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. 2018 Apr 11;32(9):5051–5062. doi: 10.1096/fj.201701543RR

Angiotensin II increases angiogenesis by NF-κB–mediated transcriptional activation of angiogenic factor AGGF1

Wenxia Si *,†,1, Wen Xie *,1, Wenbing Deng *, Yi Xiao ‡, Sadashiva S Karnik §,¶,‖,#, Chengqi Xu *, Qiuyun Chen §,¶,‖,#,2, Qing Kenneth Wang *,‡,§,¶,‖,#,**,3
PMCID: PMC6103173  PMID: 29641288

Abstract

Angiogenic factor with G-patch and FHA domains 1 (AGGF1) is involved in vascular development, angiogenesis, specification of hemangioblasts, and differentiation of veins. When mutated, however, it causes Klippel-Trenaunay syndrome, a vascular disorder. In this study, we show that angiotensin II (AngII)—the major effector of the renin–angiotensin system and one of the most important regulators of the cardiovascular system—induces the expression of AGGF1 through NF-κB, and that AGGF1 plays a key role in AngII-induced angiogenesis. AngII significantly up-regulated the levels of AGGF1 mRNA and protein in HUVECs at concentrations of 10–40 μg/ml but not >60 μg/ml. AngII type 1 receptor (AT1R) inhibitor losartan inhibited AngII-induced up-regulation of AGGF1, whereas AT2R inhibitor PD123319 further increased AngII-induced up-regulation of AGGF1. Up-regulation of AGGF1 by AngII was blocked by NF-κB inhibitors, and p65 binds directly to a binding site at the promoter/regulatory region of AGGF1 and transcriptionally activates AGGF1 expression. AngII-induced endothelial tube formation was blocked by small interfering RNAs (siRNAs) for RELA (RELA proto-oncogene, NF-κB subunit)/p65 or AGGF1, and the effect of RELA siRNA was rescued by AGGF1. AngII-induced angiogenesis from aortic rings was severely impaired in Aggf1+/− mice, and the effect was restored by AGGF1. These data suggest that AngII acts as a critical regulator of AGGF1 expression through NF-κB, and that AGGF1 plays a key role in AngII-induced angiogenesis.—Si, W., Xie, W., Deng, W., Xiao, Y., Karnik, S. S., Xu, C., Chen, Q., Wang, Q. K. Angiotensin II increases angiogenesis by NF-κB–mediated transcriptional activation of angiogenic factor AGGF1.

Keywords: AT1R, AT2R, p65

Angiogenic factor with G-patch and FHA domains 1 (AGGF1) was originally cloned by our laboratory and found to encode a potent angiogenic factor with a G-patch domain and a forkhead-associated domain responsible for the vascular disease Klippel-Trenaunay syndrome (1). In zebrafish, Aggf1 was found to be involved in the specification of vein differentiation and embryonic angiogenesis by activating Akt signaling as well as the specification of hemangioblasts, multipotent progenitor cells responsible for differentiation of endothelial cells and all types of blood cells (2–4). In a mouse limb ischemia model, overexpression of AGGF1 promoted therapeutic angiogenesis with intramuscular administration of AGGF1 DNA (5). Aggf1+/− knockout (KO) mice showed reduced physiologic angiogenesis due to reduced PI3K-Akt signaling and decreased tumor growth and angiogenesis (6). AGGF1 can also induce autophagy by activating JNK signaling, and it promotes therapeutic angiogenesis, a robust treatment for coronary artery disease and myocardial infarction in mice (7). Importantly, we showed that AGGF1-induced autophagy was required for therapeutic angiogenesis (7). We also demonstrated that purified AGGF1 blocked neointimal formation, proliferation, and migration of vascular smooth muscle cells (VSMCs) and promoted phenotypic switching of VSMCs from a synthetic phenotype to the contractile phenotype after vascular injury, and Aggf1+/− mice showed increased neointimal formation and increased proliferation and migration of VSMCs (8). Moreover, AGGF1 expression was reduced in patients with dilated cardiomyopathy and heart failure, and cellular and animal models for cardiac hypertrophy (9). AGGF1 protein therapy successfully treated cardiac hypertrophy and heart failure by blocking the ER stress signaling via a novel noncanonical pathway (9). AGGF1 was also found to be an anti-inflammatory factor, antagonizing the ERK/NF-κB pathway in endothelial activation in response to TNF-α (10).

AngII is generated by the activation of angiotensin I through the angiotensin II converter enzyme (11). AngII participates in neoplastic and nonneoplastic tissue angiogenesis and modulates contraction, cell growth, apoptosis, cell differentiation, cell migration, extracellular matrix conformation, and inflammation (12–15). The effects of AngII are mediated by 4 different cell surface receptors: AngII type 1, 2, 3, and 4 receptors (AT1R, AT2R, AT3R, and AT4R). AT1R and AT2R are the 2 most well-studied receptors for AngII and display opposite effects. AngII was shown to regulate the expression levels of various growth factors such as VEGF, PDGF, TGF-β, EGF, and IGF as well as their receptors (16–19), but it remains to be determined whether AngII induces angiogenesis through these factors.

