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Biomarkers in Bronchopulmonary Dysplasia
Paediatric Respiratory Reviews 14 (2013) 173–179
Contents lists available at SciVerse ScienceDirect
Paediatric Respiratory Reviews
CME Review
Biomarkers in Bronchopulmonary Dysplasia Anita Bhandari 1,*, Vineet Bhandari 2 1 2
Division of Pediatric Pulmonology, Connecticut Children’s Medical Center, Hartford, CT Division of Perinatal Medicine, Yale University School of Medicine, New Haven, CT
EDUCATIONAL AIMS To provide an overview of the definition, pathology and aetipathogenesis of bronchopulmonary dysplasia (BPD). To familiarize the reader with different biomarkers of BPD. To review research literature in the area of biomarkers as it pertains to BPD.
A R T I C L E I N F O
S U M M A R Y
Keywords: Cytokines Lung Premature Newborn Growth factors
Bronchopulmonary dysplasia (BPD) is a complex disorder secondary to gene-environment interactions, and is the commonest chronic lung disease in infancy. There is no specific or effective treatment available to date for BPD. Since the aetiopathogenesis of BPD is multifactorial, involving diverse molecular signaling pathways, a variety of biomarkers detected in biological fluids have been proposed for early identification of infants predisposed to BPD. This review will be restricted to biomarker studies in human infants, conducted mostly in the last decade. The majority of the studies have been conducted using blood, urine or tracheal aspirate samples. Despite the multitude of biomarkers proposed, most studies have been conducted in small numbers of infants, with few being replicated by independent investigators. Confirmatory studies with adequate sample sizes and assessment of the role of putative biomarkers in the aetiology of BPD in developmentally appropriate animal models and human lungs with BPD will enhance the potential for therapeutic interventions. Genomic and proteomic approaches have the greatest potential to significantly advance the field of biomarkers in BPD. ß 2013 Published by Elsevier Ltd.
INTRODUCTION Bronchopulmonary dysplasia (BPD) is the commonest chronic lung disease of infancy.1 While many definitions of BPD exist,2 the most commonly accepted definitions include the need for oxygen supplementation at 28 days of life and/or 36 weeks postmenstrual age (PMA).1,2 The incidence of BPD has remained stable3 or increased slightly over the last decade.1,2 The incidence of BPD is variable among centers, and was reported to range from 12% to 32% among infants <32 weeks of gestation at birth.2 Infants with birth weight (BW)<1250 g account for the vast majority (97%) of all BPD patients.1 With the survival of younger premature infants, and changes in
* Corresponding author. Associate Professor of Pediatrics, Division of Pediatric Pulmonology, Connecticut Children’s Medical Center, 282 Washington Street, Hartford, CT 06106, USA. Tel.: +860 545 9679; fax: +860 545 9445. E-mail address: [email protected] (A. Bhandari). 1526-0542/$ – see front matter ß 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.prrv.2013.02.008
management style, there has been a shift in the clinical and pathological phenotype – designated as the ‘‘new’’ BPD.1 PATHOLOGY Over the past decade changes in neonatal ventilation strategies, widespread use of antenatal steroids, aggressive nutritional intervention and fluid restriction have led to a change in lung histopathology of premature babies.1 The new BPD has some unique features that distinguish it from the older forms of BPD.4,5 Lungs of patients with new BPD have fewer, larger and simplified alveoli, and along with dysmorphic vasculature4,6 are considered the pathognomic features of the new BPD. These findings suggest a disruption of pulmonary development in that the vascular and alveolar growth parameters are both impaired.1 Lungs from infants with new BPD have been reported to have more uniform inflation and less regional heterogeneity of lung disease. Airway smooth muscle is only mildly thickened and rare epithelial lesions and fibroproliferative changes may be occasionally seen.1
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PATHOGENESIS Over the last 5 years, an improved understanding has evolved regarding the pathogenesis of BPD.7 For decades, an immature lung upon which a variety of ‘‘environmental’’ factors (infection, hyperoxia, baro/volu-trauma) acted upon to cause injury, has been the cornerstone of the pathogenesis of BPD. Recent publications by independent investigators have confirmed the significant genetic contribution to the pathogenesis of new BPD.8,9 This has transformed BPD to a condition with significant genetic-environmental interactions,10 thus enabling potentially novel approaches for understanding the pathogenesis of this condition.11 Inflammation, contributed to by antenatal (chorioamnionitis) and postnatal (local or systemic infections, hyperoxia, ventilator-induced injury) factors, initiates and modifies the process of lung injury in the developing lung.7 This inflammatory process is dependent upon the effective release and balance of cytokines. An imbalance in these mediators leads to activation of the cellular death pathways in the lung.7 This is followed by healing (resolution of injury to normal lung architecture) or repair.7,12 The latter is characterised by impaired alveolarisation and dysregulated angiogenesis leading to fewer, larger simplified alveoli.7 It is obvious from the above description that a sufficient length of time is required for the interactive genetic-environmental factors to act on the immature lung to progress to the clinical-pathological phenotype of BPD. We have proposed that BPD be assessed in early, evolving and established phases, in relation to postnatal age, and have suggested management strategies for the same.1,7 In terms of postnatal preventive approaches, early recognition of infants at high risk of developing BPD would be critical to plan out therapeutic strategies. Use of clinical factors to develop scoring systems and predictive modeling utilizing multiple variables has been reported with modest success,2,13,14 with increasing capability at or after the first week of life.15–18 However, we believe that the identification of these infants needs to occur within the first 3 days of the early phase of BPD, for potential maximal impact.19 Early detection of biochemical biomarkers, especially if they are also integral in the pathogenesis of BPD, could provide an enhancement of the capability to identify and potentially target the infants predisposed to BPD.19 In addition, biomarkers could be combined with clinical parameters to further increase the ability to identify and prognosticate the outcome of high-risk infants to develop BPD and perhaps predict the severity of BPD. BIOMARKERS IN BPD Given the multifactorial aetiopathogenesis of BPD involving diverse molecular signaling pathways, a variety of biomarkers detected in different biological fluids have been proposed for early identification of infants predisposed to BPD. The majority of the studies have been conducted using blood, urine or tracheal aspirate (TA) samples.7 This review will be restricted to biomarker studies in human infants, conducted mostly in the last decade. Blood Biomarkers In the second-trimester maternal serum levels of alphafetoprotein and/or human chorionic gonadotropin above the 95th percentile and/or levels of unconjugated estriol <5th percentile, were associated with increased risk of BPD (n = 82 vs. 164 with no BPD).20 Having a combination of 2 or more of these biomarkers further increased the risk.20 Cord blood is probably one of the easiest and earliest samples to get access to in premature neonates predisposed to BPD. Expectedly, the focus has been on markers involved in angiogenesis, given the dysregulated vasculature in BPD lungs. Using the
older definition of 28-days supplemental oxygen for BPD, using a cut-off value of 17 mg/dl, cord blood placental growth factor (PlGF) was noted to have 95% specificity, 53% sensitivity with positive and negative predictive values of 83% and 82%, respectively in 63 preterm infants.21 Another molecular marker that is involved in angiogenesis is endostatin, an antiangiogenic factor. In very low birth weight (VLBW) infants, a higher circulating concentration of endostatin was noted in cord blood in those who developed BPD (n = 19, 100.7 29.7 vs. those who did not, n = 73, 85.6 28.7 ng/mL; p = 0.029).22 This finding was recently independently confirmed.23 In addition, the same authors also noted low Angiopoietin-1 cord blood levels to predict the subsequent development of BPD (n = 28, vs. 78 with no BPD).23 Endothelial colony-forming cells in cord blood were significantly lower in infants who later developed BPD (n = 24), though the endothelial and hematopoietic cell subsets were comparable in those with or without BPD (n = 74).24 These data have been recently confirmed in an independent study.25 Among other markers, type IV collagen, CD9, C-terminal fragment of Type I collagen, and soluble L-selectin have been shown to be decreased, while eosinophilic cationic protein, 8isoprostanes, and soluble E-selectin are all increased in infants developing BPD.7 Plasma concentrations of N-terminal pro-B-type natriuretic peptide (BNP) were found to be significantly higher at 4 weeks of age in patients with BPD (n = 11), and decreased at 6 and 8 weeks of age.26 Another focus of the investigative groups has been pulmonary epithelial cell markers as well as extracellular matrix proteins which could be involved in impaired alveolarisation, a key pathologic hallmark of BPD. Clara cells are non-ciliated epithelial cells which line the respiratory and terminal bronchioles. Low cord blood clara cell protein (CC10 or CC secretory protein/CCSP or CC16) was reported in cord blood plasma of 79 preterm infants, of which 17 developed BPD.27 Interestingly, serum CC10 has also been reported to be persistently low in infants who developed BPD (n = 8).28 In striking contrast, serum CC10 levels within 2 h of life and at post natal day 14 [PN14] were significantly higher in preterm neonates (n = 35, with gestational age 31 weeks) who were mechanically ventilated and later developed BPD (n = 7) vs. those who were not ventilated (n = 12).29 Matrix metalloproteinase-9 (MMP-9)/tissue inhibitor of metalloproteinase-1 (TIMP-1) ratios in cord blood were significantly higher in those infants who developed moderate/severe (n = 9) BPD versus those who had mild/no BPD (n = 20).30 Granulocyte-specific S100A12, a marker for inflammatory disorders, was not found to be a useful marker for BPD (n = 10 vs. 18 with no BPD), when assayed on PN1-7.31 Cord blood KL-6 (a lung injury marker) was increased in infants with BPD (n = 50, vs. non-BPD n = 24),32 and was also useful at 1 week of life as a predictor of moderate/severe BPD.33 In an interesting study, ‘‘old’’ BPD (n = 11, compared to n = 22) was associated with significantly increased proinflammatory (interleukin-6 or IL-6, IL-8, monocyte chemoattractant protein-1 or MCP-1) and antiinflammatory IL-10 levels, but with no significant difference in profibrotic/angiogeneic cytokines (transforming growth factor-beta 1 or TGFb1, platelet-derived growth factor BB fraction or PDGF BB, vascular endothelial growth factor or VEGF) on postnatal (PN) days 3 or 5.34 In contrast, when the authors used the ‘‘new’’ BPD definition (n = 22, compared to n = 9), the samples were characterized with increases only in the latter category on PN5.34 This highlights the fact the aetiology of new BPD is markedly different from that of the earlier forms of BPD.1 With the advent of multiplex assays, researchers have attempted to simultaneously evaluate multiple cytokines, and assess their association to BPD. Out of 11 cytokines evaluated (n = 128), higher IL-8, IL-10 and granulocyte colony stimulating factor (G-CSF) levels were found to predict BPD in 1-day old infants.35 Blood samples were collected from infants within 4 h of birth, and on days 3, 7, 14
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and 21 and concentrations of 25 cytokines were assayed.36 Using multiple logistic regression analyses, combining results from all models, BPD/death (n = 606, out of 1062) was associated with higher levels of IL-1b, 6, 8, 10 and interferon gamma (IFNg), but lower concentrations of IL-17, regulated on activation normal T-cell expressed and secreted (RANTES), and tumor necrosis factor beta (TNFb).36 These have been summarised in Table 1. The major limitation of these studies is the fact that cord or infant blood concentrations of the various molecules could be influenced by a variety of conditions, and are not necessarily reflective of what is going on in the lung. While earlier studies also suffer from the drawback of small sample sizes, the latter ones do have sufficient number of infants and simultaneously measure multiple cytokines at sequential time points to give a more comprehensive picture. At least among the cytokines, it is important to note consistent results with increased blood levels of IL-6, 8 and IL-10 being associated with BPD. This is supported by increased concentrations of IL-6 and -8 in TA obtained from human infants developing BPD. In addition, developmentally-appropriate animal models overexpressing IL-1b,37 IL-638 and IFNg39 in the lung provide supportive evidence of these molecules being involved in the pathogenesis of BPD.
