Activating CD137 Signaling Promotes Sprouting Angiogenesis via Increased VEGFA Secretion and the VEGFR2/Akt/eNOS Pathway

Combination of antiangiogenesis and immunotherapy may be an effective strategy for treatment of solid tumors. Our previous work reported that activation of CD137 signaling promotes intraplaque angiogenesis. A number of studies have demonstrated that vascular endothelial growth factor receptor 2 (VEGFR2) is a key target for angiogenesis. However, it is unknown whether CD137-mediated angiogenesis is related to VEGFR2. In this study, we investigated the effect of CD137 on the VEGFR2 expression and explored the underlying mechanisms of CD137-mediated angiogenesis. Knock-out of CD137 in ApoE−/− mice significantly decreased neovessel density in atherosclerotic plaques. CD137 silencing or inhibition attenuated endothelial cell (ECs) proliferation, migration, and tube formation. We found activation of CD137 signaling for increased VEGFR2 transcription and translation steadily. Moreover, CD137 signaling activated phosphorylated VEGFR2 (Tyr1175) and the downstream Akt/eNOS pathway, whereas neutralizing CD137 signaling weakened the activation of VEGFR2 and the downstream Akt/eNOS pathway. The aortic ring assay further demonstrated that CD137 signaling promoted ECc sprouting. Inhibition of VEGFR2 by siRNA or XL184 (cabozantinib) and inhibition of downstream signaling by LY294002 (inhibits AKT activation) and L-NAME (eNOS inhibitor) remarkably abolished proangiogenic effects of CD137 signaling both in vitro and ex vivo. In addition, the condition medium from CD137-activated ECs and vascular endothelial growth factor A (VEGFA) had similar effects on ECs that expressed high VEGFR2. Additionally, activating CD137 signaling promoted endothelial secretion of VEGFA, while blocking CD137 signaling attenuated VEGFA secretion. In conclusion, activation of CD137 signaling promoted sprouting angiogenesis by increased VEGFA secretion and the VEGFR2/Akt/eNOS pathway. These findings provide a basis for stabilizing intraplaque angiogenesis through VEGFR2 intervatioin, as well as cancer treatment via combination of CD137 agonists and specific VEGFR2 inhibitors.


Introduction
Angiogenesis is an intricate process involving basement membrane degradation, as well as endothelial cell activation, proliferation, and migration to form new vessels [1]. Neovessels are essential for providing sufficient amount of nutrients and oxygen for proliferating cells in hyperplastic diseases [2,3]. As the main cause of atherosclerotic plaque hemorrhage and rupture, angiogenesis promotes adverse cardiovascular events, such as myocardial infarction, which is a serious issue during treatment [4]. Although many angiogenesis inhibitors have been developed, the process of pathological angiogenesis is quite complex and involves multiple regulatory factors. Therefore, further research is needed to identify other potential therapeutic targets for angiogenesisrelated diseases.
CD137 (also known as ILA/4-1BB/TNFRSF9) is an important immune checkpoint molecule, which is expressed on immune cells, myeloid cells, and vascular cells, such as ECs in an activation-dependent manner. In addition, CD137 is a well-known T cell costimulatory molecule [5,6]. There is accumulating evidence for the expression of CD137 in human atherosclerotic plaque lesions [7]. The CD137 expression in inflamed tissues was found to be induced in mural cells, such as vascular ECs and smooth muscle cells, after stimulation with proinflammatory cytokines [7][8][9]. The ligand of the CD137 receptor, CD137L (4-1BBL), is mainly expressed by antigen-presenting cells, such as dendritic cell macrophages and activated T cells [3]. CD137L binding with CD137 on ECs could trigger a cascade of inflammation and immune responses in atherosclerotic plaque [7,10,11]. We previously reported that activation of CD137 signaling promoted angiogenesis in the plaque of ApoE -/mice through different pathways [12,13]. However, it remains unclear whether CD137 plays a key role in angiogenesis.
