Endothelin-1 overexpression in mice increases vascular Vegfa 164 and Vegfa 188 alternative splice isoforms via enhanced Khdrbs3 expression

OBJECTIVES: Mice with tamoxifen-inducible endothelium-restricted human endothelin-1 (ET-1) overexpression (ieET-1) exhibited blood pressure elevation after 3 weeks and 3 months, and vascular injury after 3 months. We hypothesised that ET-1 overexpression-induced vascular injury is mediated by gene expression dysregulation. METHODS: Male ieET-1 mice and mice expressing a tamoxifen-inducible Cre recombinase in endothelial cells were induced with tamoxifen and studied 3-week and 3-month post-treatment. MicroRNA and gene expression were profiled in mesenteric arteries by RNA-sequencing and validated by RT-qPCR. Aorta was used for immunofluorescence microscopy. RESULTS: KH domain containing RNA binding signal transduction associated 3 (Khdrbs3) was the top upregulated gene identified by RNA-sequencing (P = 8x10-12) validated by RT-qPCR (P < 0.001) after 3-week and 3-month ET-1 overexpression. KHDRBS3 protein was increased in aortic endothelial and vascular smooth muscle cells (VSMCs) after 3-month ET-1 overexpression. KHDRBS3 is a splicing regulator of various mRNAs, including human vascular endothelial growth factor A (VEGFA) 165 isoform with exon 7 retention. Three-month ET-1 overexpression upregulated isoforms Vegfa164 containing exon 7 and Vegfa188 retaining exons 6 and 7, and downregulated Vegfa120 isoform lacking both exons (P < 0.05). Analysis of RNA-immunoprecipitation-sequencing of myocardium of patients with dilated cardiomyopathy identified VEGFA and four key VSMC-specific pre-mRNAs as KHDRBS3 targets. Reanalysis of RNA-sequencing data of angiotensin II-infused mice revealed a high correlation between ET-1 (Edn1) and Khdrbs3 mRNAs in mesenteric arteries (r2 = 0.82, P = 3.7 x 10-9). CONCLUSION: Endothelial human ET-1 overexpression induced RNA splicing via enhanced Khdrbs3 expression, which may play a role in vascular injury, and could represent a therapeutic target in hypertension.