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8-iso-PGF2a was evaluated in the urine in early PN life, but was not found to correlate with the development of BPD.42 In another study, 8-hydroxydeoxyguanosine (8-OHdG, also an oxidant-injury marker) urinary levels on PN7, but not LTE4, were an independent risk factor for developing moderate/severe BPD.43 The most promising urinary biomarker for BPD has been bombesin-like peptide, with concentrations >20,000 pg/mg creatinine on PN1-4 occurred among 54% of the infants who developed BPD, vs. 10% among BPD controls.44 The latter data is supported by animal studies.45 These have been summarised in Table 2. Pulmonary Biomarkers
Urinary Biomarkers
Not surprisingly, most of the work on biomarkers has been done using TA samples, allowing direct access to the pulmonary compartment. While TA samples are a suitable substitute for bronchoalveolar lavage specimens in preterm neonates, there is some controversy about the need to ‘‘correct’’ for dilution. In addition, such samples can only be collected from intubated infants, and hence samples from appropriately matched (healthy and by extension, non-intubated) controls are not available.7,11 Since the topic of pulmonary biomarkers has been recently reviewed by us7,11 and others,46 we will summarize the major findings, and refer the reader to those publications for additional details.
With inflammation playing a major role in the pathogenesis of BPD, investigators evaluated urinary levels of leukotriene E4 (LTE4) in the first month of life, but did not find any association with development of BPD.40 Since exposure to hyperoxia is another critical factor in BPD pathogenesis,41 the oxidant injury marker,
Cytokines Among these, the traditionally considered proinflammatory IL-1b, -6, -16, and TNFa have been shown to be increased in TA in earlier studies.7,11,46 A recent addition has been increased IFNg.39,47 Regarding anti-inflammatory cytokines, most of the
Table 1 Blood biomarkers in BPD. Source: Biomarker
Description/Physiologic Role
Levels associated with Increased BPD Risk refs
Maternal blood: AFP and/or HCG Maternal blood: unconjugated estriol Cord blood: PlGF
>95th centile20 <5th centile20 Higher21
Cord blood: Endostatin Cord blood: Angiopoietin 1 Cord blood: Endothelial colony-forming cells Infant blood: C-IV Infant blood: PICP Infant blood: soluble L-selectin Infant blood: eosinophilic cationic protein Cord and Infant blood: soluble E-selectin Infant blood: N-terminal pro-B-type natriuretic peptide Cord and Infant blood: Clara cell secretory protein
AFP: Unknown; HCG: Maintains pregnancy Maintains pregnancy Proangiogenic. It is postulated that PlGF boosts angiogenesis by binding to VEGFR1, leaving a higher concentration of free VEGF-A that can bind to VEGFR2 Antiangiogenic Proangiogenic Proangiogenic Serum antigen of basement membrane component Serum antigen of extracellular matrix component Adhesion molecule Marker of eosinophil activation Adhesion molecule Regulation of extracellular fluid volume and blood pressure Non-ciliated epithelial cells lining the respiratory and terminal bronchioles
Cord blood: MMP 9/TIMP 1 Infant blood: S100A12 Cord and Infant blood: KL-6 Infant blood: IL-6 Infant blood: IL-8 Infant blood: IL-10 Infant blood: MCP-1 Infant blood: TGFb1 Infant blood: PDGF-BB Infant blood: VEGF Infant blood: G-CSF Infant blood: IL-1b Infant blood: IFNg Infant blood: IL-17 Infant blood: RANTES Infant blood: TNFb
Protease/antiprotease of extracellular matrix Granulocyte-specific proinflammatory molecule, interacts with RAGE Lung epithelial cellular injury marker Proinflammatory cytokine Proinflammatory cytokine Antiinflammatory cytokine Chemokine Profibrotic Proangiogenic Proangiogenic Proinflammatory cytokine Proinflammatory cytokine Proinflammatory cytokine Proinflammatory cytokine Chemokine Proinflammatory cytokine
Higher22,23 Lower23 Lower24,25 Lower7 Lower7 Lower7 Higher7 Higher7 Higher26 Lower27,28 Higher29 Higher30 No difference31 Higher32,33 Higher36 (old BPD)34 Higher35,36 (old BPD)34 Higher35,36 (old BPD)34 Higher (old BPD)34 Higher (new BPD)34 Higher (new BPD)34 Higher (new BPD)34 Higher35 Higher36 Higher36 Lower36 Lower36 Lower36
AFP: alphafeto protein; HCG: human chorionic gonadotropin; PlGF: placental growth factor; C-IV: type IV collagen; PICP: C-terminal fragment of Type I collagen; MMP: matrix metalloproteinase; TIMP: tissue inhibitor of metalloproteinase; IL: interleukin; MCP: monocyte chemoattractant protein; TGFb: transforming growth factor beta; PDGF-BB: platelet derived growth factor-BB isoform; VEGF: vascular endothelial growth factor; R1/2: receptor 1/2; RAGE: receptor for advanced glycation end products; G-CSF: granulocyte colony stimulating factor; IFNg: interferon gamma; RANTES: regulated on activation normal T-cell expressed and secreted; TNFb: tumor necrosis factor beta.
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176 Table 2 Urinary biomarkers in BPD. Biomarker Leukotriene E4 8-iso-PGF2a 8-OHdG Bombesin-like peptide
Description/Physiologic Role
Levels associated with Increased BPD Risk
Marker of inflammation Marker of oxidant injury Marker of oxidant injury Role in lung development; pulmonary injury
refs
40,43
No difference No difference42 Higher43 Higher45
8-iso-PGF2a: 8-iso-prostaglandin F2 alpha; 8-OHdG: 8-hydroxydeoxyguanosine.