The expression level of CD137 in tumor vessels highly correlates with the tumor's degree of malignancy [14]. Therefore, the strategy for cancer should include a combination of antiangiogenesis and immunotherapy [15,16]. The experience of application of CD137 agonists and angiogenesis inhibitors in tumor therapy [17][18][19][20][21][22] may suggest that inhibition of angiogenesis might be useful also for CD137expressing stable atherosclerotic plaque. Thus, it is becoming increasingly important to further explore how CD137 signaling regulates angiogenesis in atherosclerosis.
Angiogenesis encompasses several stages, including basement membrane degradation, ECs migration, and proliferation, as well as recruitment of mural cells. Migrating tip cells along with proliferating stalk cells form a vascular sprout, and endothelial tip cells of two sprouts converge to form a neovessel [23,24]. The process of sprouting involves sensing and guiding roles of endothelial tip cells with long, dynamic filopodia. Specifically, VEGFR2, mainly expressed in filopodia, can sense environmental stimulators, such as VEGFA. Migrating tip cells are followed closely by proliferative stalk cells that extend the sprouts together [23]. Zarkada et al. reported that VEGFR2 is indispensable for postnatal angiogenesis, and even a little VEGFR2 is able to sustain angiogenesis to some extent [25].
Here, we detected angiogenesis-related factors and found that VEGFR2 increased steadily in both transcription and translation levels after activation of CD137 signaling. These findings prompted us to explore the underlying mechanisms of CD137 regulation of the VEGFR2 expression.

Materials and Methods
2.1. Ethics Statement and Animals. Animal experiments conducted in this study were reviewed and approved by the Animal Care and Use Committee of Jiangsu University. Six-to eight-week-old, male, wide-type C57BL/6 J mice, weight 20-22 g, were purchased from the Animal Center of Jiangsu University. Six-to eight-week-old, male, 20-22 g, ApoE -/and ApoE -/-CD137 -/mice were obtained from the Nanjing Bio-medical Research Institute of Nanjing University (Nanjing, China). C57BL/6 J mice were fed with a normal diet, and ApoE -/mice were fed with a high fat diet. All of the mice were provided with water ad libitum.

Cell
Culture. Human umbilical vein endothelial cells (HUVECs) applied in the tube formation assay were maintained in ECM (Endothelial Cell Medium, ScienCell) supplemented with 10% FBS, 100 U/mL 100 U/mL penicillin, and 100 mg/mL. Mouse brain microvascular endothelial cells (MBVECs) were cultured in Dulbecco's Modified Eagles Medium (DMEM) (Sigma-Aldrich) supplemented with 10%FBS (Sigma-Aldrich), 100 U/mL penicillin, and 100 mg/mL streptomycin (Sigma-Aldrich). Cells were maintained in an incubator at 37°C in humidified atmosphere with 5% CO 2 and subjected to serum starvation overnight before different treatments.
2.5. Immunohistochemistry Staining. At the age of 19 weeks, ApoE -/and ApoE -/-CD137 -/mice were euthanized by cervical dislocation, and thoracic aorta was removed. Serial sections on the same paraffin blocks were applied to perform immunohistochemistry staining by SP Rabbit & Mouse HRP Kit (DAB) (CWbiotech). The slides were boiled for 10 minutes with EDTA (ethylene diamine tetraacetic acid) antigen retrieval buffer, blocked with normal goat serum, and incubated with primary antibody for CD31 (1 : 100, Abcam) at 4°C overnight. The following day, the slides were treated 2 Mediators of Inflammation with biotin-linked goat anti-rabbit/mouse IgG, followed by Streptavidin-HRP. DAB chromogen solution was used to develop the color (positive cells stained brown), while hematoxylin was applied to counterstain cell nuclei. Brown areas were calculated using Image-Pro Plus 8.0 (Media Cybernetics).
