Activation of CD137 Signaling Promotes Angiogenesis in Atherosclerosis via Modulating Endothelial Smad1/5‐NFATc1 Pathway
Background Excessive angiogenesis is a key feature of vulnerable atherosclerotic plaques, and is considered an independent predictor of cardiovascular risk. CD137 signaling has previously been shown to be involved in atherosclerosis. However, the possible role of CD137 signaling in regulating angiogenesis has not been reported.
Methods and Results Apolipoprotein E‐deficient (ApoE−/−) mice were used as the in vivo model of atherosclerosis. Masson and immunohistochemical analysis of atherosclerotic plaques and Matrigel plug assay were used to evaluate the angiogenesis. Human umbilical vein endothelial cells and mouse brain microvascular endothelial cells were used as in vitro and ex vivo models to study how CD137 signaling affects angiogenesis. Matrigel tube formation assay, mouse aortic ring angiogenesis assay, and migration and proliferation assay were employed to assess angiogenesis. Western blot was used to detect protein expression. We found increased neovessel formation in atherosclerotic plaques of ApoE−/− mice treated with agonist anti‐CD137 antibody. Activation of CD137 signaling induced angiogenesis, endothelial proliferation, and endothelial cell migration. CD137 signaling activates the pro‐angiogenic Smad1/5 pathway, induces the phosphorylation of Smad1/5 and nuclear translocation of p‐Smad1/5, which in turn promotes the expression and translocation of NFATc1. Blocking CD137 signaling with inhibitory anti‐CD137 antibody could inhibit this activation and attenuated agonist anti‐CD137 antibody‐induced angiogenesis.
Conclusions These findings suggest that CD137 signaling is a new regulator of angiogenesis by modulating the Smad1/5‐NFATc1 pathway.
Angiogenesis, a process involving endothelial cell proliferation, migration, and tube formation, is a characteristic component of atherosclerosis. Studies have shown that extensive angiogenesis and microvessel formation are associated with intraplaque hemorrhage, suggesting that neovascularization may contribute to lesion progression and rupture, leading to adverse clinical outcome.1, 2 However, the mechanisms underlying excessive angiogenesis associated with atherosclerosis are not fully understood.
Evidence indicates that the TNFR superfamily, including OX40, CD40, and CD137, plays important roles in atherosclerosis. CD137 (4‐1BB), a well‐known costimulatory molecule, has been demonstrated to express in both human atherosclerotic plaques3 and the endothelial cells of mouse atherosclerotic lesions upon stimulation by tumor necrosis factor‐α (TNF‐α).4 In our previous studies, we found that the expression of both soluble and membrane‐bound CD137 was significantly elevated in patients with acute coronary syndromes, suggesting that the increased CD137 expression may contribute to the instability of atherosclerotic lesions.5 We also demonstrated that the nuclear factor of activated T cells 1 (NFATc1) is the critical factor linking CD137 signaling to the progression of atherosclerotic plaques.6 NFAT family consists of 5 isoforms (NFATc1 to NFATc5), with NFATc1 being expressed outside of the immune system, including the cardiovascular system.7 In the cardiovascular system, NFATc1 contributes to cell proliferation, calcification, and remodeling of smooth muscle cells,8 and controls multiple steps during cardiovascular system development and angiogenesis.9, 10 NFATc1 plays a critical role in angiogenesis,9 and can be activated in endothelial cells in response to inflammatory factors.11 Evidence proved that the NFATc1 axis can be activated by the TNF superfamily members, such as OX40 and CD137 in various cells. However, the critical factor that transmits the signal from CD137 to NFATc1 remains unclear.
Eapen et al clearly showed activation of Smad1 resulted in the expression of NFATc1 in osteoblast differentiation.12 Smad proteins are intracellular mediators of the transforming growth factor‐β family and consist of 3 types: receptor‐regulated (R‐Smad), common‐mediator (Co‐Smad), and inhibitory (I‐Smad). R‐Smad1,5,8 play an important role in angiogenesis.13 Phosphorylation and activation of the transcription factors Smad1/5/8 result in the promotion of pro‐angiogenesis. The Smad1/5/8 signaling pathway, which inhibits extracellular matrix deposition, promotes endothelial cell proliferation and migration.14 Once phosphorylated, p‐Smad1/5/8 interact with Smad4 (Co‐Smad) to form heteromeric complexes and then enter the nucleus to bind DNA and regulate the expression of target genes.15
Overall, previous studies have shown that both CD137 signaling and angiogenesis contribute to vulnerable atherosclerotic plaques, and NFATc1 is the critical factor between CD137 signaling and atherosclerosis. Smad1/5 proteins play a crucial role in angiogenesis and may be the upstream of NFATc1 protein. Here, we explore whether CD137 signaling regulates angiogenesis by modulating endothelial Smad1/5‐NFATc1 signaling.
