Published ahead of print on May 31, 2007, doi:10.1165/rcmb.2006-0349OC
© 2007 American Thoracic Society DOI: 10.1165/rcmb.2006-0349OC Embryonic Essential Myosin Light Chain Regulates Fetal Lung Development in RatsLife and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal; Proteomics Core Facility, Biocenter Oulu, Department of Biochemistry, University of Oulu, Finland; and Division of Pediatric Surgery, Hospital S João, Porto, Portugal Correspondence and requests for reprints should be addressed to Prof. Jorge Correia-Pinto, M.D., Ph.D., Escola de Ciências da Saúde, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal. E-mail: jcp{at}ecsaude.uminho.pt
Congenital diaphragmatic hernia (CDH) is currently the most life-threatening congenital anomaly the major finding of which is lung hypoplasia. Lung hypoplasia pathophysiology involves early developmental molecular insult in branching morphogenesis and a late mechanical insult by abdominal herniation in maturation and differentiation processes. Since early determinants of lung hypoplasia might appear as promising targets for prenatal therapy, proteomics analysis of normal and nitrofen-induced hypoplastic lungs was performed at 17.5 days after conception. The major differentially expressed protein was identified by mass spectrometry as myosin light chain 1a (MLC1a). Embryonic essential MLC1a and regulatory myosin light chain 2 (MLC2) were characterized throughout normal and abnormal lung development by immunohistochemistry and Western blot. Disruption of MLC1a expression was assessed in normal lung explant cultures by antisense oligodeoxynucleotides. Since early stages of normal lung development, MLC1a was expressed in vascular smooth muscle (VSM) cells of pulmonary artery, and MLC2 was present in parabronchial smooth muscle and VSM cells of pulmonary vessels. In addition, early smooth muscle differentiation delay was observed by immunohistochemistry of  -smooth muscle actin and transforming growth factor- 1. Disruption of MLC1a expression during normal pulmonary development led to significant growth and branching impairment, suggesting a role in branching morphogenesis. Both MLC1a and MLC2 were absent from hypoplastic fetal lungs during pseudoglandular stage of lung development, whereas their expression partially recovered by prenatal treatment with vitamin A. Thus, a deficiency in contractile proteins MLC1a and MLC2 might have a role among the early molecular determinants of lung hypoplasia in the rat model of nitrofen-induced CDH.
Key Words: congenital diaphragmatic hernia • lung hypoplasia • proteomics • myosin light chains • smooth muscle
Prenatal development of a diaphragmatic defect, intrathoracic herniation of abdominal content, and lung hypoplasia characterize at the most basic level congenital diaphragmatic hernia (CDH). CDH remains the most life-threatening congenital pathology with an incidence of 1 in 2,500 newborns (1). Lung hypoplasia and pulmonary hypertension associated with this disorder are the major causes of respiratory distress and common failure of the currently available treatments. Lung development involves complex processes that may be divided into two distinctively regulated cellular phenomena: early branching morphogenesis and late maturation and differentiation processes (2, 3). In CDH, regulation of both cellular phenomena seems impaired, resulting in lower number of airways and vascular generations and increased smooth muscle (SM) cells layer in pulmonary arteries (4, 5). Furthermore, CDH lung development appears to be stalled at canalicular or early saccular stage (6). Only a better understanding of the molecular mechanisms involved in normal and in abnormal CDH-associated lung development can contribute to the disentanglement of CDH etiology and pathophysiology, allowing clinically pertinent novel perspectives concerning lung hypoplasia treatment and modulation of lung repair. In this regard, it is currently believed that the prenatal period might be the opportunity window in which targeting key molecules may possibly result in improvement of lung hypoplasia. Proteomics allows the analysis of the protein content of a biological system, also termed the proteome (7–9). Several attempts to apply proteomic methodologies to pulmonary medicine have been made. Evaluation of bronchoalveolar lavage fluid has been useful in diagnosis and research of several inflammatory lung diseases, including emphysema, pulmonary fibrosis, cystic fibrosis, pulmonary transplantation, and acute lung injury (9). However, a proteomic approach has not yet been reported in the study of CDH. The current knowledge regarding the complex molecular and genetic mechanisms involved in CDH pathophysiology has met improvements with the establishment of CDH animal models. Particularly, the nitrofen-induced CDH rat model has been widely used in the study of CDH because it is the one that best mimics the human disease (10–13). The right dose administration of the teratogen 2,4-dichorophenyl-p-nitrophenylether (nitrofen), during a precise developmental window, leads to an equal growth interference in both lungs before the diaphragm defect occurs (14). According to the dual-hit hypothesis, the pathophysiology of lung hypoplasia associated with this CDH model possesses distinct early (acting essentially before 18 d post-conception [dpc]) and late (acting essentially after 18 dpc) gestational determinants (13, 14). Early determinants appear to be secondary to retinoid pathway disturbances during precocious stages and disrupt mainly branching morphogenesis (15), whereas late determinants seem to be primarily related to mechanical compression mediated by visceral thoracic herniation affecting essentially lung maturation and differentiation (13, 14). The most promising targets for envisioning new clinically relevant, minimally invasive, prenatal treatment modalities are inherent to early determinants of CDH-associated pulmonary hypoplasia.
Proteomic analyses were performed to identify the major early determinants of fetal lung hypoplasia in the nitrofen-induced CDH model. Total protein profiles from control and nitrofen-exposed 17.5 dpc hypoplastic lungs were determined by two-dimensional gel electrophoresis. The comparison of control group versus nitrofen and CDH groups revealed a protein with an all-or-nothing expression, which was identified by mass spectrometry as myosin light chain 1a (MLC1a), an embryonic essential myosin light chain. Expression of MLC1a, as well as MLC2 (regulatory myosin light chain),
Animal Model and Experimental Design Animal experiments were performed according to the Portuguese law for animal welfare. Animals were housed in an accredited mouse house and treated as specified in the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85–23, revised 1996). Protocol 1. Sprague-Dawley female rats (225 g; Charles River, Barcelona, Spain) were maintained in appropriate cages under controlled conditions and fed with commercial solid food. The rats were mated and checked daily for vaginal plugging. The day of plugging was defined as Gestational Day 0.5 for time dating purposes. Dams, at Day 9.5 of gestation, randomly received either a single dose of 100 mg of nitrofen dissolved in 1 ml of olive oil, or an equal volume of vehicle by gavage. Control fetuses were removed by caesarian section at 13.5, 15.5, 17.5, 19.5, and 21.5 dpc. Nitrofen-exposed fetuses were killed at 17.5, 19.5, and 21.5 dpc because only at these gestational ages an unambiguous distinction of diaphragmatic hernia is achieved. This protocol resulted in three experimental groups: (1) control group, vehicle-exposed fetus with normal lungs; (2) nitrofen group, nitrofen-exposed fetus with hypoplastic lungs but without diaphragmatic defect; and (3) CDH group, nitrofen-exposed fetus with hypoplastic lungs and with left diaphragmatic defect. Control fetuses, as well as the fetal lungs of all studied gestational ages, were processed for paraffin embedding, whereas hypoplastic nitrofen-induced fetal lungs of 17.5, 19.5, and 21.5 dpc were collected and processed for immunohistochemistry. Twenty-one and a half dpc lungs were inflation fixed. Protein extraction for Western analysis was performed in 15.5, 17.5, 19.5, and 21.5 dpc control fetal lungs and in 17.5, 19.5, and 21.5 dpc hypoplastic fetal lungs. Seventeen and a half dpc lung samples (n = 9 control, n = 9 nitrofen, and n = 9 CDH group) were collected and processed for protein extraction and two-dimensional gel electrophoresis as described in the next section. Protocol 2. Vitamin A administration was performed as described by Baptista and coworkers (15). Briefly, nitrofen-exposed dams were treated at 9.5 dpc with vitamin A (15,000 IU; A-Vite; J Neves, Paio Pires, Portugal) diluted in 1 ml of olive oil by oral administration. The dosage used was within therapeutical rage. Fetuses were collected by caesarian section and killed at 17.5 dpc. Lung samples were collected and processed for protein extraction, whereas fetuses were processed for paraffin embedding.
