Abstract
Purpose: :
Vascular development requires endothelial cells (EC) to migrate, differentiate, and assemble. We sought to define the effects of high oxygen levels, 95% oxygen/5% carbon dioxide, on functional aspects of vascular development in vivo and in vitro in the dog.
Methods: :
Dogs were exposed to hyperoxia and normoxia from postnatal day (P) 1 to P5 and then sacrificed. TdT–mediated dUTP Nick End Labeling (TUNEL) and von Willebrand factor (vWF) immunostaining were performed on retinas from P5 dogs to identify apoptotic EC. Adult dog retinal EC migration in hyperoxia was evaluated with an in vitro wound healing assay described by Lutty et al. (1998). Secretion of alkaline phosphatase (SEAP) driven by NFkB, CREB, and constitutively active promoters was determined by transfecting EC with the plasmids and quantified with pNPP substrate at 405nm.
Results: :
In hyperoxia–exposed retinas, vaso–obliteration was evident. Apoptotic EC were observed in whole mount retinas using immunofluorescence. The vast majority of apoptotic cells were found in peripheral microvessels. In vitro studies showed that 24 hours oxygen exposure greatly increased apoptosis and significantly decreased EC migration into the wound by 683um or 39%, when compared to normoxia control cells (p < 0.0001). Protein levels (BioRad) in ADRECs were increased by 2.7 fold after 24 hours oxygen exposure compared to that in normoxia control cells. Cells transfected with a constitutively expressed secreted SEAP showed significantly decreased SEAP activity when exposed to hyperoxia for 12 and 24 hours [34.5%, p = 0.0035; and 38.5%, p = 0.0084, respectively] compared to the normoxic controls. Increased NFkB and PKA/CREB pathway activities were detected after 24 hours oxygen exposure.
Conclusions: :
In vivo data supports the in vitro evidence that vaso–obliteration occurs through apoptosis. In vitro evidence suggests that vascular development in hyperoxia is inhibited through decreased migration and disruption of EC function. This may be due to intracellular signaling changes that occur in less than 12 hours, ultimately resulting in decreased secretion and increased protein expression; indicating a substantial change in cellular function.
Keywords: retinopathy of prematurity • retinal development • signal transduction