Materials and Methods
Ethics Statement and Animals
The study protocol was reviewed and approved by the Animal Care and Use Committee of the Jiangsu University. Eight‐week‐old male wide‐type C57BL/6J mice were purchased from the animal center of Jiangsu University. ApoE−/− mice were purchased from Vital River Laboratories (Distributor of Jackson Laboratory, Beijing, China). All mice were fed with a high fat diet and water ad libitum. Each group had 5 mice.
Mouse Model of Atherosclerosis
Fifteen ApoE−/− mice were divided into 3 groups randomly: Control group (isotype matched IgG), agonist anti‐CD137 antibody group, and agonist anti‐CD137 antibody group+inhibitory anti‐CD137 antibody group. ApoE−/− mice were injected with isotype‐matched IgG (eBioscience), agonist anti‐CD137 antibody (R&D), or inhibitory anti‐CD137 antibody (eBioscience) (50 μg/kg body weight), respectively, intraperitoneally at 10, 13, and 16 weeks of age. At 19 weeks of age, the mice were euthanized and the aortas were dissected from connective tissue.
Immunostaining and Masson's Trichrome Assay
Cells and mouse tissue were both fixed in 4% (wt/vol) paraformaldehyde in PBS. We performed immunohistochemical staining on 6‐μm‐thick cryosections. Serial sections were stained using the Immunohistochemistry kit (Cwbiotech, China). For tissue staining, the following primary antibodies were used: rabbit anti‐NFATc1 (Santa Cruz) (1:400), rat monoclonal antibodies against CD31 (Abcam) (1:30), and biotin‐labeled anti‐CD68 (Santa Cruz) (1:500). For cell immunofluorescence, after blocking with rabbit serum, cells were incubated overnight with rabbit anti‐NFATc1 (Santa Cruz) (1:400). After incubation with primary antibody, goat anti‐rabbit dylight594 was applied in the second step to visualize the antigen.
Masson's trichrome kit (Sigma) was used to detect the fibrous cap.
Human umbilical vein endothelial cells (HUVECs), mouse brain microvascular endothelial cells (MBVECs), and HEK293T cells were purchased from the Cell Bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Wisent) at 37°C with 5% CO2 in a humidified atmosphere. The cells were passaged at the density of 70% to 80%. ECs used in the experiments were pretreated by recombinant human TNF‐α (10 ng/mL) (Peprotech, USA).
SDS‐PAGE and Western Blotting
Total or nucleus proteins of cells and tissue samples were prepared using a protein extraction reagent (Pierce, USA). The protein concentration was measured with the BCA protein assay reagent kit (Thermo, Fisher). An equal amount of protein was separated by 10% SDS‐polyacrylamide gel electrophoresis and transferred to poly(vinylidene fluoride) (PVDF) membranes. After blocking with 5% nonfat milk for 1 hour at room temperature, blots were probed with primary antibodies against NFATc1, p‐Smad1/5, β‐actin (Cell Signaling Technology), Smad1/5 (Abcam), and proliferating cell nuclear antigen (Immunoway) overnight at 4°C. Next day, the membranes were washed with Tris‐Buffered Saline and Tween 20 (TBST) and incubated with an HRP‐conjugated secondary antibody (Cell Signaling Technology) for 1 hour at room temperature. Bound antibody was detected with an Image Quant LAS 4000 Imager, and densitometry was performed using the Image J image processing program.
Matrigel HUVEC Tube Formation Assay
Tube formation assay was performed as described.16 HUVECs were grown on growth factor‐reduced Matrigel (Trevigen). The 96‐well plates were coated with Matrigel containing PBS (control) or agonist anti‐CD137 antibody (5 μg/mL), inhibitory anti‐CD137 antibody (5 μg/mL), and were incubated for 30 minutes in 37°C to form gel. 1.5×104/mL cells/well were seeded in each well and the plates were incubated at 37°C for 24 hours. Tube formation was observed and photographed with a microscope (Olympus) and analyzed with Image J. Three independent experiments were carried out and each was performed in triplicate.
