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* Department of Pediatrics and
Internal Medicine, National Jewish Medical and Research Center, Denver, Colorado, USA;
University of Texas Health Science Center and Southwest Foundation for Biomedical Research, San Antonio, Texas, USA;
SRI International, Menlo Park, and Cardiovascular Research Institute, University of California San Francisco, California, USA;
|| Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, USA; and
¶ Division of Neonatology, Vanderbilt University Medical Center, Nashville, Tennessee, USA
1Correspondence: Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson St., Rm. J-318, Denver, CO 80206, USA. E-mail: whitec{at}njc.org
SPECIFIC AIMS
Advances in perinatal care have lowered mortality rates associated with bronchopulmonary dysplasia (BPD) but have introduced a new form of BPD characterized by structural immaturity of the lung in surviving babies. None of the new, clinically available experimental treatment strategies, with the possible exception of inhaled NO, have successfully restored impaired lung development and lung function in ventilated preterm infants with evolving BPD. This chronic lung disease is characterized by arrested development of microvasculature and distal air spaces. Since lung branching morphogenesis depends on vascular growth, and lung vascular development and expression of hypoxia-inducible factor (HIF) are disrupted by preterm birth and ventilatory therapy, we hypothesized that enhancement of angiogenesis by HIF stimulation could have advantageous effects on lung growth in prematurely born neonates. Our aims were to investigate lung growth and function in preterm baboons (125 days+14 days pro re nata O2 model, corresponds to 27 human gestational weeks) treated with continuous, intravenous (i.v.) FG-4095, an inhibitor of prolyl hydroxylase domain-containing proteins (PHDs) that degrade HIFs.
PRINCIPAL FINDINGS
1. Clinical characteristics were similar in FG-4095-treated and control baboons
General criterion for inclusion was birth between 11/2003 and 2/2005 ± 1 month (15 FG-4095-treated and 26 control baboons). To assess lung growth and function, 14 days survival was required. Common reasons for nonsurvival were early cardiovascular instability, sepsis, and/or iatrogenic causes. Following exclusions (6 FG-4095-treated animals and 8 controls) according to a priori criteria, 9 FG-4095-treated and 18 control baboons remained. For data analyses, these animals were separated into 14 day survivor FG-4095-treated (n=5) and control (n=12) subgroups, and early death (ED) FG-4095-treated (n=4) and control (n=6) subgroups. Unless otherwise indicated, results depict analyses of the included 14 day survivor groups.
Plasma FG-4095 accumulated for the first 3 to 4 days, remained high until age 5 to 6 days, and declined thereafter. The plasma levels obtained corresponded to concentrations that are effective for HIF stabilization in lung cells and explants in vitro. Clinical characteristics, including mortality, gender, birth weight, lung weight, incidence of sepsis, and blood chemistry parameters were similar in untreated and FG-4095-treated animals.
2. Lung growth is enhanced in FG-4095-treated animals
Lung morphometry by digital image analysis of untreated controls relative to fetal age-matched controls (140 day gestational controls) revealed many abnormalities, including loss of total alveolar surface area, and decreased number of internodal segments (branch points) and of end segments (secondary crests) suggestive of alveolar simplification and arrested alveolar septal budding (formation of new alveoli) (Table 1
). Notably, several of these abnormalities were improved or normalized in FG-4095-treated baboons. Specifically, surface density and area of primary septae were greater in FG-4095-treated baboons than in untreated newborns (Table 1)
. Moreover, 77% of diminished total alveolar surface area in untreated controls was recovered by FG-4095 treatment (Table 1)
.
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3. Lung function is improved by FG-4095 treatment
Alveolar-arterial O2 gradient and oxygenation index were improved (50–60% and 30–40% reduction, respectively) for the last 5 to 6 days by FG-4095 treatment (Fig. 1
A, B). Ventilation index appeared improved in FG-4095-treated animals as compared with controls, but the difference was not statistically significant (Fig. 1C
). Dynamic lung compliance was enhanced 20–40% during the last 4 days by FG-4095 treatment (Fig. 1D
).
