Selective Targeting of a Novel Epsin-VEGFR2 Interaction Promotes VEGF-Mediated Angiogenesis

Abstract

Rationale: We previously reported that vascular endothelial growth factor (VEGF)-induced binding of VEGF receptor 2 (VEGFR2) to epsins 1 and 2 triggers VEGFR2 degradation and attenuates VEGF signaling. The epsin ubiquitin interacting motif (UIM) was shown to be required for the interaction with VEGFR2. However, the molecular determinants that govern how epsin specifically interacts with and regulates VEGFR2 were unknown.

Objective: The goals for the present study were as follows: (1) to identify critical molecular determinants that drive the specificity of the epsin and VEGFR2 interaction and (2) to ascertain whether such determinants were critical for physiological angiogenesis in vivo.

Methods and results: Structural modeling uncovered 2 novel binding surfaces within VEGFR2 that mediate specific interactions with epsin UIM. Three glutamic acid residues in epsin UIM were found to interact with residues in VEGFR2. Furthermore, we found that the VEGF-induced VEGFR2-epsin interaction promoted casitas B-lineage lymphoma-mediated ubiquitination of epsin, and uncovered a previously unappreciated ubiquitin-binding surface within VEGFR2. Mutational analysis revealed that the VEGFR2-epsin interaction is supported by VEGFR2 interacting specifically with the UIM and with ubiquitinated epsin. An epsin UIM peptide, but not a mutant UIM peptide, potentiated endothelial cell proliferation, migration and angiogenic properties in vitro, increased postnatal retinal angiogenesis, and enhanced VEGF-induced physiological angiogenesis and wound healing.

Conclusions: Distinct residues in the epsin UIM and VEGFR2 mediate specific interactions between epsin and VEGFR2, in addition to UIM recognition of ubiquitin moieties on VEGFR2. These novel interactions are critical for pathophysiological angiogenesis, suggesting that these sites could be selectively targeted by therapeutics to modulate angiogenesis.

Keywords: VEGFR2 protein, mouse; epsin; neovascularization, physiologic; ubiquitin; ubiquitination.

Figure 1. The Epsin UIM mediates VEGFR2 signaling and interacts with the VEGFR2 kinase domain

(A) Ribbon representation and (B) stick representation of epsin UIM (green) docked into the putative hairpin-shaped binding pocket of VEGFR2 KD (blue). VEGFR2 KD crystal structure (3U6J) was obtained from the Protein Data Bank. Ribbon diagram for epsin UIM was predicted using PEP-FOLD. ClusPro and PyMol software were used for the docking; highest scoring model with good topologies is shown. (C) Alignment of epsin UIM sequences from mouse, rat and human epsins 1 and epsin 2. Note: E183 (corresponding to E3 in UIM peptide), E184 (corresponding to E4 in UIM peptide) and E185 (corresponding to E5 in UIM peptide) are highly conserved. (D) Western blot analysis of VEGFR2 co-immunoprecipitation by epsin 1 in HUVEC cells overexpressing wild type VEGFR2, or VEGFR2 with the indicated substitutions, and stimulated with VEGF (50 ng/mL) for 2 min. (E) Western blot analysis of VEGFR2 co-immunoprecipitation by epsin 1 in HUVEC cells overexpressing wild type VEGFR2 and either HA-tagged full-length epsin 1 (HA-Epsin 1), HA-Epsin 1^{E183A, E184A, E185A} or HA-Epsin 1-ΔUIM, and stimulated with 50 ng/mL VEGF for 2 min. All representative Western blots were selected from n=3. Error bars indicate the mean ± s.e.m. *P<0.05.