2.6. Mouse Aortic Ring Assay. Aortic rings of C57BL/6 J mice were obtained in line with the protocol [34]. Briefly, after euthanasia, thoracic aorta was exposed and removed. We peeled the aortic adventitia and cut the rest of aorta into 1 mm wide rings. The rings were treated with different treatments according to the grouping after overnight serum starvation and transferred into a 96-well plate which preembedded with type I collagen (1 mg/mL, Millipore) containing rmCD137L, p-Akt inhibitor (LY294002), p-eNOS inhibitor (L-NAME), or/and VEGFR2 inhibitor (XL184, cabozantinib   2.14. Statistical Analysis. Each experiment was performed at least three times. All of the analyses were performed using SPSS 23 software. The data were reported as mean ± standard deviation (SD). To compare between control and treated groups, we used two-tailed Student's t-test or oneway ANOVA (LSD t-test). P values <0.05 were considered to be statistically significant.

Endothelial CD137 Is Critical to Intraplaque
Angiogenesis. Previous studies have shown that CD137 is a key regulator in the progression of atherosclerotic plaque [36,37]. Activation of CD137 signaling in ECs or macrophages has been shown to contribute to intraplaque angiogenesis [12,13]. To further investigate the effects of CD137 on the angiogenesis process, we evaluated CD31-positive microvessels in CD137 -/-ApoE -/mice and sprouting in the aortic rings in CD137 -/mice. The number of microvessels expressing CD31 was lower in the CD137 -/group compared to the control mice (Figures 1(a) and 1(b)). As shown in Figures 1(c) and 1(d), fewer sprouts were observed in the CD137 -/-ApoE -/group. In order to verify the important role of CD137 molecule, ECs were transfected with CD137 siRNA, and the silencing efficiency was detected by western blot (Figures 1(e) and 1(f)). After treatment with human siCD137, the total length of HUVEC tubes and number of sprouting branch points decreased significantly compared with the control group. Furthermore, we transfected mouse brain microvascular ECs (BMVECs) with murine siCD137 and observed a lower ratio of ECs proliferation (Figures 1(k) and 1(j)) and a reduced number of migratory cells.

Ligation of CD137 with CD137L Influences the VEGFR2
Expression and Activates Downstream Akt/eNOS. Although activation of CD137 signaling contributed to angiogenesis, the potential key molecules involved in this process are still unclear. In this context, we screened a series of angiogenesisrelated candidates and revealed that activation of CD137 signaling with recombinant human CD137L affected the mRNA levels of several molecules, including VEGFR2 ( Figure 2(a)), Neuropilin-1, EphrinB2, DLL4, and Notch-1 (Supplementary Figure 1). Among these, VEGFR2 showed the highest expression (both transcription and translation levels) 3 hours after treatment with CD137L (Figures 2(a)-(c)). VEGFR2 is essential for both proliferation and differentiation of ECs [38]. Whether the VEGFR2 signal is activated, we detected the phosphorylation level of VEGFR2 after activating CD137 signaling for a time point, 0, 2, 5, 10, 15, and 30 minutes and found that phosphorylated VEGFR2 (Tyr1173) was increased, achieving the maximum level at 10 minutes, which indicated that CD137 rapidly promoted VEGFR2   e)). Besides, activating the CD137 signal for 10 minutes promoted Akt and eNOS phosphorylation (Figures 2(f) and 2(g)) both the downstream molecules of phosphorylated VEGFR2 [39]. And blocking CD137 signaling with inhibitory anti-CD137 antibody weakened the effects of CD137 signaling on VEGFR2 and Phospho-Akt (p-Akt, Ser473) and phospho-eNOS (p-eNOS, Ser1177) (Figures 2(f) and 2(g)). These data suggest that the CD137 signal may be an additional way to affect the VEGFR2 expression and activation.
