IL-28A/IL-10Rβ axis promotes angiogenesis via eNOS/AKT signaling and AP-1/NF-κB/MMP-2 network by regulating HSP70-1 expression

Abstract

Introduction: Angiogenesis plays a significant role in the development of tumor progression and inflammatory diseases. The role of IL-28A in angiogenesis and its precise regulatory mechanisms remain rarely elucidated.

Objectives: We report the novel regulatory role of IL-28A in physiological angiogenesis. The study aimed to elucidate the regulatory mechanisms involved in IL-28A-mediated angiogenesis and identify key genes associated with IL-28A-induced angiogenic responses.

Methods: To know the effect of IL-28A on angiogenesis, HUVECs were applied to perform proliferation, migration, invasion, tube formation, immunoblot, and EMSA. Gene expression changes in HUVECs following IL-28A treatment were analyzed by NGS. The functional role of HSP70-1 and IL-10Rβ in IL-28A-induced angiogenic responses was evaluated using PCR and siRNA knockdown. Animal studies were conducted by aortic ring ex vivo assays, Matrigel plug in vivo assays, and immunochemistry using HSP70-1 knockout and transgenic mice models. The efficacy of IL-28A in angiogenesis was confirmed in a hind-limb ischemia model.

Results: Autocrine/paracrine actions in HUVECs regulated IL-28A protein expression. Exogenous IL-28A increased the proliferation of HUVECs via eNOS/AKT and ERK1/2 signaling. IL-28A treatment promoted migration, invasion, and capillary tube formation of HUVECs through induction of the AP-1/NF-κB/MMP-2 network, which was associated with eNOS/AKT and ERK1/2 signaling. The efficacy of IL-28A-induced angiogenic potential was confirmed by aortic ring and Matrigel plug assay. HSP70-1 was identified as an IL-28A-mediated angiogenic effector gene using bioinformatics. Knockdown of HSP70-1 abolished angiogenic responses and eNOS/AKT signaling in IL-28A-treated HUVECs. IL-28A-induced microvessel sprouting formation was testified in HSP70-1-deficient and HSP70-1 transgenic mice. Flow recovery in hind-limb ischemia mice was accelerated by IL-28A injection. Finally, ablation of the IL-10Rβ gene impeded the angiogenic responses and eNOS/AKT signaling stimulated by IL-28A in HUVECs.

Conclusion: HSP70-1 drives the progression of angiogenesis by the IL-28A/IL-10Rβ axis via eNOS/AKT signaling and the AP-1/NF-κB/MMP-2 network.

Keywords: Angiogenesis; HSP70-1; IL-10Rβ; IL-28A.

Graphical abstract

Fig. 1

IL-28A induced proliferation of HUVECs via eNOS/AKT/ERK1/2 signaling. A Immunofluorescence staining of IL-28A in surrounding bladder and muscle-invasive bladder cancer (MIBC) tissues. The images show DAPI staining for nuclei (blue), IL-28A staining (red), and merged images to indicate co-localization. Differential interference contrast (DIC) images provide detailed structural context. The merged images combine DAPI, IL-28A, and DIC to show the overall localization and expression of IL-28A. The scale bar represents 50 µm. B Immunohistochemical (IHC) staining for CD31 in surrounding bladder and MIBC tissues. The scale bar represents 50 µm. C RT-PCR analysis was performed to measure IL-28A mRNA expression in HUVECs treated with IL-28A compared to control. D IL-28A treatment induced production of IL-28A in HUVECs. E MTT assay was performed in IL-28A-treated HUVECs. F Phosphorylation level of eNOS, AKT, and ERK1/2 was examined using immunoblot analysis. The non-phosphorylation level of each protein was used as an internal control. G Inhibition of IL-28A-induced proliferation of HUVECs in the presence of specific signaling inhibitors, such as L-NAME (eNOS), LY294002 (AKT), and U0126 (ERK1/2). Data are presented as mean values ± SE from three independent experiments. *P<0.05 compared with control, ^#P<0.05 compared with IL-28A treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

IL-28A promoted migration, invasion, and tube formation via transcription factor-mediated MMP-2 expression in HUVECs. A Wound-healing assay was performed to determine the wound closure ability of HUVECs after IL-28A treatment for 24 h. The scale bar represents 200 µm. B Result of invasive potential induced by IL-28-A in HUVECs for 24 h. The scale bar represents 200 µm. C Colony tube formation assay in HUVECs treated with IL-28A for 24 h. The scale bar represents 200 µm. D, E MMP-2 level was examined in IL-28A-treated HUVECs using gelatin zymography and immunoblot analysis. F IL-28A-promoted binding ability of AP-1 and NF-κB was determined from the nuclear extract. G HUVECs were transfected with AP-1 siRNA or scrambled siRNA and then exposed to IL-28A for the analysis of MMP-2 levels through immunoblot. H Analysis of MMP-2 protein levels through immunoblot in HUVECs transfected with NF-κB siRNA or scrambled siRNA following IL-28A treatment. Data are presented as mean values ± SE from three independent experiments. *P<0.05 compared with control.