Figure 2. c-Cbl-dependent ubiquitination of epsin promotes the interaction with VEGFR2

(A) Western blot analysis of epsin 1 ubiquitination and VEGFR2 co-immunoprecipitation with epsin 1 in HUVECs transfected with either control or c-Cbl-targeted siRNA. Cells were serum starved overnight and stimulated with or without 50 ng/mL VEGF for 2 min prior to lysis. (B) Western blot analysis of epsin 1 ubiquitination in HUVECs overexpressing wild type VEGFR2 and either HA-Epsin 1, HA-Epsin 1^{E183A, E184A, E185A} or HA-Epsin 1-ΔUIM and stimulated with 50 ng/mL VEGF for 2 min. All representative Western blots were selected from n=3. Error bars indicate the mean ± s.e.m. *P<0.05.

Figure 3. Identification of a novel ubiquitin-binding interface within VEGFR2 that promotes an interaction with ubiquitinated epsin

(A) Ribbon representation of the predicted supercomplex between epsin ENTH (red), VEGFR2 KD (blue) and ubiquitin (pink). Ubiquitin was docked into the epsin ENTH:VEGFR2 KD structure using ClusPro and PyMol software; highest scoring model with good topologies is shown. Enlarged stick representation to the right highlights the interacting residues between ubiquitin and VEGFR2 KD. (B) Western blot analysis of VEGFR2 co-immunoprecipitation with epsin 1 in HEK 293T cells overexpressing wild type VEGFR2, VEGFR2^S1021A, VEGFR2^{S1021AK1014R, K1023R} or VEGFR2^{K1014R, K1023R, G1158A}. Cells were serum starved overnight and stimulated with 50 ng/mL VEGF for 2 min. (C) Western blot analysis of mono- or poly-ubiquitin pulldown by wild type VEGFR2 overexpressed in HEK 293T cells. (D) Western blot analysis of VEGFR2 pulldown by di-ubiquitin in HEK 293T cells overexpressing wild type VEGFR2, VEGFR2^H891A or VEGFR2^S1021A. (E) Putative interacting residues of VEGFR2 and ubiquitin with their respective hydrogen bond distances, and schematic representation of epsin 1 conjugated to wild type ubiquitin or ubiquitin with I44A, R42A or R72A and R74A substitutions. (F) Western blot analysis of epsin 1 co-immunoprecipitation with VEGFR2 in HEK 293T cells overexpressing wild type VEGFR2 and either HA-Epsin1 conjugated to wild type ubiquitin (HA-Epsin 1-Ub) or ubiquitin with I44A, R42A, R72A or R74A point substitutions. Cells were serum starved overnight and stimulated with 50 ng/mL VEGF for 2 min. All representative Western blots were selected from n=3. Error bars indicate the mean ± s.e.m. *P<0.05.

Figure 4. Internalization of VEGFR2 upon VEGF stimulation is critical for productive downstream signaling

(A) Western blot analysis of VEGF-induced phosphorylation of VEGFR2 and downstream AKT and ERK in mouse primary endothelial cells. Tamoxifen-induced the knockout of endogenous VEGFR2, expression was restored with wild type VEGFR2, VEGFR2^H891A or VEGFR2^S1021A. Cells were serum starved overnight and stimulated with 50 ng/mL VEGF for 5 min. (B) Representative immunofluorescence images of VEGF-dependent changes in the subcellular localization of VEGFR2 in HUVECs overexpressing wild type VEGFR2, VEGFR2^H891A or VEGFR2^S1021A. Cells were serum starved overnight and stimulated with 50 ng/mL VEGF for 10 min. Arrows indicate the colocaliztion of wild type VEGFR2 with EEA1. (C) Western blot analysis of VEGF-induced VEGFR2 internalization, relative to Transferrin Receptor, in mouse primary endothelial cells overexpressing wild type VEGFR2, VEGFR2^H891A or VEGFR2^S1021A using a cleavable surface biotinylation and internalization assay. Cells were serum starved overnight and stimulated with 50 ng/mL VEGF for 10 min. All representative Western blots and immunofluorescence images were selected from n=3. Error bars indicate the mean ± s.e.m. *P<0.05. Scale bar in B: 10 μm.