3.3. Inactivation of VEGFR2 Impairs CD137-Induced EC Proliferation and Migration. EC migration and proliferation contribute to angiogenic sprouting [32]. Since VEGFR2 plays an important role in angiogenesis [31,40,41], it is necessary to investigate whether the CD137-dependent enhancement of the EC angiogenic function occurred through VEGFR2 signaling. In order to observe the silencing efficiency of siVEGFR2, VEGFA165 (20 ng/mL) [42], the most studied VEGFA isoform, was used to upregulate the VEGFR2 expression at different time points. The protein level of p-VEGFR2 (Tyr1173) obviously increased after stimulation with VEGFA for 5 minutes (Figures 3(a) and 3(b)). Subsequently, siVEGFR2 (50 nM) was transfected into ECs, and the knockdown efficiency of siVEGFR2-2 was verified by western blot (Figures 3(c) and 3(d). Meanwhile, XL184 (a potent inhibitor of VEGFR2, 0.03 μM) was incubated with HUVECs and aortic rings (Figures 3(e) and 3(f)). In accordance with previous studies [8,12], we preincubated ECs with TNFα (10 ng/mL) to increase the CD137 expression on the cell surface before treatment with CD137L. As shown by western blot, activation of CD137 signaling with CD137L significantly increased phosphorylation of VEGFR2(Tyr1173), p-Akt(Ser473), and p-eNOS(Ser1177), whereas silencing of VEGFR2 abolished the effect of CD137 activation (Figures 3(g) and 3(h).
Next, we performed the EC tube formation assay to observe the endothelial function. Compared to TNFα treatment alone, capillary-like structures with a higher number of branches and greater total length of tube network were observed after incubation with additional CD137L for 3 hours. Silencing of VEGFR2 markedly weakened the angiogenic ability of ECs induced by the CD137 signaling     Mediators of Inflammation activation with less connected ECs and less well-formed tubes. In contrast, incubation with CD137L did not abrogate the effect of VEGFR2 inhibitors (Figures 3(i), 3(l), and 3(m)). In addition, the results of endothelial Edu-555 and transwell assays showed that VEGFR2 knockdown notably decreased CD137-induced endothelial proliferation and migration. Consistent with the results on tube formation, added CD137L did not reverse the EC function induced by VEGFR2 inhibitors (Figures 3(j), 3(k), 3(n), and 3(o)).

CD137
Signaling Promotes the Endothelial Function via the VEGFR2/Akt/eNOS Pathway. Phosphorylated VEGFR2 at 1173 tyrosine residue transduces the signaling of cell proliferation and migration [31]. Besides, studies have reported that the VEGFR2-dependent activation of PI3K-Akt signaling regulates cell survival [26,43]. Akt is a key molecule in that signaling pathway, which is critical for VEGFR2 functioning. Moreover, eNOS activated by Akt regulates cell permeability. From the above results, CD137 signaling activated p-VEGFR2 and p-Akt and increased the p-eNOS protein expression. Therefore, we investigated whether the VEG-FR2/Akt/eNOS pathway was indispensable for the CD137 signaling-mediated endothelial tube formation, proliferation, and migration. We applied LY294002 (an inhibitor of p-Akt) to verify the downstream signaling transduced by CD137-CD137L. First, we found that LY294002 (5 μM) obviously inhibited the p-Akt (Ser473) expression (Figures 4(a)  and 4(b)). Compared with the group without inhibitors, siVEGFR2 impaired the expression of phosphorylated VEGFR2 (Tyr1173), p-Akt (Ser473), and p-eNOS (Ser1177). Treatment of LY294002 decreased the expression of p-Akt   The tube formation was significantly impaired after treatment with siVEGFR2 or LY294002, showing less branches points and a reduced total length of tubes. Combination of VEGFR2 silencing and LY294002 could achieve a synergistic effect on angiogenesis (Figures 4(i), 4(e), and 4(f)). Similarly, either siVEGFR2 or LY294002 decreased EC proliferation and migration induced by CD137 signaling. Correspondingly, siVEGFR2 combined with LY294002 synergistically inhibited proliferation and migration (Figures 4(j), 4(k), 4(g), and 4(h)).

