Dexamethasone and Betamethasone forPrenatal Lung Maturation: Differences inVascular Endothelial Growth FactorExpression and Alveolarization in Rats
Laura San Feliciano a Ana Remesal a María Isidoro-García b Dolores Ludeña c
Departments of a Paediatrics, b
Clinical Biochemistry and c Cellular Biology and Pathology, University Hospital of
Salamanca, University of Salamanca, Salamanca , Spain
that received dexamethasone. Conclusions: Our resultssupport the notion that betamethasone could be a better choice than dexamethasone for antenatal lung maturation.
One of the most important advances in neonatal care has been the introduction of antenatal glucocorticoid therapy for preventing respiratory distress syndrome and improving the survival of preterm infants [1] . Fetal and postnatal lung development is regulated by glucocorti-coids [2] . There are many reports showing that corticoids can alter normal stages of lung development during the period of alveolarization. Corticoids are known to trigger the structural maturation of mesenchymal cells and functional maturation of the surfactant system [3] . Dexa-methasone and betamethasone are the only corticoste-roids recommended for antenatal therapy [1] , and as yet there are no indications favoring the use of one molecule over the other [4] , although there are some previous re-ports giving preference to betamethasone [5, 6] . The dos-age and number of treatment cycles in gestating women at risk of preterm delivery are still in controversy because of the long-term effects of these drugs on growth [7] .
Background: Fetal and postnatal lung development is reg-ulated by glucocorticoids. The use of antenatal corticoste-roids is reported to produce effects on vascular endothelial growth factor (VEGF), which plays a crucial role in pulmonary development. Objectives: The purpose of this study was to compare pulmonary VEGF expression in newborn rats that were exposed to antenatal betamethasone versus dexa-methasone and to evaluate its impact on the alveolarization period of rats (0–14 days of life). Methods: Betamethasone, dexamethasone or equivalent saline solution (control group) was administered to pregnant rats on 20th and 21st days of gestation. Pulmonary VEGF mRNA, VEGF protein expression, and alveolarization changes were evaluated at birth and at 14 days of life. Results: Betamethasone and dexamethasone were observed to have different actions on VEGF expression with a correlation with alveolarization on both days of study. Antenatal dexamethasone decreased VEGF expression, be-tamethasone tended to produce the induction of the ex-pression of VEGF, and moreover, betamethasone did not produce a decrease in alveolarization as seen in the animals
Received: September 6, 2010 Accepted after revision: November 30, 2010 Published online: February 9, 2011
Laura San Feliciano, MD, PhD Hospital Universitario de Salamanca Paseo de San Vicente 58-182 ES–37007 Salamanca (Spain) Tel. +34 92 329 1116, Fax +34 92 329 1452, E-Mail lausafe @ yahoo.es
The intrauterine lung development of premature ba-bies is interrupted at delivery and continues extra-utero. In consequence, alveolar architecture and vascular devel-opment may be altered [8] . The alveolarization process is complex, and not all the mechanisms and signaling path-ways that regulate normal alveolar development have been elucidated. Recent experimental studies have shown that the development of pulmonary circulation and al-veolarization is strongly coordinated by vascular growth factors and that there is a critical period for septation and the formation of pulmonary vessels [9] .
The structural features in the alveolar phase of lung development have been well described, and there are many similarities between the rat and human lung. In our study, we used Wistar rats as an experimental model. In rats, al-veolar formation occurs up to the 14th postnatal day [10] .
Vascular development of the lung is crucial in the mat-uration of lung structure [11] . The interactions between the airways and blood vessels through growth factors are critical to normal lung development, vascular endothelial growth factor (VEGF) being one of the most important factors related to this process [8, 12] . VEGF expression is regulated by several factors; among them, interactions be-tween corticoids and VEGF have been reported [13–15] . Many in vitro studies have shown the strong negative reg-ulation of dexamethasone in the expression of VEGF in different cell types, including epithelial alveolar cells [16] . There are several differences in the levels of VEGF expres-sion in bronchial aspirates from the lungs of newborn ba-bies that had received postnatal dexamethasone [17–19] . However, there are few experimental studies addressing the action of betamethasone on VEGF [15, 20] .
