Prenatal origins of human intrapulmonary arteries formation and smooth muscle maturation

Recent studies on the morphogenesis of the pulmonary arteries have focused on nonhuman species such as the chick and the mouse. Using immunohistochemical techniques, we have studied 16 lungs from human embryos and fetuses from 28 d of gestation to newborn, using serial sections stained with a panel of antibodies specific for endothelium, smooth muscle, and extracellular matrix proteins. Cell replication was also assessed. Serial reconstruction showed a continuity of circulation between the heart and the capillary plexus from at least 38 d of gestation. The intrapulmonary arteries appeared to be derived from a continuous expansion of the primary capillary plexus that is from within the mesenchyme, by vasculogenesis. The arteries formed by continuous coalescence of endothelial tubes alongside the newly formed airway. Findings were consistent with the pulmonary arterial smooth muscle cells being derived from three sites in a temporally distinct sequence: the earliest from the bronchial smooth muscle, later from the mesenchyme surrounding the arteries, and last from the endothelial cells. Despite their different origins, all smooth muscle cells followed the same sequence of expression of smooth muscle‐specific cytoskeletal proteins with increasing age. The order of appearance of these maturing proteins was from the subendothelial cells outward across the vessel wall and from hilum to periphery. The airways would seem to act as a template for pulmonary artery development. This study provides a framework for studying the signaling mechanisms controlling the various aspects of lung development. The past few years have seen advances in our understanding of pulmonary endothelial and smooth muscle cell phenotype and function, and yet our understanding of the origins of the human pulmonary arteries is still based on the reconstructions of embryos described by Congdon in 1922 (1). Later studies showed that intrapulmonary preacinar airway and arterial branching is complete by 16 wk of gestation (2), but the origins of the capillary plexus and its connection with the pulmonary arteries have remained unclear. A recent study has shown that in the mouse, the pulmonary circulation appears to derive from two sources (3). In that study, using casts and electron microscopy, segmental arteries were shown to develop by angiogenesis from the extrapulmonary circulation, and simultaneously, the peripheral capillary plexus formed by vasculogenesis from the pulmonary mesenchyme. The communication between the two was completed at 13 to 14 d of gestation (3). In the present study, we have studied the development of the capillary plexus in human embryonic and fetal lungs on serial sections, having immunostained the endothelial cells for expression of cluster of differentiation number 31 (CD31) and von Willebrand factor. Newly formed endothelial tubes in the systemic and pulmonary circulation become invested by smooth muscle cells, but the origins of these cells are not certain. In vitro studies indicate that smooth muscle cells may be recruited by the endothelium from the surrounding mesenchyme or arise by transdifferentiation from the endothelium (4). In the systemic circulation of the developing quail embryo, mesenchymal cells associating with the endothelium express smooth muscle‐specific a -actin ( a -SM-actin) and these committed cells then express more smooth muscle cell markers as they mature (5). The phenotype of vascular smooth muscle cells varies, with adult bovine and porcine pulmonary arteries containing several different cell phenotypes, differing in their cytoskeletal composition (6, 7). In the present study, we have explored the hypothesis that the different muscle phenotypes have different developmental origins. We have examined the temporal and spatial pattern of pulmonary arterial smooth muscle maturation by studying the expression of smooth muscle‐specific, contractile, and cytoskeletal proteins using immunohistochemical labeling in the human fetal lung. Cell migration during tissue morphogenesis is regulated in part by the composition of the tissue matrix. Fibronectin is a chemoattractant for mesenchymal cells (8), and in vitro migration of smooth muscle cells is dependent on fibronectin (9). Expression of tenascin mediates cell migration and proliferation (10‐12). Versican is expressed at sites of tissue interaction in the developing fetal mouse where it regulates paths of cell migration (13, 14). Therefore, we also examined the temporal and spatial distribution of these three proteins in relation to the development of the pulmonary arteries.