Fig. 3

Enos/akt/erk1/2 signaling is involved in migration, invasion, and tube formation in il-28a-treated huvecs. cells were treated with il-28a for 24 h in the presence or absence of L-NAME, LY294002, and U0126. A, D Wound-healing migration, B, e invasion, and C, F colony tube forming assay was performed in HUVECs. All scale bars represent 200 µm. Data are presented as mean values ± SE from three independent experiments. *P<0.05 compared with control, ^#P<0.05 compared with IL-28A treatment.

Fig. 4

Transcription factor-mediated MMP-2 expression is abolished following inhibition of eNOS/AKT/ERK1/2 signaling in HUVECs treated with IL-28A. After pretreatment of HUVECs with L-NAME, LY294002, and U0126 for 40 min, cells were further incubated with IL-28A for 24 h. A Enzyme activity of MMP-2 was determined by zymographic assay. B Immunoblot results of protein level of MMP-2 and phosphorylation level of signaling molecules (eNOS, AKT, and ERK1/2). C Binding ability of AP-1 and NF-κB was performed using EMSA. Data are presented as mean values ± SE from three independent experiments. *P<0.05 compared with control, ^#P<0.05 compared with IL-28A treatment.

Fig. 5

IL-28A induced microvessel outgrowth of angiogenesis ex vivo and in vivo. A Image of microvessel formation, followed by IL-28A treatment using aortic ring assay ex vivo. B Quantification of the number of vessels sprouting after IL-28A treatment. C Angiogenic effect of IL-28A in Matrigel plug in vivo assay. D Quantitative analysis of hemoglobin content. E Image of CD31 immunostaining in Matrigel plug. Effect of IL-28A antibody in IL-28A-mediated angiogenesis was examined via F, G aortic ring assay ex vivo, H, I Matrigel plug in vivo assay, and J immunostaining of CD31 antibody. The aortic ring assay ex vivo has a scale bar of 100 μm. The immunostaining of the CD31 antibody has a scale bar of 50 μm. Data are presented as mean values ± SE from three independent experiments. *P<0.05 compared with control, ^#P<0.05 compared with IL-28A treatment.

Fig. 6

Identification of HSP70-1 as a potential target gene in IL-28A-mediated angiogenesis. A Hierarchical clustering of RNA-seq expression data for IL-28A-induced genes. Red areas represent 1.5-fold upregulated DEGs, and green areas represent 1.5-fold downregulated DEGs between nontreated- and IL-28A-treated HUVECs. B Gene ontology annotations of upregulated DEGs. C Gene network analysis of 1.5-fold upregulated DEGs in IL-28A-induced HUVECs. The most significant modules of DEGs represent top three genes (red color), which were identified as HSP70-1, HSPA6, and HSP70-2. D, E Expression level of HSP70-1 was confirmed by RT-PCR and immunoblot analysis in IL-28A-treated HUVECs. Data are presented as mean values ± SE from three independent experiments. *P<0.05 compared with control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Knockdown of HSP70-1 attenuated IL-28A-induced angiogenic programs in vitro. A, D−F IL-28A-mediated proliferation, migration, invasion, and tube formation was suppressed in HUVECs transfected with HSP70-1 siRNA (si-HSP70-1). All scale bars represent 200 µm. B Phosphorylation level of eNOS, AKT, and ERK1/2 induced by IL-28A was measured by immunoblot analysis in si-HSP70-1-transfected HUVECs. C HUVECs were treated with IL-28A for 24 h. Cell lysates were immunoprecipitated with the anti-eNOS antibody or anti-AKT antibody, followed by immunoblot analysis using HSP70-specific antibody. GAPDH was used as an internal control. G, H IL-28A-induced MMP-2 level was performed in si-HSP70-1-transfected cells by analysis of gelatin zymography (enzyme activity) and immunoblot (protein expression). I EMSA experiment was performed to determine the IL-28A-promoted AP-1 and NF-κB binding activity in HUVECs transfected with si-HSP70-1. Data are presented as mean values ± SE from three independent experiments. *P<0.05 compared with control, ^#P<0.05 compared with IL-28A treatment.