In light of the foregoing, the aim of the present study was to compare the action of corticosteroids (dexametha-sone and betamethasone) administered prenatally on the expression of VEGF and its impact during the postnatal alveolarization.
Methods
Animal Protocol We evaluated pulmonary VEGF mRNA, VEGF protein expres-
sion and alveolarization in Wistar rats at 0 and 14 days of life. All procedures were approved by the Animal Health Care Committee of the University of Salamanca. All experiments were performed following the regulations of the Directive of the Council of the Eu-ropean Community (DOCE L 222; 24/08/1999). Dexamethasone (sodium phosphate) at 0.4 mg/kg/day, betamethasone (sodium phosphate) at 0.4 mg/kg/day or an equivalent saline solution (con-trol) were administered intravenously to pregnant Wistar rats on the 20th and 21st days of gestation. The rats were born on day 22
of gestation by natural delivery and were randomly mixed and dis-tributed in a litter size adjusted to 8 pups. In total, 66 animals were studied, and 11 animals were analyzed in each experimental group. This experimental model has been reported previously by our group [21, 22] . Rat pups were killed at 0 and 14 days of life by intraperitoneal administration of pentobarbital, and their lungs were processed according to the experiments to be performed.
Pulmonary VEGF mRNA To determine VEGF mRNA expression in lungs, total RNA
was isolated from the pulmonary tissue (from 6 animals/experi-mental subgroup) using the RNeasy Mini Kit (Qiagen, Valencia, Calif., USA). Following the isolation of total RNA from animal tissue, RT-PCR was performed (online suppl. material, www.karger.com/doi/10.1159/000281816). We used primers of rat glyc-eraldehyde-3-phosphate dehydrogenase (GAPDH) and rat VEGF designed from the published M32167 cDNA sequence of the VEGF gene [23] . PCR band intensities were analyzed by densi-tometry using ImageJ software (W.S. Rasband, National Insti-tutes of Health; http://rsb.info.nih.gov/ij/, 1997–2008). Measure-ments were performed in triplicate. Standardization was carried out based on the intensities of the known concentration bands of the marker (MK). The final band intensity for VEGF was ex-pressed relative to GAPDH.
VEGF Protein Expression For VEGF immunohistochemistry and morphometric stud-
ies, lungs were fixed with a 4% paraformaldehyde solution through the trachea under a constant pressure of 20 cm of water. The tra-chea was then ligated, and the lungs were immersed in 4% para-formaldehyde. The respective lungs were processed and embed-ded in paraffin before 24 h had elapsed. For VEGF mRNA assess-ments, the lungs were perfused with PBS buffer (PBS Sigma), immediately frozen in liquid nitrogen in portions of 20 mg, and stored at –80 ° C until the time of processing.
To study protein expression, three tissue microarrays were built, incorporating pulmonary samples from 5 animals per group. All samples were embedded in triplicate. Tissue cylinders with a diameter of 1 mm were then punched from lung areas from each donor tissue block and embedded in a recipient paraffin block. Four-micrometer sections of the tissue microarray block were transferred to glass slides [22] .
Immunostaining was performed automatically using theOPTIMAX PLUS processor, employing a 1: 100 dilution of rabbit anti-human VEGF-A (sc-152, Santa Cruz Biotech, Santa Cruz, Calif., USA). The immunostaining was carried out simultaneous-ly to minimize interassay variability. Image processing tech-niques were employed to perform optical density (OD) analyses of the immunostained tissue from 15 images from each group.
The images were analyzed using ImageJ software (W.S. Ras-band, National Institutes of Health, Bethesda, Md., USA; http://rsb.info.nih.gov/ij/, 1997–2008), following the steps previously re-ported by our group [22] . Negative controls for the antibody were performed using non-immune serum. The measurements of the negative controls did not reveal significant differences among groups.