Two-Dimensional Gel Electrophoresis and Protein Identification
Immunohistochemistry
Lung Explant Culture Ten cultured lungs were treated daily with 40 µM phosphorothioated oligodeoxynucleotides (Metabion International AG, Martinsried, Germany) against the translational initiation site of MLC1a in the antisense direction (Table 1). Cultured lungs were treated with the same concentration of sense orientated oligodeoxynucleotide (n = 10) (Metabion International AG, Germany) (Table 1), whereas control lungs (n = 10) did not receive any treatment. The branching morphogenesis was monitored daily by photographing the explants. Lungs were collected for protein extraction.
Western Blot Analysis Proteins were obtained according to the method of Kling and coworkers (20). Ten micrograms of protein were loaded onto 12.5% acrylamide minigels, electrophoresed at 100 V at room temperature, and then transferred to Hybond-C Extra (GE Healthcare Life Sciences). Blots were probed with monoclonal mouse anti-MLC1a (Abnova Corporation) (1:1,000) and polyclonal rabbit anti-MLC2 (Cell Signaling Technology, Inc., Danvers, MA) (1:1,000) according to the manufacturer's instructions. For loading control, blots were probed with -tubulin monoclonal antibody (1:10,000; Santa Cruz Biotechnology). Afterward, blots were incubated with a secondary horseradish peroxidase conjugate (New England Biolabs, Ipswich, MA), developed with Super Signal West Femto Substrate (Pierce Biotechnology, Rockford, IL) and exposed to Hyperfilm ECL (GE Healthcare Life Sciences).
Proteomic Analysis To better understand early determinants of CDH pathophysiology, 17.5 dpc control and hypoplastic nitrofen-exposed rat fetal lungs were processed for two-dimensional PAGE analysis. Total fetal lung crude protein content was separated by IEF in the nonlinear pH gradient 3–10, followed by SDS-PAGE. Figure 1A depicts representative examples of the typical two-dimensional gel of total rat control, nitrofen, and CDH fetal lung protein. Analysis of fetal lung proteomic signature of control group versus nitrofen and versus CDH group revealed one major protein reduction in hypoplastic fetal lungs (Figure 1B). That protein was identified by peptide fingerprinting as MLC1a, a protein encoded by the gene Myl4.
MLC1a Expression in Control Fetus The MLC1a expression was further analyzed by Western blot and immunohistochemistry (IHC) of control lungs of 13.5, 15.5, 17.5, 19.5, and 21.5 dpc fetuses (Figure 2). Western analysis detected MLC1a from 15.5 dpc onwards (Figure 2A), whereas IHC-positive MLC1a signal was clearly observed from 17.5 dpc onwards (Figures 2B–2E). MLC1a was present on 15.5 dpc control fetal lungs, and its expression was sustained and seemed to increase during the remaining studied gestational ages (Figure 2A). In fact, by 17.5 dpc mesenchymal tissue surrounding a major vessel exhibited MLC1a-positive signal (Figure 2B). In addition, during subsequent stages of lung development (19.5 and 21.5 dpc) MLC1a expression remained limited to vascular smooth muscle (VSM) cells of arteries (Figures 2C–2E). Interestingly, MLC1a appeared to be restricted to the branches of pulmonary artery (Figures 2B–2F and 2H). Thus, to verify the identity of the MLC1a-positive vessel, immunostainings in adjacent tissue sections for Jagged1 (a known marker of pulmonary artery) and for MLC1a were performed (Figures 2F–2I). Jagged1 in the lung is usually detected in endothelial cells in general and SM cells of pulmonary artery in particular (Figures 2G and 2I). MLC1a-positive mesenchymal cells also stained for Jagged1 (Figures 2F–2I). Hence, MLC1a expression appeared to be confined to the pulmonary artery VSM cells.
