© 2003 American Thoracic Society DOI: 10.1165/rcmb.F255
MidkineA Potential Bridge between Glucocorticoid and Retinoid Effects on Lung Vascular DevelopmentDivision of Cardiology, Department of Medicine, and Division of Neonatology, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Address correspondence to: Rashmin C. Savani, M.B.Ch.B., Abramson Research Center, Rm 416F, Children's Hospital of Philadelphia, 3516 Civic Center Boulevard, Philadelphia, PA 19104-4318. E-mail: rsavani{at}mail.med.upenn.edu
Abbreviations: glucocorticoid, GC • Retinoic acid, RA • Sonic Hedgehog, SHH • vascular endothelial growth factpor, VEGF Lung development is a highly ordered and coordinated process that results in the formation of an organ that contains sophisticated gas exchange units (alveoli), multiple innate and acquired host defense systems, and mechanisms to counteract the adverse effects of oxygen in the environment. Specific patterns of epithelial and vascular development occur during lung development, each under the regulation of a genetic hierarchy that includes pattern formation genes, transcription factors controlling cell type differentiation, and finally hormones and growth factors that regulate bud formation and vascularization. Interaction of growth factors with extracellular matrix components heavily influences the establishment of gradients that further regulate cell and tissue differentiation. The late stages of lung development, involving the maturation of the distal airways to generate the thin diffusible stratum for gas exchange, are essential for the ability of postnatal terrestrial animals to respire. Although starting out as a simple pouch of epithelial cells, the mature postnatal lung is ultimately comprised of many different cell types, most of which arise from either this epithelium or the surrounding mesenchyme. These cell types include the airway epithelium (which is itself a highly complex mixture of different epithelial lineages), various types of smooth muscle (including bronchial and vascular), and endothelial cells of the pulmonary circulation. The molecular mechanisms underlying these processes are only now becoming clear. Most importantly, as observed in other tissues, epithelial–mesenchymal crosstalk at the level of signal transduction plays an enormously important role at all stages of lung development. To appreciate the complex interaction of cell types in the lung, an understanding of the basic mechanisms and stages of lung development is required. In the mouse, the lung arises from the primitive foregut endoderm starting at approximately E9.5 during mouse development. Mesodermally derived mesenchymal cells—which will in time differentiate into several cell lineages, including bronchial and vascular smooth muscle, pulmonary fibroblasts, and endothelial cells of the vasculature—surround this primitive epithelium. During gestation, the airway epithelium evolves and grows through a process termed branching morphogenesis (reviewed in Ref. 1). This process results in the three-dimensional arborized network of airways required to generate sufficient surface area for postnatal respiration. Mouse lung development can be divided into at least four stages: embryonic (E9.5–E12.5), pseudoglandular (E12.5–E16.0), canalicular (E16.0–E17.5), and saccular/alveolar (E17.5–postnatal). Each one of these stages is marked by specific changes in the development and differentiation of lung epithelial and mesenchymal cells. It is in the last two stages that maturation in preparation of postnatal respiration proceeds rapidly. This maturation process involves the differentiation of distal airway type 1 epithelial cells, important for forming the thin diffusible interface between the airways and blood circulation, along with the simultaneous close apposition of the developing pulmonary vascular plexus, which differentiates from the adjacent mesenchyme and circulating endothelial precursors. As with earlier events such as branching morphogenesis, these late stages of lung development/maturation involve extensive epithelial–mesenchymal interaction. It is also clear that physiologic function is dependent upon appropriate development in the lung.
Several signaling pathways have been implicated in regulating late lung maturation and vascular development in the lung, including the vascular endothelial growth factor (VEGF), Sonic Hedgehog (SHH), glucocorticoid (GC), and retinoic acid (RA) systems. In particular, the GC and RA pathways appear to play very important roles in late lung maturation, and antenatal GC treatment is a standard of clinical practice in preterm labor that has successfully reduced the incidence of respiratory distress syndrome (2). Further, both GC receptor and RA receptor In this issue of AJRCMB, Kaplan and coworkers (10) report that midkine, an angiogenic product of an RA-responsive gene, is differentially regulated by the RA and the GC pathways, thus providing a "bridge" between these two pathways. Midkine was originally cloned as a 13-kD growth/differentiation factor that bound heparin. The first study linking midkine to lung development, performed by Kadomatsu and colleagues (11), demonstrated that during mouse embryogenesis, midkine mRNA was induced ubiquitously between d5 and d7, with expression becoming more restricted by d11–13 to sites including epithelial tissues of the lung. Further studies by Mitsiadis and coworkers (12) showed that RA induced midkine expression in mandibular arch mesenchyme and affected cell proliferation and morphogenesis during ex vivo tooth development. These results suggested that midkine may play an important role in epithelial–mesenchymal interactions during embryogenesis. Toriyama and colleagues (13) showed, in a lung explant culture system, that midkine played an important role in rescuing heparitinase inhibition of branching morphogenesis and thinned the mesenchyme of treated explants. Midkine caused mesenchymal thinning, but did not affect branching morphogenesis. Interestingly, thinning of the mesenchyme is an important and necessary process that occurs during late lung maturation and correlates with vascular development during the canalicular and saccular stages of lung development. Mesenchymal thinning is also important for the close apposition of the developing vascular bed with the airspaces for efficient gas exchange. By contrast, branching morphogenesis plays a more important role in the earlier pseudoglandular stage of lung development. Taken together, these data suggest an important role for midkine in late lung maturation, particularly in the process of mesenchymal thinning, which is necessary for proper vascular development and function. Kaplan's group identified midkine as a candidate for regulation by glucocorticoids based on microarray mRNA data showing increased (relative to wild-type controls) midkine mRNA in glucocorticoid receptor knockout mice and in mice homozygous for a hypomorphic allele that severely blunts glucocorticoid responses. These findings were confirmed by Northern blots, in situ hybridization, and immunohistochemistry. In addition, differential regulation of midkine by cortisol and RA was demonstrated in isolated primary rat epithelial cells and fibroblasts. Thus, in wild-type mice, midkine is normally downregulated in late gestation in conjunction with increased GC and decreased RA signals (Figure 1). However, midkine expression decreased by neonatal day 1 even in GC receptor–null mice, suggesting that additionally, either loss of positive or increase in negative regulators of midkine normally occurs at the time of birth.