In this study, we show that AngII regulates the expression level of AGGF1 in a dose-dependent manner. AngII up-regulates the expression of AGGF1 by AT1R-mediated NF-κB (p65) signaling. NF-κB binds directly to the AGGF1 promoter (or regulatory region) and activates the transcription of AGGF1. We also found that AGGF1 is required for AngII-induced angiogenesis.

MATERIALS AND METHODS

Cell culture

HUVECs were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA). Cells were maintained at 37°C with 5% CO2. HUVECs were transfected using Fugene HD (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The transfection efficiency was ∼40%.

Human cervical carcinoma cells (HeLa line) were purchased from China Center for Type Culture Collection (Wuhan, China) and cultured in RPMI-1640 medium supplemented with 10% FBS in a humidified incubator with 5% CO2 at 37°C. Cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. The transfection efficiency was ∼60%. HeLa cells were used for luciferase assays, which require transfection of multiple plasmids.

Plasmids and other reagents

The full-length RELA (RELA proto-oncogene, NF-κB subunit) cDNA was obtained from RT-PCR analysis using total RNA isolated from HeLa cells. The PCR product was digested with restriction enzymes Hind III and Kpn I (Takara Bio, Kusatsu, Japan) and was then subcloned into the p3-Flag-CMV-7.1 vector cut with the same enzymes. This generates a mammalian expression plasmid for RELA encoding p65 (pFlag-p65).

The wild-type (WT) AGGF1-luc luciferase reporter with the AGGF1 promoter/regulatory region fused to the luciferase reporter gene was described by us previously (20). The NF-κB binding site was mutated in AGGF1-WT-luc using PCR-based site-directed mutagenesis resulting in the AGGF1-MUT-luc luciferase reporter. The primers used for mutagenesis included a forward primer of 5′-GCACGTACACTGTCAGATCGTACCGCTTT-3′ and a reverse primer of 5′-CGGAAAGCGGTACGATCTGACAGTGTA-3′.

AngII was purchased from MilliporeSigma (Burlington, MA, USA). PD98059, SB203580, SP600125 and PDTC were also from MilliporeSigma. Losartan and PD123319 were from MedChem Express (Monmouth Junction, NJ, USA). Quinacrine was kindly provided by Dr. George Stark (Cleveland Clinic).

The siRNAs for RELA and AGGF1 were synthesized by RiboBio (Guanzhou, China), and their sequences are shown in Supplemental Table 1. The negative control siRNA (siNC) does not have homology to any sequences in the human genome.

RNA extraction and real-time quantitative PCR

Total RNA was extracted from HUVECs using the Trizol Reagent Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. A Promega reverse-transcription kit was used to reverse-transcribe 2 μg of total RNA. Quantitative real-time PCR was performed using the ABI 7900HT system (Thermo Fisher Scientific). PCR reactions were carried out in 10 μl volumes using SYBR Green PCR Master Mix (Thermo Fisher Scientific) and 0.2 μM specific primers. Primer sequences for specific genes are described in Supplemental Table 1.

Western blot analysis

Total protein extracts were extracted from HUVECs and HeLa cells using lysis buffer (Beyotime Biotechnology, Haimen, China) with 1-time protease and phosphatase inhibitors (Roche, Basel, Switzerland), and used for Western blot analysis as described previously (21). The antibodies used include a polyclonal antibody for AGGF1 from Proteintech (Rosemont, IL, USA); polyclonal antibodies for phospho-p65, total p65, phospho-ERK1/2, phospho-p38, total p38, phospho-JNK, and total JNK from Cell Signaling Technology (Danvers, MA, USA); and an anti-FLAG antibody, mouse IgG, and rabbit IgG from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Goat anti-rabbit and goat anti-mouse horseradish peroxidase–conjugated secondary antibodies were purchased from MilliporeSigma.

Matrigel-based capillary tube formation assay

HUVECs were cultured in 12-well plates and transfected with AGGF1 small interfering RNA (siRNA), RELA siRNAs, and control siRNA (RiboBio, Guangzhou, China). HUVECs were then treated with AngII (10 μg/ml) after starvation without FBS for 12 h. The treated HUVECs were trypsinized and resuspended in reduced serum medium. Resuspended HUVECs were added to 48-well plates coated with growth factor–reduced Matrigel basement membrane matrix (Corning, Corning, NY, USA), and incubated for 6 h. The number of capillary tubes was quantified under a microscope (Nikon Celipse Ti-S, Tokyo, Japan). To quantify the tubes, we counted the number of complete rings in all images for each well using the angiogenesis analysis tool of ImageJ (National Institutes of Health, Bethesda, MD, USA), and then calculated the total number of rings in each well.

