MO725DAPT ALLEVIATES CKD-RELATED CAC BY MODULATING PTH-INDUCED ENDOTHELIAL-TO-OSTEOBLAST TRANSITION VIA NOTCH1 PATHWAY ACTIVATION

Abstract

Abstract Background and Aims Coronary artery calcification (CAC)-induced myocardial infarction (MI) is an important cause of death in patients with chronic kidney disease (CKD). However, effective treatment for CAC is lacked at present. Previous studies have shown that endothelial cells (ECs) participated in vascular calcification through endothelial-to-osteoblast transition. DAPT, N-[N-(3,5-difluorophenacetyl)-l-alanyl]- S-phenylglycine t-butyl ester, could inhibit the activity of γ-secretase and block the activation of the Notch1 pathway. In this study, we investigated the function of DAPT in alleviating the CAC process by blocking endothelial-to-osteoblast transition via inhibition of the Notch1 pathway. Method We administered 5/6 subtotal nephrectomy and a 10-week high-phosphate diet (P, 2.0%) to construct a rat model of CKD. DAPT and AAV-129-5p was administered orally and injected abdominally to rats respectively in the treatment groups at the beginning of the high-phosphate diet. In vivo, it was performed to detect the expression levels of EndMT and Notch1 pathway markers in the coronary arteries. In vitro, the effect of high PTH levels on the endothelial-to-osteoblast transition and the role of the miR-129-5p/Notch1 signaling pathway were studied in human coronary artery endothelial cells (HCAECs). Results In vivo, endothelial-to-osteoblast transition accompanied with the Notch1 pathway activation was found in HCAECs upon stimulation of PTH, characteristic with up-regulated endothelial markers (CD31, CD34) and down-regulated mesenchymal markers (CD44, CD10, α-SMA, FSP1) and ostoblast markers (Runx2, Osterix). miR-129-5p was responsible for regulating Notch1; γ-secretase was time-dependently and concentration-dependently activated by PTH, which further affected the transcription of downstream regulators (HES1, HEY1). DAPT arrested HCAECs migration through decreasing γ-secretase activity, thus inhibiting endothelial-to-osteoblast transition. In vivo data showed that serum γ-secretase activity decreased in rats intraperitoneally injected with DAPT (10mg/kg) once a week after 5/6 nephrectomy. DAPT intervention or overexpression of mir-129-5p inhibited coronary endothelial-to-osteoblast transition by blocking the activation of the Notch1 pathway. Notably, DAPT retarded CAC and MI without obvious negative effects on rats heart function. Conclusion DAPT is a promising agent for protecting against PTH-induced endothelial-to-osteoblast transition via inhibiting the Notch1 pathway in HCAECs, thus alleviating CAC.