KIF13B regulates VEGF-induced endothelial permeability
KIF13B transports newly synthesized VEGFR2 from Golgi to the cell surface [15], and the VEGFR2 trafficking is essential for angiogenesis [5, 6, 15]. We showed that KIF13B also plays an important role in VEGF-induced vascular leakage, as VEGF-A fails to induce vascular leakage in Kif13bKOor when KIF13B-mediated VEGFR2 trafficking is blocked by KAI [16]. However, VEGF-induced endothelial permeability is an early response of ECs to VEGF-A, occurring within 15 min. It cannot be explained by the known function of KIF13B, which is the release of synthesized VEGFR2 from Golgi and its trafficking to the cell surface, taking several hours after VEGF-A stimulation [15]. To explore whether KIF13B has any role in the relatively rapid response to VEGF-A, we used lentivirus-based shRNA-KIF13B to knockdown [15], and tested VEGF-induced endothelial permeability in human retina endothelial cells (hREC) using transendothelial electric resistance (TEER) assay. VEGF-A transiently reduced TEER at 15 min after VEGF-A stimulation in control hREC, transduced with scrambled shRNA (Fig. 1A, B), whereas knockdown of KIF13B suppressed VEGF-induced endothelial permeability compared to controls (Fig. 1A, B). After initial permeability, the endothelial barrier function recovered in both scrambled and shRNA-KIF13B treated cells (Fig. 1A, C).
Next, we tested whether the inhibition of VEGFR2 trafficking also affects endothelial permeability (Fig. 1D, E, F). KAI is a synthetic peptide designed to disrupt the interaction between VEGFR2 and KIF13B [5]. After pretreatment with the control peptide or KAI (10 µM) for 2 hours, the TEER of confluent hREC was monitored before and after VEGF-A stimulation (Fig. 1D). KAI treatment prevented VEGF-induced endothelial permeability, compared to no-peptide control and control peptide treated hREC (Fig. 1D). KAI-treated hREC showed higher electrical resistance values than both no peptide and control peptide-treated cells 15 min and 12 hr after stimulation with VEGF-A (Fig. 1D, E, F). These data demonstrate that KIF13B is required for VEGF-induced endothelial permeability.
KIF13B regulates VEGFR2 signaling pathways mediating endothelial permeability
To examine how KIF13B regulates endothelial permeability, we examined the activation of the signaling pathway regulating endothelial permeability and tested the effect of knockdown of KIF13B (Fig. 2A, B). VEGF-A treatment increased phosphorylation of VEGFR2 (at Y951 and Y1175), Src (at Y416), and VE-cadherin (at Y658 and Y685) in scrambled shRNA transduced control hREC. Compared to scrambled shRNA transduced cells, shRNA-KIF13B transduced cells showed significantly lower phosphorylated/total protein ratio in p-VEGFR2 (Y951 and Y1175), p-Src (Y416), and p-VE-cadherin (Y658 and Y685) through 3-15 min of VEGF-A treatment (Fig. 2A, B).
Next, we tested the effect of KAI compared with the control peptide (Fig. 2C, D). Similar to scrambled shRNA treated control above, VEGF-A induced p-VEGFR2 (at Y951 and Y1175), p-Src (at Y416), and p-VE-cadherin (at Y658 and Y685) in control peptide treated hREC (Fig. 2C, D). KAI treatment significantly decreased phosphorylation of VEGFR2 (Y951), Src (Y416), and VE-cadherin (Y685) compared with control peptide (Fig. 2C, D). KAI treatment also decreased p-VEGFR2 (Y1175) and pY658-VE-cadherin, although the difference was not statistically significant. Together, these data suggest the important role of KIF13B in regulating VEGF/VEGFR2 signaling to regulate endothelial permeability.
KIF13B regulates translocation of internalized VEGFR2
As KIF13B mediates VEGFR2 trafficking to the cell surface [15], we hypothesized that KIF13B tunes the degree of VEGF/VEGFR2 signaling by regulating the amount of VEGFR2 on the cell surface. We examined the effect of the knockdown of KIF13B on cell surface VEGFR2 before and after VEGF-A stimulation (Fig. 3). First, hREC were transduced with scrambled shRNA or shRNA-KIF13B, and stimulated with VEGF-A for indicated times. Cell surface proteins were biotinylated and precipitated with streptavidin beads, and biotinylated cell surface VEGFR2 was detected by western blotting (Fig. 3A, B). Cell surface VEGFR2 was decreased at 15-30 min after VEGF-A stimulation in both control and shRNA-KIF13B transduced hRECs, indicating VEGF-induced internalization (Fig. 3A, B). Interestingly, the knockdown of KIF13B further decreased the amount of VEGFR2 on the cell surface (Fig. 3B).