MLC1a Expression in Hypoplastic Nitrofen-Induced Fetal Lungs To characterize MLC1a levels throughout gestation in the disease context, WB was performed in hypoplastic nitrofen-induced fetal lungs and matched with control lungs from normal pups (Figure 3A). In 17.5 dpc hypoplastic fetal lungs MLC1a was absent; nonetheless, at 19.5 and 21.5 dpc hypoplastic fetal lungs synthesized MLC1a (Figure 3A).
To determine the role of MLC1a during hypoplastic nitrofen-induced fetal lung development, MLC1a expression pattern was studied by IHC during pseudoglandular, canalicular and saccular stages. As expected, 17.5 dpc rat hypoplastic fetal lungs (nitrofen and CDH groups) were negative for MLC1a (Figure 3B). Comparisons of 19.5 and 21.5 dpc fetal lungs revealed that, although hypoplastic lungs displayed arteries with increased thickness of tunica adventitia and media, like control lungs they presented MLC1a-positive VSM cells in the tunica media (Figure 3B). Thus, MLC1a was not missexpressed throughout gestation in hypoplastic nitrofen-induced fetal lungs, with the exception of 17.5 dpc, at which point this protein was undetectable (Figures 3A and 3B).
Lung Explant Cultures in the Presence of Antisense Oligodeoxynucleotides for MLC1a
MLC2 Expression in Control Fetus Since the embryonic essential myosin light chain was expressed in VSM cells of pulmonary arteries, the regulatory myosin light chain (MLC2), a key component of SM myosin, was also studied. Expression of MLC2 was determined by WB and IHC of control fetuses throughout development (Figure 5). Western blot revealed that control fetal lungs expressed MLC2 during all studied gestational ages (15.5, 17.5, 19.5, and 21.5 dpc) (Figure 5A). IHC revealed that MLC2 was expressed as early as 13.5 dpc, with positive cells first appearing in the mesenchymal tissue around the bifurcation of the trachea (Figures 5B and 5C). This layer of MLC2-positive cells adjoining the pulmonary epithelium are known as parabronchial smooth muscle (PBSM) cells (Figures 5B and 5C). During the remaining studied gestational ages (15.5, 17.5, 19.5, and 21.5 dpc), besides PBSM cell expression, MLC2 was also expressed in pulmonary VSM cells (Figures 5D, 5E, and 5G–5J). Furthermore, MLC2 expression in PBSM cells throughout gestation was established in a proximal-distal manner, in agreement with PBSM cell differentiation. During the earliest studied gestational ages (13.5 and 15.5 dpc), only the most proximal mesenchyme surrounding the primitive epithelium displayed MLC2-positive cells (Figures 5B–5E). At 17.5 dpc, distal mesenchymal cells started to express MLC2, however, the adjoined mesenchyme at the most distal budding epithelium remained MLC2 negative (Figure 5G). Moreover, at 17.5 dpc MLC2 expression could be correlated with PBSM cell differentiation since, on branching epithelium, the MLC2-positive cell layer is merely around the central mature epithelial core, whereas budding epithelium seemed deprived of the MLC2-positive cell layer (Figure 5F). At 19.5 and 21.5 dpc, all PBSM cells and pulmonary VSM cells express MLC2 following a centrifugal pattern (Figures 5H–5J).
MLC2 Expression in Hypoplastic Nitrofen-Induced Fetal Lungs Western blots and IHC for MLC2 were performed to analyze MLC2 expression in control and hypoplastic nitrofen-induced fetal lungs throughout gestation (Figures 6A and 6B). Hypoplastic fetal lungs lost MLC2 expression at 17.5 dpc, whereas for the remaining studied gestational ages (19.5 and 21.5 dpc) MLC2 was present in similar expression pattern and levels in control and hypoplastic nitrofen-induced fetal lungs (Figures 6A and 6B).