Ex vivo mouse aortic ring assay for angiogenesis

Mouse aortic ring assays were performed as previously described (7). Thoracic aortas were removed from WT mice and Aggf1+/− KO mice and were then cut into 2-mm-long aortic rings and embedded in Matrigel. AngII (10 μg/ml) or AGGF1 protein (5 μg/ml) was then added to the medium. After 4 d of incubation in 37°C with 5% CO2, newly formed microvessels were analyzed under a microscope (Nikon Celipse Ti-S).

Luciferase assays

The luciferase assay was performed using the Dual-Luciferase Reporter Assay System (Promega) as previously described (22). AGGF1-WT-luc or AGGF1-MUT-luc was cotransfected with pRL-TK expressing renilla luciferase (Promega) into HeLa cells for 48 h. The cells were lysed using 1 time lysis buffer (Promega) and used for luciferase assays using the Dual-Luciferase Reporter Assay System (Promega). The fluorescence values of firefly luciferase and Renilla luciferase were read using Promega GloMaxTM 20/20 Luminometer, and the ratio was considered as the luciferase activity.

Chromatin immunoprecipitation analysis

Chromatin immunoprecipitation (ChIP) analysis was performed using the MilliporeSigma EZ-ChIP kit. Briefly, HeLa cells were cultured in 10-ml (100 × 20 mm) dishes (Thermo Fisher Scientific) and transfected with the mammalian expression plasmid for p65 (10 μg) or a control plasmid for 48 h. The transfected cells were then used for ChIP following the manufacturer’s protocol. Immunoprecipitation was performed with an anti-p65 antibody. The primers used for qPCR were shown in Supplemental Table 2.

Electrophoretic mobility shift assays

Nuclear extracts were prepared from HeLa cells with overexpression of p65 using the Nuclear and Cytoplasmic Protein Extraction kit (P0028; Biotime, Alameda, CA, USA). Nuclear extract (10 μg) was mixed with 6 ng of DNA probe and incubated for 20 min. The protein–DNA complex was separated on 5% polyacrylamide gels. For competition analysis, 200-fold excess of unlabeled DNA probe was added to the mixture. For the supershift electrophoretic mobility shift assay (EMSA), 1 μl of anti-p65 antibody (Cell Signaling Technology) was also added.

Statistical analysis

The data come from 3 independent experiments and are presented as means ± sd. We utilized SPSS v.16.0 software (IBM, Armonk, NY, USA) to perform Student’s t test for comparing 2 groups of means, or GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA) for calculating 1-way ANOVA with Tukey’s post hoc test for comparing more than 2 groups of means. Values of P < 0.05 were considered statistically significant.

RESULTS

AngII affects the expression level of AGGF1 in HUVECs

AngII is involved in the development of various cardiovascular diseases (23). The expression level of AGGF1 varies under different physiologic and pathologic conditions (8, 9, 20, 21). Therefore, we examined whether AngII affects the expression level of AGGF1. HUVECs were starved for 12 h and then were treated with different concentrations of AngII, after which Western blot analysis was performed to measure the level of AGGF1. As shown in Fig. 1A, B, AngII induced AGGF1 expression at the concentrations from 10 to 40 μg/ml, however, the induction was lost at concentrations of 60 and 80 μg/ml. Real-time RT-PCR analysis also showed that AngII increased the expression level of AGGF1 mRNA at concentrations of 5–30 μg/ml. These data suggest that AngII significantly up-regulates the expression of AGGF1 in a concentration-dependent manner.

Figure 1.

Figure 1

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AngII up-regulates the expression of AGGF1 in HUVECs. A) HUVECs were starved for 12 h and subsequently treated with AngII for 12 h at different concentrations: 10 μg/ml (9.56 μM), 20 μg/ml (19.12 μM), 40 μg/ml (38.24 μM), 60 μg/ml (57.36 μM), and 80 μg/ml (76.48 μM). Cells were harvested and used for Western blot analysis with an anti-AGGF1 antibody or control anti-GAPDH. B) HUVECs were starved for 12 h and then treated with AngII for 12 h at different concentrations: 5 μg/ml (4.78 μM), 10 μg/ml (9.56 μM), 15 μg/ml (14.34 μM), 20 μg/ml (19.12 μM), and 30 μg/ml (28.68 μM). Cells were harvested and used for Western blot analysis. C) HUVECs were treated as in B and used for real-time RT-PCR analysis. D) HUVECs were starved for 12 h, treated with 10 μg/ml (9.56 μM) of AngII with or without AT1R inhibitor losartan (IC50 = 20 nM) or AT2R inhibitor PD123319 (IC50 = 34 nM) for 12 h, and were subsequently used for Western blot analysis. *P < 0.05, **P < 0.01, ****P < 0.0001 (n = 3).