earlier TA studies have failed to detect IL-10, and researchers have suggested that those infants who went on to develop BPD have a reduced ability to generate adequate levels of IL-10.7,11,46 Decreased levels of macrophage migration inhibitory factor (MIF) have been associated with increased risk of BPD.48 Chemokines IL-8 (CXCL8), MCP-1, -2 and -3 TA levels were significantly increased in infants developing BPD.7,11,46 Proteases/Antiproteases Increased levels of MMP8 and 9 and decreased TA levels of TIMP2 have been reported in infants developing BPD.7,11,46 Higher trypsin-2 levels may predispose to BPD.7,11,46 Cathepsin K expression has been noted to be decreased in infants with BPD.49 Adhesion molecules Soluble intercellular adhesion molecule-1 and L-selectin are increased in TA samples in the first PN week of infants developing BPD.11,46 Oxidative Injury Elevated levels of 3-cholrotyrosine and malondialdehyde were correlated with development of BPD.11 Breast regression protein (BRP)–39 and its human homolog, YKL-40 (also called chitinase-3– like protein 1 and human cartilage glycoprotein–39) has been implicated in hyperoxia-induced lung injury.50 Low levels of YKL40 have been reported in infants subsequently developing BPD.50 Peptide Growth Factors Among these, TGFb1 has been consistently shown to be increased in human TA samples,7,11,46 and has been borne out in animal models of BPD.51 Keratinocyte and pulmonary hepatocyte growth factors are decreased in TA samples of infants predisposed to BPD.7,11 Among angiogenic agents, Angiopoietin2, endothelin-1, and fibroblast growth factor are increased in infants developing BPD.7,11 Multiple studies have evaluated VEGF TA levels.7,11,46 In a recent study, VEGF TA levels on day 1 were lower, while soluble VEGF receptor 1 (sVEGFR1) levels were higher in infants who developed BPD (n = 31, vs. 34 with no BPD).52 Subsequently, on days 3 and 7, the levels of VEGF continued to rise, while that of sVEGFR1 declined.52 In a recent review, it was summarized that VEGF levels in humans tend to be low to normal in early BPD stages.53 Though VEGF levels tend to increase with time in some studies, others point toward lower relative levels in infants developing BPD at various chronological points. One study reported a bimodal distribution of VEGF levels with proportionately higher levels measured within the first 12 hours of life and by 3–4 weeks PN.54 To discern the true relative quantity of VEGF right after birth, before the effects of surfactant replacement have occurred, more studies looking at neonates within the first hours of life would be helpful.53 While lower VEGFR2 staining has been noted in the lungs of human infants with BPD (n = 6),55 increased expression of endoglin (a TGFb co-receptor; an important regulator of angiogenesis) has been shown in another study.56 In another study, there was increased expression of inducible nitric oxide synthase (iNOS) and endothelial NOS as well as nitrotyrosine
staining in BPD lungs (n = 7), compared to controls (n = 7).57 Since there is a close interaction between VEGF,53 NOS57 and Angiopoietins,58,59 all being implicated in human BPD, the above data suggests that there is a disruption in the normal angiogenic signaling pathways, with some factors being suppressed and other enhanced,56 leading to the dysregulated vasculature in BPD lungs. Others Lower levels of CC10 have been associated with an increased risk of BPD,7,11,46 as also noted in 2 studies using blood samples.27,28 Nuclear factor-kappa B TA levels were increased, while parathyroid hormone-related protein TA levels were decreased in infants predisposed to BPD.7,11 TA levels of fibronectin and plasminogen activator inhibitor-1 were increased, while those of lysozyme decreased in those developing BPD.11 On day 1, TA levels of polyunsaturated fatty acids and dimethylacetals (representing plasmalogens) were significantly lower in those infants who developed BPD (n = 10), versus those who did not (n = 15); these differences did not persist, however, in the subsequent PN days.60 TA levels of neutrophil-gelatinase-associated lipocalin (NGAL), which is included in larger macromolecular complex together with MMP9, was significantly increased in infants with BPD.61 TA pepsin concentrations, which was secondary to gastric aspiration (and not by hematogenous spread or local synthesis in the lungs), was significantly higher in those who developed BPD (n = 31) and/or died (n = 16), versus those with no BPD (n = 12), in the first week and month of life.62 These have been summarized in Table 3. Using cell pellets from TA, investigators noted a high IL-10 and IFNg, but not TGFb1 or PDGF-B mRNA expression in infants developing BPD (n = 13 vs. 13 with no BPD).63 Lower Sirtuin1 in TA leukocytes was associated with the development of BPD or death in premature infants.64 Genome-wide expression analysis was applied to define pathways affected in BPD lungs (n = 11 vs. 17 matched controls).65 The investigators identified 159 genes differentially expressed in BPD tissues. Interestingly, 3 of the 5 most highly induced genes turned out to be mast cell-specific markers. The investigators confirmed an increased accumulation of connective tissue mast cell (chymase expressing) cells in human BPD tissues, and in an animal model.65 Miscellaneous Tissue Biomarkers Placental IL-10 was less prominent in those infants who subsequently developed BPD (n = 49) vs. those who did not (n = 49), with no differences in IL-6 expression.66 Once again highlighting the contribution of angiogenic factors, use of oral spectrophotometry (reflectance measurements of lower gingival and vestibular oral mucosa) revealed lower light reflectance values in the red, but higher values in the blue and blue-green sections of the spectrum in infants who went on to develop BPD (n = 25, out of 75), on PN1.67 Among breath condensate biomarkers, increased end-tidal carbon monoxide (ETCO) and exhaled nitric oxide (NO) levels were significantly higher in infants with BPD on PN14 (n = 39, out of 78)18 and (n = 46, out of 80) PN28,68 respectively.
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Table 3 Pulmonary (tracheal aspirates) biomarkers in BPD. Biomarker Cytokines IL-1b, -6, -16, TNFa IFNg IL-10 MIF Chemokines IL-8, MCP-1, -2, -3 Proteases/Antiproteases MMP 8 and 9 TIMP 2 Trypsin -2 Cathepsin K Adhesion Molecules sICAM-1, L-selectin Oxidative Injury 3-cholorotyrosine, malondialdehyde YKL-40 Peptide Growth Factors TGFb1 KGF, Pulmonary HGF Angiopoietin 2, Endothelin-1, FGF2 VEGF sVEGFR1 Others Clara cell secretory protein Nuclear factor-kappa B PTHrP Fibronectin PAI-1 Lysozyme PUFA and DMA NGAL Pepsin
Description/Physiologic Role
Levels associated with Increased BPD Risk
Proinflammatory Proinflammatory Antiinflammatory Proinflammatory
Higher7,11,46 Higher39,47 Lower7,11,46 Lower48
Proinflammatory
Higher7,11,46
Protease of extracellular matrix Antiprotease of extracellular matrix Protease of extracellular matrix Protease of extracellular matrix
Higher7,11,46 Lower7,11,46 Higher7,11,46 Lower49
Stabilizing cell-cell interactions and facilitating leukocyte transmigration
Higher11,46
Marker of neutrophil oxidation, oxidative byproduct Chitinase-like proteins
Higher11 Lower50
Profibrotic Alveolar epithelial proliferation, repair Proangiogenic Proangiogenic Antiangiogenic
Higher7,11,46 Lower7,11 Higher7,11 Variable7,11,46,52,53 Biphasic54 Variable52
Non-ciliated epithelial cells lining the respiratory and terminal bronchioles Transcription factor Alveolar growth/development Extracellular matrix formation Fibrinolysis Bactericidal Substrates for lipid peroxidation in pulmonary surfactant Antimicrobial, binds to MMP 9 inhibiting its degradation Protein degrading enzyme
Lower7,11,46 Higher7,11 Lower7,11 Higher11 Higher11 Lower11 Lower60 Higher61 Higher62
refs
IL: interleukin; b: beta; TNFa: tumor necrosis factor alpha; IFNg: interferon gamma; MIF: macrophage migration inhibitory factor; MCP: monocyte chemoattractant protein; MMP: matrix metalloproteinase; TIMP: tissue inhibitor of metalloproteinase; sICAM: soluble intercellular adhesion molecule; YKL-40: also known as chitinase-3 like protein 1, human homologue of breast regression protein 39 (BRP39); TGFb: transforming growth factor beta; KGF: keratinocyte growth factor; HGF: hepatocyte growth factor; FGF: fibroblast growth factor; VEGF: vascular endothelial growth factor; sVEGFR1: soluble VEGF receptor 1; PTHrP: parathyroid hormone related protein; PAI: plasminogen activator inhibitor; PUFA: polyunsaturated fatty acid; DMA: dimethylacetals; NGAL: neutrophil gelatinase associated lipocalin. Table 4 Miscellaneous tissue biomarkers in BPD Source: Biomarker
Description/Physiologic Role
Levels associated with Increased BPD Risk
Placenta: IL-10 Placenta: IL-6 Oral mucosa: spectrophotometry
Antiinflammatory cytokine Proinflammatory cytokine Reflectance of lower gingival and vestibular oral mucosa
Breath: Carbon monoxide Breath: Nitric oxide Gastric fluid: Microbes Chest radiograph: Thymus
Marker of inflammation and oxidative stress Marker of inflammation Presence of microbes, including Ureaplasma species Assessment of size of thymus
refs
66
Lower No difference66 Decreased reflectance values in red, but increased in blue and blue-green sections of the spectrum. May signify alterations in microvascular and extracellular matrix networks73 Higher18 Higher68 Increased69 Smaller70
IL: interleukin.
Presence of gastric fluid microbes (42 out of 103 specimens, including Ureaplsama species) collected within 3 h of birth correlated well with severe BPD (n = 1 vs. 14 infants) and higher plasma KL-6 levels (a lung injury marker).69 A prospective study of 400 VLBW infants noted that the presence of a small thymus at birth on a standard chest radiograph could accurately identify those who developed BPD (n = 51), with 94% sensitivity and 98% specificity.70 These have been summarised in Table 4. BPD has a significant genetic component.8,9 While a detailed description of the genetic biomarkers is outside the scope of this review, significant progress continues to be made in this area.10,71,72 It is important that reproducible results with large sample sizes be available for further evaluation as biomarkers and potential therapeutic targeting.7,10,71,72
CONCLUSIONS Despite the multitude of biomarkers proposed, most studies have been conducted in small numbers of infants, with few being replicated by independent investigators. The biomarkers that have shown consistent results include: IL-6, -8, -10, MCP-1, CC10, TIMP 2, soluble E-selectin, KGF, TGFb1, Angiopoietin 2 and IFNg. VEGF has been extensively studied, with reasonably consistent results if the temporal sequence is taken into consideration. It is important to clarify that these biomarkers only show an association with BPD. To establish a causal relationship, studies in developmentally-appropriate animal models and human lungs with BPD need to be conducted. Among the biomarkers discussed, the following are the strongest candidates, with published data
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fulfilling the criteria mentioned above: IL-1b, IL-6, MCP-1, TGFb1, VEGF, pulmonary HGF, KGF, bombesin-like peptide, MMP 9, PTHrP, MIF, Angiopoietin 2, and IFNg. Confirmatory studies with adequate sample sizes and assessment of the role of putative biomarkers in the aetiopathogenesis of BPD in developmentally appropriate animal models and human lungs with BPD will enhance the potential for therapeutic interventions. Genomic and proteomic approaches have the greatest potential to significantly advance the field of biomarkers in BPD.
PRACTICE POINTS Incidence of bronchopulmonary dysplasia (BPD) has remained unchanged despite changes in neonatal therapies. The routine use of surfactant and gentler noninvasive ventilatory techniques has led to ‘‘New’’ BPD with pathological findings distinct from ‘‘Old BPD’’ Biomarkers of BPD can be identified in various biological fluids including blood, tracheal aspirates, urine and breath condensate. Biomarkers of BPD are not routinely used in clinical practice.
RESEARCH DIRECTIONS Genomic and proteomic approaches for early identification of infants predisposed to BPD. Use of developmentally appropriate animal models to correlate with molecular biomarkers in human lungs with BPD. Targeted therapeutic options to ameliorate early, evolving and established phases of BPD to improve long term pulmonary and neurodevelopment outcomes.