CD137
Signaling Enhances Aortic Sprouting through the VEGFR2/Akt/eNOS Pathway. VEGFR2 is highly expressed on tip cell filopodia which is responsible for guiding and migrating to an avascular region during neovessel formation [26,27,31,44,45]. Since VEGFR2 controls the migration of tip cells, it is important to figure out whether CD137 signaling induces VEGFR2 and downstream pathway implicated in aortic ring sprouts. We performed the mouse aortic ring assay, which revealed that CD137 signaling promoted budding of aortic rings as evidenced by a higher number of sprouts. Inhibition of VEGFR2 with XL184 impaired the proangiogenic sprouting mediated by CD137 signaling (Figures 5(a) and 5(b)). Additionally, inhibition of VEGFR2 and/or Akt decreased the number of sprouts, suggesting weakened sprouting capability of aortic rings (Figures 5(a)  and 5(b)). Moreover, the capability of the aortic ring sprouting was attenuated considerably in the XL184 plus LY294002 group (Figures 5(c) and 5(d)), suggesting that sprouts were significantly reduced after pretreatment with XL184, LY294002, or both, before activation of CD137 signaling.
3.6. Inhibition of eNOS Weakens Angiogenesis Mediated by CD137 Signaling. To explore the potential effects of eNOS activation on angiogenesis mediated by CD137 signaling, we applied L-NAME (eNOS inhibitor) to inhibit the eNOS activity (Figure 6(a)). Specifically, we pretreated the ECs and aortic rings with L-NAME (100 μM) before activating CD137 signaling. In vitro, both Edu-555 proliferation and transwell assay showed that L-NAME decreased the proliferative and migratroy cells compared with the control group (Figures 6(d) and 6(f)). Besides, the endothelial tube formation assay displayed the same tendency, showing a decreased

11
Mediators of Inflammation total length and branch points of tubes in the L-NAMEtreated group (Figures 6(h) and 6(i)). Moreover, the aortic ring assay indicated fewer sprouts compared with controls ( Figure 6(k)).

Activation of CD137
Signaling Increases the EC Secretion of VEGFA. VEGFR2 is a member of VEGF receptors (VEGFR-1, -2, -3), highly expressed in migratory ECs (tip cells) filopodia, and senses VEGF [46]. VEGFA/VEGFR-2 is likely the prominent signaling that mediates cellular responses involved in angiogenesis [31]. VEGF binding to VEGFR2 initiates VEGFR2 dimerization and autophosphorylation at tyrosine kinase residues [47,48], which is essential for downstream signaling transduction. To figure out whether CD137-mediated increase in VEGFR2 was related to VEGF, we incubated CD137-activated ECs either with   (Figure 6(a)). Since VEGF is a secreted protein, we speculated that CD137-dependent secretion of VEGFA would be verified by the similar effect of VEGFA on ECs (Figures 6(b) and 6(c)). Furthermore, the VEGFA expression of EC extracts was obviously decreased in the CD137L group (Figures 6(d) and 6(e)), while neutralized the CD137 signaling with inhibitory antibody increased the VEGFA level. The concentration of VEGF in concentrated condition medium of the CD137L group was significantly increased (Figures 6(f)-(h)), while anti-CD137 signaling decreased the VEGFA concentration. Meanwhile, the VEGFA expression in exosomes (extracted as the representatives from the condition medium as previously described [24]) was obviously lower in the CD137L group in comparison with the controls (Figures 6(i) and 6(j)). Inhibition of the CD137 signaling reversed this decrease. These results indicated that activation of CD137 signaling with CD137L promoted endothelial VEGFA secretion.
Taken together, this study indicated that CD137 signaling promoted sprouting angiogenesis through activation of the VEGFR2/Akt/eNOS pathway.

Discussion
Targeting intraplaque angiogenesis may be an important strategy in slowing down the progression of atherosclerotic diseases. Here, we investigated mechanisms of CD137mediated angiogenesis. Specifically, this study showed that activation of CD137 signaling promoted sprouting angiogenesis in vitro and ex vivo, suggesting that blockage of CD137 may reduce angiogenesis through decreased VEGFA/-VEGFR2 signaling. Therefore, combination of CD137 inhibitor and antiangiogenic agents could be an attractive strategy for treating atherosclerosis.