The effects of AngII are mediated primarily by AT1R or AT2R (24). To determine whether AT1R or AT2R mediates the effect of AngII on the expression of AGGF1, we analyzed the effects of losartan and PD123319, specific inhibitors for AT1R and AT2R, respectively. As shown in Fig. 1D, at a concentration of 10 μg/ml, AngII induced the expression of AGGF1, but the effect was significantly inhibited by losartan. Losartan reduced the expression level of AGGF1 in a dose-dependent manner although the effect remained constant after reaching 10 nM (Supplemental Fig. 1). The data suggest that AngII induces the AGGF1 expression through the AT1R. Interestingly, the AT2R inhibitor PD123319 further enhanced the effect of AngII on AGGF1 up-regulation (Fig. 1D). These data suggest that AT1R and AT2R play opposing roles in the up-regulation of AGGF1 by AngII.

AngII up-regulates the expression of AGGF1 via the NF-κB signaling pathway

AngII can modulate gene expression via the activation of multiple downstream signaling pathways, such as PI3K, NF-κB (p65), and MAPK, through AT1R or AT2R (24–26). To identify the signaling pathway by which AngII up-regulates the expression of AGGF1, we studied PD98059, SB203580, SP600125, and PDTC, which specifically inhibit the phosphorylation of ERK1/2, p38, JNK, and p65, respectively. As shown in Fig. 2A–D, AngII can activate the phosphorylation of JNK, p38, p65, and ERK1/2 and enhance the expression of AGGF1 in HUVECs; however, the effect on AGGF1 ended only when p65 phosphorylation was blocked with PDTC. These data suggest that AngII up-regulates the expression of AGGF1 via the NF-κB signaling pathway.

Figure 2.

Figure 2

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AngII regulates the expression of AGGF1 through the NF-κB pathway. A) Effect of JNK inhibitor SP600125 (20 μM) on Ang II-induced expression of AGGF1. B) Effect of p38 MAPK inhibitor SB203580 (10 μM) on Ang II-induced expression of AGGF1. C) Effect of NF-κB inhibitor PDTC (10 μM) on Ang II-induced expression of AGGF1. D) Effect of ERK inhibitor PD98059 (20 μM) on Ang II-induced expression of AGGF1. E) Effect of NF-κB inhibitor PDTC (10 μM) and AT1R inhibitor losartan (IC50 = 20 nM) on increased p65 phosphorylation and Ang II-induced expression of AGGF1. NS, not significant; PDTC, pyrrolidine dithiocarbamate. **P < 0.01, ***P < 0.001, ****P < 0.001 (n = 3).

Treatment of HUVECs with AT1R inhibitor losartan showed a similar effect to PDTC, which significantly inhibited the phosphorylation of p65, and reduced the expression level of AGGF1 (Fig. 2E). These data suggest that AT1R activates the NF-κB signaling pathway, which mediates AngII–up-regulated expression of AGGF1.

NF-κB activates the transcription of AGGF1

Quinacrine (QC), an antimalaria drug that blocks the activity of NF-κB (27, 28), is reportedly an inhibitor for the interaction between p65 and its DNA binding sequence because it blocks p65 transcription activity (29). In addition, QC appeared to decrease the level of p65 protein (Supplemental Fig. 2A, B), which is consistent with the data in Fig. 2C by Jani et al. (28). In order to test the role of NF-κB on AngII-induced AGGF1 expression, we treated HUVECs with QC and AngII, then performed Western blot to analyze the level of AGGF1 expression. AngII failed to increase AGGF1 expression in the presence of QC (Supplemental Fig. 2A, C).

To test whether NF-κB can transcriptionally activate AGGF1, HUVECs were treated with QC. Real-time RT-PCR and Western blot analyses showed that QC significantly reduced the expression levels of p65 and AGGF1 both at the mRNA and protein expression levels (Fig. 3A, B).

Figure 3.

Figure 3

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NF-κB inhibitor QC inhibits the expression of AGGF1. A) HUVECs were treated with different concentrations of QC for 12 h, lysed, and used for Western blot analysis along with a phosphorylated p65 antibody, an anti-AGGF1 antibody, and a control anti-GAPDH antibody. B) HUVECs were treated as in A and used for real-time RT-PCR analysis to determine RELA and AGGF1 expression. C). HeLa cells were transfected with an AGGF1-promoting luciferase reporter (pGL3-AGGF1p) for 24 h, treated with different concentrations of QC for 12 h, and used for luciferase assays. The AGGF1 promoter luciferase reporter (pGL3-AGGF1p) contains the AGGF1 promoter and regulatory region (−636 bp from the translation start site) fused to the luciferase gene. ***P < 0.001 (n = 3).

To test the direct effect of NF-κB on transcriptional activation of AGGF1, we cloned the AGGF1 promoter and regulatory region (−636 to +1 from the translation start codon) to the luciferase reporter gene in the pGL3-Basic vector (AGGFp-636-Luc reporter). The reporter was transfected into HeLa cells treated with different concentrations of QC and luciferase activities were measured. As shown in Fig. 3C, luciferase assays showed that QC significantly reduced the transcriptional activation of the AGGF1 promoter. Those data suggest that NF-κB directly activates the AGGF1 promoter.

