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 5

ROS-induced intracellular phosphorylation of VEGFR3 is c-Src dependent and ligand independent.

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ROS-induced intracellular phosphorylation of VEGFR3 is c-Src dependent a...
(A) WT LECs were treated with control or c-Src siRNA followed by treatment with H2O2 and analysis via Western blot (n = 3). (B) Quantification of activation percentage of VEGFR3 and phosphorylation of VEGFR3 in A. (C) WT and LEC-iDKO LECs were treated with H2O2 to induce phosphorylation of VEGFR3, PP2 to inhibit c-Src, and 22 mM glucose followed by analysis using Western blot (n = 3). (D) Quantification of activation percentage of VEGFR3 and phosphorylation of VEGFR3 in C. (E) Western blot analysis of phosphorylation of VEGFR3 abundance in LECs of WT and LEC-iDKO treated with 5 mM N-acetyl-L-cysteine (NAC) for 30 minutes, and then treated with 500 μM hydrogen peroxide (H2O2) for 15 minutes (n = 7). (F) Quantification of activation percentage of VEGFR3 and phosphorylation of VEGFR3 in E. (G) Western blot analysis of H2O2-induced phosphorylation of VEGFR3 was protected from trypsin digestion. WT and LEC-iDKO LECs were briefly exposed to 0.25% trypsin for cell-surface proteolysis, and then treated with 500 μM H2O2 or 200 ng/mL VEGF-C for 15 minutes prior to lysis. VE-cadherin is shown as membrane control. VE-cadherin and tubulin were run on the same gel but were noncontiguous (n = 7). (H) Quantification of activation percentage of phosphorylation of VEGFR3 in G. Quantification was done with NIH ImageJ. Data are mean ± SEM. *P < 0.05; **P < 0.01, by 2-way ANOVA (B, D, and F) or 3-way ANOVA (H) followed by Tukey’s post hoc test.