As a component of internalized VEGFR2 is recycled back to the cell surface [7], the reduction of cell-surface VEGFR2 may be the result of increased internalization or less recycling. Thus, we used another biotinylation assay [21] to quantify internalized VEGFR2 (Fig. 3C, D). hRECs were transduced with scrambled shRNA or shRNA-KIF13B. After the serum-starvation of cells, cell surface proteins were biotinylated. Then, cells were stimulated with VEGF-A to let VEGFR2 internalize. After removal of cell surface biotin, internalized VEGFR2 was detected by western blotting in streptavidin precipitates (Fig. 3C, D). After VEGF-A stimulation, VEGFR2 was internalized in both control and shRNA-KIF13B transduced cells without any difference (Fig. 3C, D), suggesting the possibility of KIF13B mediating recycling of internalized VEGFR2.
To further examine the question of localization of VEGFR2 and p-VEGFR2, we stained hRECs with their respective antibodies (Fig. 4). Before VEGF-A stimulation, the majority of VEGFR2 was localized near nuclei as in [13, 15]. And a portion of VEGFR2 was also observed at cell-cell junctions in both control peptide-treated and KAI-treated cells (Fig. 4A). Three min after VEGF-A stimulation, phosphorylation of VEGFR2 (Y1175) was mainly observed at cell-cell junctions (Fig. 4A). KAI treatment did not alter the initial area where VEGFR2 is phosphorylated at 3 min. In control peptide-treated hRECs, p-VEGFR2 was rapidly internalized and distributed in the cells at 15 min after VEGF-A stimulation. However, p-VEGFR2 accumulated in KAI-treated hRECs at 15 min and 30 min after VEGF-A stimulation. The number of cells showing p-VEGFR2 accumulation was significantly increased in KAI-treated cells at 15 min and 30 min after VEGF-A stimulation (Fig. 4D). The fluorescence intensity of p-VEGFR2 and VEGFR2 were not different between the two groups (Fig. 4B, C), indicating that a similar amount of p-VEGFR2 was distributed in control and accumulated in KAI-treated hREC. Total VEGFR2 was also largely distributed throughout control cells, whereas total VEGFR2 remained near nuclei in KAI-treated cells at 15 min and 30 min after VEGF-A stimulation (Fig. 4A). These results together showed that VEGFR2 is first phosphorylated at cell-cell junction, then phosphorylated VEGFR2 is internalized to induce signaling, which releases of VEGFR2 from Golgi [15]. Thus both phosphorylated and unphosphorylated VEGFR2 were distributed throughout the cells in control. KAI treatment inhibited the release of unphosphorylated VEGFR2 from Golgi, consistent with the effects of knockdown of KIF13B [15]. The distribution of p-VEGFR2 was also inhibited by KAI treatment, resulting in accumulation of p-VEGFR2. Phosphorylated VEGFR2 is either dephosphorylated and recycled to the cell surface or undergoes degradation [7]. Thus, the question arises about the disposition of VEGFR2 during the inhibition of VEGFR2 trafficking.
Inhibition of VEGFR2 recycling localizes VEGFR2 in late endosomes
Rab family GTPases localize to specific intracellular compartments where they regulate cargo trafficking [22]. Internalized VEGFR2 localizes at Rab5A positive early endosome, then post-dephosphorylation, VEGFR2 enters either Rab11-mediated recycling pathway or Rab7-mediated degradation pathway. To determine how KIF13B contributes to VEGFR2 trafficking, we performed co-immunoprecipitation with Rab family proteins, Rab5A, Rab11, and Rab7, and analyzed binding proteins by western blotting (Fig. 5). In scrambled peptide-treated control hRECs, VEGFR2 and KIF13B were co-immunoprecipitated with Rab5A (Fig. 5A). KAI treatment decreased KIF13B on Rab5A-positive vesicles, whereas VEGFR2 remained co-precipitated with Rab5A (Fig. 5A, B). KAI treatment reduced VEGFR2 associated with Rab11-positive recycling vesicle (Fig 5C, D), suggesting reduced recycling in KAI-treated cells. The result consistent with the biotinylation assay (Fig. 3) shows the crucial role of KIF13B in mediating recycling of internalized VEGFR2. Upon inhibition of VEGFR2 trafficking by KAI treatment, we observed accumulation of p-VEGFR2 near nuclei (Fig. 4A). Inhibition of VEGFR2 recycling may thus traffic it to the degradation pathway.
