Abstract 500: Endothelial Specific Meis1 Knockout Protects Cells from Doxorubicin-Induced Apoptosis

Abstract

Introduction: Meis1 belongs to the TALE (three amino-acid loop extension) subclass of the homeobox gene families. It is a highly conserved transcription factor in all eukaryotes and plays a crucial role in anatomical development during embryogenesis and maintaining homeostasis after birth. However, little is known about the role of Meis1 gene on regulating functions of endothelial cells (ECs). Our preliminary study found that EC-specific knockout (KO) of Meis1 showed protective benefits in response to hindlimb ischemia, such as increased blood flow and decreased toe apoptosis. We hypothesize that deletion of Meis1 may enhance EC viability or resistance to harsh environment. The present study aims to explore the potential underlying mechanisms using an in vitro apoptotic condition induced by doxorubicin (Dox). Methods: EC-specific deletion of Meis1 gene was generated by crossbreeding Meis1 flox/flox mice with Tie2-Cre mice. Following confirmation of genotyping, ECs were isolated from the lungs of 3-5 WT and KO newborn pups using CD31 microbeads. Cells were then plated and treated with 0, 0.5, and 1 μM Dox for 24 hours prior to various assays. Results: Our qPCR data indicated that KDR expression was ~100-times more in CD31 + fraction than in CD31 - fraction both in WT and KO groups (n=4); while in KO group, Meis1 expression was significantly decreased to 20% of WT control, suggesting the successful isolation of Meis1-KO ECs. Further analysis with immunocytochemistry indicated that 83.1±2.3% CD31 + cells were also KDR + , compared to 8.3±1.2% KDR + cells in CD31 - fraction (n=7-8). Using EC-specific dye uptake assay, we found that 84.6±2.4% CD31 + cells were positive for Dil-Ac-LDL, compared to 7.9±3.2% in CD31 - cells (n=6), which appears to correspond to more tube formation observed in KO than WT groups. As we expected, Meis1-KO ECs demonstrated greater cell viability (81.2±6.1%) than WT cells (58.0±1.6%) under 0.5 μM Dox treatment (n=5-6). The increased viability in KO ECs may be due to the decreased expression of Bax , a pro-apoptotic factor. Conclusions: We conclude that activation of Meis1 gene may facilitate EC death under ischemic condition while deletion of Mei1 gene promote protective effects on ECs.