A highly conserved p65 binding region at the AGGF1 promoter is responsible for the transcriptional activation of AGGF1 by NF-κB

To further show that p65 mediates the transcriptional activation of AGGF1, we used bioinformatic analysis to analyze the sequences at the promoter and regulatory region of AGGF1. Our previous study identified the transcriptional start site (TSS) of AGGF1 at −219 bp from the ATG (20). Using the JASPAR database (http://jaspar.genereg.net), we identified a p65 binding site upstream of the TSS (Fig. 4A). The p65 binding site is located between −278 and −265 bp from the start codon ATG and highly conserved across different species during evolution (Fig. 4B, C). We constructed an AGGF1-luc WT reporter gene in which the AGGF1 promoter/regulatory region −636 to +6 bp from the ATG was cloned upstream of the luciferase gene (Fig. 4B, C). Cotransfection of the AGGF1-luc WT reporter and an expression plasmid for p65 (pFlag-RELA) significantly increased the AGGF1 promoter activity (Fig. 4E). To show that the p65 binding site is necessary for p65-induced AGGF1 promoter activity, we mutated the p65-binding site, resulting in a mutant AGGF1-luc MUT reporter gene (Fig. 4D). Compared to the AGGF1-luc WT reporter, the mutant AGGF1-luc MUT reporter failed to be activated by overexpression of p65 (Fig. 4E). Those data suggest that the p65 binding site at the AGGF1 promoter or regulatory region is directly involved in the transcriptional activation of the AGGF1 promoter.

Figure 4.

Figure 4

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Identification of a conserved p65 binding site at the AGGF1 promoter and regulatory region and overexpression of p65 activates the AGGF1 transcription. A) The position weight matrix of the p65 binding site at the AGGF1 promoter and regulatory region from the JASPAR Database. B) The p65 binding site is located in the region from −278 to −266 bp from the AGGF1 translation start site. C) The p65 binding site at the AGGF1 promoter (or regulatory region) shows high evolutionary conservation among different species. D) Schematic diagram of the AGGF1 promoter luciferase reporter (AGGF1-LUC WT) with the p65 binding site. A mutant report was created by mutating the p65 site from 5′-GGTCCTTCCC-3′ to 5′-GATCGTACCG-3′, resulting in a mutant luciferase reporter AGGF1-LUC MUT. E) Overexpression of p65 activates AGGF1-LUC WT, but not AGGF1-LUC MUT. HeLa cells were cotransfected with pFlag-RELA as well as the WT or MUT luciferase reporter and subsequently used for luciferase assays. ***P < 0.001 (n = 3).

Effect of overexpression and knockdown of p65 on AGGF1 gene expression

Western blot analysis showed that overexpression of p65 significantly increased the expression level of AGGF1, whereas knockdown of RELA/p65 expression by 3 different siRNAs significantly decreased the expression level of AGGF1 (Fig. 5A, B). Similar results were obtained with the expression level of the AGGF1 mRNA by real-time RT-PCR analysis (Fig. 5C). Luciferase assays also showed that overexpression of p65 increased AGGF1 promoter activity, but knockdown of RELA expression significantly decreased AGGF1 promoter activity (Fig. 5D). These data suggest that p65 transcriptionally regulates AGGF1 expression.

Figure 5.

Figure 5

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Transcriptional factor p65 regulates transcriptional activation of AGGF1. A, B) HUVECs were transfected with pFlag-RELA (p65) for overexpression of p65 or siRNAs specific for the RELA gene for 48 h and used for Western blot analysis to determine the expression levels of p65 and AGGF1. C) HUVECs were treated as in A and B and used for real-time RT-PCR analysis to measure the expression level of AGGF1. D) HeLa cells were cotransfected with the AGGF1 promoter reporter AGGF1-LUC MUT, pRL-TK, pFlag-RELA (p65), or RELA siRNAs for 48 h, and then used for luciferase assays. **P < 0.01, ***P < 0.001 (n = 3).

Transcriptional factor p65 directly binds to the AGGF1 promoter/regulatory region

We performed ChIP analysis using HeLa cells transfected with a p65 overexpression plasmid, a pair of primers spanning the NF-κB binding site, and a pair of control primers 10 kb upstream of the AGGF1 TSS (Fig. 6A). HeLa cell lysates were subject to ultrasonication, and the protein–DNA complexes (p65 and its binding site) were immunoprecipitated with an anti-p65 antibody or control anti–rabbit IgG. The identity of the DNA in immunoprecipitates was identified by real-time PCR analysis (Fig. 6B). A positive PCR signal was identified for the p65 binding, but not with the control site (Fig. 6B, C). These data demonstrate specific binding of p65 to the NF-κB binding site upstream of the AGGF1 transcription start site.

Figure 6.