To test address this concept, we performed co-immunoprecipitation with a late endosome marker, Rab7 (Fig. 5E, F). In control hRECs, p-VEGFR2 (Y1175) was detected in Rab7-positive late endosome at 3 min after VEGF-A stimulation and dephosphorylated at 15 min (Fig. 5E). Interestingly, in KAI-treated hREC, accumulation of p-VEGFR2 was observed in Rab7-positive late endosome (Fig. 5E).
To confirm the western blotting results, we stained hREC with Rab11 and VEGFR2 (Fig. 6). In control peptide-treated cells, colocalization of VEGFR2 with recycling vesicle marker Rab11 was observed, but KAI treatment significantly reduced colocalization at 3 min and 15 min after VEGF-A stimulation (Fig. 6A, B). In contrast, p-VEGFR2 (Y1175) was highly colocalized with the late endosome marker Rab7 in KAI-treated cells (Fig. 7). At 3 min after VEGF-A stimulation, phosphorylation of VEGFR2 was observed at cell-cell junctions in both control and KAI-treated hREC, consistent with Fig. 4. KAI treatment induced accumulation of p-VEGFR2, which was colocalized with Rab7 (Fig. 7A, B). We further analyzed colocalization of p-VEGFR2 (Y1175) and lysosome marker LAMP2 (Fig. 8). KAI induced accumulation of p-VEGFR2 in LAMP2 positive lysosomes at 15 min and 30 min after VEGF-A stimulation, compared with control (Fig. 8A, B). Together, these data show that inhibition of VEGFR2 trafficking by KAI treatment traffics VEGFR2 to late endosome and lysosome for degradation.
We also quantified the total amount of VEGFR2 after VEGF-A stimulation in control peptide-treated hREC and KAI-treated hREC (Fig. 9A, B). Compared to control, KAI treatment reduced the total amount of VEGFR2 at 15 min after VEGF-A stimulation (Fig. 9A, B). Degradation of VEGFR2 was also tested in scrambled shRNA-transduced control and shRNA-KIF13B-transduced cells (Fig. 9C, D). Knockdown of KIF13B tended to decrease the total amount of VEGFR2, although it was not statistically significant (Fig. 9C, D). These results together show that KIF13B contributes recycling of internalized VEGFR2. Inhibition of KIF13B traffics VEGFR2 to lysosomal degradation instead of recycling and limits the amount of VEGFR2 on the cell surface. Overall, the results show that VEGFR2 recycling is essential for VEGF-A signaling and endothelial permeability.
Inhibition of VEGFR2 trafficking ameliorates pathological vascular leakage
We examined whether inhibition of VEGFR2 trafficking is an effective strategy to inhibit vascular leakage in wet AMD. We tested the efficacy of KAI on laser-induced vascular leakage in the mouse model (Fig. 10). C57BL/6 mice received laser burns on Bruch’s membrane, which mimics disruption of Bruch’s membrane in wet AMD, and induces vascular leakage and neovascularization [23, 24]. Mice were treated with eyedrop of either control peptide or KAI (5 µg/eye in 5 µl in PBS) once a day for 3 days. Three days after laser burns, Evans blue was injected i.p. to examine the Evans blue extravasation on the following day. The control peptide-treated mice showed extravasation of Evans blue at the site with laser burn (Fig. 10A top panel). KAI treatment reduced the extravasation of Evans blue (Fig. 10A bottom panel and 10B). Laser-induced neovascularization at day 4 was also analyzed by staining with isolectin B4 (ILB4), an endothelial marker. Compared to the control mice, laser-induced neovascularization was also reduced by daily treatment with KAI, but it was not statistically significant (Fig. 10A, C). Note, neovascularization at the late stage was significantly reduced by continuous KAI treatment for 14 days [6]. As laser-induced vascular leakage also facilitated macrophages recruitment [23], we observed macrophages by staining with marker CD68 (Fig. 10A, D). Thus, it appears that KAI treatment also inhibited macrophage recruitment.