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4. Surfactant expression did not differ between FG-4095-treated baboons and controls
Lung SP-B and -C mRNA did not differ between treated and untreated neonates. Bronchoalveolar lavage fluid (BALF) or lung saturated phosphatidylcholine (Sat PC) content was similar in FG-4095-treated and control animals. BALF and lung SP-B and -C proteins by Western blot and/or ELISA were unaltered in FG-4095-treated relative to control baboons.
5. FG-4095 elicited positive hemodynamic changes
Left ventricular function was identical for control and FG-4095-treated baboons. Pulmonary to systemic blood flow ratio was lower in FG-4095 baboons than controls. Systemic diastolic and mean arterial blood pressure were higher in baboons receiving FG-4095 than in controls. Further, incidence of spontaneous ductus arteriosus (DA) closure, assessed by daily echocardiography, was higher in FG-4095-treated 14-day animals than controls.
6. FG-4095 treatment was associated with possible untoward effects
FG-4095 generally was well tolerated and elicited some beneficial lung outcomes in 14 day survivors. However, when early death subgroups were examined, several troublesome findings were identified. Early death FG-4095 animals exhibited a skin rash never before observed in this model. In addition, early death FG-4095 baboons had more moderate to severe pulmonary microscopic extravasation of red blood cells when compared to early death controls, and at necropsy gross examination showed more moderate to severe bleeding in lungs and other organs compared to early death controls that had none to mild grades. Of note, rash or bleeding were not noted in baboons completing 14 day treatment with FG-4095.
CONCLUSIONS AND SIGNIFICANCE
By lung morphometry, untreated control baboons had decreased total alveolar surface area, alveolar simplification as demonstrated by reduced numbers of branch points, and arrested growth of new alveoli as implicated by fewer secondary crests. Remarkably, total alveolar surface area and number of branch points were normalized in FG-4095-treated baboons vs. untreated newborn and fetal controls, suggesting improved alveolar growth. We recently found that mRNA and/or protein for several angiogenic factors, including HIF-1
, platelet-endothelial cell adhesion molecule 1 (PECAM-1), and vascular endothelial growth factor (VEGF) are up-regulated in lungs of FG-4095-treated baboons as compared with untreated controls. Further, PECAM-1 expressing capillary endothelial cells are augmented by FG-4095. These data are supported by recent investigations by others showing that VEGF treatment, especially if combined with angiopoietin 1, preserves alveolar development in term newborn rodents with O2-induced lung injury. Collectively, the data suggest that both vascular and alveolar growth are enhanced by FG-4095.
To assess whether the lung morphometric findings in FG-4095-treated newborns were associated with improved lung function, dependence on O2 and ventilatory support was assessed. Both control and FG-4095-treated baboons had typical worsening of oxygenation on days 2 to 5 due to severe respiratory distress. However, at age 8 to 9 days oxygenation improved in FG-4095-treated relative to untreated baboons. The 2-fold reduction in oxygenation index and alveolar-arterial O2 gradient likely is physiologically significant. In addition, lung dynamic compliance increased on the final 4 days of life. These data indicate that lung structural changes in FG-4095-treated baboons improved gas exchange.
Possible mechanisms for improved oxygenation and lung compliance in FG-4095-treated animals were investigated. Since HIF-VEGF axis has been implicated in surfactant production, this process was investigated. We measured Sat PC and mRNA and protein for SP-B and -C in BALF and lung tissue. None were altered by FG-4095 treatment. Hence, improved oxygenation and lung compliance were not due to changes in surfactant production or secretion detectable at necropsy.
Hypotension is common in preterm baboons. Hemodynamically, FG-4095-treated baboons had moderately increased diastolic blood pressure. Moreover, pulmonary to systemic flow ratio was reduced in the FG-4095-treated relative to control baboons. These positive hemodynamic effects likely resulted from increased DA closure in FG-4095-treated animals.
Untoward effects, including skin rash and bleeding, were noted in FG-4095-treated early death baboons. Potential mechanisms, including drug-specific effects, potentiation of inflammation and/or of VEGF, warrant further investigation, as do alternate routes and/or timing of delivery.
To summarize, treatment of preterm baboons with HIF PHD inhibitor is associated with enhanced lung growth, improved oxygenation and lung compliance, and increased ductus arteriosus closure (Fig. 2)
. Thus, HIF stimulation ameliorates pathological and physiological consequences of BPD.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5887fje
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