SM Differentiation in Developing Nitrofen-Induced Hypoplastic Fetal Lungs Aiming to correlate the absence of contractile proteins MLC1a and MLC2 in 17.5 dpc hypoplastic nitrofen-induced fetal lungs with a SM cell differentiation delay, several markers were studied by IHC throughout gestation (Figure 7). -SMA is the first SM differentiation marker expressed in the developing SM of fetal lung. Either control or nitrofen-exposed hypoplastic lungs presented a centrifugal expression pattern (Figure 7A). Interestingly, at 17.5 and 19.5 dpc -SMA staining in PBSM cells of nitrofen-exposed hypoplastic lungs was decreased when compared with the control group (Figure 7A). However, in late gestation (21.5 dpc) no significant differences were observed. Regarding TGF-1 that has a negative effect on cell proliferation and promotes SM cell differentiation during lung development via transcription regulation of SM markers, our IHC studies revealed that 17.5 dpc nitrofen-exposed hypoplastic lungs presented decreased TGF-1 signal in comparison with age-matched controls (Figure 7B). Platelet-derived growth factor BB (PDGF-BB) promotes cell proliferation and is a potent negative regulator of SM cell differentiation. Conversely to -SMA and TGF-1, PDGF-BB expression had no differences between control and nitrofen-exposed hypoplastic lungs throughout gestation (Figure 7C).
Vitamin A Administration to Nitrofen-Exposed Dams In CDH, retinoic acid metabolism is compromised and could constitute the cause of MLC1a and MLC2 down-regulation at 17.5 dpc. Therefore, to investigate whether vitamin A could modulate MLC expression, nitrofen-exposed dams were treated with vitamin A in a therapeutical dose and its effects on MLC1a and MLC2 in 17.5 dpc hypoplastic fetal lungs were determined by Western blot and IHC. Vitamin A administration was able to partially recover MLC1a and MLC2 protein synthesis in either 17.5 dpc nitrofen or CDH fetal lungs (Figure 8).
The present study employed proteomics aiming to characterize major early determinants of pulmonary hypoplasia associated with CDH. As major findings, we detected that embryonic essential myosin light chain (MLC1a) and regulatory myosin light chain (MLC2) were absent in rat hypoplastic nitrofen-induced fetal lungs during pseudoglandular stage of development. We also found that MLC1a was expressed only in VSM cells of pulmonary artery, whereas MLC2 was present in PBSM cells and VSM cells of pulmonary vessels. MLC expression is most likely regulated by retinoic acid metabolism, whereas disruption of MLC1a expression during early pulmonary development led to growth and branching impairment, entailing an important role in normal lung branching morphogenesis. The above-mentioned results and the demonstration of an overall delay in SM differentiation during pseudoglandular stage in nitrofen-induced hypoplastic fetal lungs suggest an involvement of SM cells in CDH pathophysiology. Proteomics is a promising approach toward the elucidation of fundamental cell and disease biology. The unraveling of biochemical processes and pathways implicated in disease provides insights into the pathophysiology, allows detection of novel biomarkers for early diagnoses and defines new therapeutic targets (21). Currently, proteome analysis has been applied successfully to several lung diseases including cancer, emphysema, pulmonary fibrosis, cystic fibrosis, pulmonary transplantation, and acute lung injury (9). However, to the best of our knowledge, elucidation of proteomic signature of CDH fetal lungs was never attempted. In the ignorance of CDH etiology, clinically relevant CDH treatment modalities that rely on prenatal modulation of lung hypoplasia aiming to increase lung parenchyma and/or modulation of pulmonary hypertension remain the most appealing therapeutic approaches for CDH. In our opinion, early determinants of CDH comprise the most promising targets for envisaging such new clinically relevant prenatal therapeutics. Thus, the primary goal of the present study was to identify the major altered early determinants of CDH-associated hypoplastic lung development. To achieve such aim the nitrofen-induced CDH rat model was employed and proteomic analysis was performed on crude protein extracts of control and nitrofen-induced hypoplastic lungs at 17.5 dpc (end-time for early determinants action) (13, 15). Interestingly, proteomic analysis revealed that MLC1a was strongly down-regulated during early hypoplastic fetal lung development. Myosin, the main component of muscles, consists of a hexameric molecule comprising two myosin heavy chain molecules (MHC) and four myosin light chains (MLC). Different muscle types contain myosins composed of different MHC and MLC isoforms. Two types of MLC are present in any myosin molecule: phosphorylatable regulatory light chain (MLC2-type), and alkali essential light chain (MLC1-type) (22). Regulatory MLC has a catalytic function: phosphorylation of a single serine residue (Ser-19) in the N-terminus of this protein switch on actin-activated ATPase and hence the contraction of SM (22). Essential MLC is involved in actomyosin interaction, influencing MgATPase kinetics and thus affecting myosin motor (23). MLC1a is the embryonic myosin light chain isoform and has been described in fetal atrium, ventricle, and skeletal muscle (24, 25).
