Introduction
Angiogenesis refers to the process of generating new blood vessels and is strictly regulated by angiogenic factors and cytokines. Angiogenesis plays a central role in tissue repair, wound healing, and organ development [
1]. Abnormal angiogenesis status is associated with many diseases [
2,
3]. Excessive angiogenesis aggravates the progression of tumours, diabetic retinopathy, arthritis, and other diseases, while insufficient angiogenesis is detrimental to recovery from diseases such as peripheral arterial disease (PAD), myocardial infarction, and stroke [
4,
5].
Hypoxia is characteristic of many ischemic diseases and tumours and is a strong stimulus for angiogenesis [
6]. Hypoxia drives angiogenesis primarily by inducing the release of proangiogenic growth factors [
4,
7]. Vascular endothelial growth factor (VEGF) is considered a major angiogenic growth factor and the main target of antiangiogenic therapy [
8]. However, VEGF pathway inhibitors are failing to produce enduring clinical responses in most patients and many patients with metastatic disease are refractory or resistant to VEGF inhibitors [
5,
9‐
12]. For ischemic disease, there are few effective treatments other than endovascular therapy [
13‐
15]. Therefore, it is of great importance to identify the key regulators of angiogenesis and therapeutic target pairs in this process.
Karyopherin subunit alpha (KPNA) is a nuclear transport protein family that can bind cargo proteins containing a nuclear localization signal (NLS) and transport them into the nucleus by interacting with karyopherin subunit beta 1 (KPNB1). However, an increasing number of studies have indicated that KPNA has multiple functions beyond nuclear transport [
16]. Previous studies suggest that the KPNA family play a role in the angiogenesis of ageing myocardial microvascular endothelial cells and ageing gastric mucosal endothelial cells [
17,
18]. Karyopherin subunit alpha 2 (KPNA2), a member of the KPNA family, has been implicated in tumorigenesis and progression in previous studies [
19]. KPNA2 can be induced by hypoxia in various tumour cell types [
20,
21], and its knockdown inhibits osteosarcoma cell proliferation and reduces blood flow [
22]. Therefore, KPNA2 may be a key molecule in hypoxia-induced endothelial angiogenesis, and this hypothesis deserves further study.
In this study, we found that the binding of KPNA2 to signal transducer and activator of transcription 3 (STAT3) was increased under hypoxia, and KPNA2 promoted angiogenesis in vivo and in vitro by regulating the phosphorylation of STAT3 under hypoxia.
Materials and methods
Animals
Adult male C57BL/6 J mice (8 weeks), obtained from the Laboratory Animal Center at the Huazhong University of Science and Technology were used in this study. All animal protocols were approved by the Institutional Animal Care and Use Committee of Ethics of Tongji Medical College, Huazhong University of Science and Technology.
Murine hindlimb ischemia model
As described previously [
23], male c57Bl mice (8 weeks) were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight). After skin preparation, the superficial skin was incised to expose the femoral artery. The femoral artery was separated from the accompanying vein and nerve. The proximal and distal ends of the femoral artery were ligated, and the middle segment was cut to induce hindlimb ischemia.
Detection of perfusion recovery
Blood perfusion before and after surgery was monitored with a laser Doppler system (Perimed, Sweden). In detail, the mice were anaesthetized by intraperitoneal injection of pentobarbital sodium, and then the hindquarters were imaged by laser Doppler imaging. The reduction in and recovery of blood perfusion were obtained by calculating the ratio of perfusion in ischemic to nonischemic limbs.
Cell culture
Human umbilical vein endothelial cells (HUVEC) were isolated from fresh umbilical cords as described previously [
24]. The cells were cultured in Endothelial Cell Medium (ECM) (ScienCell) at 37 °C in a humidified incubator (Thermo Scientific, 3111) with 5% CO
2. Hypoxia exposure was applied by culturing the cells in an incubator (Thermo Scientific, 3131) with 1% O
2 and 5% CO
2. For endothelial infection, empty adenoviral vector (Ad-Vector) and adenoviruses vector encoding KPNA2 (Ad-KPNA2), scramble short hairpin RNA (Ad-NC), and KPNA2 shRNA (Ad-shKPNA2) were applied to infect HUVEC
in vitro. Viral fluid volume = number of cells × multiplicity of infection (MOI) /viral titer. Cells were lysed 48 h after infection.