Morphometric Study All morphometric assessments were made by two independent
observers in a double-blind manner. Lung sections stained with
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Neonatology 2011;100:105–110 107
hematoxylin-eosin were examined at ! 200 with a microscope, holding the sample over a square grid (model CPLW 1018, Zeiss Optical, Hannover, Md., USA) in the eyepiece [24] . Each line had test points positioned to form a square lattice; the distance be-tween points was 60 � m at a magnification of ! 200. The total length of the test line was 2,520 � m. Mean alveolar linear length (Lm) was calculated as an indication of mean alveolar diameter by dividing the total length (2,520 � m) by the number of inter-cepts of the septal wall. The same measurements were performed on at least 20 different areas of the lung section (superior and in-ferior sections of each lung). The internal surface area (ISA) of the lung was calculated using the formula (4 ! lung volume)/Lm. ISA data were normalized to 100 g body weight and were used as spe-cific ISA [25] .
Statistical Analyses Results are expressed as means 8 SD. ANOVA was used to
compare continuous variables across the levels of each condition. The homogeneity of variance was analyzed previously. p ! 0.05 was considered statistically significant. Particular attention was paid to sample size, depending on the statistical power of this variable. Statistical power was calculated when required (http://www.dssresearch.com/toolkit). All statistical analyses were per-formed using the SPSS software, version 12.0 (Chicago, Ill., USA).
PCR for VEGF mRNA transcripts in the lungs of the rat groups. The mRNA expression level of GAPDH was used to normalize the data for VEGF. Mean values and SD are shown in table 1 .
VEGF mRNA expression tended to be higher in the animals receiving betamethasone than those receiving dexamethasone, but not significantly. These findings were observed on day 0 and day 14.
VEGF Protein Expression The distribution of VEGF immunostaining was abun-
dant in epithelial cells of the distal airway and alveolar cells (pneumocytes) and was also observed in endothelial cells, the muscle cells of the vascular wall, and interstitial cells ( fig. 1 ).
The OD values, expressed as means and standard de-viations, of the different groups and study days are shown in table 1 . The group that received prenatal dexametha-sone had a lower expression of VEGF protein, with statis-tically significant differences in comparison with the controls on day 0 (p = 0.02) values. On day 14, no signifi-cant differences (NS) were observed upon comparing the dexamethasone group with the control group. There were no significant differences (NS) in the protein expression of VEGF between the betamethasone group and the con-trol group on day 0 of the study. On day 14, the protein expression of VEGF was significantly higher (p ! 0.0001) in the betamethasone group as compared to the controls. The group that received antenatal betamethasone treat-ment had significantly increased (p ! 0.0001) VEGF pro-tein expression as compared to the group receiving pre-natal dexamethasone on both 0 and 14 study days.
Morphometry Figure 2 shows the changes in the septation between
the different groups of animals. The values concerning Lm and specific alveolar exchange surface (ISA) of the different groups and study days are shown in table 1 .
The group that received prenatal dexamethasone had significantly increased (p ! 0.01) Lm in comparison with the control group. The ISA was lower in the group that received prenatal dexamethasone compared with the control group on both days of the study, with significant differences (p ! 0.05) at birth (day 0). There were no sig-nificant differences (NS) in the Lm or the ISA between
Table 1. Summary of overall changes in VEGF mRNA, VEGF protein, Lm and ISA
Groups VEGF mRNA VEGF protein, OD Lm, � m ISA, cm 2 /100 g b.w.
* p < 0.05 versus control group, ** p < 0.01 versus dexamethasone group. Dex = Dexamethasone; Beta = betamethasone; b.w. = body weight.
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Neonatology 2011;100:105–110108
C0
C14 D14 B14
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Fig. 1. Immunohistochemistry. Lung sections from each group of the study. C = Control; D = dexamethasone; B = betamethasone;0 = at birth; 14 = at the age of 14 days. Calibration 50 μm.
Fig. 2. Morphometric study of the lungs. Hematoxilin eosin. Lung sections from each group of the study. C = Control; D = dexameth-asone; B = betamethasone; 0 = at birth; 14 = at the age of 14 days. Calibration 50 μm.
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sion
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Neonatology 2011;100:105–110 109
the group that received antenatal betamethasone and the control group. The Lm of the prenatal betamethasone group of animals was lower, and the ISA was higher than in the prenatal dexamethasone group, and the differenc-es were statistically significant (p ! 0.01) in the Lm at birth. All animals in the study showed higher average al-veolar length (Lm) and a less specific ISA on day 0, with significant differences in comparison with day 14 (p ! 0.0001).
