling pathway in vivo, we were able to completely block the nicotine-induced increase in alveolar type II cell proliferation, once again underpinning the critical role of PTHrP/PPARg signaling in nicotine-induced lung injury.[27]
Significance of PTHRP Signaling in Human BPD
Since PTHrP secreted by pulmonary alveolar type II cells is a key physiologic paracrine factor in maintaining alveolar homeostasis, we hypothesized that its levels in the tracheal aspirates (TA) of ventilated Very Low Birth Weight Infants (VLBWI) would correlate with the development of BPD.[28] The authors examined whether TA PTHrP content during the first week of life correlated with the later development of BPD. Forty VLBWIs (birth weight 943 ± 302 grams (mean ± SD); gestational age 27 ± 2 weeks; 21 males and 19 females), who were ventilated for Respiratory Distress Syndrome, were studied. The TAs were collected once daily until the infants were extubated and assayed for PTHrP. The levels of TA PTHrP were correlated with the later development of BPD. PTHrP in the TA during the first week of life was significantly lower in those infants who developed BPD (12/40) than among those who did not (28/40). The PTHrP levels also correlated with the duration of mechanical ventilation needed in these infants. A PTHrP level of ￡ 1.32 pg/mg protein predicted the later development of BPD maximally [84.6% correct classifications (true positives + true negatives)], with a sensitivity = 76.9% and specificity = 88.5%. Using a TA PTHrP level of 1.32 pg/mg protein as the cutoff, the authors constructed Kaplan-Meier curves to compare the duration of ventilation needed between the two groups, i.e., ￡ and > than 1.32 pg/mg protein TA PTHrP level. Infants with TA PTHrP levels greater than 1.32 pg/mg protein were off ventilatory support significantly earlier. To determine how PTHrP levels compared with the other known predictors of BPD, such as birth weight (< 1000 g), GA (< 28 wk), and sex (male gender), we performed multivariate logistic regression analysis for these 4 variables in predicting the development of BPD. Of these variables, a PTHrP level of ￡ 1.32 pg/mg protein was the strongest predictor of BPD and remained so after adjusting for the other 3 variables, i.e., birth weight of < 1000 g, GA of < 28 wk, and male gender. From these data, we concluded that lower TA PTHrP content during the first week of life in ventilated VLBWI inversely correlates with prolonged ventilation and the later development of BPD.
Prevention of Molecular Lung Injuries Leading to BPD with PTHrP/PPARγ Agonists
It is clear from the work outlined above that we have systematically demonstrated the central role of PTHrP-driven epithelial-mesenchymal signaling in maintaining alveolar homeostasis in barotrauma, oxotrauma, infection and nicotine injury. The lipofibroblast expresses both ADRP and leptin in response to PTHrP signaling from the alveolar type II cell, resulting in direct protection of the mesoderm against oxidant injury, and protection against atelectasis by augmenting surfactant phospholipid and protein synthesis. Disruption of PTHrP signaling down-regulates the adipogenic signaling and up-regulates the myogenic signaling pathways, causing myofibroblast transdifferentiation.[11],[22],[23],[24],[25],[26],[27] Unlike lipofibroblasts, myofibroblasts cannot promote AT II cell growth and differentiation,[11] leading to the failed alveolarization characteristic of BPD. A variety of factors associated with failed alveolarization- barotrauma, oxotrauma, infection and nicotine- all cause myofibroblast transdifferentiation in vitro[11],[22],[23],[25],[26] and in vivo.[24], [27] More importantly, the authors have shown that PTHrP/PPARg signaling pathway agonists such as PGJ 2 and RGZ can prevent or rescue myofibroblast transdifferentiation effectively, therefore, preventing the inhibition of alveolarization in the developing lung.
In summary, we have outlined a novel and innovative molecular approach to prevent BPD that is based upon sound understanding of the molecular processes involved in its pathogenesis. Therefore, after trying a host of different approaches that have been utilized so far and that have essentially been futile in preventing BPD, we might have discovered a rationale approach that works. However, it still needs some fine tuning before it is ready for clinical use. In the mean time, we need to exploit every little opportunity to adopt and refine NICU care practices that minimize lung damage. These include but are not limited to maximizing the administration of antenatal steroids and early postnatal surfactant, avoidance or minimization of ventilatory support, and acceptance of lower target range for pulse oximetry during oxygen supplementation.
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