Figure 6

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Transcriptional factor p65 binds directly to the AGGF1 promoter. A) Schematic diagram of the 5′-UTR of the human AGGF1 gene. B) ChIP assays. The interaction between p65 and its binding site was detected using qPCR analysis with agarose gel electrophoresis. C) The data from B were quantified and plotted. D) EMSA. The p65–DNA complex can be detected when p65 is overexpressed (compare lanes 1 and 2). The p65–DNA complex was eliminated by addition of 200-fold excess of the EMSA competitor (lane 3). The p65–DNA complex can be supershifted by a specific anti-p65 antibody (lane 4). For probe design and preparation for EMSA, a pair of primers labeled with biotin (5′-TGCCGCTGGCGCCGTTGTTT-3′, 5′-TACAGAGACAGAGGGAGGAG–biotin-3′) were used to amplify a 200 bp DNA fragment containing the p65 binding site. The same pair of primers, unlabeled, was used to prepare the unlabeled DNA fragment containing the p65 binding site, which was used as the competitor in EMSA. SS complex, supershifted protein–DNA complex. NS, not significant. **P < 0.01 (n = 3).

The ChIP data were confirmed using EMSA analysis with a biotin-labeled DNA probe containing the p65 binding site (Fig. 6D). We used PCR with biotin-labeled primers to generate a 200 bp biotin-labeled double-stranded DNA probe for EMSA. Incubation of the biotin-labeled probe with protein extracts from HeLa cells transfected with the p65 expression plasmid resulted in a protein–DNA complex (Fig. 6D, lane 2). The protein complex formation was disrupted with addition of a 200-fold, unlabeled excess of the same probe (Fig. 6D, lane 3). The p65 DNA complex disappeared with addition of an anti-p65 antibody in EMSA and a supershifted protein–DNA complex was observed (Fig. 6D, lane 4). These data confirm the ChIP results that p65 can directly bind to the AGGF1 promoter or regulatory region.

AGGF1 is involved in AngII-induced angiogenesis

AngII induces angiogenesis (30). As AGGF1 is an angiogenic factor that promotes angiogenesis as potently as VEGF-A, we studied the effect of AGGF1 in AngII-induced angiogenesis. First, we observed the effect of AGGF1 on AngII-induced capillary tube formation by HUVECs in a Matrigel assay. HUVECs treated with AngII showed a significant increase in tube formation (Fig. 7A, B, F). The AngII-induced tube formation was halted by knockdown of AGGF1 or RELA/p65 expression by siRNAs (Fig. 7C, D, F). The effect of p65 siRNA was restored by AGGF1 treatment (Fig. 7E, F), suggesting that AGGF1 acts downstream of p65.

Figure 7.

Figure 7

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AngII induces capillary tube formation by AGGF1. A) Capillary tube formation by HUVECs transfected with siNC. B) Capillary tube formation by HUVECs transfected with control siNC and treated with AngII (10 μg/ml or 9.56 μM). C) Capillary tube formation by HUVECs transfected with siRELA and treated with AngII (10 μg/ml or 9.56 μM). D) Capillary tube formation by HUVECs transfected with siAGGF1 and treated with AngII (10 μg/ml or 9.56 μM). E) AGGF1 restores the effect of siRELA on AngII-induced capillary tube formation. Capillary tube formation was observed in HUVECs transfected with siRELA and treated with AngII (10 μg/ml or 9.56 μM) and AGGF1. F) The images in (A-E) were quantified and plotted. Scale bars, 200 μm. **P < 0.01, ***P < 0.001 (n = 3).

Second, we characterized the role of AGGF1 in AngII-induced angiogenesis using an ex vivo mouse aortic ring assay. The WT aortic rings treated with AngII showed a significantly higher number of sprouting neovessels than the control did (Fig. 8A, B, F). The stimulating effect of AngII on neovessel sprouting was, however, dramatically reduced in Aggf1+/− aortic rings (Fig. 8B, D, F). When the aortic rings from Aggf1+/− KO mice were treated with purified AGGF1 protein, AngII was able to induce neovessel sprouting again (Fig. 8D, E, F). Together, these data suggest that AGGF1 plays an important role in AngII-induced angiogenesis.

Figure 8.

Figure 8

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AngII induces neovessels to sprout by activating AGGF1. A) Neovessels sprout from aortic rings of WT mice treated with control water. B) Neovessels sprout from aortic rings of WT mice treated with AngII (10 μg/ml or 9.56 μM). C) Neovessels sprout from aortic rings of Aggf1+/− mice treated with control elution buffer. D) Neovessels sprout from aortic rings of Aggf1+/− mice treated with AngII (10 μg/ml or 9.56 μM). E) Neovessels sprout from aortic rings of Aggf1+/− mice treated with AngII (10 μg/ml or 9.56 μM) and AGGF1 (5 μg/ml). Scale bar, 200 μm . F) Quantitative data for sprout areas. ***P < 0.001, ****P < 0.0001 (n = 3).

