CXCL8-induced endothelial permeability - PubMed (original) (raw)

Transactivation of vascular endothelial growth factor receptor-2 by interleukin-8 (IL-8/CXCL8) is required for IL-8/CXCL8-induced endothelial permeability

Melissa L Petreaca et al. Mol Biol Cell. 2007 Dec.

Abstract

Interleukin-8 (IL-8/CXCL8) is a chemokine that increases endothelial permeability during early stages of angiogenesis. However, the mechanisms involved in IL-8/CXCL8-induced permeability are poorly understood. Here, we show that permeability induced by this chemokine requires the activation of vascular endothelial growth factor receptor-2 (VEGFR2/fetal liver kinase 1/KDR). IL-8/CXCL8 stimulates VEGFR2 phosphorylation in a VEGF-independent manner, suggesting VEGFR2 transactivation. We investigated the possible contribution of physical interactions between VEGFR2 and the IL-8/CXCL8 receptors leading to VEGFR2 transactivation. Both IL-8 receptors interact with VEGFR2 after IL-8/CXCL8 treatment, and the time course of complex formation is comparable with that of VEGFR2 phosphorylation. Src kinases are involved upstream of receptor complex formation and VEGFR2 transactivation during IL-8/CXCL8-induced permeability. An inhibitor of Src kinases blocked IL-8/CXCL8-induced VEGFR2 phosphorylation, receptor complex formation, and endothelial permeability. Furthermore, inhibition of the VEGFR abolishes RhoA activation by IL-8/CXCL8, and gap formation, suggesting a mechanism whereby VEGFR2 transactivation mediates IL-8/CXCL8-induced permeability. This study points to VEGFR2 transactivation as an important signaling pathway used by chemokines such as IL-8/CXCL8, and it may lead to the development of new therapies that can be used in conditions involving increases in endothelial permeability or angiogenesis, particularly in pathological situations associated with both IL-8/CXCL8 and VEGF.

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Figures

Figure 1.

IL-8 induces endothelial permeability in a receptor-dependent manner. hMVECs were plated for the permeability assay as described in Materials and Methods. (A) The transwell cultures were treated with 50 ng/ml IL-8 or 100 ng/ml VEGF for multiple times, as indicated. IL-8 stimulates endothelial permeability over time, similarly to VEGF. Each treatment group was performed in duplicate; data are shown as means ± SE. Statistics are shown as comparisons between the treatment and control (*p < 0.05, **p < 0.01, ***p < 0.001. (B) Cultures either left untreated or treated with 50 ng/ml IL-8 or 100 ng/ml VEGF, as indicated, were immunostained with PECAM-1 to visualize paracellular gap formation, and then they were visualized by fluorescence microscopy. Arrows indicate the position of certain paracellular gaps. Like VEGF, IL-8 increased gap formation in hMVECs. (C) Confluent endothelial cells were treated with IL-8 or with VEGF for 1 h, followed by immunoblot analysis of cell lysates by using an antibody that specifically recognizes phosphorylated VE cadherin at Y731 (top). This blot was stripped an reprobed with a VE cadherin antibody to ensure equal loading (bottom). Like VEGF, IL-8 increased phosphorylation of VE cadherin. Irrelevant lanes were removed, as indicated by dotted lines within the blots. (D) Transwell cultures were pre-incubated with repertaxin, an inhibitor of CXCR1 and CXCR2, for 30 min before IL-8 treatment, then the permeability assay was conducted as described, using 50 ng/ml IL-8. Repertaxin blocked IL-8–induced permeability. Each treatment group was performed in triplicate; data are shown as the mean ± SE. Statistics are shown as comparisons of the IL-8–treated group with the control and inhibitor groups (*p < 0.05, **p < 0.01).

Figure 2.

VEGFR2 is important in IL-8–induced permeability both in vitro and in vivo. (A) hMVECs were plated for the permeability assay as described in Materials and Methods. Before treatment with 50 ng/ml IL-8 or 100 ng/ml VEGF, the transwell cultures were incubated with 400 nM VEGFR inhibitor for 1 h. The VEGFR inhibitor prevented the permeability induced by both IL-8 (A) and VEGF (data not shown). Each treatment group was performed in triplicate; data are shown as means ± SE. Statistics are shown as comparisons of the IL-8–treated group with the control and inhibitor groups (**p < 0.01, ***p < 0.001). (B) Mice were injected with Evans blue via the tail vein, and they were then subcutaneously injected with either vehicle (PBS) or with 1 μg IL-8, in the presence and absence of prior treatment with 8 μM VEGFR inhibitor. Thirty minutes after IL-8 treatment, mice were killed and perfused with PBS, followed by removal of treated regions of skin via 7-mm punch biopsy. Skin punches were photographed to visualize Evans blue extravasation into treated or untreated tissues, as indicated. IL-8 treatment increased endothelial permeability in vivo, as shown by increased Evans blue extravasation into the tissue; this was blocked in tissues pretreated with the VEGFR inhibitor. (C) Mice were treated and skin punches were isolated as described in B. Evans blue was then extracted from the skin punches using formamide, quantified with a spectrophotometer, and calculated relative to the appropriate control (PBS treatment for the IL-8-treated regions; VEGFR inhibitor only for the IL-8 + VEGFR inhibitor-treated regions). Data are shown as mean ± SD. Differences were found to be significant using ANOVA and the Tukey–Kramer multiple comparisons post test (*p <0.01, n = 2).