References 1. Bhandari A, Bhandari V. ‘‘New’’ Bronchopulmonary Dysplasia: A clinical review. Clin Pulm Med 2011;18:137–43. 2. Trembath A, Laughon MM. Predictors of bronchopulmonary dysplasia. Clin Perinatol 2012;39:585–601. Epub 2012/09/08. 3. Smith VC, Zupancic JA, McCormick MC, Croen LA, Greene J, Escobar GJ, et al. Trends in severe bronchopulmonary dysplasia rates between 1994 and 2002. J Pediatr 2005;146:469–73. Epub 2005/04/07. 4. Coalson JJ. Pathology of bronchopulmonary dysplasia. Semin Perinatol 2006;30:179–84. 5. Philip AG. Chronic lung disease of prematurity: a short history. Semin Fetal Neonatal Med 2009;14:333–8. 6. Baraldi E, Filippone M. Chronic lung disease after premature birth. N Engl J Med 2007;357:1946–55. 7. Bhandari A, Bhandari V. Pitfalls, problems, and progress in bronchopulmonary dysplasia. Pediatrics 2009;123:1562–73. 8. Bhandari V, Bizzarro MJ, Shetty A, Zhong X, Page GP, Zhang H, et al. Familial and genetic susceptibility to major neonatal morbidities in preterm twins. Pediatrics 2006;117:1901–6. 9. Lavoie PM, Pham C, Jang KL. Heritability of bronchopulmonary dysplasia, defined according to the consensus statement of the national institutes of health. Pediatrics 2008;122:479–85. 10. Bhandari V, Gruen JR. The genetics of bronchopulmonary dysplasia. Semin Perinatol 2006;30:185–91. 11. Thompson A, V.B. Pulmonary biomarkers of bronchopulmonary dysplasia. Biomarker Insights 2008;3:361–73. 12. Bhandari A, Bhandari V. Pathogenesis, pathology and pathophysiology of pulmonary sequelae of bronchopulmonary dysplasia in premature infants. Front Biosci 2003;8:e370–80.
13. Ambalavanan N, Walsh M, Bobashev G, Das A, Levine B, Carlo WA, et al. Intercenter differences in bronchopulmonary dysplasia or death among very low birth weight infants. Pediatrics 2011;127:e106–16. Epub 2010/12/15. 14. Srisuparp P, Marks JD, Khoshnood B, Schreiber MD. Predictive power of initial severity of pulmonary disease for subsequent development of bronchopulmonary dysplasia. Biol Neonate 2003;84:31–6. Epub 2003/08/02. 15. Bhering CA, Mochdece CC, Moreira ME, Rocco JR, Sant’Anna GM. Bronchopulmonary dysplasia prediction model for 7-day-old infants. J Pediatr (Rio J) 2007;83:163–70. Epub 2007/03/24. 16. Cunha GS, Mezzacappa-Filho F, Ribeiro JD. Risk factors for bronchopulmonary dysplasia in very low birth weight newborns treated with mechanical ventilation in the first week of life. J Trop Pediatr 2005;51:334–40. Epub 2005/06/02. 17. Oh W, Poindexter BB, Perritt R, Lemons JA, Bauer CR, Ehrenkranz RA, et al. Association between Fluid Intake and Weight Loss during the First Ten Days of Life and Risk of Bronchopulmonary Dysplasia in Extremely Low Birth Weight Infants. J Pediatr 2005;147:786–90. 18. May C, Patel S, Kennedy C, Pollina E, Rafferty GF, Peacock JL, et al. Prediction of bronchopulmonary dysplasia. Arch Dis Child Fetal Neonatal Ed 2011;96:F410–6. Epub 2011/03/03. 19. Schmidt HJ, Bhandari V, Bhandari A, Davies J, Marshall BC, Praud JP, et al. The future in paediatric respirology. Respirology 2010. 20. Jelliffe-Pawlowski LL, Shaw GM, Stevenson DK, Oehlert JW, Quaintance C, Santos AJ, et al. Risk of bronchopulmonary dysplasia by second-trimester maternal serum levels of alpha-fetoprotein, human chorionic gonadotropin, and unconjugated estriol. Pediatr Res 2012;71:399–406. Epub 2012/03/07. 21. Tsao PN, Wei SC, Su YN, Lee CN, Chou HC, Hsieh WS, et al. Placenta growth factor elevation in the cord blood of premature neonates predicts poor pulmonary outcome. Pediatrics 2004;113:1348–51. 22. Janer J, Andersson S, Kajantie E, Lassus P. Endostatin concentration in cord plasma predicts the development of bronchopulmonary dysplasia in very low birth weight infants. Pediatrics 2009;123:1142–6. Epub 2009/04/ 02. 23. Mohamed WA, Niyazy WH, Mahfouz AA. Angiopoietin-1 and endostatin levels in cord plasma predict the development of bronchopulmonary dysplasia in preterm infants. J Trop Pediatr 2011;57:385–8. Epub 2010/12/07. 24. Borghesi A, Massa M, Campanelli R, Bollani L, Tzialla C, Figar TA, et al. Circulating endothelial progenitor cells in preterm infants with bronchopulmonary dysplasia. Am J Respir Crit Care Med 2009;180:540–6. Epub 2009/07/04. 25. Baker CD, Balasubramaniam V, Mourani PM, Sontag MK, Black CP, Ryan BS, et al. Cord blood angiogenic progenitor cells are decreased in bronchopulmonary dysplasia. Eur Respir J 2012. Epub 2012/04/13. 26. Joseph L, Nir A, Hammerman C, Goldberg S, Ben Shalom E, Picard E. N-terminal pro-B-type natriuretic peptide as a marker of bronchopulmonary dysplasia in premature infants. Am J Perinatol 2010;27:381–6. Epub 2009/12/17. 27. Schrama AJ, Bernard A, Poorthuis BJ, Zwinderman AH, Berger HM, Walther FJ. Cord blood Clara cell protein CC16 predicts the development of bronchopulmonary dysplasia. Eur J Pediatr 2008;167:1305–12. Epub 2008/06/04. 28. Loughran-Fowlds AS, Lin JW, Oei J, Michalowski J, Henry R, Lui K. Early postnatal surge of serum Clara cell secretory protein in newborn infants. Neonatology 2012;101:125–31. Epub 2011/09/29. 29. Sarafidis K, Stathopoulou T, Diamanti E, Soubasi V, Agakidis C, Balaska A, et al. Clara cell secretory protein (CC16) as a peripheral blood biomarker of lung injury in ventilated preterm neonates. Eur J Pediatr 2008;167:1297–303. Epub 2008/06/04. 30. Fukunaga S, Ichiyama T, Maeba S, Okuda M, Nakata M, Sugino N, et al. MMP-9 and TIMP-1 in the cord blood of premature infants developing BPD. Pediatr Pulmonol 2009;44:267–72. Epub 2009/02/11. 31. Loughran-Fowlds A, Leach S, Lin J, Oei J, Henry R, Day AS, et al. Respiratory disease and early serum S100A12 changes in very premature infants. Acta Paediatr 2011;100:1538–43. Epub 2011/06/16. 32. Kim DH, Kim HS, Shim SY, Lee JA, Choi CW, Kim EK, et al. Cord blood KL-6, a specific lung injury marker, correlates with the subsequent development and severity of atypical bronchopulmonary dysplasia. Neonatology 2008;93:223–9. Epub 2007/11/21. 33. Ogihara T, Hirano K, Morinobu T, Kim HS, Ogawa S, Hiroi M, et al. Plasma KL-6 predicts the development and outcome of bronchopulmonary dysplasia. Pediatr Res 2006;60:613–8. Epub 2006/09/22. 34. Vento G, Capoluongo E, Matassa PG, Concolino P, Vendettuoli V, Vaccarella C, et al. Serum levels of seven cytokines in premature ventilated newborns: correlations with old and new forms of bronchopulmonary dysplasia. Intensive Care Med 2006;32:723–30. Epub 2006/03/22. 35. Paananen R, Husa AK, Vuolteenaho R, Herva R, Kaukola T, Hallman M. Blood cytokines during the perinatal period in very preterm infants: relationship of inflammatory response and bronchopulmonary dysplasia. J Pediatr 2009; 154:39–43. e3 Epub 2008/09/02. 36. Ambalavanan N, Carlo WA, D’Angio CT, McDonald SA, Das A, Schendel D, et al. Cytokines associated with bronchopulmonary dysplasia or death in extremely low birth weight infants. Pediatrics 2009;123:1132–41. 37. Hogmalm A, Backstrom E, Bry M, Lappalainen U, Lukkarinen HP, Bry K. Role of CXC Chemokine Receptor 2 in a Mouse Model of Bronchopulmonary Dysplasia. Am J Respir Cell Mol Biol 2012. Epub 2012/08/07. 38. Choo-Wing R, Nedrelow JH, Homer RJ, Elias JA, Bhandari V. Developmental differences in the responses of IL-6 and IL-13 transgenic mice exposed to hyperoxia. Am J Physiol Lung Cell Mol Physiol 2007;293:L142–50. 39. Harijith A, Choo-Wing R, Cataltepe S, Yasumatsu R, Aghai ZH, Janer J, et al. A role for matrix metalloproteinase 9 in IFNgamma-mediated injury in developing
