Endothelial-specific Tie2 knockout mouse model - Tie2fl/fl;Cdh5-creERT2(+/−): a potent tool for inducible Tie2 deletion in vasculature across multiple organs

Abstract Background TEK receptor tyrosine kinase (Tie2) is predominantly expressed on endothelial cells and is known to play a critical role in vascular development and stability. Due to its role in vascular development, germline knockout of Tie2 is embryonically lethal. In this study we aimed to generate an inducible Tie2 knockout mouse model that specifically targets the mature endothelium. Methods Mice with floxed Tie2 exon 9 alleles ( Tie2 fl/fl ) were crossed with heterozygous Cdh5-creERT2 (+/−) mice to generate Tie2 fl/fl ; Cdh5-creERT2 (+/−) (cTie2-KO) mice. Deletion of Tie2 in the endothelium of adult cTie2-KO mice was induced by intraperitoneal injection of tamoxifen, where Tie2 fl/fl /Cre- ( Tie2 fl/fl ) mice served as controls. Five weeks after the start of treatment, organs were evaluated for Tie2 deletion at the DNA, RNA, and protein level. Another set of organs were collected for immunocytochemical analysis, to visually assess the expression levels of Tie2 within the tissue vasculature. The functional effect of the loss of Tie2 on the vasculature was evaluated by measuring vascular permeability in all organs in a separate set of mice. Results Tie2 exon 9 was deleted at the DNA level in the brains of cTie2-KO mice. All organs in cTie2-KO mice showed a significant reduction in expression of Tie2 mRNA at multiple exon levels as compared to Tie2 fl/fl controls. Total protein evaluation via ELISA showed a significant reduction of Tie2 in all tissue homogenates of cTie2-KO mice. Co-localization of Tie2 with a pan-endothelial marker demonstrated that Tie2 expression was lost in the microvascular segments of all organs evaluated, as compared to control mice. Notably, vascular permeability as measured by dextran extravasation was increased in the brain parenchyma of cTie2-KO mice as compared to control mice. Deletion of Tie2 did not affect peripheral organ permeability. Conclusions We have successfully generated a Tie2 conditional knockout mouse with significant endothelial specific deletion across multiple organs. Furthermore, at baseline we see functional deficits in cTie2-KO mice that are related specifically to blood brain barrier permeability independent of permeability in peripheral organs. This mouse model holds promise for opportunities to investigate the role of Tie2 in multiple organ vasculatures in health and disease conditions.