Figure 3.

IL-8 transactivates VEGFR2. (A) Confluent hMVECs were treated with IL-8 at various concentrations, for 30 min, followed by immunoblot analysis of cell lysates using a phospho-VEGFR2 antibody (top). Extracts were also analyzed for total VEGFR2 content as a loading control (bottom). IL-8 increased phospho-VEGFR2 levels in a dose-dependent manner, with 100 ng/ml inducing maximum phosphorylation. (B) hMVECs were preincubated with with 8.3 μM VEGF inhibitor for 30 min, followed by treatment with 100 ng/ml IL-8 for 5 min. Protein extracts were analyzed as in A. IL-8–induced VEGFR2 phosphorylation was not eliminated by the VEGF inhibitor. (C) Confluent hMVECs were treated with 100 ng/ml IL-8 for various times. Protein extracts were analyzed as described in A. IL-8 increased phospho-VEGFR2 levels over time in a biphasic manner, with peaks at 5 and 60 min. (D) Confluent HMEC-1 cells were treated with 100 ng/ml IL-8 for various times. Protein extracts were analyzed as described in A. IL-8 increased phospho-VEGFR2 levels over time in a biphasic manner, with peaks at 1 and 60 min. (E) Endothelial cells were preincubated with 600 nM VEGFR inhibitor for 15 min, followed by treatment with 100 ng/ml IL-8 for 1 min. Protein extracts were analyzed as described in A. IL-8–induced VEGFR2 phosphorylation was abolished by the VEGFR inhibitor. (F) HMEC-1 cells were treated as in (D), with cell extracts analyzed by immunoblotting with a phospho-specific antibody directed against VEGFR2 phosphotyrosines 1054 and 1059 (top). The blot was stripped and reprobed with anti-VEGFR2 as a loading control (bottom). VEGFR2 was phosphorylated at Y1054/Y1059 in a biphasic manner similar to what was seen with total phosphotyrosine blots.

Figure 4.

IL-8–induced VEGFR2 phosphorylation requires the activity of its receptors, CXCR1 and CXCR2. (A) Endothelial cells were preincubated with the CXCR1 and CXCR2 inhibitor repertaxin, for 30 min, and then they were then treated with 100 ng/ml IL-8 for 1 min. (B) Endothelial cells were preincubated for 15 min with 3 μg of neutralizing antibodies against CXCR1, CXCR2, or CXCR1 and CXCR2, and then they were treated with 100 ng/ml IL-8 for 1 min. In addition, IL-8 was preincubated with 3 μg of IL-8 antibody for 30 min, and then they were used to treat HMEC-1 cells for 1 min. Equal protein concentrations of the extracts were subjected to immunoblot analysis using a phospho-VEGFR2 antibody (top). Extracts were also analyzed for total VEGFR2 content as a loading control (bottom). IL-8–induced phosphorylation of VEGFR2 was prevented by the CXCR1/CXCR2 inhibitor (A) and CXCR1 and CXCR2 neutralizing antibodies (B), alone or in combination.

Figure 5.

IL-8 promotes the physical interaction of VEGFR2 with CXCR1 and CXCR2. Confluent HMEC-1 cells were treated with 100 ng/ml IL-8 for various times. (A) Equal amounts of cleared HMEC-1 extracts were immunoprecipitated using the VEGFR2/Flk-1 antibody, and then they were analyzed by immunblotting with the CXCR1 antibody. The blot was stripped and reprobed with a VEGFR2 antibody to determine equal immunoprecipitate (IP) and loading (bottom). CXCR1 coimmunoprecipitated with VEGFR2 after IL-8 treatment over time (top), with the time of maximal interaction similar to the time of maximal VEGFR2 phosphorylation (compare with Figure 3D). (B) Confluent HMEC-1 cells were treated with 100 ng/ml IL-8 for various times, and then they were subjected to immunoprecipitation as described in A, followed by immunoblotting with the CXCR2 antibody. The blot was stripped and reprobed with a VEGFR2 antibody to determine equal IP and loading (bottom). Like CXCR1, CXCR2 coimmunoprecipitated with VEGFR2 after IL-8 treatment over time (top), with the time of maximal interaction similar to the time of maximal VEGFR2 phosphorylation (compare with Figure 3D).

