Abstract P312: Three-Month Human Endothelin-1 Endothelial Overexpression Up-Regulated Khdrbs3 And Caused Vegfa And Nrp2 Alternative Splicing In Mesenteric Arteries Of Male Mice

Abstract

Background: Transgenic mice with tamoxifen-inducible endothelium-restricted human endothelin-1 overexpression (ieET-1) exhibited blood pressure (BP) elevation 3 weeks or 3 months after induction, and vascular injury was observed after 3 months. We hypothesized that 3-week or 3-month exposure to ET-1 overexpression leads to gene dysregulation in mesenteric arteries (MAs). Methods: Ten to 12-week old male ieET-1 mice and control ieCre mice expressing a tamoxifen-inducible Cre recombinase under the control of the endothelium-specific Tie2 promoter were treated with tamoxifen (1 mg/kg/day, s.c.) for 5 days and euthanized 16 days or 3 months later. RNA was extracted from MAs and used for total RNA-sequencing using Illumina HiSeq-2500. Differentially expressed (DE) genes were identified with fold change >1.3 and P <0.005. DE genes were validated by reverse transcription-quantitative PCR (RT-qPCR) using another set of mice. Alternative splicing was revealed by Spladder and validated by RT-qPCR in MAs. Results: RNA-sequencing revealed DE genes after 3-week (54) and 3-month ET-1 overexpression (7). Alternative splicing changes in mRNAs were revealed after 3-week (39) and 3-month ET-1 overexpression (21). One of the 3 genes validated by RT-qPCR, Khdrbs3, encodes KH domain containing RNA binding signal transduction associated 3 that regulates exon retention of some mRNAs including exon 7 of vascular endothelial growth factor A ( Vegfa ). Khdrbs3 was up-regulated after 3-week (fold change: 2.4±0.1 vs 1.0±0.1, P <0.05) and 3-month ET-1 overexpression (2.3±0.3 vs 1.3±0.1, P <0.05). Vegfa exon 7 retention was validated by demonstration of up-regulation of Vegfa 164 (1.3±0.1 vs 0.9±0.1, P<0.05) and Vegfa 188 isoforms (1.1±0.1 vs 0.9±0.1, P<0.05) after 3-month ET-1 overexpression ( P <0.05). Neuropilin 2 ( Nrp2 ) that interacts with VEGFA receptors was one of DE genes with alternative splicing in exon 17 ( Nrp2 a isoforms). Predicted Nrp2 a down-regulation ( P <0.05) was validated by RT-qPCR showing a decrease after 3-month ET overexpression (0.6±0.1 vs 1.1±0.2, P <0.05). Conclusions: This study demonstrated that 3-month ET-1 overexpression up-regulated Khdrbs3 and alternative splicing of Vegfa and Nrp2 , which may play a role in vascular injury in hypertension.