M. Shah1, M. R. Phillips1, T. Rao1, L. J. Edwards1, Y. Z. Lee1, S. E. McLean1 1University Of North Carolina At Chapel Hill,Chapel Hill, NC, USA
Introduction:
Congenital diaphragmatic hernia (CDH) is a structural birth defect associated with pulmonary hypoplasia and pulmonary arterial hypertension (PAH). Impaired angiogenesis is thought to play a role in the pathophysiology of PAH in CDH. We hypothesize that decreased angiogenesis in Slit3 knockout (KO) mice with CDH and PAH results in attenuated pulmonary arterial (PA) branching patterns.
Methods:
Slit3 wild-type (WT) and KO mice were harvested at 2-weeks, 4-weeks, and 2-3 months (adult) of age. The pulmonary arterial system was perfused with Microfil casting agent and scanned with MicroCT (Scanco). Vascular tree analysis was performed using AMIRA software, based on size and degree of vessel branching (rank) from the main pulmonary artery. Lungs were harvested for RNA isolation. Real-time PCR was performed for angiogenesis markers. Lungs were also lysed in RIPA buffer for protein isolation for use in Western Blot analysis.
Results:
There was a 2-fold decrease in FGF1 expression in KO vs. WT mice at 4-weeks, and a 2-fold decrease in HIF1A and eNOS expression in adult KO vs. WT mice. There was a 28% decrease in eNOS protein levels in KO vs. WT mice based on Western Blot. There was a trend towards fewer number of vessels in 2-week, 4-week, and adult KO vs. WT mice, with a more pronounced difference in adult mice (5404 vs. 7247, p=0.32, 8989 vs. 14229, p=0.12, 11293 vs. 21292 vessels, p=0.06, respectively). Number of ranks were similar in KO vs. WT mice at 2-weeks (61-70 vs. 51-60 ranks), with a trend towards decreased number of ranks in 4-week and adult KO vs. WT mice (61-70 vs. 81-90, 61-70 vs. 91-100 ranks, respectively). There were also fewer vessels per rank in KO vs. WT mice at 2-weeks, 4-weeks, and adult (1185 vs. 1902, 2186 vs. 3675, 2660 vs. 5311, respectively for ranks 31-40, p<0.05).
Conclusion:
Slit3 KO mice with CDH and PAH have decreased angiogenesis over time with decreased pulmonary arterial branching patterns. Further elucidation of the mechanisms of decreased angiogenesis is needed in order to further understand the mechanisms leading to PAH in CDH.