Figure 5. UIM peptide, but not UIM^E3,4,5A mutant peptide promotes VEGF-dependent VEGFR2 signaling and in vitro angiogenesis

(A) Western blot analysis of VEGFR2 co-immunoprecipitation with epsin 1 in HEK 293T cells overexpressing wild type VEGFR2 and HA-Epsin 1 and treated with 12.5 μM control, full length UIM or E3A, E4A and E5A UIM mutant (UIM^E3,4,5A) peptide for 16 hrs. Cells were stimulated with 50 ng/mL VEGF for 2 min prior to lysis. (B) Western blot analysis of VEGF-VEGFR2 signaling in WT primary MEC treated with 12.5 μM control, UIM or UIM^E3,4,5A peptide for 16 hrs. Cells were stimulated with 50 ng/mL VEGF for 5 min prior to lysis. (C) Quantification of EdU labeling for MEC cell proliferation, (D,E) scratch “wound” analysis of cell migration in MEC and (G-I) Matrigel culture analysis of tube formation in HUVECs treated with 12 μM control, UIM or UIM^E3,4,5A peptide for 16 hrs in the presence of 50 ng/mL VEGF. Representative images selected from n=5 in Online Figure VIII, D, F. Quantification of scratch wound and tube formation are shown in E,G and H, respectively. All representative Western blots were selected from n=3. Scale bar in D and F: 50 μm. Error bars indicate the mean ± s.e.m. *P<0.05.

Figure 6. Physiological angiogenesis is increased by administration of UIM, but not UIM^E3,4,5A peptides

(A, B) Representative montage images with entire retina review of whole-mount retinas isolated from P6 pups after intraperitoneal injection with control, UIM or UIM^E3,4,5A mutant peptide and immunofluorescently labeled with biotinylated isolectin B4. Respective quantifications for A are shown in B, including vascular progression length and vasculature density. (C, D) Representative images of whole-mount retinas isolated from P6 pups after intraperitoneal injection with control, UIM or UIM^E3,4,5A mutant peptide and immunofluorescently labeled with biotinylated isolectin B4. Respective quantification for C is shown in D. (E) Representative images of subcutaneous Matrigel plugs supplemented with 200 ng/mL VEGF and either control, UIM or UIM^E3,4,5A peptide isolated 7 days post-implantation from WT mice. (F,G) Representative image (F) and quantification (G) of CD31-positive vessels in cryopreserved, sectioned and immunofluorescently stained Matrigel plugs from (E). (H) Representative images of subcutaneous Matrigel plugs supplemented with 200 ng/mL VEGF and either control or UIM peptide isolated 7 days post-implantation from EC-iDKO mice. (I, J) Representative image (I) and quantification (J) of CD31-positive vessels in cryopreserved, sectioned and immunofluorescently stained Matrigel plugs supplemented with either control or UIM peptide and isolated from (H). Representative images selected from n=6. Scale bar in A:1000 μm; C, F and I: 50 μm. ON, a and b in A represented optic nerve center, distance from ON to retina vasculature edge and distance from ON to retina edge. Error bars indicate the mean ± s.e.m. *P<0.05.

Figure 7. UIM peptide administration increases physiological angiogenesis in wild type mice

(A) Representative images of dermal wound healing 0, 1, 3 and 7 days after dermal biopsy of wild type mice receiving control or UIM peptide by intraperitoneal injection. (B) Quantification of wound area shown in (A) and reported as a wound-healing curve. (C) Representative images of CD31-positive blood vessels in cryopreserved, sectioned and immunfluorescently stained dermal wounds from control or UIM peptide treated wild type mice isolated 7 days after dermal biopsy. (D) Quantification of CD31-positive vessel area relative to total vessel area of immunofluorescently stained dermal wounds from (C) using SlideBook software. All representative dermal wound and immunofluorescence images were selected from n=3 mice. Scale bars: A, 2 mm; C, 50 μm. Error bars indicate the mean ± s.e.m. *P<0.05; **P<0.01.