Discussion
We have examined the influence of antenatal cortico-steroid administration (betamethasone vs. dexametha-sone) on the expression of VEGF and its influence on the alveolarization period in lung maturation in rats. Both drugs are currently used in pregnant women at risk of preterm delivery [1, 6] . In previous studies, our group found differences in the action of betamethasone and dexamethasone on the expression of VEGF at the begin-ning of the period of alveolarization in rats [15] . The pres-ent paper was conceived to assess the difference between the two steroids from the saccular phase (0 days of age) until the end of the alveolar phase (14 days of age).
At birth, we observed a significant decrease in pulmo-nary VEGF expression in the prenatal dexamethasone group versus the control group. In agreement with our findings, several in vitro studies have shown that dexa-methasone inhibits the expression of VEGF in different cellular lines [16, 26, 27] . In contrast, in vivo, there are several discrepancies regarding the effect of dexametha-sone on VEGF expression. Some reports have shown that dexamethasone decreases VEGF expression [15, 17, 28] , while other have reported no change [18, 29] and even increases [30] . These differences in the effects of dexa-methasone on the expression of VEGF can be attributed to the different models used.
In our study, decreased pulmonary VEGF expression was correlated with a decrease in alveolarization. This finding has been described previously by our group [15, 21] . Such an effect of dexamethasone on VEGF and al-veolarization was not found in the animals that received antenatal betamethasone. The VEGF expression in these animals was similar to that observed for the control group. Furthermore, we failed to find significant differ-ences in the alveolarization between the betamethasone and control groups. Reporting findings similar to ours, Roubliova [20] observed that VEGF expression after pre-natal exposure to betamethasone was increased in lungs.
On the 14th day of our study, we found a higher ex-pression of VEGF in the groups of animals exposed to antenatal steroids compared to the controls. We believe this may have been due to a certain degree of recovery in lung maturation when the action of corticosteroids had ceased. This long-term recovery was proposed previously by Tschanz et al. [31] , who described the microvascular maturation of the lungs of rats after ceasing exposure to corticosteroids.
Although we have not detected publications by other authors comparing the effects of betamethasone or dexa-methasone on VEGF in lung development, our results sug-gest that the compounds have different actions. In a previ-ous study by our group [15] , we reported that dexametha-sone decreased VEGF expression at the beginning of the alveolarization period (4 days of age), and this was not ob-served in animals that had received betamethasone. These changes were correlated with the lower degree of alveolar-ization observed in animals exposed to prenatal dexa-methasone. With this present study, we can confirm that in rats these differences, which exist since birth, are main-tained until the end of the alveolarization period. Our results also suggest that antenatal betamethasone would have a positive effect on the expression of VEGF, or at least would not have an inhibitory effect, and that it does not alter long-term alveolarization, unlike dexamethasone.
Both betamethasone and dexamethasone are recom-mended for use in pregnant women at risk of preterm birth [1, 4] , and no recommendations for the choice of one over another have been recently proposed [7] . Clinical studies and trials have revealed several biological effects between the two corticosteroids. Better outcomes in mortality and neurological and pulmonary diseases are being obtained with the use of prenatal betamethasone [5, 32, 33] . Our ex-perimental results also support the notion that betameth-asone may be the best choice for treating pregnant women at risk of preterm delivery because of the negative effect on lung maturation and VEGF expression caused by prenatal dexamethasone until the end of the alveolarization period.
Acknowledgements
We thank Prof. Bancalari for useful suggestions. We also thank Dr. Horta-Júnior for his directions in the densitometry analysis and Dr. Sanz for her help in the RT-PCR technics. Finally, we wish to dedicate this article to the memory of Prof. Carmen Pedraz.
This study was supported in part by a grant of the Junta de Castilla y León (grant No. HUS01A09), a SCCALP Society re-search grant, and the Department of Paediatrics and Department of Cellular Biology and Pathology, School of Medicine, Univer-sity of Salamanca (Spain).
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References
1 Effect of corticosteroids for fetal maturation on perinatal outcomes. NIH Consensus De-velopment Panel on the Effect of Corticoste-roids for Fetal Maturation on Perinatal Out-comes. JAMA 1995; 273: 413–418.