Unresolved by the studies of Kaplan and coworkers (because the mice studied die at birth or soon thereafter) is how the dynamic signaling through GC and RA influences midkine gene expression during postnatal lung development in the mouse. Alveolization and the development of the alveolar vascular plexus are postnatal events in the rodent (14). Saccular walls contain two layers of endothelial cells. Secondary septation involves the invagination of the inner endothelial cell layer to initiate secondary crest formation. The invaginating layer of endothelial cells finally fuses such that mature alveolar septae contain single endothelial cells, epithelial cells, stromal components, and elastin at the tips (reviewed in Ref. 15). The result of secondary septation is a dramatic increase in alveolar surface area and decreased distances for gas diffusion. A number of observations have implicated vascular differentiation as a key component of alveolar development. For instance, fibroblast growth factor receptors 3 and 4 (FGFR3/4) double knockout mice (16), and neonatal rat pups treated with anti-angiogenic drugs such as thalidomide, fumagillin, or a VEGF receptor blocker fail to develop alveoli and retain a simplified peripheral lung architecture, which in adulthood is associated with pulmonary hypertension (17, 18). In addition, failure of alveogenesis in PDGF-A knockout animals is thought to be due to the inability of smooth muscle cells to migrate to the appropriate sites of septal formation (19). The first evidence of postnatal changes in midkine expression and the influence of RA have come from a study by Matsuura and colleagues (20) in which expression of midkine was evaluated in the first 14 d after birth in control mice and those maintained in 95% oxygen. Midkine expression increased from Day 2 to Day 7 in control mice and was decreased by hyperoxia. Hyperoxia per se was not responsible for decreased midkine expression, because midkine expression was not decreased in adult animals subjected to the same hyperoxic conditions. Interestingly, RA-treated control 14-d-old mice had increased lung midkine expression, thereby confirming the regulation of midkine by RA. However, regulation of midkine by GC and RA during postnatal alveolar development remains to be examined. Like midkine, many genes important for lung development are expressed in a defined temporal manner, including important signaling molecules such as WNTs, SHH, BMPs, FGFs (21–26), and transcription factors important for lung development such as Foxa2, TTF-1, and GATA6 (27–30). Many of these genes, like midkine, are expressed robustly in the embryonic and pseudoglandular stages of lung development and decrease during the canalicular and saccular stages. Several experiments have demonstrated that persistent expression of many of these factors results in aberrant lung development. This suggests that the precise temporal and spatial regulation of gene expression for these factors is important so as to allow distinct signaling and transcriptional pathways to initiate the necessary programs for late-stage maturation. Midkine may also act in this manner. Through its precise temporal regulation by both the GC and RA, midkine may provide an important signal through which the lungs begin to initiate the maturation process. Important experiments still remain to be performed. Muramatsu's group has generated midkine null mice that have been used to examine inflammatory processes regulating responses to injury in kidney ischemic and carotid artery endothelial injury models (31, 32). At present, it is unclear what (if any) the lung phenotype in midkine-null mice is and whether these mice have appropriate embryonic and postnatal lung vasculature. Because these mice are viable and fertile, it is likely that the phenotype is subtle. Unanswered questions include whether these mice exhibit lung developmental abnormalities associated with hormonally regulated mesenchymal differentiation/development, in particular during the postnatal period of alveolar development. Further, if the hypothesis that downregulation of midkine expression is important for lung epithelial maturation, what is the effect of forced persistent expression of midkine in the canalicular/saccular stages of lung development? These questions will need to be answered before a direct connection between midkine expression and activity and lung maturation can be drawn. Thus, we come full circle: the development of the lung is exquisitely sensitive to the appropriate temporal expression of the transcriptional and signaling molecules that regulate its development. The finding that midkine is tightly regulated in an opposing manner by the GC and RA pathways supports this notion and reiterates the important connection between development and physiologic function in the lung.
The authors thank Dr. Susan H. Guttentag for helpful comments. This work was funded by NIH grants HL64632 to E.E.M., and HL62868 and HL62472 to R.C.S. Received in original form November 4, 2002
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knockout mice develop defects consistent with defects in late lung maturation (
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