During embryogenesis, the lung develops from an avascular bud into a complex organ with two complete circulatory systems. Although extracellular matrices (ECMs) and growth factors have been implicated in vascular formation in vitro, the mechanisms and molecules involved in the vascularization of the lung are largely unknown. Preliminary observations in our laboratory revealed that cells expressing markers for endothelial cells (EC) are present in prevascular murine lungs during the early pseudoglandular stage of lung development. These cells increased in number with maturation, became coalescent with other EC, and organized into vascular structures at 16 days of gestation. Thus, lung vasculogenesis appears to involve the differentiation of mesenchymal cells into EC during the pseudoglandular stage, and the proliferation and organization of these cells into vascular structures during the latter stages of lung development. These events are likely to be driven by compositional changes in the ECM. This is supported by the following observations: 1) The differentiation and organization of ECs in developing lungs coincide with dramatic alterations in ECM composition characterized by, among other things, an increase in fibronectin expression during the pseudoglandular stage followed by a decrease thereafter. 2) Fibronectin induces the expression of markers of EC differentiation (i.e., von Willebrand's Factor, FKL-1) in lung mesenchymal cells. 3) Matrigel (a complex matrix containing large concentrations of laminin) stimulates mesenchymal cells obtained from pseudoglandular stage lungs to form primitive capillary-like structures. This stimulatory effect is inhibited in a dose-dependent manner by fibronectin. 4) Matrigel-induced vasculogenesis can also be inhibited by the pretreatment of EC with antisense oligos to beta1 matrix-binding integrin receptors. Overall, our observations have led us to hypothesize that increased concentrations of fibronectin in pseudoglandular stage lungs drive the differentiation (and proliferation) of mesenchymal cells into EC, but prevent their organization into vascular structures. The subsequent decrease in fibronectin allows for the organization of EC into vascular structures during late lung development. This proposal is designed to investigate this hypothesis in a novel model of murine lung vasculogenesis. Moreover, we intend to further develop this model in a rotating bioreactor that provides reduced hydrodynamic shear stress, high mass transfer of nutrients and wastes, optimal oxygen transfer, and growth of cells to extremely high cell densities in three dimensions.
| Walia, Baljit; Castaneda, Florencia E; Wang, Lixin et al. (2004) Polarized fibronectin secretion induced by adenosine regulates bacterial-epithelial interaction in human intestinal epithelial cells. Biochem J 382:589-96 |
