Extended Data Fig. 10: Postnatal persistent PDA is induced by inhibition of SPHK1 signaling, which is independent of prostaglandin E pathway.
From: Sphingosine kinase 1 is integral for elastin deficiency-induced arterial hypermuscularization
(a) Experimental strategy for (b, c). Pregnant dams bearing wild-type embryos were injected daily from E13.5-17.5 with SPHK1 inhibitor (PF-543, 20 mg/kg) or vehicle and subjected to cesarean section at E18.5 to standardize the time of delivery. Neonates were kept alive and warm on a heating pad and harvested at postnatal ages up to 6 h as indicated to evaluate the kinetics of postnatal DA closure. Graphical objects were created with BioRender (https://biorender.com/). (b) Representative images of transverse sections of DA at postnatal 6 h stained for markers of SMCs (SMA), ECs (CD31), and nuclei (DAPI). Scale bar, 200 μm. (c) Medial wall and lumen area of DA at indicated time points after cesarean section. For the medial wall area, at sequential timepoints from 0 min to 6 h, n = 10, 8, 8, 7 mice with vehicle; n = 10, 10, 10, 10 mice with PF-543. For the lumen area, at sequential timepoints from 0 min to 6 h, n = 10, 8, 8, 6 mice with vehicle; n = 10, 10, 10, 10 mice with PF-543. (d, e) Descending aortic and DA SMCs were isolated from wild-type mice at P0.5. In d, SMCs were treated with either vehicle (DMSO), PGE2 (10−6 M), or indomethacin (10−5 M), and lysates were subjected to qRT–PCR for fibulin 1 (Fbln1), Egr1, and Sphk1. For Fbln1 expression, n = 7 aortic SMCs per group, n = 6 DA SMCs per group, biological replicates. For Egr1 expression, n = 7 aortic SMCs per group, n = 7 DA SMCs per group, biological replicates. For Sphk1 expression, n = 7 aortic SMCs with vehicle; n = 6 aortic SMCs with PGE2; n = 6 aortic SMCs with indomethacin; n = 7 DA SMCs with vehicle; n = 7 DA SMCs with PGE2; n = 6 DA SMCs with indomethacin, biological replicates. In e, SMCs were treated with Scr RNA or siSphk1, and lysates were subjected to qRT–PCR for Sphk1, Fbln1, and cyclooxygenase 2 (Cox2). For Sphk1 expression, n = 5 aortic SMCs with Scr RNA; n = 6 aortic SMCs with siSphk1; n = 5 DA SMCs with Scr RNA; n = 6 DA SMCs with siSphk1, biological replicates. Bar graphs are shown as mean ± SD. P values were determined by two-tailed unpaired Student’s t test (c) or one-way ANOVA with Tukey’s post hoc test (d, e).
