Pulmonary vascular remodeling is a process generally associated with pulmonary hypertension that involves intimal thickening, medial hyperthrophy, and plexiform lesions. Morphological studies during pulmonary hypertension have indicated that intimal thickening consists of immature smooth muscle cells (SMCs) associated with determined extracellular matrix components, suggesting an important role for these cells in vascular lesions. Controversy exists regarding the nature and origin of the cells conforming the intimal thickenings. In this study, the authors characterized the in vivo phenotype of the cells located in the pulmonary artery wall during the advanced stages of chicken embryo development and examined whether intimal thickenings are present in such stages. Immunolabeling of cryosections demonstrated presence of intimal thickenings composed of mesenchymal cells that may arise from the endothelium. These cells persist either as nonmuscle throughout the development, or possibly convert to cells expressing alpha -smooth muscle actin (alpha-SM actin). To determine whether pulmonary endothelial cells undergo a transition to mesenchymal cells, the authors used pulmonary artery explants from 10- to 11-day-old chicken embryos and found that explanted endothelial cells detached from the monolayer and acquired mesenchymal characteristics. Some of these cells maintained immunoreactivity for von Willebrand factor (vWF), whereas other jointly lost vWF and gained alpha -SM actin expression (transitional cells), suggesting conversion to SMCs. Therefore, these findings strongly support the authors' in vivo observations and demonstrate that embryonic pulmonary endothelial cells undergo a transition to mesenchymal cells and participate in intimal thickening formation and pulmonary vascular remodeling.

Members of the family of large chondroitin sulfate proteoglycans (CSPGs), such as versican and aggrecan, are involved in early heart development, and in the development and progression of atherosclerosis and restenosis. Given the important roles played by versican and aggrecan in such processes, we sought to determine whether these molecules are present in the aortic wall during the advanced stages of chicken embryo development and the endothelial-mesenchymal transformation (EMT). Immunolabeling of serial cryosections revealed versican immunoreactivity around the cells within the intimal thickening, and the cells organized in lamellar and interlamellar cell layers. In contrast, a weak aggrecan immunoreactivity was limited to the cells arranged into lamellar and interlamellar cell layers. Immunolabeling also demonstrated that V2 is the main versican isoform present at the intimal thickening. According to immunoblotting analysis, the aggrecan content was very low in all stages examined, and two versican isoforms (V0 and V2) were present at day 14 of development. We also investigated whether versican isoforms were present during EMT in vitro. Versican immunoreactivity was detected in patches of endothelial cells; in the detaching and migrating cells, and the extracellular matrix (ECM) deposited by them; and in cells that had acquired mesenchymal characteristics. These data indicate that versican and aggrecan have different spatial and temporal patterns of expression, and they have different functions during remodeling of the aortic wall. Also, the different immunoreactivity and immunolocalization patterns observed for versican both in vivo and in vitro, in addition to being associated with the presence of different versican isoforms, may be related to the predominance of the V2 isoform during intimal thickening formation and EMT. © 2004 Wiley-Liss, Inc. Key words: aorta; remodeling; endothelial-mesenchymal transformationEndothelial-mesenchymal transformation (EMT) is believed to play a crucial role in embryonic and adult vascular development, and the development of intimal lesions

The serine protease thrombin, independently of its participation in hemostasis and thrombosis, has been involved in tissue repair and remodeling, embryogenesis, angiogenesis, and development and progression of atherosclerosis. Many of these functions appear to be mediated by specific thrombin receptors, particularly the protease-activated receptor-1 (PAR1). In this study, we investigated whether both thrombin and PAR1 were present in the aortic wall of chicken embryos at days 11 and 12 of development. We found that PAR1 was limited to some cells of the intimal thickening and the inner media, whereas thrombin appeared distributed accross the aortic wall. We also investigated whether PAR1 was present during endothelial-mesenchymal transdifferentiation (EMT) in vitro. A moderate immunoreactivity was detected in the monolayer of endothelial cells. In contrast, a strong cytoplasmic immunoreactivity was observed in the detaching and migrating cells and those that had acquired mesenchymal characteristics. This PAR1 expression was confirmed by flow cytometry. In this study, the addition of thrombin to arrested endothelial cell cultures was assessed. We found that thrombin stimulated endothelial cell spreading and migration, as no migrating cells were observed in serum-free medium (SFM) condition. Immunolocalization of PAR1 in the thrombin-treated cultures showed strong cytoplasmic immunoreactivity in the monolayers and in spreading and migrating cells, whereas in the SFM condition undetectable PAR1 immunoreactivity was observed. Flow cytometry of these cultures revealed an elevated expression of PAR1 in the presence of thrombin, in contrast to that detected in SFM and complete medium. These data indicate that both thrombin and PAR1 are involved in the remodeling of the aortic wall and intimal thickening formation, and in the endothelial-mesenchymal transdifferentiation process. 815

The serine protease thrombin, independently of its participation in hemostasis and thrombosis, has been involved in tissue repair and remodeling, embryogenesis, angiogenesis, and development and progression of atherosclerosis. Many of these functions appear to be mediated by specific thrombin receptors, particularly the protease-activated receptor-1 (PAR1). In this study, we investigated whether both thrombin and PAR1 were present in the aortic wall of chicken embryos at days 11 and 12 of development. We found that PAR1 was limited to some cells of the intimal thickening and the inner media, whereas thrombin appeared distributed across the aortic wall. We also investigated whether PAR1 was present during endothelial-mesenchymal transdifferentiation (EMT) in vitro. A moderate immunoreactivity was detected in the monolayer of endothelial cells. In contrast, a strong cytoplasmic immunoreactivity was observed in the detaching and migrating cells and those that had acquired mesenchymal characteristics. This PAR1 expression was confirmed by flow cytometry. In this study, the addition of thrombin to arrested endothelial cell cultures was assessed. We found that thrombin stimulated endothelial cell spreading and migration, as no migrating cells were observed in serum-free medium (SFM) condition. Immunolocalization of PAR1 in the thrombin-treated cultures showed strong cytoplasmic immunoreactivity in the monolayers and in spreading and migrating cells, whereas in the SFM condition undetectable PAR1 immunoreactivity was observed. Flow cytometry of these cultures revealed an elevated expression of PAR1 in the presence of thrombin, in contrast to that detected in SFM and complete medium. These data indicate that both thrombin and PAR1 are involved in the remodeling of the aortic wall and intimal thickening formation, and in the endothelial-mesenchymal transdifferentiation process.