Sprouting angiogenesis is crucial both in physiological and in pathological circumstances, such as ischemia, inflammation, diabetes, and cancer [49]. Interaction of various factors and cells takes place in pathological angiogenesis that occurs in pathogenesis of atherosclerosis. Apart from providing nutrients and oxygen needed for the thickened arterial intima caused by excessive cellular proliferation, angiogenesis also provides a pathway for inflammatory mediators to enter the plaque, thereby reducing the plaque's stability, and increasing the risk of rupture and consequent adverse cardiovascular events [50,51].
As an immunostimulatory receptor and inflammation regulator, CD137 plays a critical role in pathogenesis of atherosclerosis [10]. CD137 is activated and highly expressed on ECs under inflammatory conditions of atherosclerosis [7]. Its ligand (CD137L), which is expressed by activated antigenpresenting cells (APCs), binds to CD137 on ECs and promotes angiogenesis. We previously demonstrated that the CD137 signaling activation promoted angiogenesis in plaque [12,13]. CD137 knock-out mice showed the essential effect of CD137 on sprouting angiogenesis as evidenced by a significantly decreased number of microvessels in the plaque and sprouts in aortic rings. Assays on ECs in vitro also suggested the importance of CD137 for endothelial proliferation and migration, as well as for the tube formation ( Figure 1). Together, these data showed that CD137 was critical for angiogenesis.
Next, we intended to explore the mechanisms of CD137mediated angiogenesis in vascular inflammatory milieu. Olofsson [7] reported that TNFα alone induced the strongest mRNA expression of endothelial CD137, which was also verified in our previous study [12]. In this context, we used TNFα as the basic treatment for ECs to improve the CD137 protein expression before stimulation with CD137L. Although the relationship between CD137 signaling and angiogenesis has been poorly studied, activation of CD137 signaling could promote angiogenesis in atherosclerosis through modulating the endothelial smad1/5-NFATc1 pathway [12]. Moreover, in an indirect way, CD137 signaling mediates phenotypic conversion of macrophages to influence angiogenesis [13]. It remains unknown whether there is an obvious factor involved in CD137 signaling-mediated angiogenesis. We screened lots of angiogenesis-related candidates   groups. * p < 0:05, * * p < 0:01, * * * p < 0:001, n = 3. The CD137 agonist group was stimulated with TNFα for 24 hours and then CD137L for 3 hours. The Anti-CD137 group was treated with inhibitory CD137 signaling antibody for 30 minutes then CD137L for 3 hours. Condition medium contained the supernatant after the activation of endothelial CD137 signaling for 3 hours.
at the transcriptional level and found that VEGFR2, the critical regulator of angiogenesis, was obviously increased both at transcriptional and translational levels. In addition, considering the complex role of VEGFR2 in atherosclerosis, regulation of VEGFR2 through CD137 signaling might be crucial for the neovessel formation. The high expression of VEGFR2 on EC surfaces provided broader access for VEGF binding [52], although some studies reported that VEGFR2, if retained on cell membrane for a long time, inhibited VEGF's binding and inactivated the downstream transduction. The VEGFR2 protein expression measured in ECs which treated with CD137L for a shorter time course, as indicated in Figure S2 (Supplementary materials), suggested a substantial upregulation of VEGFR2 which may contribute to activation VEGFR2 signaling [51]. CD137 activated the phosphorylated VEGFR2 at Tyr1173 (Figure 2). Additionally, p-Akt and p-eNOS changed synchronously with p-VEGER2. Moreover, our group has demonstrated that the CD137 signaling activated Akt in ECs in atherosclerosis [53]. Moreover, endothelial NO synthase (eNOS) is produced by ECs and reflects endothelial activities. A low level of eNOS is considered essential for maintaining the endothelial functions [54]. The deficiency in eNOS markedly decreased retinal neovascularization in a mouse model [55]. Additionally, Huang et al. reported that soluble delta-like 1 homolog (DLK1) stimulated angiogenesis through Notch1/Akt/e-NOS signaling in ECs [39]. CD137 signaling activating eNOS conforms to the studies on CD137 proangiogenesis effects [12,13].
