Angiogenesis and morphogenesis of murine fetal distal lung in an allograft model

Abstract

Neovascularization is crucial to lung morphogenesis; however, factors determining vessel growth and formation are poorly understood. The goal of our study was to develop an allograft model that would include maturation of the distal lung, thereby ultimately allowing us to study alveolar development, including microvascular formation. We transplanted 14-day gestational age embryonic mouse lung primordia subcutaneously into the back of nude mice for 3.5-14 days. Lung morphogenesis and neovascularization were evaluated by light microscopy, in situ hybridization, and immunohistochemical techniques. Embryonic 14-day gestational age control lungs had immature structural features consistent with pseudoglandular stage of lung development. In contrast, 14 days after subcutaneous transplantation of a 14-day gestational age lung, the allograft underwent significant structural morphogenesis and neovascularization. This was demonstrated by continued neovascularization and cellular differentiation, resulting in mature alveoli similar to those noted in the 2-day postnatal neonatal lung. Confirmation of maturation of the allograft was provided by progressive type II epithelial cell differentiation as evidenced by enhanced local expression of mRNA for surfactant protein C and a threefold (P < 0.008) increase in vessel formation as determined by immunocytochemical detection of platelet endothelial cell adhesion molecule-1 expression. Using the tyrosine kinase Flk-1 receptor (flk-1) LacZ transgene embryos, we determined that the neovascularization within the allograft was from the committed embryonic lung endothelium. Therefore, we have developed a defined murine allograft model that can be used to study distal lung development, including neovascularization. The model may be useful when used in conjunction with an altered genetic background (knockout or knock in) of the allograft and has the further decided advantage of bypassing placental barriers for introduction of pharmacological agents or DNA directly into the lung itself.

Differentiation of xenografted human fetal lung parenchyma

“…Saccular stage-like changes were observed in 11 weeks post-engraftment as compared to 3 weeks in our study. The accelerated timing observed in the present study is comparable to the subcutaneous mouse allograft model, where E14.5 mouse fetal lungs required two weeks to differentiate to the saccular stage (Schwarz et al, 2000). On the other hand, both of these are in contrast to the renal subcapsular mouse allograft model (Vu et al, 2003), where timing of developmental stages of E12.5 mouse embryonic lungs closely follows that of in utero development.…”

“…Various xenograft models have been used extensively in cancer research to study mechanisms of carcinogenesis and the effects of different pharmacological agents on human tumor growth (Giovanella and Fogh, 1985;Malkinson, 2001), as well as in studies of transmission of infection in various tissues by pathogens (Howett et al, 1997;Howett et al, 1990;Howett et al, 1999;Kish et al, 2001;Kreider et al, 1985). With respect to lung embryogenesis, immunocompromised mice have been used to study whole organ lung development in mice (Schwarz et al, 2000;Vu et al, 2003), as well as lower (Groscurth and Tondury, 1982;Peault et al, 1994) and upper airway development in humans (Delplanque et al, 2000;Deutsch, 1997;Engelhardt et al, 1993;Filali et al, 2002;Pilewski et al, 1994;Puchelle and Peault, 2000). Allograft models have been used where whole embryonic mouse lungs were grafted into severe-combined immunodeficient (SCID) mice (Schwarz et al, 2000;Vu et al, 2003).…”

“…With respect to lung embryogenesis, immunocompromised mice have been used to study whole organ lung development in mice (Schwarz et al, 2000;Vu et al, 2003), as well as lower (Groscurth and Tondury, 1982;Peault et al, 1994) and upper airway development in humans (Delplanque et al, 2000;Deutsch, 1997;Engelhardt et al, 1993;Filali et al, 2002;Pilewski et al, 1994;Puchelle and Peault, 2000). Allograft models have been used where whole embryonic mouse lungs were grafted into severe-combined immunodeficient (SCID) mice (Schwarz et al, 2000;Vu et al, 2003). Progression of structural development as well as appearance of mature alveoli was observed 14 days after grafting.…”

“…Based on the knowledge available from a number of lung xenograft models (Cobb, 1975;Delplanque et al, 2000;Deutsch, 1997;Engelhardt et al, 1993;Filali et al, 2002;Groscurth and Tondury, 1982;Peault et al, 1994;Phillips and Gazet, 1969;Pilewski et al, 1994;Puchelle and Peault, 2000;Schwarz et al, 2000;Vu et al, 2003), we hypothesized that human fetal lung parenchyma grafted into immunocompromised mice will undergo changes that mimic the stages of in utero lung development in many respects. The assumption was that although grafted tissue may undergo accelerated maturation, this acceleration would not be as rapid as that seen in fetal lung explants, enabling one to study stage-specific processes of lung development.…”

Pulmonary vascular development in congenital diaphragmatic hernia

“…We and others have shown that very early in lung development pulmonary vessels are present and connected to the systemic circulation. The vasculature develops in close relation with the airways and is a rate-limiting factor in branching morphogenesis [2][3][4]. This implies that the pulmonary vasculature plays an important role in lung development.…”