2 Speirs HJ, Seckl JR, Brown RW: Ontogenyof glucocorticoid receptor and 11beta-hy-droxysteroid dehydrogenase type-1 gene ex-pression identifies potential critical periods of glucocorticoid susceptibility during de-velopment. J Endocrinol 2004; 181: 105–116.
3 Jobe AH: Glucocorticoids, inflammation and the perinatal lung. Semin Neonatol 2001; 6: 331–342.
5 Lee BH, Stoll BJ, McDonald SA, Higgins RD: Adverse neonatal outcomes associated with antenatal dexamethasone versus antenatal betamethasone. Pediatrics 2006; 117: 1503–1510.
6 Miracle X, Di Renzo GC, Stark A, Fanaroff A, Carbonell-Estrany X, Saling E: Guideline for the use of antenatal corticosteroids for fe-tal maturation. J Perinat Med 2008; 36: 191–196.
7 Sweet DG, Carnielli V, Greisen G, Hallman M, Ozek E, Plavka R, Saugstad OD, Simeoni U, Speer CP, Halliday HL: European consen-sus guidelines on the management of neona-tal respiratory distress syndrome in preterm infants – 2010 update. Neonatology 2010; 97: 402–417.
8 Thebaud B: Angiogenesis in lung develop-ment, injury and repair: implications for chronic lung disease of prematurity. Neona-tology 2007; 91: 291–297.
9 Massaro D, Massaro GD: Critical period for alveologenesis and early determinants of adult pulmonary disease. Am J Physiol Lung Cell Mol Physiol 2004; 287:L715–L717.
10 Burri PH: Structural aspects of postnatal lung development – alveolar formation and growth. Biol Neonate 2006; 89: 313–322.
11 Voelkel NF, Vandivier RW, Tuder RM: Vas-cular endothelial growth factor in the lung. Am J Physiol Lung Cell Mol Physiol 2006; 290:L209–L221.
12 Thebaud B, Ladha F, Michelakis ED, Sawicka M, Thurston G, Eaton F, Hashimoto K, Har-ry G, Haromy A, Korbutt G, Archer SL: Vas-cular endothelial growth factor gene therapy increases survival, promotes lung angiogen-esis, and prevents alveolar damage in hyper-oxia-induced lung injury: evidence that an-giogenesis participates in alveolarization. Circulation 2005; 112: 2477–2486.
13 Lassus P, Heikkila P, Andersson LC, von BK, Andersson S: Lower concentration of pul-monary hepatocyte growth factor is associ-ated with more severe lung disease in pre-term infants. J Pediatr 2003; 143: 199–202.
14 Vento G, Matassa PG, Capoluongo E, Tor-torolo L, Romagnoli C, Ameglio F, Lassus P, Andersson S: Glucocorticoids in preterm in-fants and discrepancies of vascular endothe-lial growth factor. Am J Respir Crit Care Med 2003; 168: 501–502.
15 Remesal A, San Feliciano L, Isidoro-Garcia M, Ludena D: Effects of antenatal betameth-asone and dexamethasone on the lung ex-pression of vascular endothelial growth fac-tor and alveolarization in newborn rats ex-posed to acute hypoxia and recovered in normoxia or hyperoxia. Neonatology 2010; 98: 313–320.
16 Nauck M, Karakiulakis G, Perruchoud AP, Papakonstantinou E, Roth M: Corticoste-roids inhibit the expression of the vascular endothelial growth factor gene in human vascular smooth muscle cells. Eur J Pharma-col 1998; 341: 309–315.
17 Vento G, Matassa PG, Ameglio F, Capoluon-go E, Tortorolo L, Romagnoli C: Effects of early dexamethasone therapy on pulmonary fibrogenic mediators and respiratory me-chanics in preterm infants. Eur Cytokine Netw 2002; 13: 207–214.
18 Lassus P, Turanlahti M, Heikkila P, Anders-son LC, Nupponen I, Sarnesto A, Andersson S: Pulmonary vascular endothelial growth factor and Flt-1 in fetuses, in acute and chronic lung disease, and in persistent pul-monary hypertension of the newborn. Am J Respir Crit Care Med 2001; 164: 1981–1987.