The present study revealed that MLC1a was present from 15.5 until 21.5 dpc of normal fetal lung development. Remarkably, MLC1a was present in VSM pulmonary cells surrounding arteries. Until now, there was no previous evidence of mammalian MLC1a expression in SM. However, the avian embryonic counterpart (L23) is mainly expressed in fetal smooth (gizzard) muscle, although it is also present in early fetal skeletal and cardiac muscle (26). Curiously, mRNA of other atrial myocardium makers such as Throughout hypoplastic nitrofen-induced fetal lung development, MLC1a was absent during early stages (17.5 dpc), while during late stages (19.5 and 21.5 dpc) MLC1a expression was recovered. The late expression of MLC1a in the pulmonary artery of hypoplastic lungs suggests a delay in vessel maturation. Moreover, CDH-associated pulmonary hypertension results from decreased number of vascular branches and increased adventitia and medial thickness of pulmonary arterial walls (5). Due to the early down-regulation of MLC1a expression in the pulmonary artery of CDH lungs, a role of MLC1a in the pathophysiology of CDH-associated vessel underdevelopment and pulmonary hypertension has to be considered. Furthermore, disruption of MLC1a expression via specific antisense oligodeoxynucleotides led to and impairment of lung growth and branching. Since MLC1a expression was restricted to pulmonary artery, the observed abrogation of lung growth is most likely related to defective vascular development, which in turn has been reported to determine the outcome of lung development (31). Furthermore, the aforementioned results suggest an involvement of MLC1a in pathophysiology of lung hypoplasia presented in CDH. To better understand the role of MLC during normal and abnormal lung development, and hypothesizing pulmonary contractibility impairment related to MLC deregulation during CDH lung development, the regulatory myosin light chain MLC2 was studied. During normal lung development MLC2 was initially observed in PBSM cells (13.5 and 15.5 dpc) surrounding the most proximal primitive epithelium. Later in gestation (17.5, 19.5, 21.5 dpc), besides PBSM cells, VSM cells presented MLC2. MLC2 centrifugal expression pattern throughout gestation is concomitant with SM differentiation during pulmonary development (32). Hypoplastic nitrofen-induced fetal lungs presented a MLC2 down-regulation at 17.5 dpc. This significant MLC2 down-regulation was not detected in the present proteomic analysis probably due to technical aspects intrinsic to this kind of assay, including sample preparation. Regarding the technical aspects of proteomic analysis, refinement of this assay for the study of CDH is currently underway. MLC2 expression was recovered during subsequent studied gestational ages (19.5 and 21.5 dpc) and remained restricted to PBSM cells and VSM cells. The deficiency of MLC1a and MLC2 in the VSM cells of the pulmonary artery during the pseudoglandular stage of lung development indicates a strong deregulation of the SM layer of the tunica media of this artery. The relevance of these findings in pulmonary hypertension associated with CDH remains to be elucidated. In vitro abrogation of MLC2 by antisense oligodeoxynucleotides was attempted. However, despite the dysplastic lung explants, protein down-regulation or total abrogation was never detected by Western blot (data not shown). One possible explanation can be the half-life and abundance of MLC2, which may not be suitable for this kind of assay. PBSM cells have an important role during lung branching morphogenesis (33). A recent report has suggested that fibroblast growth factor 10 (FGF10)-expressing cells in distal lung mesenchyme are PBSM cell progenitors that are relocated during epithelial outgrowth to the areas surrounding the proximal epithelial tubes to form PBSM cells (34). Furthermore, it