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lungs: relevance to bronchopulmonary dysplasia. Am J Respir Cell Mol Biol 2011;44:621–30. Sheikh S, Null D, Gentile D, Bimle C, Skoner D, McCoy K, et al. Urinary leukotriene E(4) excretion during the first month of life and subsequent bronchopulmonary dysplasia in premature infants. Chest 2001;119: 1749–54. Bhandari V. Hyperoxia-derived lung damage in preterm infants. Semin Fetal Neonatal Med 2010. Reuter SD, O’Donovan DJ, Hegemier SE, Smith EO, Heird WC, Fernandes CJ. Urinary F2-isoprostanes are poor prognostic indicators for the development of bronchopulmonary dysplasia. J Perinatol 2007;27:303–6. Joung KE, Kim HS, Lee J, Shim GH, Choi CW, Kim EK, et al. Correlation of urinary inflammatory and oxidative stress markers in very low birth weight infants with subsequent development of bronchopulmonary dysplasia. Free radical research 2011;45:1024–32. Epub 2011/06/10. Cullen A, Van Marter LJ, Allred EN, Moore M, Parad RB, Sunday ME. Urine bombesin-like peptide elevation precedes clinical evidence of bronchopulmonary dysplasia. Am J Respir Crit Care Med 2002;165:1093–7. Subramaniam M, Bausch C, Twomey A, Andreeva S, Yoder BA, Chang L, et al. Bombesin-like peptides modulate alveolarization and angiogenesis in bronchopulmonary dysplasia. Am J Respir Crit Care Med 2007;176:902–12. Bose CL, Dammann CE, Laughon MM. Bronchopulmonary dysplasia and inflammatory biomarkers in the premature neonate. Arch Dis Child Fetal Neonatal Ed 2008;93:F455–61. Epub 2008/08/05. Aghai ZH, Saslow JG, Mody K, Eydelman R, Bhat V, Stahl G, et al. IFN-gamma and IP-10 in tracheal aspirates from premature infants: Relationship with bronchopulmonary dysplasia. Pediatr Pulmonol 2012. Epub 2012/03/21. Kevill KA, Bhandari V, Kettunen M, Leng L, Fan J, Mizue Y, et al. A role for macrophage migration inhibitory factor in the neonatal respiratory distress syndrome. J Immunol 2008;180:601–8. Knaapi J, Lukkarinen H, Kiviranta R, Steiner A, Lassus P, Andersson S, et al. Cathepsin K expression is diminished in infants with bronchopulmonary dysplasia. Acta Paediatr 2006;95:1298–300. Sohn MH, Kang MJ, Matsuura H, Bhandari V, Chen NY, Lee CG, et al. The chitinase-like proteins breast regression protein-39 and YKL-40 regulate hyperoxia-induced acute lung injury. Am J Respir Crit Care Med 2010; 182:918–28. Epub 2010/06/19. Li Z, Choo-Wing R, Sun H, Sureshbabu A, Sakurai R, Rehan VK, et al. A potential role of the JNK pathway in hyperoxia-induced cell death, myofibroblast transdifferentiation and TGF-beta1-mediated injury in the developing murine lung. BMC cell biology 2011;12:54. Epub 2011/12/17. Hasan J, Beharry KD, Valencia AM, Strauss A, Modanlou HD. Soluble vascular endothelial growth factor receptor 1 in tracheal aspirate fluid of preterm neonates at birth may be predictive of bronchopulmonary dysplasia/chronic lung disease. Pediatrics 2009;123:1541–7. Epub 2009/06/02. Meller S, Bhandari V. VEGF levels in humans and animal models with RDS and BPD: temporal relationships. Experimental lung research 2012;38:192–203. Epub 2012/03/08. Bhandari V, Choo-Wing R, Lee CG, Yusuf K, Nedrelow JH, Ambalavanan N, et al. Developmental regulation of NO-mediated VEGF-induced effects in the lung. Am J Respir Cell Mol Biol 2008;39:420–30. Janer J, Andersson S, Haglund C, Karikoski R, Lassus P. Placental growth factor and vascular endothelial growth factor receptor-2 in human lung development. Pediatrics 2008;122:340–6. Epub 2008/08/05. De Paepe ME, Patel C, Tsai A, Gundavarapu S, Mao Q. Endoglin (CD105) Upregulation in Pulmonary Microvasculature of Ventilated Preterm Infants. Am J Respir Crit Care Med 2008.
57. Davis CW, Gonzales LW, Ballard RA, Ballard PL, Guo C, Gow AJ. Expression of nitric oxide synthases and endogenous NO metabolism in bronchopulmonary dysplasia. Pediatr Pulmonol 2008;43:703–9. Epub 2008/05/27. 58. Aghai ZH, Faqiri S, Saslow JG, Nakhla T, Farhath S, Kumar A, et al. Angiopoietin 2 concentrations in infants developing bronchopulmonary dysplasia: attenuation by dexamethasone. J Perinatol 2008;28:149–55. 59. Bhandari V, Choo-Wing R, Lee CG, Zhu Z, Nedrelow JH, Chupp GL, et al. Hyperoxia causes angiopoietin 2-mediated acute lung injury and necrotic cell death. Nat Med 2006;12:1286–93. 60. Rudiger M, von Baehr A, Haupt R, Wauer RR, Rustow B. Preterm infants with high polyunsaturated fatty acid and plasmalogen content in tracheal aspirates develop bronchopulmonary dysplasia less often. Crit Care Med 2000;28:1572– 7. Epub 2000/06/02. 61. Capoluongo E, Vento G, Lulli P, Di Stasio E, Porzio S, Vendettuoli V, et al. Epithelial lining fluid neutrophil-gelatinase-associated lipocalin levels in premature newborns with bronchopulmonary dysplasia and patency of ductus arteriosus. International journal of immunopathology and pharmacology 2008;21:173–9. Epub 2008/03/14. 62. Farhath S, He Z, Nakhla T, Saslow J, Soundar S, Camacho J, et al. Pepsin, a marker of gastric contents, is increased in tracheal aspirates from preterm infants who develop bronchopulmonary dysplasia. Pediatrics 2008;121:e253–9. Epub 2008/ 02/05. 63. Hikino S, Ohga S, Kinjo T, Kusuda T, Ochiai M, Inoue H, et al. Tracheal aspirate gene expression in preterm newborns and development of bronchopulmonary dysplasia. Pediatr Int 2012;54:208–14. Epub 2011/11/10. 64. Mody K, Saslow JG, Kathiravan S, Eydelman R, Bhat V, Stahl GE, et al. Sirtuin1 in tracheal aspirate leukocytes: possible role in the development of bronchopulmonary dysplasia in premature infants. J Matern Fetal Neonatal Med 2012;25:1483–7. Epub 2012/01/26. 65. Bhattacharya S, Go D, Krenitsky DL, Huyck HL, Solleti SK, Lunger VA, et al. Genome-wide transcriptional profiling reveals connective tissue mast cell accumulation in bronchopulmonary dysplasia. Am J Respir Crit Care Med 2012;186:349–58. Epub 2012/06/23. 66. McGowan EC, Kostadinov S, McLean K, Gotsch F, Venturini D, Romero R, et al. Placental IL-10 dysregulation and association with bronchopulmonary dysplasia risk. Pediatr Res 2009;66:455–60. Epub 2009/07/08. 67. De Felice C, Parrini S, Barducci A, Chitano G, Tonni G, Latini G. Abnormal oral mucosal light reflectance in bronchopulmonary dysplasia. Early Hum Dev 2006;82:273–8. Epub 2005/12/13. 68. May C, Williams O, Milner AD, Peacock J, Rafferty GF, Hannam S, et al. Relation of exhaled nitric oxide levels to development of bronchopulmonary dysplasia. Arch Dis Child Fetal Neonatal Ed 2009;94:F205–9. Epub 2009/04/23. 69. Oue S, Hiroi M, Ogawa S, Hira S, Hasegawa M, Yamaoka S, et al. Association of gastric fluid microbes at birth with severe bronchopulmonary dysplasia. Arch Dis Child Fetal Neonatal Ed 2009;94:F17–22. Epub 2008/08/05. 70. De Felice C, Latini G, Del Vecchio A, Toti P, Bagnoli F, Petraglia F. Small thymus at birth: a predictive radiographic sign of bronchopulmonary dysplasia. Pediatrics 2002;110:386–8. Epub 2002/08/08. 71. Lavoie PM, Dube MP. Genetics of bronchopulmonary dysplasia in the age of genomics. Curr Opin Pediatr 2010;22:134–8. Epub 2010/01/21. 72. Cohen J, Van Marter LJ, Sun Y, Allred E, Leviton A, Kohane IS. Perturbation of gene expression of the chromatin remodeling pathway in premature newborns at risk for bronchopulmonary dysplasia. Genome biology 2007;8:R210. Epub 2007/10/06. 73. de Felice C, Latini G, Parrini S, Bianciardi G, Toti P, Kopotic RJ, et al. Oral mucosal microvascular abnormalities: an early marker of bronchopulmonary dysplasia. Pediatr Res 2004;56:927–31.
EDUCATIONAL QUESTIONS
a. b. c. d.
1. The most commonly accepted definition of bronchopulmonary dysplasia is: a. oxygen dependence for 28 post natal days and assessment of respiratory support at 36 weeks post menstrual age b. birth at 32 weeks of gestation c. presence of chronic lung disease d. history of respiratory distress syndrome 2. The pathogonomic finding in the lungs of patients with the BPD is: a. increased airway fibroproliferation b. marked airway smooth muscle hypertrophy c. increased lymphocytic infiltration d. alveolar simplification 3. All of the following are thought to play a role in development of BPD except:
prematurity maternal chorioamnionitis antenatal steroids genetic factors
4. Among inflammatory cytokines, which has been shown in developmentally-appropriate animal models and human tracheal aspirates to be strongly associated with BPD? a. IL-10 b. MIF c. IFNg d. IL-16 5. The source of pepsin in tracheal aspirates in infants developing BPD is the . . . a. Lung b. Gastric mucosa c. Blood d. Nasopharyngeal secretions
Contents lists available at SciVerse ScienceDirect
Paediatric Respiratory Reviews
CME Review
Biomarkers in Bronchopulmonary Dysplasia Anita Bhandari 1,*, Vineet Bhandari 2 1 2
Division of Pediatric Pulmonology, Connecticut Children’s Medical Center, Hartford, CT Division of Perinatal Medicine, Yale University School of Medicine, New Haven, CT
EDUCATIONAL AIMS To provide an overview of the definition, pathology and aetipathogenesis of bronchopulmonary dysplasia (BPD). To familiarize the reader with different biomarkers of BPD. To review research literature in the area of biomarkers as it pertains to BPD.