19 D’Angio CT, Maniscalco WM, Ryan RM, Avissar NE, Basavegowda K, Sinkin RA: Vascular endothelial growth factor in pul-monary lavage fluid from premature infants: effects of age and postnatal dexamethasone. Biol Neonate 1999; 76: 266–273.
20 Roubliova XI, Van der Biest AM, Vaast P, Lu H, Jani JC, Lewi PJ, Verbeken EK, Tibboel D, Deprest JA: Effect of maternal administra-tion of betamethasone on peripheral arterial development in fetal rabbit lungs. Neonatol-ogy 2008; 93: 64–72.
21 Pedraz C, San Feliciano L, Perez P, Revestido R, Ludena MD, Remesal A, Fernandez F: Ef-fect of prenatal dexamethasone on lung in neonatal rats exposed to hypoxia and recov-ered in oxygen or air. Pediatr Res 2003; 54: 582.
22 Remesal A, Pedraz C, San Feliciano L, Lude-na D: Pulmonary expression of vascular en-dothelial growth factor (VEGF) and alveolar septation in a newborn rat model exposed to acute hypoxia and recovered under condi-tions of air or hyperoxia. Histol Histopathol 2009; 24: 325–330.
23 Conn G, Bayne ML, Soderman DD, Kwok PW, Sullivan KA, Palisi TM, Hope DA, Thomas KA: Amino acid and cDNA se-quences of a vascular endothelial cell mito-gen that is homologous to platelet-derived growth factor. Proc Natl Acad Sci USA 1990; 87: 2628–2632.
24 Blanco LN, Frank L: Development of gas-ex-change surface area in rat lung. The effect of alveolar shape. Am J Respir Crit Care Med 1994; 149: 759–766.
25 Weibel ER, Kistler GS, Scherle WF: Practical stereological methods for morphometric cy-tology. J Cell Biol 1966; 30: 23–38.
26 Tanabe K, Tokuda H, Takai S, Matsushima-Nishiwaki R, Hanai Y, Hirade K, Katagiri Y, Dohi S, Kozawa O: Modulation by the ste-roid/thyroid hormone superfamily of TGF-beta-stimulated VEGF release from vascular smooth muscle cells. J Cell Biochem 2006; 99: 187–195.
27 Gille J, Reisinger K, Westphal-Varghese B, Kaufmann R: Decreased mRNA stability as a mechanism of glucocorticoid-mediated in-hibition of vascular endothelial growth fac-tor gene expression by cultured keratino-cytes. J Invest Dermatol 2001; 117: 1581–1587.
28 Hewitt DP, Mark PJ, Waddell BJ: Glucocor-ticoids prevent the normal increase in pla-cental vascular endothelial growth factorexpression and placental vascularity during late pregnancy in the rat. Endocrinology 2006; 147: 5568–5574.
29 Lassus P, Ristimaki A, Ylikorkala O, Viinik-ka L, Andersson S: Vascular endothelial growth factor in human preterm lung. Am J Respir Crit Care Med 1999; 159: 1429–1433.
30 Bhatt AJ, Amin SB, Chess PR, Watkins RH, Maniscalco WM: Expression of vascular en-dothelial growth factor and Flk-1 in develop-ing and glucocorticoid-treated mouse lung. Pediatr Res 2000; 47: 606–613.
31 Tschanz SA, Makanya AN, Haenni B, Burri PH: Effects of neonatal high-dose short-term glucocorticoid treatment on the lung: a mor-phologic and morphometric study in the rat. Pediatr Res 2003; 53: 72–80.
32 Figueras-Aloy J, Serrano MM, Rodriguez JP, Perez CF, Serradilla VR, Jimenez JQ, Gonza-lez RJ: Antenatal glucocorticoid treatment decreases mortality and chronic lung disease in survivors among 23- to 28-week gesta-tional age preterm infants. Am J Perinatol 2005; 22: 441–448.
33 Feldman D, Carbone J, Belden L, Borgida A, Herson V: Bethametasone vs dexametha-sone for the prevention of morbidity in very-low-birthweight neonates. Am J Obstet Gy-necol 2007; 197: 284.e1–284.e4.
