Abstract 12253: Occludin as a Key Mediator in Adiponectin-Mediated Diabetic Vascular Protection

Abstract

Background: APN exerts its vascular protective effects through APPL1. However, the downstream target genes that mediate the protective effects of this signaling axis on diabetic vascular endothelium remain unclear. Methods and Results: APPL1knock-out mice were fed normal or high-fat-diet (HFD) for 10 weeks. 8 weeks after HFD, mice were randomized to receive vehicle or APN treatment for another two weeks. Aortic vessels were prepared for transcriptome analysis. Bioinformatics analysis indicates that the Tight Junction (TJ) pathway exhibited the most prominent changes. Using a global signaling transduction network showed that TJ-related genes had the highest degree values. Among these genes, PCR results indicated that Occludin was the sole gene inhibited in endothelial cells treated by HG/HL and promoted by APN treatment in an APPL1-dependent manner. The protein level of Occludin was reduced in diabetic condition, and APN treatment upregulated the expression of Occludin in APPL1-mediated manner in vivo and vitro. Endothelial specific OCLN knockdown blunted APN-mediated blood flow recovery after femoral artery ligation in vivo , and abolished APN’s effects upon HG/HL-induced permeability, and apoptosis in vitro . Mechanically, transcription factor expression and activity assay kits, and mass spectrometry techniques were employed to identify transcription factors involved in mediating the APN/APPL1 signaling axis. Through a comprehensive analysis, we discovered MYB, HDAC1, and FOXO1 play a role in the regulation of Occludin. This finding was validated by CHIP-QPCR. Western blot analysis revealed that the APN-induced increase in Occludin protein level was abolished after knockdown of MYB and FOXO1. However, knockdown of HDAC1 in endothelial cells did not alter the expression of Occludin mediated by APN. Finally, CHIP-qPCR showed that both MYB and FOXO1 participate in the enrichment of Occludin promoters mediated by APN. Conclusion: Our findings firstly indicate that the APN/APPL1 signaling axis promotes the expression of Occludin through MYB and FOXO1, ultimately exerting a protective effect on vascular endothelium.