A R T I C L E I N F O
S U M M A R Y
Keywords: Cytokines Lung Premature Newborn Growth factors
Bronchopulmonary dysplasia (BPD) is a complex disorder secondary to gene-environment interactions, and is the commonest chronic lung disease in infancy. There is no specific or effective treatment available to date for BPD. Since the aetiopathogenesis of BPD is multifactorial, involving diverse molecular signaling pathways, a variety of biomarkers detected in biological fluids have been proposed for early identification of infants predisposed to BPD. This review will be restricted to biomarker studies in human infants, conducted mostly in the last decade. The majority of the studies have been conducted using blood, urine or tracheal aspirate samples. Despite the multitude of biomarkers proposed, most studies have been conducted in small numbers of infants, with few being replicated by independent investigators. Confirmatory studies with adequate sample sizes and assessment of the role of putative biomarkers in the aetiology of BPD in developmentally appropriate animal models and human lungs with BPD will enhance the potential for therapeutic interventions. Genomic and proteomic approaches have the greatest potential to significantly advance the field of biomarkers in BPD. ß 2013 Published by Elsevier Ltd.
INTRODUCTION Bronchopulmonary dysplasia (BPD) is the commonest chronic lung disease of infancy.1 While many definitions of BPD exist,2 the most commonly accepted definitions include the need for oxygen supplementation at 28 days of life and/or 36 weeks postmenstrual age (PMA).1,2 The incidence of BPD has remained stable3 or increased slightly over the last decade.1,2 The incidence of BPD is variable among centers, and was reported to range from 12% to 32% among infants <32 weeks of gestation at birth.2 Infants with birth weight (BW)<1250 g account for the vast majority (97%) of all BPD patients.1 With the survival of younger premature infants, and changes in
* Corresponding author. Associate Professor of Pediatrics, Division of Pediatric Pulmonology, Connecticut Children’s Medical Center, 282 Washington Street, Hartford, CT 06106, USA. Tel.: +860 545 9679; fax: +860 545 9445. E-mail address: [email protected] (A. Bhandari). 1526-0542/$ – see front matter ß 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.prrv.2013.02.008
management style, there has been a shift in the clinical and pathological phenotype – designated as the ‘‘new’’ BPD.1 PATHOLOGY Over the past decade changes in neonatal ventilation strategies, widespread use of antenatal steroids, aggressive nutritional intervention and fluid restriction have led to a change in lung histopathology of premature babies.1 The new BPD has some unique features that distinguish it from the older forms of BPD.4,5 Lungs of patients with new BPD have fewer, larger and simplified alveoli, and along with dysmorphic vasculature4,6 are considered the pathognomic features of the new BPD. These findings suggest a disruption of pulmonary development in that the vascular and alveolar growth parameters are both impaired.1 Lungs from infants with new BPD have been reported to have more uniform inflation and less regional heterogeneity of lung disease. Airway smooth muscle is only mildly thickened and rare epithelial lesions and fibroproliferative changes may be occasionally seen.1
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PATHOGENESIS Over the last 5 years, an improved understanding has evolved regarding the pathogenesis of BPD.7 For decades, an immature lung upon which a variety of ‘‘environmental’’ factors (infection, hyperoxia, baro/volu-trauma) acted upon to cause injury, has been the cornerstone of the pathogenesis of BPD. Recent publications by independent investigators have confirmed the significant genetic contribution to the pathogenesis of new BPD.8,9 This has transformed BPD to a condition with significant genetic-environmental interactions,10 thus enabling potentially novel approaches for understanding the pathogenesis of this condition.11 Inflammation, contributed to by antenatal (chorioamnionitis) and postnatal (local or systemic infections, hyperoxia, ventilator-induced injury) factors, initiates and modifies the process of lung injury in the developing lung.7 This inflammatory process is dependent upon the effective release and balance of cytokines. An imbalance in these mediators leads to activation of the cellular death pathways in the lung.7 This is followed by healing (resolution of injury to normal lung architecture) or repair.7,12 The latter is characterised by impaired alveolarisation and dysregulated angiogenesis leading to fewer, larger simplified alveoli.7 It is obvious from the above description that a sufficient length of time is required for the interactive genetic-environmental factors to act on the immature lung to progress to the clinical-pathological phenotype of BPD. We have proposed that BPD be assessed in early, evolving and established phases, in relation to postnatal age, and have suggested management strategies for the same.1,7 In terms of postnatal preventive approaches, early recognition of infants at high risk of developing BPD would be critical to plan out therapeutic strategies. Use of clinical factors to develop scoring systems and predictive modeling utilizing multiple variables has been reported with modest success,2,13,14 with increasing capability at or after the first week of life.15–18 However, we believe that the identification of these infants needs to occur within the first 3 days of the early phase of BPD, for potential maximal impact.19 Early detection of biochemical biomarkers, especially if they are also integral in the pathogenesis of BPD, could provide an enhancement of the capability to identify and potentially target the infants predisposed to BPD.19 In addition, biomarkers could be combined with clinical parameters to further increase the ability to identify and prognosticate the outcome of high-risk infants to develop BPD and perhaps predict the severity of BPD. BIOMARKERS IN BPD Given the multifactorial aetiopathogenesis of BPD involving diverse molecular signaling pathways, a variety of biomarkers detected in different biological fluids have been proposed for early identification of infants predisposed to BPD. The majority of the studies have been conducted using blood, urine or tracheal aspirate (TA) samples.7 This review will be restricted to biomarker studies in human infants, conducted mostly in the last decade. Blood Biomarkers In the second-trimester maternal serum levels of alphafetoprotein and/or human chorionic gonadotropin above the 95th percentile and/or levels of unconjugated estriol <5th percentile, were associated with increased risk of BPD (n = 82 vs. 164 with no BPD).20 Having a combination of 2 or more of these biomarkers further increased the risk.20 Cord blood is probably one of the easiest and earliest samples to get access to in premature neonates predisposed to BPD. Expectedly, the focus has been on markers involved in angiogenesis, given the dysregulated vasculature in BPD lungs. Using the
older definition of 28-days supplemental oxygen for BPD, using a cut-off value of 17 mg/dl, cord blood placental growth factor (PlGF) was noted to have 95% specificity, 53% sensitivity with positive and negative predictive values of 83% and 82%, respectively in 63 preterm infants.21 Another molecular marker that is involved in angiogenesis is endostatin, an antiangiogenic factor. In very low birth weight (VLBW) infants, a higher circulating concentration of endostatin was noted in cord blood in those who developed BPD (n = 19, 100.7 29.7 vs. those who did not, n = 73, 85.6 28.7 ng/mL; p = 0.029).22 This finding was recently independently confirmed.23 In addition, the same authors also noted low Angiopoietin-1 cord blood levels to predict the subsequent development of BPD (n = 28, vs. 78 with no BPD).23 Endothelial colony-forming cells in cord blood were significantly lower in infants who later developed BPD (n = 24), though the endothelial and hematopoietic cell subsets were comparable in those with or without BPD (n = 74).24 These data have been recently confirmed in an independent study.25 Among other markers, type IV collagen, CD9, C-terminal fragment of Type I collagen, and soluble L-selectin have been shown to be decreased, while eosinophilic cationic protein, 8isoprostanes, and soluble E-selectin are all increased in infants developing BPD.7 Plasma concentrations of N-terminal pro-B-type natriuretic peptide (BNP) were found to be significantly higher at 4 weeks of age in patients with BPD (n = 11), and decreased at 6 and 8 weeks of age.26 Another focus of the investigative groups has been pulmonary epithelial cell markers as well as extracellular matrix proteins which could be involved in impaired alveolarisation, a key pathologic hallmark of BPD. Clara cells are non-ciliated epithelial cells which line the respiratory and terminal bronchioles. Low cord blood clara cell protein (CC10 or CC secretory protein/CCSP or CC16) was reported in cord blood plasma of 79 preterm infants, of which 17 developed BPD.27 Interestingly, serum CC10 has also been reported to be persistently low in infants who developed BPD (n = 8).28 In striking contrast, serum CC10 levels within 2 h of life and at post natal day 14 [PN14] were significantly higher in preterm neonates (n = 35, with gestational age 31 weeks) who were mechanically ventilated and later developed BPD (n = 7) vs. those who were not ventilated (n = 12).29 Matrix metalloproteinase-9 (MMP-9)/tissue inhibitor of metalloproteinase-1 (TIMP-1) ratios in cord blood were significantly higher in those infants who developed moderate/severe (n = 9) BPD versus those who had mild/no BPD (n = 20).30 Granulocyte-specific S100A12, a marker for inflammatory disorders, was not found to be a useful marker for BPD (n = 10 vs. 18 with no BPD), when assayed on PN1-7.31 Cord blood KL-6 (a lung injury marker) was increased in infants with BPD (n = 50, vs. non-BPD n = 24),32 and was also useful at 1 week of life as a predictor of moderate/severe BPD.33 In an interesting study, ‘‘old’’ BPD (n = 11, compared to n = 22) was associated with significantly increased proinflammatory (interleukin-6 or IL-6, IL-8, monocyte chemoattractant protein-1 or MCP-1) and antiinflammatory IL-10 levels, but with no significant difference in profibrotic/angiogeneic cytokines (transforming growth factor-beta 1 or TGFb1, platelet-derived growth factor BB fraction or PDGF BB, vascular endothelial growth factor or VEGF) on postnatal (PN) days 3 or 5.34 In contrast, when the authors used the ‘‘new’’ BPD definition (n = 22, compared to n = 9), the samples were characterized with increases only in the latter category on PN5.34 This highlights the fact the aetiology of new BPD is markedly different from that of the earlier forms of BPD.1 With the advent of multiplex assays, researchers have attempted to simultaneously evaluate multiple cytokines, and assess their association to BPD. Out of 11 cytokines evaluated (n = 128), higher IL-8, IL-10 and granulocyte colony stimulating factor (G-CSF) levels were found to predict BPD in 1-day old infants.35 Blood samples were collected from infants within 4 h of birth, and on days 3, 7, 14
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and 21 and concentrations of 25 cytokines were assayed.36 Using multiple logistic regression analyses, combining results from all models, BPD/death (n = 606, out of 1062) was associated with higher levels of IL-1b, 6, 8, 10 and interferon gamma (IFNg), but lower concentrations of IL-17, regulated on activation normal T-cell expressed and secreted (RANTES), and tumor necrosis factor beta (TNFb).36 These have been summarised in Table 1. The major limitation of these studies is the fact that cord or infant blood concentrations of the various molecules could be influenced by a variety of conditions, and are not necessarily reflective of what is going on in the lung. While earlier studies also suffer from the drawback of small sample sizes, the latter ones do have sufficient number of infants and simultaneously measure multiple cytokines at sequential time points to give a more comprehensive picture. At least among the cytokines, it is important to note consistent results with increased blood levels of IL-6, 8 and IL-10 being associated with BPD. This is supported by increased concentrations of IL-6 and -8 in TA obtained from human infants developing BPD. In addition, developmentally-appropriate animal models overexpressing IL-1b,37 IL-638 and IFNg39 in the lung provide supportive evidence of these molecules being involved in the pathogenesis of BPD.