has been proposed that PBSM cells participate in lung development via a stretch-induced signaling mechanism (35). The spontaneous contractile nature and rhythmic mechanical activity of PBSM cells is thought to induce stretching of pulmonary epithelium and mesenchyme (36). Moreover, this PBSM-promoted airway peristalsis was reported to a pacemaker region of the rat fetal lung and was related with pulmonary growth (37). It has been proposed that the airway peristalsis may result in the release of growth factors, signaling molecules, or regulation of gene expression with concomitant fetal lung growth and differentiation (38). In addition, PBSM cells have been proposed to be important players in the fundamental epithelial–mesenchymal signaling that regulates branching morphogenesis (33). Interestingly, FGF10, sonic hedgehog (SHH), bone morphogenic protein 4 (BMP4), retinoic acid, and extracellular matrix proteins laminin and fibronectin, besides modulating pulmonary morphogenesis, also regulate PBSM myogenesis (33).
Pulmonary hypoplasia is characterized by a decreased number of airways with smaller airspaces (4, 5). The present study provided evidence for SM disturbance during hypoplastic CDH-associated lung development. During the pseudoglandular stage of lung development, hypoplastic lungs display major interruption of MLC1a and MLC2 expression in VSM cells of the pulmonary artery and in PBSM cells, respectively. Concurrently, SM differentiation marker Several reports emphasize the association of disruption of retinoic acid metabolism and CDH pathophysiology. Maternal vitamin A deficiency induces the development of fetal diaphragmatic and pulmonary deficiencies, which can be corrected by retinoid rescue (42). Moreover, retinoic acid effects in the nitrofen-exposed rat model include decreased CDH incidence (15, 43) and pulmonary hypoplasia (15). Interestingly, in a moderate maternal vitamin A deficiency model, neonatal decreased expression of myogenic regulators Myf5 and myogenin, as well as myosin heavy chain, was reported (44). Moreover, treatment with high doses of retinoic acid induces the surrounding of distal epithelial lung tips with smooth muscle cell layer, suggesting an induction of PBSM cell differentiation (33). The above-mentioned observations proposed an expression regulation of MLC1a and MLC2 by retinoic acid. Therefore, vitamin A was administered to nitrofen-exposed dams in a therapeutical dose, and was shown to promote partial recovery of MLC1a and MLC2 expression in 17.5 dpc nitrofen-induced hypoplastic lungs. In conclusion, disruption of MLC1a expression during normal pulmonary development leads to growth and branching impairment. Furthermore, MLC1a and MLC2 expression appears to be regulated by retinoic acid. Pseudoglandular hypoplastic lungs present SM differentiation delay translated into a deficiency in contractile protein content, which might have a role among the early molecular determinants of lung hypoplasia in a rat model of nitrofen-induced CDH.
The authors thank Lucília Goreti Ribeiro Pinto and Luís Filipe Forte Oliveira Martins for technical assistance with histologic procedures.
This project was funded by Fundação para a Ciência e a Tecnologia (POCI/SAU-OBS/56428/2004). M.S. was supported by Fundação para a Ciência e a Tecnologia (reference SFRH/BD/9631/2002) through the G.A.B.B.A. Program, University of Porto, Portugal. R.S.M. was supported by Fundação para a Ciência e a Tecnologia (reference SFRH/BPD/15408/2005). S.G. was supported by Fundação para a Ciência e a Tecnologia (reference SFRH/BD/15260/2004). Originally Published in Press as DOI: 10.1165/rcmb.2006-0349OC on May 31, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form September 18, 2006 Accepted in final form April 18, 2007
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Abstract
CLINICAL RELEVANCE