Mouse Aortic Ring Angiogenesis Assay
The mouse aortic ring angiogenesis assay was performed using a modified method provided by Baker et al.17 The aortas from 8‐week‐old male C57BL/6J mice were cut into sections 0.5 mm in width. After they had been serum‐starved overnight, aortic rings were then embedded in the 96‐well plate coated with type I collagen (1 mg/mL, Millipore) containing PBS or agonist anti‐CD137 antibody, inhibitory anti‐CD137 antibody. The growth medium contained 2.5% (vol/vol) fetal bovine serum and was replaced every other day. At day 7, the microvessels sprouting from explants were photographed using a microscope (Olympus) and the number of sprouts was counted manually. Three independent experiments were carried out with a mean of ≥10 aortic rings being analyzed for each treatment.
In Vivo Matrigel Plug Assay
Matrigel plug angiogenesis assay was adapted as described earlier.18 C57BL/6J mice were subcutaneously injected with 700 μL of Matrigel® Matrix (Corning) supplemented with mouse vascular endothelial growth factor (VEGF) (R&D, USA) (100 ng/mL), mouse VEGF plus agonist anti‐CD137 antibody (100 μg/mL), or mouse VEGF plus inhibitory anti‐CD137 antibody (100 μg/mL). After 2 weeks, mice were euthanized and plugs were removed and photographed with a camera. The hemoglobin content of the Matrigel plugs was quantified using QuantiChrom™ Hemoglobin.
Migration and Proliferation Assay
Cell migration was performed by the endothelial transwell assays. Endothelial cell (EC) proliferation was determined by the CCK‐8 assay using the CCK‐8 kit (Vazyme Biotech).
Flow Cytometry Assay
HUVECs and MBVECs were stained with a phycoerythrin‐conjugated anti‐CD137 monoclonal antibody (BD Bioscience) or a relevant isotype control IgG (BD Bioscience). Cells were analyzed by flow cytometry (BD ACCURIC6).
Molecular Biological Methods
The coding sequence of human shNFATc1 was cloned into a eukaryotic expression vector PLKO.1‐puro (Sigma) at the EcoRI/AgeI site, namely PLKO.1‐shNFATc1. PLKO.1‐shGFP (Sigma) was used as a control for PLKO.1‐shNFATc1. Short interfering RNA (siRNA) oligonucleotides (Ribobio) were used for Smad1/5 genes knockdown in HUVEC and MBVEC, and nontargeting siRNA (Ribobio) was used as a control for knockdown in HUVEC and MBVEC. Lipofectamine 2000 transfection reagent (Invitrogen) was used for transfection of cells and lentivirus package. HUVEC and MBVEC infected the lentivirus to establish the stable cell line in the presence of polybrene (2.5 g/mL).
Data were expressed as the mean±SD of at least 3 independent experiments. The Wilcoxon rank sum test or the Kruskal–Wallis test was used to compare data using SPSS version 12.0 software (SPSS, Chicago, IL). A 2‐tailed P<0.05 was considered statistically significant.
CD137 Signaling Induces NFATc1 Expression and Contributes to Angiogenesis in Atherosclerotic Plaques in ApoE−/− Mice
To investigate the potential role of CD137 in atherosclerotic angiogenesis, we activated the CD137 signaling by agonist anti‐CD137 antibody and inhibited it by inhibitory anti‐CD137 antibody. We found that atherosclerotic plaques in the agonist anti‐CD137 antibody group have a thin fibrous cap and less lesion size but more necrotic core (Figure 1A). Immunohistochemistry revealed that CD31 and CD68 expression were significantly higher in the plaque of the agonist anti‐CD137 antibody group than that of the inhibitory anti‐CD137 antibody group and control group (Figure 1B and 1C). These findings indicated that CD137 signaling triggered infiltration by monocytes/macrophages, angiogenesis, and fibrous cap thinning, all of which lead to plaque instability.
Both immunohistochemical and Western blot analysis showed significant increase of NFATc1 expression in the plaques when CD137 signaling is activated, while inhibition of CD137 signaling decreased the expression of NFATc1 (Figure 1B through 1D). It implies that activation of CD137 signaling induces NFATc1 expression and may contribute to angiogenesis in atherosclerotic plaques of ApoE−/− mice.
TNF‐α Stimulates CD137 Expression in ECs
Since the expression of CD137 was low in normal ECs, we first stimulated CD137 expression in ECs by TNF‐α (10 ng/mL). Our data show that expression of membrane CD137 and protein expression were significantly upregulated in both HUVEC and MBVEC at 24 hours after TNF‐α stimulation (Figure 2A and 2B).