AngII increases the expression level of AGGF1 independent of VEGF-A and VEGFR2

As both VEGF-A and AGGF1 were involved in AngII-induced angiogenesis, we determined whether the 2 key angiogenic factors cross-regulate each other upon AngII treatment. HUVECs were transfected with VEGF receptor 2 (VEGFR2) and control siRNA (siNC) for 24 h, starved for 12 h, and then treated with AngII (10 μg/ml) for 12 h. Western blot and real-time RT-PCR analyses showed that AngII increased the expression levels of AGGF1 mRNA and protein and that knockdown of VEGFR2 expression did not have any significant effect on AngII (Fig. 9A, B). Real-time RT-PCR analysis showed that AngII or AGGF1 siRNA did not have any effect on the expression level of VEGFR2 (Fig. 9C). AngII treatment significantly increased the expression level of VEGFA mRNA, and the effect was not affected by AGGF1 siRNA. These data suggest that that there is no cross-regulation between AGGF1 and VEGF-A/VEGFR2 in AngII functions.

Figure 9.

Figure 9

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AGGF1 signaling does not crosstalk with VEGF-A signaling. A) HUVECs were transfected with siRNA for VEGFR2 (siVEGFR2), treated with AngII (10 μg/ml/9.56 μM), and used for Western blot analysis to measure the level of AGGF1. B) HUVECs were transfected with siRNA for VEGFR2 (siVEGFR2), treated with AngII (10 μg/ml or 9.56 μM), and used for real-time RT-PCR analysis to measure the level of AGGF1 mRNA. C) HUVECs were transfected with siRNA for AGGF1 (siAGGF1), treated with AngII (10 μg/ml or 9.56 μM), and used for real-time RT-PCR analysis to measure the level of VEGFR2 mRNA. D) HUVECs were transfected with siRNA for AGGF1 (siAGGF1), treated with AngII (10 μg/ml or 9.56 μM), and used for real-time RT-PCR analysis to measure the level of VEGFA mRNA. NS, not significant. *P < 0.05, ***P < 0.001 (n = 3).

DISCUSSION

Here we show that AngII, the major effector of the renin–angiotensin system and one of the most important regulators of the cardiovascular system, can induce the expression of the AGGF1 gene at low concentrations of 10–40 μg/ml (Fig. 1). We further demonstrated that up-regulation of AGGF1 expression by AngII occurred through the activation of the NF-κB pathway via AT1R (Figs. 2 and 3). We found that p65 bound directly to the AGGF1 promoter or regulatory region and activated the transcription of AGGF1 (Figs. 4–6). Interestingly, we showed that AngII-induced AGGF1 expression was functionally relevant because knockdown of the expression of AGGF1 stopped AngII-induced capillary tube formation by HUVECs (Fig. 7). Moreover, knockdown of p65 expression also abolished AngII-induced capillary tube formation by HUVECs, however, the effect was restored after treatment with recombinant AGGF1 protein (Fig. 7). Similarly, our mouse aortic ring assays showed that AGGF1 plays an important role in AngII-induced angiogenesis (Fig. 8). These data identify a novel regulatory system for the expression of AGGF1. Our data also identify the angiogenic factor AGGF1 as a novel regulator for AngII-induced angiogenesis.

Previous studies showed that AngII up-regulated other angiogenic factors, including VEGF-A, bFGF (basic fibroblast growth factor), platelet-derived growth factor, IGF-1, EGF (epidermal growth factor), and TGF-β (16–19, 31). However, it is unknown whether 1 or more of these angiogenic factors is required for AngII-induced angiogenesis. Buharalioglu et al. (32) reported that AngII induced tube formation in endothelial cells and capillary sprouting in rat aortic rings by transactivation of EGFR, which, promoted the phosphorylation of VEGF receptor Flt-1 (fms-related tyrosine kinase 1), and a Flt-1 inhibitor blocked the proliferation and tube formation of HUVECs and capillary sprouting in aortic rings induced by AngII. However, these studies did not provide direct evidence for involvement of VEGF or EGF in AngII-induced angiogenesis. In our study here, we showed that knockdown of AGGF1 expression abolished AngII-induced tube formation by HUVECs, suggesting that AGGF1 plays an important role in AngII-induced angiogenesis (Fig. 7). Our study, therefore, provides important insights into the angiogenic process induced by AngII. We also investigated whether AGGF1 crosstalks with the VEGF signaling pathway but found no evidence of crosstalk between the 2 angiogenic signaling pathways (Fig. 8). In AngII-treated HUVECs with knockdown of AGGF1, other angiogenic factors such as VEGFA, bFGF, EGF and others are also up-regulated, but AngII-induced angiogenesis was abolished. Therefore, it should be interesting to further study the roles of VEGF-A, bFGF, EGF and other angiogenic factors in AngII-induced angiogenesis in the future.