Figure 6.

IL-8 stimulates c-Src activation. (A) Confluent HMEC-1 cells were treated with 100 ng/ml IL-8 for various times, as indicated. After treatment, proteins were extracted, and equal protein concentrations of the cleared extracts were separated by SDS-PAGE and subjected to immunoblotting by using an antibody that specifically recognizes Src phosphorylated at tyrosine 419, indicative of active Src (top). The blot was stripped and reprobed with an antibody that recognizes total Src to determine equal loading (bottom). IL-8 induced rapid biphasic phosphorylation of Src in a way that is temporally similar to that of VEGFR2 phosphorylation. (B) Confluent HMEC-1 cells were treated with 100 ng/ml IL-8 for 1 min, ± preincubation with 5 nM repertaxin for 30 min. After treatment, proteins were prepared, separated, and detected as described in A. IL-8–induced Src phosphorylation was blocked by the the CXCR1 and CXCR2 inhibitor repertaxin.

Figure 7.

IL-8–induced permeability, receptor complex formation, and VEGFR2 phosphorylation are dependent upon Src family kinases. (A) hMVECs were plated for permeability assays as described in Materials and Methods. Before treatment with 50 ng/ml IL-8, the transwell cultures were incubated with 10 μM SU6656, an inhibitor of Src family kinases, for 30 min. The Src inhibitor abolished IL-8–induced permeability. Each treatment group was performed in triplicate; data are shown as means ± SE. Statistics are shown as comparisons of the IL-8–treated group with the control and inhibitor groups (*p < 0.05, ***p < 0.001). (B and C) Confluent HMEC-1 cells ± preincubation with 1.1 μM SU6656 were treated with 100 ng/ml IL-8 for 3 min (for CXCR1; B) or 1 min (for CXCR2; C); equal amounts of cleared HMEC-1 extracts were immunoprecipitated using the VEGFR2/Flk-1 antibody, and precipitates were analyzed by Western blot with the CXCR1 (B) and CXCR2 (C) antibodies (top). Blots were stripped and reprobed with a VEGFR2 antibody to determine equal IP and loading (Bottom). Interactions between CXCR1 and CXCR2 and VEGFR2 were abrogated in the presence of the Src inhibitor SU6656. (D) Confluent HMEC-1 cells were preincubated with 1.1 μM SU6656 for 5 min, followed by treatment with 100 ng/ml IL-8 for 1 min, and then protein extracts prepared and analyzed for VEGFR2 phosphorylation as in Figure 4. IL-8–induced VEGFR2 phosphorylation was eliminated by the Src inhibitor.

Figure 8.

IL-8–induced VEGFR2 transactivation is required for RhoA activation. HMEC-1 cells treated with IL-8 in the presence and absence of inhibitors were lysed and then subjected to GTP-Rho pull-down assays by using GST-RBD beads. Precipitates were separated by SDS-PAGE and Western blotting by using the RhoA antibody (top). Crude lysates were also analyzed by SDS-PAGE and Western blotting to ensure equal RhoA input (bottom). (A and B) Cells were preincubated with 600 nM VEGFR inhibitor for 15 min, followed by treatment with 100 ng/ml IL-8 for 5 min (A) or 50 ng/ml IL-8 for 3 h (B). IL-8–induced RhoA activation was abolished by the VEGFR inhibitor, at early and late time points. Irrelevant lanes were removed, as indicated by dotted lines within the blots. (C) Cells were either transfected with the pEGFP-C1 empty vector or with pEGFP-C1 containing exoenzyme C3, an inhibitor of Rho GTPase, followed by treatment with 100 ng/ml IL-8 for 30 min, and then they were stained with rhodamine-phalloidin. IL-8–induced gap formation was prevented by transfection with C3. (D) Cells were preincubated with 560 nM Y-27632, an inhibitor of ROCK, for 30 min, followed by treatment with 100 ng/ml IL-8 for 30 min and staining with rhodamine-phalloidin. F-actin staining and gap formation were visualized by fluorescence microscopy. IL-8–induced actin reorganization and gap formation were prevented with the ROCK inhibitor. (E) Cells were preincubated with 600 nM VEGFR inhibitor for 15 min, followed by treatment with 100 ng/ml IL-8 for 30 min and staining with rhodamine-phalloidin. F-actin staining and gap formation were visualized by fluorescence microscopy. IL-8–induced actin reorganization and gap formation were prevented with the VEGFR inhibitor.

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Transactivation of vascular endothelial growth factor receptor-2 by interleukin-8 (IL-8/CXCL8) is required for IL-8/CXCL8-induced endothelial permeability - PubMed (original) (raw)