293 T cells with STR identification were obtained from the American Type Culture Collection. They were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco) with 10% FBS (Gibco) and 1% volume of 100 × penicillin/streptomycin solution (Beyotime Biotechnology, C0222). The final concentration of penicillin is 100 U/ml and the final concentration of streptomycin is 0.1 mg/ml. The cells were cultured at 37 °C in a humidified incubator with 5% CO2.
Western blotting
Western blotting was performed as described previously [
25]. Tissue or cells were lysed using RIPA lysis buffer (Beyotime Biotechnology, P0013C) supplemented with PMSF (Thermo Scientific, 36978), protease inhibitors (Roche, 04693132001) and phosphatase inhibitors (Roche, 04906837001). Protein concentrations were determined using a Pierce BCA protein assay kit (Thermo Scientific, cat No. 23225). Western blotting was performed with antibodies against the following: KPNA2 (1:1000, Abclonal, A5012), KPNA1 (1:1000, Abclonal, A1742), KPNA3 (1:1000, Abclonal, A8347), KPNA4 (1:1000, Abclonal, A2026), KPNB1 (1:1000, Abclonal, A8610), β-ACTIN (1:1000, CST, 3700S), STAT3 (1:1000, Abclonal, A19566), P-STAT3 (1:1000, Abclonal, AP0705), VEGF (1:1000, Proteintech, 66828–1-Ig), P-VEGFR2 (1:1000, Cell Signaling Technology, #3770), GAPDH (1:1000, Abclonal, AC001), Laminb1 (1:1000, Cell Signaling Technology, #13435), ANGPT2 (1:1000, Abclonal, A11306), Flag (1:1000, Sigma, F2555), JAK1 (1:1000, Abclonal, A18323), JAK2 (1:1000, Abclonal, A7694), SRC (1:1000, Abclonal, A0324), TYK2 (1:1000, Abclonal, A2128), P-JAK1 (1:1000, Abclonal), GPX1 (1:1000, Abclonal, A11166), and MOV10 (1:1000, Abclonal, A7227).
Relative quantitative analysis of protein expression levels was performed using Image Lab software (Bio-Rad).
Immunofluorescence analysis
Tissue/cells on coverslips were fixed with 4% paraformaldehyde and deparaffinized. Antigen retrieval was carried out by heating. Then, the tissue sections/cells were incubated with donkey serum to block background signal, followed by the detection of specific antigens with primary and fluorescent secondary antibodies. The nuclei were stained with DAPI. Then the tissue sections/cells were observed and photographed under a fluorescence microscope (Olympus). Antibodies against the following were used: CD31 (1:200, Abcam, ab76533), KPNA2 (1:200, Abclonal, A5012), and P-STAT3 (1:200, Abclonal, AP0705). The fluorescent secondary antibodies used are as follows: Alexa Fluor488 donkey anti-rabbit lgG (H + L) (1:400, Life Technologies, A21206), Alexa Fluor555 donkey anti-rabbit lgG (H + L) (1:400, Life Technologies, A31572).
Recombinant adenovirus production
Recombinant adenovirus was constructed using Gateway cloning according to the manufacturer’s protocol. The entry vector for overexpression is the pENTY-ccDB-T2-pcdh vector with the CMV promoter, and the entry vector for knockdown is the PEnty-kd-ccDb2 vector with the U6 promoter. The Entry clone with the gene of interest flanked by attL sequences was produced using the restriction enzyme and ligase cloning method. Expression clones were generated with Gateway LR Clonas II enzyme mix (Thermo) catalysed in vitro by recombination of the entry vector (containing the gene of interest flanked by the attL sequences) with the destination vector (containing the attR sequences) pDEST (Thermo Scientific). Recombinant adenoviral plasmids were linearized with PacI (Thermo Scientific) and transfected into HEK293A cells for adenoviral packaging and amplification. Then, the adenovirus was purified by CsCl gradient centrifugation.
Tubule formation assays were performed as described previously [
26]. A 96-well plate was coated with 50 µl of Matrigel per well, and then HUVEC were seeded in the Matrigel-coated 96-well plate at a density of 1.5 × 10
4 cells per well. After 6 h, pictures were taken with a light microscope (Olympus, Tokyo, Japan), and the Angiogenesis Analyser plugin of ImageJ was used to count the total length.