CD137 Signaling Promotes Angiogenesis In Vivo, In Vitro, and Ex Vivo
In cultured EC assays, we observed that activation of CD137 signaling could increase proliferation and migration of MBVECs, and which can be inhibited by inhibitory anti‐CD137 antibody (Figure 3A and 3B). In the Matrigel HUVEC tube formation assay, we also found that activation of CD137 signaling significantly increased the tube formation and number of branching points, which was attenuated by inhibitory anti‐CD137 antibody (Figure 3C). Next, we used mouse aortic ring angiogenesis assay to investigate whether CD137 signaling promotes outgrowth of microvessels ex vivo. Outgrowth of microvessels in the aortic ring assay was remarkably enhanced when CD137 signaling was activated by agonist anti‐CD137 antibody, but inhibited by inhibitory anti‐CD137 antibody (Figure 3D). These data demonstrated that activating of CD137 signaling promotes angiogenesis in vitro and ex vivo.
Then we investigated whether CD137 signaling promotes angiogenesis in an in vivo model of angiogenesis. C57BL/6J mice were used to perform a Matrigel plug assay. No significance in angiogenic response was observed with agonist anti‐CD137 antibody. However, we found that agonist anti‐CD137 antibody enhanced VEGF‐mediated angiogenesis, and the enhancement was significantly attenuated in the presence of inhibitory anti‐CD137 antibody, which is consistent with the former observation (Figure 3E). It may be associated with lower expression of CD137 in normal ECs and upregulation in response to inflammation stimulation such as VEGF and TNF‐α.
CD137 Signaling Regulates Phosphorylation of Smad1/5 and NFATc1 Expression in MBVEC
As mentioned in the Introduction, Smad1/5 and NFATC1 play important roles in angiogenesis. To further investigate the mechanism that is involved in CD137 signaling and angiogenesis, we determined how Smad1/5 and NFATc1 respond to CD137 signaling. In this study, we found that the phosphorylation of Smad1/5 and NFATc1 expression are dramatically increased and reached the peak at 1 and 12 hours, respectively, after treating with agonist anti‐CD137 antibody in MBVEC (Figure 4A). Both the increased Smad1/5 phosphorylation and NFATc1 expression were inhibited by inhibitory anti‐CD137 antibody (Figure 4B).
When NFATc1 was activated, it dephosphorylated and then translocated into the nucleus. Immunofluorescence was employed to detect nuclear protein. Our result indicated that NFATc1 was activated by dephosphorylation and translocated to the nucleus after stimulation with agonist anti‐CD137 antibody, but was inhibited by inhibitory anti‐CD137 antibody in MBVECs (Figure 4C).
siRNASmad1/5 Attenuates the Expression of NFATc1 in HUVEC and Angiogenesis In Vitro Induced by CD137 Signaling
NFATc1 dephosphorylation, which is induced by agonist anti‐CD137 antibody, was inhibited by siRNASmad1/5 in HUVECs (Figure 5A). As expected, silencing of Smad1/5 blocked the pro‐angiogenic state induced by agonist anti‐CD137 antibody and resulted in a significant reduction of HUVECs tube formation and branching in the Matrigel HUVECs tube‐formation assay (Figure 5B). In accordance with these results, aortic ring assay also showed a significant reduction in the number of microvessels after knocking down of the Smad1/5 gene (Figure 5C). Therefore, siRNASmad1/5 attenuates NFATc1 expression in HUVEC and angiogenesis in vitro induced by activation of CD137 signaling.
NFATc1 Gene Suppressing Inhibited Angiogenesis Induced by CD137 Signaling In Vitro
To examine the role of NFATc1 in angiogenesis of CD137 signaling, we evaluated the effect of suppressed NFATc1 endothelial cell line (PLKO.1‐shNFATc1) and control endothelial cell line (PLKO.1‐shGFP) on the ability to form tubes and branching of HUVEC under stimulation of agonist anti‐CD137 antibody. In Figure 6A, we observed that the PLKO.1‐shNFATc1 group significantly inhibited the total and nuclear protein levels of NFATc1. As we expected, compared with the PLKO.1‐shGFP group, the tube formation and branching of HUVEC in the PLKO.1‐shNFATc1 group were dramatically decreased (Figure 6B). Meanwhile, compared with the PLKO.1‐shGFP group, we also found that the numbers of microvessels outgrowth in aortic rings were dramatically reduced in the PLKO.1‐shNFATc1 group (Figure 6C). These data suggest that suppressing NFATc1 gene in HUVECs inhibited angiogenesis induced by CD137 signaling in vitro.
