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. 2015 Apr;77(4):511-9.
doi: 10.1038/pr.2015.13. Epub 2015 Jan 12.

Altered pulmonary artery endothelial-smooth muscle cell interactions in experimental congenital diaphragmatic hernia

Shannon N Acker  1 , Gregory J Seedorf  2 , Steven H Abman  2 , Eva Nozik-Grayck  3 , Katherine Kuhn  4 , David A Partrick  5 , Jason Gien  2
Affiliations

Altered pulmonary artery endothelial-smooth muscle cell interactions in experimental congenital diaphragmatic hernia

Shannon N Acker et al. Pediatr Res. 2015 Apr.

Abstract

Background: Pulmonary hypertension (PH) secondary to vascular remodeling contributes to poor outcomes in congenital diaphragmatic hernia (CDH), however mechanisms responsible are unknown. We hypothesized that pulmonary artery endothelial cell (PAEC) dysfunction contributes to smooth muscle cell (SMC) hyperplasia in experimental CDH.

Methods: PAEC and SMC were isolated from fetal sheep with experimental CDH and controls. SMC growth was assessed alone and with SOD plus catalase and during coculture with control or CDH PAEC with and without ET-1 siRNA transfection. ET-1 protein was measured in PAEC and SMC lysates and supernatant. ROS production was measured in normal and CDH PAECs with and without ET-1 siRNA. PAEC growth and tube formation were measured with SOD plus catalase.

Results: CDH SMC growth was decreased and increased with coculture with CDH PAEC more than control PAEC. Treatment of CDH PAEC with SOD plus catalase or ET-1 siRNA prevented the increase in SMC growth seen with coculture. ET-1 protein was increased in CDH PAEC and SMC. ROS production was increased in CDH PAEC and decreased with ET-1 SiRNA. SOD plus catalase restored CDH PAEC growth and tube formation.

Conclusion: PAEC dysfunction in experimental CDH increases SMC proliferation via ET-1 induced ROS production by PAEC.

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Figures

Figure 1
Figure 1
PASMC growth in experimental CDH. In comparison with cells from control animals, PASMC from right CDH animals grew significantly slower. PASMC from left grew slower than those from the right in CDH animals (Panel A). The addition of SOD plus catalase had no effect on the growth of PASMC from either control or right CDH PASMC (Panel B; p not significant for all comparisons noted)). Bars represent standard error from the mean. Bosentan treatment had no effect on PASMC growth from either control or right CDH PASMC (Panel C; p not significant for all comparisons noted). *p<0.05.
Figure 2
Figure 2
Effect of PAEC on PASMC growth in co-culture conditions. Co-culture of CDH PAEC with control PASMC increases normal and CDH PASMC growth more than measured during co-culture with control PAEC. Bars represent standard error from the mean. *p<0.05.
Figure 3
Figure 3
Measurement of ET-1 protein levels in cellular lysates and cell culture media. ET-1 protein levels were increased in CDH PAEC (Panel A) and PASMC (Panel B) whole cell lysates when compared to controls. ET-1 protein levels in the supernatant from CDH PAEC (Panel C) and CDH PASMC (Panel D) were increased when compared to media from control cells. PPHN PASMC were used as a control as PPHN PASMC are known to produce increased levels of ET-1 compared to control PASMC. PPHN – persistent pulmonary hypertension). Bars represent standard error from the mean. *p<0.01.
Figure 4
Figure 4
SOD and catalase restores CDH PAEC growth and tube formation. CDH PAEC growth was decreased by 38% in comparison with control PAEC (Panel A). The addition of SOD plus catalase restores growth of CDH PAEC to control values. Tube formation is reduced in CDH PAEC by 18% when compared to controls. The addition of SOD plus catalase restores tube structure formation of CDH PAEC to levels of non-stimulated control PAEC (Panel B). Bars represent standard error from the mean. hpf=high power field. *p<0.05.
Figure 5
Figure 5
ROS production in PAEC. At baseline, ROS production is increased in CDH PAEC (Panel A). Transfection of CDH PAEC with ET-1 siRNA decreased ROS production to baseline levels (Panel B). Transfection with either a negative control or control siRNA did not decrease ROS production. Bars represent standard error from the mean. *p<0.05.
Figure 6
Figure 6
Role of ROS on PAEC-PASMC signaling during co-culture. SOD plus catalase treatment prevented CDH PAEC from stimulating control PASMC to grow faster than during co-culture with control PAEC (Panel A). Transfection of CDH PAEC with ET-1 siRNA also prevented CDH PAEC from stimulating control PASMC growth (Panel B). Bars represent standard error from the mean. *p<0.05.
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