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8-iso-PGF2a was evaluated in the urine in early PN life, but was not found to correlate with the development of BPD.42 In another study, 8-hydroxydeoxyguanosine (8-OHdG, also an oxidant-injury marker) urinary levels on PN7, but not LTE4, were an independent risk factor for developing moderate/severe BPD.43 The most promising urinary biomarker for BPD has been bombesin-like peptide, with concentrations >20,000 pg/mg creatinine on PN1-4 occurred among 54% of the infants who developed BPD, vs. 10% among BPD controls.44 The latter data is supported by animal studies.45 These have been summarised in Table 2. Pulmonary Biomarkers
Urinary Biomarkers
Not surprisingly, most of the work on biomarkers has been done using TA samples, allowing direct access to the pulmonary compartment. While TA samples are a suitable substitute for bronchoalveolar lavage specimens in preterm neonates, there is some controversy about the need to ‘‘correct’’ for dilution. In addition, such samples can only be collected from intubated infants, and hence samples from appropriately matched (healthy and by extension, non-intubated) controls are not available.7,11 Since the topic of pulmonary biomarkers has been recently reviewed by us7,11 and others,46 we will summarize the major findings, and refer the reader to those publications for additional details.
With inflammation playing a major role in the pathogenesis of BPD, investigators evaluated urinary levels of leukotriene E4 (LTE4) in the first month of life, but did not find any association with development of BPD.40 Since exposure to hyperoxia is another critical factor in BPD pathogenesis,41 the oxidant injury marker,
Cytokines Among these, the traditionally considered proinflammatory IL-1b, -6, -16, and TNFa have been shown to be increased in TA in earlier studies.7,11,46 A recent addition has been increased IFNg.39,47 Regarding anti-inflammatory cytokines, most of the
Table 1 Blood biomarkers in BPD. Source: Biomarker
Description/Physiologic Role
Levels associated with Increased BPD Risk refs
Maternal blood: AFP and/or HCG Maternal blood: unconjugated estriol Cord blood: PlGF
>95th centile20 <5th centile20 Higher21
Cord blood: Endostatin Cord blood: Angiopoietin 1 Cord blood: Endothelial colony-forming cells Infant blood: C-IV Infant blood: PICP Infant blood: soluble L-selectin Infant blood: eosinophilic cationic protein Cord and Infant blood: soluble E-selectin Infant blood: N-terminal pro-B-type natriuretic peptide Cord and Infant blood: Clara cell secretory protein
AFP: Unknown; HCG: Maintains pregnancy Maintains pregnancy Proangiogenic. It is postulated that PlGF boosts angiogenesis by binding to VEGFR1, leaving a higher concentration of free VEGF-A that can bind to VEGFR2 Antiangiogenic Proangiogenic Proangiogenic Serum antigen of basement membrane component Serum antigen of extracellular matrix component Adhesion molecule Marker of eosinophil activation Adhesion molecule Regulation of extracellular fluid volume and blood pressure Non-ciliated epithelial cells lining the respiratory and terminal bronchioles
Cord blood: MMP 9/TIMP 1 Infant blood: S100A12 Cord and Infant blood: KL-6 Infant blood: IL-6 Infant blood: IL-8 Infant blood: IL-10 Infant blood: MCP-1 Infant blood: TGFb1 Infant blood: PDGF-BB Infant blood: VEGF Infant blood: G-CSF Infant blood: IL-1b Infant blood: IFNg Infant blood: IL-17 Infant blood: RANTES Infant blood: TNFb
Protease/antiprotease of extracellular matrix Granulocyte-specific proinflammatory molecule, interacts with RAGE Lung epithelial cellular injury marker Proinflammatory cytokine Proinflammatory cytokine Antiinflammatory cytokine Chemokine Profibrotic Proangiogenic Proangiogenic Proinflammatory cytokine Proinflammatory cytokine Proinflammatory cytokine Proinflammatory cytokine Chemokine Proinflammatory cytokine
Higher22,23 Lower23 Lower24,25 Lower7 Lower7 Lower7 Higher7 Higher7 Higher26 Lower27,28 Higher29 Higher30 No difference31 Higher32,33 Higher36 (old BPD)34 Higher35,36 (old BPD)34 Higher35,36 (old BPD)34 Higher (old BPD)34 Higher (new BPD)34 Higher (new BPD)34 Higher (new BPD)34 Higher35 Higher36 Higher36 Lower36 Lower36 Lower36
AFP: alphafeto protein; HCG: human chorionic gonadotropin; PlGF: placental growth factor; C-IV: type IV collagen; PICP: C-terminal fragment of Type I collagen; MMP: matrix metalloproteinase; TIMP: tissue inhibitor of metalloproteinase; IL: interleukin; MCP: monocyte chemoattractant protein; TGFb: transforming growth factor beta; PDGF-BB: platelet derived growth factor-BB isoform; VEGF: vascular endothelial growth factor; R1/2: receptor 1/2; RAGE: receptor for advanced glycation end products; G-CSF: granulocyte colony stimulating factor; IFNg: interferon gamma; RANTES: regulated on activation normal T-cell expressed and secreted; TNFb: tumor necrosis factor beta.
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176 Table 2 Urinary biomarkers in BPD. Biomarker Leukotriene E4 8-iso-PGF2a 8-OHdG Bombesin-like peptide
Description/Physiologic Role
Levels associated with Increased BPD Risk
Marker of inflammation Marker of oxidant injury Marker of oxidant injury Role in lung development; pulmonary injury
refs
40,43
No difference No difference42 Higher43 Higher45
8-iso-PGF2a: 8-iso-prostaglandin F2 alpha; 8-OHdG: 8-hydroxydeoxyguanosine.
earlier TA studies have failed to detect IL-10, and researchers have suggested that those infants who went on to develop BPD have a reduced ability to generate adequate levels of IL-10.7,11,46 Decreased levels of macrophage migration inhibitory factor (MIF) have been associated with increased risk of BPD.48 Chemokines IL-8 (CXCL8), MCP-1, -2 and -3 TA levels were significantly increased in infants developing BPD.7,11,46 Proteases/Antiproteases Increased levels of MMP8 and 9 and decreased TA levels of TIMP2 have been reported in infants developing BPD.7,11,46 Higher trypsin-2 levels may predispose to BPD.7,11,46 Cathepsin K expression has been noted to be decreased in infants with BPD.49 Adhesion molecules Soluble intercellular adhesion molecule-1 and L-selectin are increased in TA samples in the first PN week of infants developing BPD.11,46 Oxidative Injury Elevated levels of 3-cholrotyrosine and malondialdehyde were correlated with development of BPD.11 Breast regression protein (BRP)–39 and its human homolog, YKL-40 (also called chitinase-3– like protein 1 and human cartilage glycoprotein–39) has been implicated in hyperoxia-induced lung injury.50 Low levels of YKL40 have been reported in infants subsequently developing BPD.50 Peptide Growth Factors Among these, TGFb1 has been consistently shown to be increased in human TA samples,7,11,46 and has been borne out in animal models of BPD.51 Keratinocyte and pulmonary hepatocyte growth factors are decreased in TA samples of infants predisposed to BPD.7,11 Among angiogenic agents, Angiopoietin2, endothelin-1, and fibroblast growth factor are increased in infants developing BPD.7,11 Multiple studies have evaluated VEGF TA levels.7,11,46 In a recent study, VEGF TA levels on day 1 were lower, while soluble VEGF receptor 1 (sVEGFR1) levels were higher in infants who developed BPD (n = 31, vs. 34 with no BPD).52 Subsequently, on days 3 and 7, the levels of VEGF continued to rise, while that of sVEGFR1 declined.52 In a recent review, it was summarized that VEGF levels in humans tend to be low to normal in early BPD stages.53 Though VEGF levels tend to increase with time in some studies, others point toward lower relative levels in infants developing BPD at various chronological points. One study reported a bimodal distribution of VEGF levels with proportionately higher levels measured within the first 12 hours of life and by 3–4 weeks PN.54 To discern the true relative quantity of VEGF right after birth, before the effects of surfactant replacement have occurred, more studies looking at neonates within the first hours of life would be helpful.53 While lower VEGFR2 staining has been noted in the lungs of human infants with BPD (n = 6),55 increased expression of endoglin (a TGFb co-receptor; an important regulator of angiogenesis) has been shown in another study.56 In another study, there was increased expression of inducible nitric oxide synthase (iNOS) and endothelial NOS as well as nitrotyrosine
staining in BPD lungs (n = 7), compared to controls (n = 7).57 Since there is a close interaction between VEGF,53 NOS57 and Angiopoietins,58,59 all being implicated in human BPD, the above data suggests that there is a disruption in the normal angiogenic signaling pathways, with some factors being suppressed and other enhanced,56 leading to the dysregulated vasculature in BPD lungs. Others Lower levels of CC10 have been associated with an increased risk of BPD,7,11,46 as also noted in 2 studies using blood samples.27,28 Nuclear factor-kappa B TA levels were increased, while parathyroid hormone-related protein TA levels were decreased in infants predisposed to BPD.7,11 TA levels of fibronectin and plasminogen activator inhibitor-1 were increased, while those of lysozyme decreased in those developing BPD.11 On day 1, TA levels of polyunsaturated fatty acids and dimethylacetals (representing plasmalogens) were significantly lower in those infants who developed BPD (n = 10), versus those who did not (n = 15); these differences did not persist, however, in the subsequent PN days.60 TA levels of neutrophil-gelatinase-associated lipocalin (NGAL), which is included in larger macromolecular complex together with MMP9, was significantly increased in infants with BPD.61 TA pepsin concentrations, which was secondary to gastric aspiration (and not by hematogenous spread or local synthesis in the lungs), was significantly higher in those who developed BPD (n = 31) and/or died (n = 16), versus those with no BPD (n = 12), in the first week and month of life.62 These have been summarized in Table 3. Using cell pellets from TA, investigators noted a high IL-10 and IFNg, but not TGFb1 or PDGF-B mRNA expression in infants developing BPD (n = 13 vs. 13 with no BPD).63 Lower Sirtuin1 in TA leukocytes was associated with the development of BPD or death in premature infants.64 Genome-wide expression analysis was applied to define pathways affected in BPD lungs (n = 11 vs. 17 matched controls).65 The investigators identified 159 genes differentially expressed in BPD tissues. Interestingly, 3 of the 5 most highly induced genes turned out to be mast cell-specific markers. The investigators confirmed an increased accumulation of connective tissue mast cell (chymase expressing) cells in human BPD tissues, and in an animal model.65 Miscellaneous Tissue Biomarkers Placental IL-10 was less prominent in those infants who subsequently developed BPD (n = 49) vs. those who did not (n = 49), with no differences in IL-6 expression.66 Once again highlighting the contribution of angiogenic factors, use of oral spectrophotometry (reflectance measurements of lower gingival and vestibular oral mucosa) revealed lower light reflectance values in the red, but higher values in the blue and blue-green sections of the spectrum in infants who went on to develop BPD (n = 25, out of 75), on PN1.67 Among breath condensate biomarkers, increased end-tidal carbon monoxide (ETCO) and exhaled nitric oxide (NO) levels were significantly higher in infants with BPD on PN14 (n = 39, out of 78)18 and (n = 46, out of 80) PN28,68 respectively.