Here, we present several novel important findings as follows: First, activation of CD137 signaling induces NFATc1 expression and promotes angiogenesis in vivo, in vitro, and ex vivo. Second, CD137 signaling regulates phosphorylation of Smad1/5 and NFATc1 expression in ECs. Silencing of Smad1/5 attenuates the expression of NFATc1 and angiogenesis in vitro induced by CD137 signaling. Suppressing NFATc1 gene could inhibit angiogenesis induced by CD137 signaling in vitro. Therefore, CD137 signaling regulates angiogenesis by modulating Smad1/5‐NFATc1 signaling.
Atherosclerosis, a disease of chronic inflammation, is the primary cause of heart disease and stroke. As an important member of the TNFR superfamily, CD137‐CD137L interaction promotes inflammation. Evidence suggests that activation of CD137‐CD137L signaling may be a significant triggering event for the process of forming atherosclerotic plaque, and contributes to the plaque vulnerability.4 However, whether activation of CD137 has the same effect in atherosclerosis remains unknown. Previous studies have reported that CD137 is expressed in both human atherosclerotic plaques and endothelial cells in inflammation sites of mouse atherosclerotic lesions.3 In the atherosclerotic plaque model, our data revealed that activation of CD137 signaling not only induced the expression of NFATc1 and CD31, but also significantly contributes to neointimal formation with less fibrous cap, more necrotic core, and more serious inflammatory infiltration, which represented vulnerable plaques prone to rupture. As reported, vulnerable atherosclerotic plaques are characterized by inflammatory infiltration and neovascularization,19, 20 and the increased number of vasa vasorum from the media may provide a conduit for the entry of inflammatory cells contributing to the recruitment of monocytes and lymphocytes.21 Cytokines secreted by inflammatory corpuscles such as TNF‐α and interferon‐γ may trigger the secretion of a number of pro‐inflammatory molecules from ECs.22 The consequence of these processes results in smooth muscle cell proliferation and extracellular matrix degradation, which support plaque development and contribute to the formation of advanced lesions prone to rupture.
In searching for mediators of angiogenesis in the atherosclerosis plaques in ApoE−/− mice, we have discovered CD137 as the new regulator of angiogenesis. CD137 expression is rare in normal endothelial cells, yet can be induced by proinflammatory cytokines.3, 4 In the current assay, TNF‐α was used to induce the expression of CD137. To further investigate the function of CD137, we used cultured EC assays and in vitro and ex vivo and in vivo models of angiogenesis. Our results supported the hypothesis that CD137 signaling contributed to angiogenesis of in vitro and ex vivo models of angiogenesis. However, in vivo data showed that the addition of agonist anti‐CD137 antibody alone did not induce an obvious angiogenic response, whereas in combination with VEGF, agonist anti‐CD137 antibody could enhance the angiogenesis. This result suggests a synergistic effect between CD137 and VEGF in angiogenesis. It may be associated with lower expression of CD137 in normal ECs. It also implies that CD137 angiogenic activity is not restricted to the atherosclerosis plaques.
To investigate the mechanism of CD137 signaling in promoting angiogenesis, we treated mouse brain ECs with agonist anti‐CD137 antibody in vitro. Data showed that upon activation and phosphorylation of Smad1/5, NFATc1 was dephosphorylated and translocated to the nucleus. To confirm that the pro‐angiogenic effect of CD137 signaling was mediated through the Smad1/5‐NFATc1 pathway, Smad1/5 was silenced with siRNA and we found that the pro‐angiogenic effect of CD137 and CD137/NFATc1 signaling pathway in ECs were blocked. When CD137 signaling was depressed or Smad1/5 phosphorylation was inhibited, NFATc1 expression was downregulated and HUVEC tube formation and branching was reduced. In addition, when NFATc1 was inhibited, the ability of tube formation and branching induced by agonist anti‐CD137 antibody was attenuated significantly.
Overall, CD137 signaling can promote angiogenesis through Smad1/5‐NFATc1 signaling. CD137 signaling may be a highly promising therapeutic target for controlling angiogenesis in atherosclerosis, and in other diseases related to angiogenesis such as age‐related macular degeneration, proliferative diabetic retinopathy, and cancer.
Sources of Funding
This project was supported by the National Natural Science Foundation of China (81670405, 81370409 and 81400269), the Natural Science Foundation of Jiangsu Province (BK20161355) and the social development Foundation of Zhenjiang (SH2015041).
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