105 - Decreased Micro-RNA 30b Impairs Angiogenesis in Fetal Lambs with Persistent Pulmonary Hypertension Through Altered Notch Signaling

Poster Number: 105
Publication Number: 105.426

Sunil K. Sati, Medical College of Wisconsin, Brookfield, WI, United States; Ujala Rana, Medical College of Wisconsin, Milwaukee, WI, United States; Teresa Michalkiewicz, Medical College of Wisconsin, Waukesha, WI, United States; Chintamani Joshi, Medical College of Wisconsin, Milwaukee, WI, United States; Ru-Jeng Teng, Medical College of Wisconsin, Milwaukee, WI, United States; girija G. konduri, medical college of Wisconsin, Milwaukee, WI, United States

Background: Persistent pulmonary hypertension of the newborn (PPHN) is characterized by sustained elevation in pulmonary vascular resistance (PVR). Impaired angiogenesis contributes to increased PVR. Notch signaling pathway is critical to lung angiogenesis in late gestation and is modulated by micro-RNAs (miR). We recently reported that increased miR-34c in PPHN downregulates Jag1-Notch1 signaling in PPHN. RNA sequencing of PAEC revealed additional differentially regulated miR with a significant decrease in levels of miR-30b in PPHN. Targetscan prediction tool revealed delta like 4 (Dll4), a notch ligand to be a target for miR-30b. We previously reported an imbalance of notch ligand expression in PPHN with excess levels of Dll4 and decreased levels of Jag1.

Objective: We investigated whether decreased levels of miR-30b affect angiogenesis by facilitation of excess Dll4 in endothelial cells.

Design/Methods: We obtained pulmonary artery endothelial cells (PAEC) from a fetal lamb model of PPHN induced by prenatal ductus arteriosus constriction for 7 days at 80% of term gestation and sham ligation as controls. We transfected human umbilical venous endothelial cells (HUVEC) and PAEC (control and PPHN) with miR-30b mimic at 70% confluency. We confirmed transfection efficiency by PCR. We assessed protein levels by immunoblotting and in vitro angiogenesis by tube formation in Matrigel.

Results: PPHN PAECs transfected with miR-30b mimic had significant decreases in Dll4 protein and transcript compared to PAECs with scrambled sequence miR (p value < 0.05). Control HUVECs and control lamb PAECs transfected with miR-30b mimic showed similar trend for decreases in Dll4. Jag1 was increased by miR-30b mimic in control lamb PAECs but did not reach significance (p = 0.058). PCR for mRNA in HUVECs transfected with miR-30b mimic showed decreased Dll4 mRNA and increased Jag1 mRNA, consistent with protein expression changes by immunoblotting. Preliminary data on tube formation assay using Matrigel showed improved tube formation in PPHN cells transfected with miR-30b mimic compared to control cells.

Conclusion(s): Our data together suggest that a decrease in miR-30b in PPHN facilitates overexpression of Dll4, which contributes to impaired angiogenesis we observed in PPHN. MiR-30b decreases Dll4 levels and improves Jag1 levels and angiogenesis in PPHN, suggesting an important epigenetic role in PPHN.