Tissue-type plasminogen activator contributes to remodeling of the rat ductus arteriosus

Abstract

AimsThe ductus arteriosus (DA) closes after birth to adapt to the robust changes in hemodynamics, which require intimal thickening (IT) to occur. The smooth muscle cells of the DA have been reported to play important roles in IT formation. However, the roles of the endothelial cells (ECs) have not been fully investigated. We herein focused on tissue-type plasminogen activator (t-PA), which is a DA EC dominant gene, and investigated its contribution to IT formation in the DA.Methods and resultsECs from the DA and aorta were isolated from fetal rats using fluorescence-activated cell sorting. RT-PCR showed that the t-PA mRNA expression level was 2.7-fold higher in DA ECs than in aortic ECs from full-term rat fetuses (gestational day 21). A strong immunoreaction for t-PA was detected in pre-term and full-term rat DA ECs. t-PA-mediated plasminogen-plasmin conversion activates gelatinase matrix metalloproteinases (MMPs). Gelatin zymography revealed that plasminogen supplementation significantly promoted activation of the elastolytic enzyme MMP-2 in rat DA ECs. In situ zymography demonstrated that marked gelatinase activity was observed at the site of disruption in the internal elastic laminae (IEL) in full-term rat DA. In a three-dimensional vascular model, EC-mediated plasminogen-plasmin conversion augmented the IEL disruption. In vivo administration of plasminogen to pre-term rat fetuses (gestational day 19), in which IT is poorly formed, promoted IEL disruption accompanied by gelatinase activation and enhanced IT formation in the DA. Additionally, experiments using five human DA tissues demonstrated that the t-PA expression level was 3.7-fold higher in the IT area than in the tunica media. t-PA protein expression and gelatinase activity were also detected in the IT area of the human DAs.Conclusiont-PA expressed in ECs may help to form IT of the DA via activation of MMP-2 and disruption of IEL.

Role of Tissue-Type Plasminogen Activator in Remodeling of the Ductus Arteriosus

“…44 We also confirmed the expression of t-PA on quantitative polymerase chain reaction using isolated DA endothelial cells and aortic endothelial cells from full-term rat fetuses (embryonic day 21). 43 In accordance with our previous microarray data, t-PA (Plat) mRNA was highly expressed in DA endothelial cells compared with aortic endothelial cells (Figure 2A). 43 We further examined the expression of t-PA in human DA.…”

“…43 In accordance with our previous microarray data, t-PA (Plat) mRNA was highly expressed in DA endothelial cells compared with aortic endothelial cells (Figure 2A). 43 We further examined the expression of t-PA in human DA. 43 Although RNA sequencing of the human DA was recently reported, 46 there has been no transcriptional profiling of the human DA intimal thickening region that contains DA endothelial cells.…”

“…43 We further examined the expression of t-PA in human DA. 43 Although RNA sequencing of the human DA was recently reported, 46 there has been no transcriptional profiling of the human DA intimal thickening region that contains DA endothelial cells. We collected human DA from neonatal patients with congenital heart disease and examined the expression of t-PA in the intimal thickening region compared with the tunica media.…”

“…Neither DA endothelial cells nor aortic endothelial cells had detectable MMP-9 expression. 43 This suggested that the conversion of plasminogen to plasmin in DA endothelial cells promotes the activation of MMP-2. 43 Next, we confirmed the gelatinase activity in the DA tissue of rats and humans.…”

“…43 This suggested that the conversion of plasminogen to plasmin in DA endothelial cells promotes the activation of MMP-2. 43 Next, we confirmed the gelatinase activity in the DA tissue of rats and humans. In addition to gelatin zymography, prominent gelatinase activity was seen on in situ zymography in the intimal thickening region, which contains endothelial cells, and in the disrupted IEL of the DA (Figure 3C,D).…”