Epsin deficiency promotes lymphangiogenesis through regulation of VEGFR3 degradation in diabetes
Hao Wu, H.N. Ashiqur Rahman, Yunzhou Dong, Xiaolei Liu, Yang Lee, Aiyun Wen, Kim H.T. To, Li Xiao, Amy E. Birsner, Lauren Bazinet, Scott Wong, Kai Song, Megan L. Brophy, M. Riaj Mahamud, Baojun Chang, Xiaofeng Cai, Satish Pasula, Sukyoung Kwak, Wenxia Yang, Joyce Bischoff, Jian Xu, Diane R. Bielenberg, J. Brandon Dixon, Robert J. D’Amato, R. Sathish Srinivasan, Hong Chen
Hao Wu, H.N. Ashiqur Rahman, Yunzhou Dong, Xiaolei Liu, Yang Lee, Aiyun Wen, Kim H.T. To, Li Xiao, Amy E. Birsner, Lauren Bazinet, Scott Wong, Kai Song, Megan L. Brophy, M. Riaj Mahamud, Baojun Chang, Xiaofeng Cai, Satish Pasula, Sukyoung Kwak, Wenxia Yang, Joyce Bischoff, Jian Xu, Diane R. Bielenberg, J. Brandon Dixon, Robert J. D’Amato, R. Sathish Srinivasan, Hong Chen
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Research Article Angiogenesis Vascular biology

Epsin deficiency promotes lymphangiogenesis through regulation of VEGFR3 degradation in diabetes

Abstract

Impaired lymphangiogenesis is a complication of chronic complex diseases, including diabetes. VEGF-C/VEGFR3 signaling promotes lymphangiogenesis, but how this pathway is affected in diabetes remains poorly understood. We previously demonstrated that loss of epsins 1 and 2 in lymphatic endothelial cells (LECs) prevented VEGF-C–induced VEGFR3 from endocytosis and degradation. Here, we report that diabetes attenuated VEGF-C–induced lymphangiogenesis in corneal micropocket and Matrigel plug assays in WT mice but not in mice with inducible lymphatic-specific deficiency of epsins 1 and 2 (LEC-iDKO). Consistently, LECs isolated from diabetic LEC-iDKO mice elevated in vitro proliferation, migration, and tube formation in response to VEGF-C over diabetic WT mice. Mechanistically, ROS produced in diabetes induced c-Src–dependent but VEGF-C–independent VEGFR3 phosphorylation, and upregulated epsins through the activation of transcription factor AP-1. Augmented epsins bound to and promoted degradation of newly synthesized VEGFR3 in the Golgi, resulting in reduced availability of VEGFR3 at the cell surface. Preclinically, the loss of lymphatic-specific epsins alleviated insufficient lymphangiogenesis and accelerated the resolution of tail edema in diabetic mice. Collectively, our studies indicate that inhibiting expression of epsins in diabetes protects VEGFR3 against degradation and ameliorates diabetes-triggered inhibition of lymphangiogenesis, thereby providing a novel potential therapeutic strategy to treat diabetic complications.

Authors

Hao Wu, H.N. Ashiqur Rahman, Yunzhou Dong, Xiaolei Liu, Yang Lee, Aiyun Wen, Kim H.T. To, Li Xiao, Amy E. Birsner, Lauren Bazinet, Scott Wong, Kai Song, Megan L. Brophy, M. Riaj Mahamud, Baojun Chang, Xiaofeng Cai, Satish Pasula, Sukyoung Kwak, Wenxia Yang, Joyce Bischoff, Jian Xu, Diane R. Bielenberg, J. Brandon Dixon, Robert J. D’Amato, R. Sathish Srinivasan, Hong Chen

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Figure 3

AP-1 drives upregulation of epsin expression via c-Src activation and VEGFR3 downregulation under diabetic conditions.

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AP-1 drives upregulation of epsin expression via c-Src activation and VE...
(A) RT-qPCR for epsins 1 and 2 in LECs isolated from diabetic patients compared with nondiabetic persons (left panel) and in primary LECs from WT/STZ/HFD mice compared with WT (right panel). (B) RT-qPCR of VEGFR3 in both WT and LEC-iDKO primary LECs. (C) Western blot of epsins 1 and 2 in primary LECs from STZ-induced diabetic mice and normal WT mice. (D) Schematic diagram of the upregulation of epsins through c-Src under diabetic conditions. (E) Schematic diagram showing the transcription factor AP-1 binding sequence located at human and mouse epsin 1 promoter regions. (F) Western blot of WT mouse LECs with c-Src knockdown by c-Src siRNA (upper panel); c-Src and phosphorylated c-Src (p-c-Src) for levels in WT and WT/STZ/HFD LEC cells (lower panel). (G) AP-1 expression vector was cotransfected with reporter plasmid pGL3-200 containing WT or mutant AP-1 binding sites into mouse LECs. (H) AP-1 expression vector was cotransfected with reporter plasmid pGL3-200 containing WT AP-1 binding sites into mouse LECs followed by treatment with an AP-1–blocking peptide. Blank vectors were used as the control. The luciferase activities of the WT and mutant reporter plasmids were measured and quantified. (I) ChIP assay to determine AP-1 binding to the epsin 1 promoter in LECs from STZ-induced diabetic mice and WT normal mice with and without c-Src knockdown (KD). Quantifications by using NIH ImageJ, n = 8. Data are mean ± SEM. *P < 0.05; ***P < 0.001, by 2-tailed Student’s t test (A and B), 1-way ANOVA followed by Tukey’s post hoc test (H), or 2-way ANOVA followed by Tukey’s post hoc test (G and I).