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177
Table 3 Pulmonary (tracheal aspirates) biomarkers in BPD. Biomarker Cytokines IL-1b, -6, -16, TNFa IFNg IL-10 MIF Chemokines IL-8, MCP-1, -2, -3 Proteases/Antiproteases MMP 8 and 9 TIMP 2 Trypsin -2 Cathepsin K Adhesion Molecules sICAM-1, L-selectin Oxidative Injury 3-cholorotyrosine, malondialdehyde YKL-40 Peptide Growth Factors TGFb1 KGF, Pulmonary HGF Angiopoietin 2, Endothelin-1, FGF2 VEGF sVEGFR1 Others Clara cell secretory protein Nuclear factor-kappa B PTHrP Fibronectin PAI-1 Lysozyme PUFA and DMA NGAL Pepsin
Description/Physiologic Role
Levels associated with Increased BPD Risk
Proinflammatory Proinflammatory Antiinflammatory Proinflammatory
Higher7,11,46 Higher39,47 Lower7,11,46 Lower48
Proinflammatory
Higher7,11,46
Protease of extracellular matrix Antiprotease of extracellular matrix Protease of extracellular matrix Protease of extracellular matrix
Higher7,11,46 Lower7,11,46 Higher7,11,46 Lower49
Stabilizing cell-cell interactions and facilitating leukocyte transmigration
Higher11,46
Marker of neutrophil oxidation, oxidative byproduct Chitinase-like proteins
Higher11 Lower50
Profibrotic Alveolar epithelial proliferation, repair Proangiogenic Proangiogenic Antiangiogenic
Higher7,11,46 Lower7,11 Higher7,11 Variable7,11,46,52,53 Biphasic54 Variable52
Non-ciliated epithelial cells lining the respiratory and terminal bronchioles Transcription factor Alveolar growth/development Extracellular matrix formation Fibrinolysis Bactericidal Substrates for lipid peroxidation in pulmonary surfactant Antimicrobial, binds to MMP 9 inhibiting its degradation Protein degrading enzyme
Lower7,11,46 Higher7,11 Lower7,11 Higher11 Higher11 Lower11 Lower60 Higher61 Higher62
refs
IL: interleukin; b: beta; TNFa: tumor necrosis factor alpha; IFNg: interferon gamma; MIF: macrophage migration inhibitory factor; MCP: monocyte chemoattractant protein; MMP: matrix metalloproteinase; TIMP: tissue inhibitor of metalloproteinase; sICAM: soluble intercellular adhesion molecule; YKL-40: also known as chitinase-3 like protein 1, human homologue of breast regression protein 39 (BRP39); TGFb: transforming growth factor beta; KGF: keratinocyte growth factor; HGF: hepatocyte growth factor; FGF: fibroblast growth factor; VEGF: vascular endothelial growth factor; sVEGFR1: soluble VEGF receptor 1; PTHrP: parathyroid hormone related protein; PAI: plasminogen activator inhibitor; PUFA: polyunsaturated fatty acid; DMA: dimethylacetals; NGAL: neutrophil gelatinase associated lipocalin. Table 4 Miscellaneous tissue biomarkers in BPD Source: Biomarker
Description/Physiologic Role
Levels associated with Increased BPD Risk
Placenta: IL-10 Placenta: IL-6 Oral mucosa: spectrophotometry
Antiinflammatory cytokine Proinflammatory cytokine Reflectance of lower gingival and vestibular oral mucosa
Breath: Carbon monoxide Breath: Nitric oxide Gastric fluid: Microbes Chest radiograph: Thymus
Marker of inflammation and oxidative stress Marker of inflammation Presence of microbes, including Ureaplasma species Assessment of size of thymus
refs
66
Lower No difference66 Decreased reflectance values in red, but increased in blue and blue-green sections of the spectrum. May signify alterations in microvascular and extracellular matrix networks73 Higher18 Higher68 Increased69 Smaller70
IL: interleukin.
Presence of gastric fluid microbes (42 out of 103 specimens, including Ureaplsama species) collected within 3 h of birth correlated well with severe BPD (n = 1 vs. 14 infants) and higher plasma KL-6 levels (a lung injury marker).69 A prospective study of 400 VLBW infants noted that the presence of a small thymus at birth on a standard chest radiograph could accurately identify those who developed BPD (n = 51), with 94% sensitivity and 98% specificity.70 These have been summarised in Table 4. BPD has a significant genetic component.8,9 While a detailed description of the genetic biomarkers is outside the scope of this review, significant progress continues to be made in this area.10,71,72 It is important that reproducible results with large sample sizes be available for further evaluation as biomarkers and potential therapeutic targeting.7,10,71,72
CONCLUSIONS Despite the multitude of biomarkers proposed, most studies have been conducted in small numbers of infants, with few being replicated by independent investigators. The biomarkers that have shown consistent results include: IL-6, -8, -10, MCP-1, CC10, TIMP 2, soluble E-selectin, KGF, TGFb1, Angiopoietin 2 and IFNg. VEGF has been extensively studied, with reasonably consistent results if the temporal sequence is taken into consideration. It is important to clarify that these biomarkers only show an association with BPD. To establish a causal relationship, studies in developmentally-appropriate animal models and human lungs with BPD need to be conducted. Among the biomarkers discussed, the following are the strongest candidates, with published data
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fulfilling the criteria mentioned above: IL-1b, IL-6, MCP-1, TGFb1, VEGF, pulmonary HGF, KGF, bombesin-like peptide, MMP 9, PTHrP, MIF, Angiopoietin 2, and IFNg. Confirmatory studies with adequate sample sizes and assessment of the role of putative biomarkers in the aetiopathogenesis of BPD in developmentally appropriate animal models and human lungs with BPD will enhance the potential for therapeutic interventions. Genomic and proteomic approaches have the greatest potential to significantly advance the field of biomarkers in BPD.
PRACTICE POINTS Incidence of bronchopulmonary dysplasia (BPD) has remained unchanged despite changes in neonatal therapies. The routine use of surfactant and gentler noninvasive ventilatory techniques has led to ‘‘New’’ BPD with pathological findings distinct from ‘‘Old BPD’’ Biomarkers of BPD can be identified in various biological fluids including blood, tracheal aspirates, urine and breath condensate. Biomarkers of BPD are not routinely used in clinical practice.
RESEARCH DIRECTIONS Genomic and proteomic approaches for early identification of infants predisposed to BPD. Use of developmentally appropriate animal models to correlate with molecular biomarkers in human lungs with BPD. Targeted therapeutic options to ameliorate early, evolving and established phases of BPD to improve long term pulmonary and neurodevelopment outcomes.
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EDUCATIONAL QUESTIONS
a. b. c. d.
1. The most commonly accepted definition of bronchopulmonary dysplasia is: a. oxygen dependence for 28 post natal days and assessment of respiratory support at 36 weeks post menstrual age b. birth at 32 weeks of gestation c. presence of chronic lung disease d. history of respiratory distress syndrome 2. The pathogonomic finding in the lungs of patients with the BPD is: a. increased airway fibroproliferation b. marked airway smooth muscle hypertrophy c. increased lymphocytic infiltration d. alveolar simplification 3. All of the following are thought to play a role in development of BPD except:
prematurity maternal chorioamnionitis antenatal steroids genetic factors
4. Among inflammatory cytokines, which has been shown in developmentally-appropriate animal models and human tracheal aspirates to be strongly associated with BPD? a. IL-10 b. MIF c. IFNg d. IL-16 5. The source of pepsin in tracheal aspirates in infants developing BPD is the . . . a. Lung b. Gastric mucosa c. Blood d. Nasopharyngeal secretions