To further investigate whether the effects of CD137 signaling on EC functions are mediated by VEGFR2, we transfected ECs with siVEGFR2 or XL184 to block VEGFR2. VEGFA165 was used as an inducer of VEGFR2 before siVEGFR2 transfection. Inhibition of VEGFR2 weakened the Akt and eNOS activation expression and reduced endothelial proliferation and migration mediated by CD137 signaling ( Figure 3).
Decreased p-eNOS and unchanged VEGFR2 protein level after inactivating Akt during the CD137 signaling activation suggest that Akt and eNOS are the downstream molecules of VEGFR2 signaling (Figure 4). Likewise, the proliferative and migratory functions were dampened when inhibiting Akt under the CD137 signaling activation condition [56]. These results are consistent with the previous studies [26,31,57,58].
VEGFR2 is critical for migratory ECs that sense proangiogenic factors. Both Akt and eNOS are essential for survival and migration of ECs. In line with in vitro results, the aortic ring assay showed that the number of sprouts per ring increased obviously when treated with CD137L. 16

Mediators of Inflammation
The prosprouting effect was significantly impaired after inhibition of VEGFR2 or/and Akt ( Figure 5). Besides, inhibition the activity of eNOS weakened the effects of CD137 on angiogenesis, suggesting that CD137-mediated angiogenesis is not only eNOS-related ( Figure 6). Together, these results demonstrated the key role of the VEGFR2/-Akt/eNOS pathway in CD137 signaling-mediated sprouting angiogenesis. Signal transduction networks initiated by VEGFA/-VEGFR2 lead to EC proliferation, migration, survival, and new vessel formation. Cocultured CD137 signaling-activated ECs with VEGFA165 or the condition medium, which was from VEGFR2 highly expressing ECs, showed the activation of the VEGFR2/Akt/eNOS pathway. Preactivating CD137 signaling of cultured ECs could explain the expression of p-VEGFR2 and p-Akt in the control group. We subsequently analyzed whether CD137 signaling influenced the expression of VEGFA and showed that VEGFA in EC extracts was lower after activating CD137 signaling. However, the VEGFA protein level was higher in the condition medium as detected by western blot and ELISA, implying that VEGFA was secreted to the extracellular space. To verify whether VEGFA was carried to the extracellular space via vesicles, we examined VEGFA protein in endothelial exosomes and found that iy\t was significantly diminished after activating CD137 signaling ( Figure 7). Furthermore, blocking the CD137 signaling reversed its effects on the VEGFA expression and secretion. Furthermore, previous studies demonstrated that PlGF potentiated the effects of VEGFA binding with VEGFR2 by stimulating VEGFA secretion [59,60], thus exerting a proangiogenic and proatherogenic effect [61]. However, whether increased VEGFA secretion mediated by CD137 signaling that influences VEGFR2 signaling transduction needs more investigation.
In addition, the expression of CD137 was reported in a wide range of tumor cells [62] and tumor vessel walls [14]. While CD137 agonist immunotherapy is beneficial for cancer control and treatment [63,64], the accompanying proangiogenic effects of CD137 activation may weaken anticancer therapy. Therefore, combining antiangiogenesis agents and immune checkpoint blockers would be an attractive strategy for cancer treatment [15], considering that the CD137 activation together with antiangiogenesis enhance the antitumor effects.
Here, we revealed that the CD137/CD137L costimulatory molecule activates VEGFR2 and influences downstream signaling transduction, which provides an avenue for combined immunological antiangiogenic therapy in angiogenic diseases. However, further studies are necessary to unravel the mechanisms of CD137-related VEGFA secretion and the effects of CD137 agonists combined with antiangiogenic agents on angiogenesis in vivo.
In summary, the activation of CD137 signaling promotes sprouting angiogenesis by the increased VEGFA secretion and VEGFR2/Akt/eNOS pathway (Figure 8). CD137 signaling and VEGFA-VEGFR2 may be the potential targets for intraplaque angiogenesis. Moreover, combination of CD137 agonists with specific VEGFA/VEGFR2 inhibitors may be effective for angiogenesis in tumors.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
This study was approved by the Ethical Committee of Jiangsu University and conducted in agreement with the institutional guidelines.