Fig. 8

Effect of angiogenesis induced by IL-28A in HSP70-1 mutant animal models. HSP70-1 knockout mice (KO) and HSP70-1 transgenic mice (Tg) were used. A Microvessel outgrowth at 9 days using aortic ring assay in KO and wild-type mice (WT). B Quantification of microvessel sprouting. C Image of angiogenic effect from Matrigel plug in vivo assay at 7 days. D Quantitative analysis of hemoglobin content. E Image of CD31 immunostaining from Matrigel plug. F Aortic assay experiment in Tg and WT. G Quantitative analysis of microvessel sprouting. H Matrigel plug in vivo assay in Tg and WT. I Statistic analysis of hemoglobin content. J Immunostaining of Matrigel plug against CD31 antibody. The aortic ring assay ex vivo has a scale bar of 100 μm. The immunostaining of the CD31 antibody has a scale bar of 50 μm. Data are presented as mean values ± SE from three independent experiments. *P<0.05 compared with control, ^#P<0.05 compared with IL-28A treatment in WT.

Fig. 9

IL-28A/IL-10Rβ axis is essential for the potential angiogenic effect of HUVECs. A, C−E IL-10Rβ siRNA (si-IL-10Rβ) or scrambled siRNA were transfected into HUVECs, then cells were examined for extent of proliferation, migration, invasion, and tube formation stimulated by IL-28A. All scale bars represent 200 µm. B Subsequently, IL-28A-induced phosphorylation of eNOS, AKT, and ERK1/2 was investigated in cells transfected with si-IL-10Rβ or scrambled siRNA using immunoblot. Additionally, the expression of HSP70-1 was examined under the same conditions. F, G IL-28A-stimulated MMP-2 level was studied in the presence of si-IL-10Rβ or scrambled siRNA transfectants. H After transfection of cells with si-IL-10Rβ or scrambled siRNA, EMSA was performed to determine the binding ability of AP-1 and NF-κB motifs. Data are presented as mean values ± SE from three independent experiments. *P<0.05 compared with control, ^#P<0.05 compared with IL-28A treatment.

Supplementary Fig. 1

Expression level of HSP70-1, VEGF-A, and IL-10Rβ in the presence or absence of IL-28A in HUVECs. A Immunoblot of AP-1 expression in HUVECs transfected with si-AP-1. B Analysis of NF-κB expression in HUVECs transfected with si-NF-κB using immunoblotting. C After transfection of HUVECs with si-HSP70-1, HSP70 expression was examined by immunoblot. D Expression level of VEGF-A was determined in IL-28A-treated HUVECs using RT-PCR and immunoblot. E Cells were transfected with si-IL-10Rβ, and then immunoblot was performed to confirm the expression level of IL-10Rβ. Actin or GAPDH shown in this figure was used as an internal control. Data are expressed as mean values ± SE from three independent experiments. *P < 0.05 compared with control.

Supplementary Fig. 2

Quantification of tube formation in HUVECs using ImageJ.A-D Quantitative analysis of tube formation assays was performed using ImageJ software with the Angiogenesis Analyzer plugin. The software measures the extent of capillary-like structure formation with branches in green, twigs in cyan, master segments in yellow, and meshes in blue sky. All scale bars represent 200 µm.

Supplementary Fig. 3

IL-28A mediates HSP70-1 expression through ERK1/2-AP-1 pathway. A Immunoblot analysis of HSP70-1 expression in HUVECs treated with IL-28A and AKT inhibitor LY294002. B Analysis of HSP70-1 in HUVECs treated with IL-28A and the ERK1/2 inhibitor U0126 using immunoblot. C IL-28A-induced expression of HSP70-1 was investigated in cells transfected with AP-1 siRNA or scrambled siRNA using immunoblot. Data are presented as mean values ± SE from three independent experiments. *P < 0.05 compared with control, ^#P < 0.05 compared with IL-28A treatment.

Supplementary Fig. 4

The recovery of the ischemic hind-limb blood flow was enhanced in mice injected with IL-28A. A Blood flow perfusions were obtained by laser-Doppler perfusion imaging. Colors indicate lower perfusion (blue) and higher perfusion (red). B The quantification of the LDBF ratio in mice administered with 10 ng/g/day IL-28A (IL-28A) and control mice injected with PBS (PBS). All data are reported as the means ± SE from three independent experiments. *P < 0.05 compared with PBS.