The differential roles of Slit2-exon 15 splicing variants in angiogenesis and HUVEC permeability
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Slit2, a secreted glycoprotein, is down-regulated in many cancers. Slit2/Robo signaling pathway plays an important, but controversial, role in angiogenesis. We identified splicing variants of Slit2 at exon 15, Slit2-WT and Slit2-ΔE15, with differential effects on proliferation and invasive capability of lung cancer cells. The aim of this study was to elucidate the differential roles of these exon 15 splicing variants in angiogenesis. Our results revealed that both Slit2-WT and Slit2-ΔE15 inhibit motility of human umbilical vein endothelial cells (HUVECs). The conditioned medium (CM) collected from CL1-5/VC or CL1-5/Slit2-WT lung adenocarcinoma cells blocked HUVEC tube formation and angiogenesis on chorioallantoic membrane (CAM) assay when compared with untreated HUVECs and CAM, respectively. However, CM of CL1-5/Slit2-ΔE15 restored the quality of tubes and the size of vessels. Although both Slit2-WT and Slit2-ΔE15 inhibited permeability induced by CM of cancer cells, Slit2-ΔE15 exhibited stronger effect. These results suggested that Slit2-ΔE15 plays important roles in normalization of blood vessels by enhancing tube quality and tightening endothelial cells, while Slit2-WT only enhances tightening of endothelial cells. It appears that Robo4 is responsible for Slit2 isoform-mediated inhibition of permeability, while neither Robo1 nor Robo4 is required for Slit2-ΔE15-enhanced tube quality. The results of this study suggest that Slit2-ΔE15 splicing form is a promising molecule for normalizing blood vessels around a tumor, which, in turn, may increase efficacy of chemotherapy and radiotherapy.
KeywordsSlit2 splicing variants Human umbilical vein endothelial cells Angiogenesis Chicken chorioallantoic membrane assay Tube formation Permeability Robo1 Robo4
Special thanks are owed to Dr. Pinpin Lin, Dr. Jiunn-Liang Ko, Dr. Gwo-Tarng Sheu, Dr. Wen-Jun Wu, and Dr. Hui Lee for their expert consultation and advice and Jingyao Chang for financial support for this work. This project was supported by National Science Council Grant NSC 102-2320-B-040-010-MY2 (Taiwan, ROC) and an inter-institutional Grant CSMU-TTM-099004 (Taiwan, ROC) to Jinghua Tsai Chang. This research was also supported by National Science Council Grant NSC 102-2320-B040-006-MY3 to Pei-Ni Chen.
Conflict of interest
The authors declare that they have no conflicts of interest.
All procedures performed in this study comply with Taiwan legislation and were approved by the Institutional and Bioethical Use Committees (Chung Shan Medical University).
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