The function of AngII was mostly mediated by the 2 receptors: AT1R and AT2R. AT1R is widely expressed in many different types of tissues, whereas AT2R is mainly expressed in fetal tissues and in a few organs after birth (30, 33). AT1R was shown to play an important role in inflammation, hypertension, cardiac hypertrophy, and angiogenesis (24). Recent studies established a role for AT2R in inhibition of angiogenesis, differentiation, antiproliferation, apoptosis, and development (34, 35). AT2R also played a role in vascular injury and tissue remodeling (36, 37). In AngII-induced angiogenesis, AT1R and AT2R showed contrasting effects. AT1R promoted AngII-induced angiogenesis whereas AT2R inhibited the angiogenesis in chicken embryo chorioallantoic membrane angiogenesis assays and a rat cremasteric muscle angiogenesis model (38, 39). In a recent in vivo Matrigel angiogenesis model in mice, the AngII-induced angiogenesis was inhibited by the AT1R inhibitor, by not by the AT2R inhibitor (40) AT2R was found to stimulate the cGMP via inducing NO production in vascular cells (34). Later, AT2R was reported to mediate the AngII-induced angiogenesis in the adult mouse heart under hypoxia (41). In the AT2R KO mice, the AngII-induced angiogenesis was impaired (14). In this study, we showed that the angiogenic factor AGGF1 is responsible for AngII-induced angiogenesis using capillary tube formation assays on HUVECs and an ex vivo mouse aortic ring angiogenesis assay (Figs. 7 and 8). Interestingly, we found that AngII-induced up-regulation of AGGF1 was blocked by the AT1R inhibitor losartan in a dose-dependent manner, although the effect plateaus at 10 nM (Fig. 1D and Supplemental Fig. 1). Conversely, the AT2R inhibitor PD123319 further enhanced AngII-induced up-regulation of AGGF1 (Fig. 1D). These data are consistent with previous findings that AT1R and AT2R play contrasting roles in angiogenesis.

The AngII concentration we used in this study was empirically determined. There are several factors involved in why a much higher concentration than is found in a physiologic setting was warranted, including a low response from HUVECs in culture and the reduced half-life of AngII during the 12-h duration of the experiments. We showed that the observed effect on AGGF1 expression was blocked by losartan and not inhibited by the AT2R antagonist PD123319, demonstrating that the effect is specific to AT1R. Endothelial cells do respond at physiologic concentrations of AngII; however, it is suspected that the local tissue concentration of AngII may be substantially larger as angiotensin-converting enzyme 1 is present at a very high concentration on the endothelial cell surface. It is possible, therefore, that endothelial cells may have evolved to require a greater concentration of AngII in vivo than do other cell types.

Surprisingly, AngII induced the expression level of AGGF1 at concentrations of 5–30 μg/ml but suppressed AGGF1 expression at concentrations >40 μg/ml (Fig. 1A, B). AT1R and AT2R demonstrated opposing effects on AngII-induced AGGF1 expression (Fig. 1D). In 5–30 μg/ml concentrations, AngII induced the expression of AGGF1 through AT1R. At higher concentrations, however, receptors opposed to AT1R, such as AT2R, may be activated, thus contributing to suppressed expression of AGGF1.

In summary, our data suggest that AngII can induce the expression of the angiogenic factor AGGF1 at low concentrations by activating the NF-κB pathway through AT1R. We found that p65 binds directly to the promoter or regulatory region of AGGF1 and activates its transcription. Our most important finding, however, is that AGGF1 is responsible for AngII-induced angiogenesis.

Supplementary Material

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

Click here for additional data file. (240.8KB, docx)

ACKNOWLEDGMENTS

The authors thank Dr. George Stalk (Cleveland Clinic) for quinacrine. This study was supported by China National Natural Science Foundation Grants 81630002, 91439129, 31430047, and 81600263; Innovative Team Grant 2017CFA014 from Hubei Province; a 2016 Top-Notch Innovative Talent Development Project Award from the Bureau of Human Resources and Social Security of Wuhan City; the Chinese National Basic Research Program (973 Project 2013CB531101); U.S. National Institutes of Health, National Heart, Lung, and Blood Institute Grants R01 HL121358 and R01 HL126729; Hubei Province Natural Science Programs (2016CFB224 and 2014CFA074); and the Outstanding Medical Academic Leader Program of Hubei Province. The authors declare no conflicts of interest.

Glossary

AngII

angiotensin II

AT1R

angiotensin II type 1 receptor

AT2R

angiotensin II type 2 receptor

bFGF

basic fibroblast growth factor

ChIP

chromatin immunoprecipitation

EGF

epidermal growth factor

EMSA

electrophoretic mobility shift assay

FBS

fetal bovine serum

KO

knockout

QC

quinacrine

siNC

negative control siRNA

siRNA

small interfering RNA

TSS

transcriptional start site

VSMC

vascular smooth muscle cell

WT

wild type

Footnotes

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

AUTHOR CONTRIBUTIONS

W. Si designed and performed experiments, analyzed data, and drafted the manuscript; W. Xie and W. Deng performed some experiments; Y. Xiao, S. S. Karnik, C. Xu, and Q. Chen analyzed data, provided valuable assistance and critically revised the manuscript; and Q. K. Wang designed and supervised the project, assisted with data analysis and interpretation, and critically revised the manuscript.

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