Transwell assays
Migration assays were performed with a Transwell cell culture insert (3 µm, Corning, NJ). HUVEC (3 × 10
4 cells) were placed on the upper layer of a Transwell cell culture insert, and ECM was placed below the cell-permeable membrane. Following an incubation period (6 h), the cells that had migrated through the membrane were stained with 0.1% crystal violet for 20 min and counted [
27].
5-Ethynyl-2′-deoxyuridine (EdU) staining
EdU experiments were performed with a BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 555 (Beyotime Biotechnology, C0075S). Briefly, HUVEC were seeded in a 96-well plate at a density of 5000 cells per well. The cultured cells were labelled with EdU, fixed, washed, and permeabilized. Click reaction solution (Click Reaction Buffer, CuSO4, Azide 555, Click Additive Solution) was used for the EdU reaction and detection. DAPI was used to stain the nuclei. Fluorescence was observed under a fluorescence microscope (Olympus).
Extraction of cytoplasmic and nuclear proteins
HUVEC were prepared in 10 cm dishes for extraction of the cytoplasmic and nuclear proteins. Nuclear and cytoplasmic proteins were isolated using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents according to the manufacturer’s protocol (Thermo Scientific).
RNA extraction and quantitative RT‒PCR
RNA was extracted from tissues and cells using TRIzol (Takara, Japan). RNA (1000 ng) was reverse transcribed using a PrimeScript™ RT Reagent Kit (Takara, Japan). Quantitative RT‒PCR was performed with SYBR Green Master Mix (Vazyme, Nanjing, China). 18S rRNA was used as an endogenous control. Relative gene expression levels were calculated with the 2^(− ΔΔCT) method. The primers used were as follows:
KPNA2, forward 5′-CTGCCCGTCTTCACAGATTCA-3′, reverse 5′-GCGGAGAAGTAGCATCATCAGG-3′, ANGPT2, forward 5′-AACTTTCGGAAGAGCATGGAC-3′, reverse 5′-CGAGTCATCGTATTCGAGCGG-3′, VEGFA forward 5′-AGGGCAGAATCATCACGAAGT-3′, reverse 5′-AGGGTCTCGATTGGATGGCA-3′, 18S, forward 5′-TTGACGGAAGGGCACCACCAG-3′, reverse 5′-GCACCACCACCCACGGAATCG-3′.
Coimmunoprecipitation (Co-IP) assays
Co-IP assays were performed as described previously. The cells were lysed with RIPA lysis buffer (Beyotime Biotechnology, P0013C) supplemented with PMSF (Thermo Scientific) and protease inhibitors (Roche, 04693132001). The lysate was sonicated for 30 s and kept on ice for 1 h, and the supernatant was collected. Mixed 50 µl protein A/G magnetic beads (MedChemExpress, cat# HY-K0202) into the protein-antibody solution and incubated on a shaker at 4 °C for 3 h. The magnetic beads were precipitated with a magnetic stand. The magnetic beads were boiled with SDS loading buffer and subjected to immunoblotting analysis. The magnetic beads were also used for immunoprecipitation-mass spectrometry (IP-MS).
IP-MS
After sample processing, mass spectrometry data were collected using a Q Exactive Plus mass spectrometer (Thermo Scientific) in series with an EASY-nLC 1200 liquid phase LC/MS system (Thermo Scientific). The mass spectral data were searched with MaxQuant (V1.6.6) software, and the database search algorithm Andromeda was used to search the Human's Proteome Reference Database in UniProt (2020-05-10, containing 75074 protein sequences).
Statistical analysis
All data are presented as the mean ± SD. Statistical analysis between two groups was carried out by the two-tailed unpaired Student’s t test. All experiments were performed as multiple biological replicates (n = 3–6). Differences for which P < 0.05 were considered to be statistically significant. Statistical analysis was performed using GraphPad Prism 8.0.
Discussion
In this study, we demonstrate that KPNA2 promotes angiogenesis in vivo and in vitro under hypoxic conditions by promoting the phosphorylation of STAT3 and promoting expression of the angiogenic growth factors VEGF and ANGPT2.
KPNA2 has generally been described in previous studies as a transporter that mediates the nuclear transport of proteins including IRF3, P65, E2F1, and c-Myc [
28‐
31]. However, an increasing number of studies have suggested that KPNA2 is a multifunctional protein with a variety of functions in addition to its role in nuclear transport [
16]. Recent studies have found that KPNA2 is localized on the surface of various tumour cell types [
32]. KPNA2 promotes the phosphorylation of AKT and GSK-3β in tumour cells without altering the expression of total AKT or GSK3β [
33]. Our study showed that KPNA2 can regulate the phosphorylation of STAT3 without changing the total amount of STAT3, thereby promoting the nuclear entry of P-STAT3 and upregulating the downstream target genes STAT3 VEGF and ANGPT2. These studies suggest that KPNA2 is involved in regulating protein phosphorylation, which provides new ideas for future research on KPNA2.
