Abstract 14119: Fibulin-1 Plays a Role in Smooth Muscle Cell Migration and Intimal Thickening in the Ductus Arteriosus
Introduction: Closure of the ductus arteriosus (DA) consists of two processes, i.e., vasoconstriction and intimal thickening (IT). Premature infants with patent DA (PDA), in which IT is not sufficiently formed, are often resistant to the current vasoconstriction therapies. Our previous study showed that prostaglandin E2 (PGE2)-EP4 signaling plays a critical role in IT formation via inhibition of elastic lamina formation and smooth muscle cell (SMC) migration. However, the molecular mechanisms of IT formation are not fully understood. We aimed to identify a novel molecular mechanism inducing EP4-mediated IT in the DA.
Methods: Primary culture of smooth muscle cells of the DA (DASMCs) was isolated from rat fetuses at the 21th day of gestation. SMCs were stimulated with EP4 agonist for 24 h, and extracted RNAs were subjected to microarray analysis. Expression of mRNA and protein were evaluated by quantitative RT-PCR and western blotting, respectively. Human and rat DA and aorta tissue sections were stained against anti-fibulin-1, anti-ADAMTS-1, and anti-versican fragment. Cell migration was examined by a scratch assay in DASMCs treated with fibulin-1-targeted or control siRNA in the presence or absence of EP4 agonist.
Results: Microarray analysis of DASMCs revealed that fubulin-1 was the most positively regulated gene by EP4 stimulation (28-fold, n = 4, p < 0.0005). Robust increase in fibulin-1 mRNA and protein (273-and over 20-fold, n=4, p<0.001, respectively) was confirmed, which was attenuated by protein kinase A or phospholipase C inhibitors. It has been reported that fibulin-1 binds a protease ADAMTS-1 to cleave versican, resulting in fragmented versican-mediated cell migration. In rat and human DA tissues, fibulin-1 was highly expressed in the area of IT in the DA where ADAMTS-1 and fragmented versican were also abundantly expressed. Furthermore, stimulation of EP4 induced DASMC migration (n = 6, p < 0.001), which was attenuated in DASMCs treated with fibulin-1-targeted siRNA.
Conclusions: PGE2-EP4-induced fibulin-1 was suggested to play a primary role in IT in the DA via versican fragment-mediated SMC migration. Stimulation of selective signaling pathway for fibulin-1 induction may be a new therapeutic strategy for PDA.
Author Disclosures: S. Ito: None. U. Yokoyama: None. C. Yanai: None. M. Masuda: None. T. Asou: None. Y. Ishikawa: None.
- © 2016 by American Heart Association, Inc.