The relationship between KPNA2 and STAT3 is controversial. Some studies have shown that KPNA2 and STAT3 colocalize in the nucleoplasm of tumour cells [
34], and IP results in fibroblast-like synoviocytes suggest that KPNA2 binds STAT3 [
35]. Immunofluorescence analysis showed reduced STAT3 nuclear entry after KPNA2 knockdown in pancreatic ductal adenocarcinoma cells [
36]. However, some studies have also shown that STAT3 enters the nucleus by binding KPNA3 rather than KPNA2 [
37]. Our IP-MS and IP-Western blot results indicated that the binding of KPNA2 to STAT3 in endothelial cells was increased under hypoxia, but we did not examine the specific mechanism related to this change. Previous studies have shown that posttranslational modification of KPNA2, such as its phosphorylation and palmitoylation, can modulate its activity and differences in substrate binding [
23,
38,
39]. However, whether hypoxia modulates KPNA2 activity and substrate binding by altering the posttranslational modification of KPNA2 requires further study.
STAT3 plays an important role in angiogenesis [
40,
41], and its main mechanism involves the upregulated expression of VEGF and ANGPT2 [
42‐
49]. VEGF is mainly involved in endothelial tube formation through VEGFR2 [
50], and unlike VEGF, ANGPT2 functions on the stalk and tip cells among endothelial cells [
51,
52]. Inhibition of ANGPT2 and VEGF has a cumulative effect on tumour growth and angiogenesis [
50,
53]. Our study showed that KPNA2 can simultaneously upregulate the expression of VEGF and ANGPT2 by promoting STAT3 phosphorylation.
STAT3 includes an N-terminal coiled-coil domain, a DNA-binding domain, a linker, an SH2 domain, and a C-terminal transactivation domain. The SH2 domain, which is located between amino acid residues 600 and 700, is essential for the recruitment of STATs to phosphorylated receptors [
54]. Our IP results indicated that KPNA2 can bind the SH2 domain of STAT3 and promote the phosphorylation of STAT3. Previous studies have shown that cell density can affect the phosphorylation level of STAT3 [
55,
56]. Therefore, to examine the effect of KPNA2 itself on STAT3 phosphorylation, we collected cellular proteins at different cell densities. The Western blotting results showed that the STAT3 phosphorylation level was higher in the KPNA2 overexpression group than in the control group at all cell densities (Additional file
3: Fig. S3A). Moreover, as the degree of KPNA2 overexpression increased, the level of STAT3 phosphorylation also increased (Additional file
3: Fig. S3B). These results supported that KPNA2 itself promoted phosphorylation of STAT3 rather than being cell density dependent.
The binding of STAT3 to JAK1 was reduced after KPNA2 knockdown, while the binding of STAT3 to other phosphorylated kinases, such as JAK2, TYK2, and SRC, was unchanged. These results suggest that the binding of KPNA2 to the SH2 domain of STAT3 promotes the binding of STAT3 to JAK1, thereby promoting the phosphorylation and nuclear import of STAT3.
Briefly, our study shows that under hypoxic conditions, the expression of KPNA2 was upregulated in a hindlimb ischemia model and endothelial hypoxia and that the binding of KPNA2 and STAT3 was increased under hypoxic conditions, which promoted angiogenesis under hypoxic conditions. Mechanistically, we found that KPNA2 binds STAT3 and promotes the binding of JAK1 to STAT3 and the phosphorylation of STAT3 to upregulate VEGF and ANGPT2. However, our research still has some limitations. We used a murine hindlimb ischemia model for our experiments, and the results have not been validated in other models. Furthermore, we used intramuscular adenovirus injection for gene overexpression and knockdown in the animal experiments. The endothelial specificity of intramuscular adenovirus injection is insufficient, so it is impossible to determine whether the effect of KPNA2 was endothelium specific.
In general, our study has clarified the role of KPNA2 in endothelial angiogenesis and provided some insights into the mechanism of angiogenesis under hypoxic conditions and the treatment of angiogenesis-related diseases.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.