# Antiangiogenic Effect of Platelet P2Y12 Inhibitor in Ischemia-Induced Angiogenesis in Mice Hindlimb

Authors: Xiaoli Wang; Huan Zhao; Naiquan Yang; Yue Jin; Jianguo Chen
Journal: BioMed Research International
 (2021)

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## Abstract

Purpose. Postischemic inflammation induces angiogenesis, while platelet P2Y12 inhibitors can alleviate this inflammation. Therefore, we studied the potential effects of P2Y12 inhibitor, ticagrelor, on angiogenesis in a mouse model of hindlimb ischemia. Methods. Laser Doppler perfusion imaging and capillary density measurement were used for angiogenesis quantified. Immunofluorescence was used to detect the level of CD31. The mice muscle was harvested for enzyme-linked immunosorbent (ELISA) assay of interleukin- (IL) 10 activity and Western blot determination of vascular endothelial growth factor (VEGF) production. Results. Ischemic hindlimb angiogenesis was sharply decreased in IL-10+/+ mice than IL-10-/- mice. Ticagrelor inhibited angiogenesis and blood reperfusion recovery significantly elevated the levels of IL-10 and decreased the expression of VEGF in the IL-10+/+ mouse ischemic hindlimb, which were abolished in IL-10-deficient (IL-10–/–) C57BL/6J mice. Conclusion. The study underscores that the effect of ticagrelor antiangiogenic function is related with the higher IL-10 expression.


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## Body

## 1. Introduction

Pathological angiogenesis is related with many circumstance such as ischemic diseases, diabetic retinopathy, and tumor growth [1–3]. Hence, understanding the mechanism of angiogenesis is very important for the treatment of pathological angiogenesis.Ischemia-induced angiogenesis is controlled by the coordination between proangiogenic growth factors, for instance vascular endothelial growth factor (VEGF), and varieties of antiangiogenic endogenous factors. Previous study shows that VEGF is upregulated by growth factors [4], proinflammatory mediators, and tissue hypoxia [5]. Moreover, VEGF also participates in the activation of inflammatory pathways by inducing other proinflammatory cytokines [6, 7]. Therefore, angiogenesis and inflammation are interlinked.Besises, it was reported that anti-inflammatory cytokines are also secreted and modulated the inflammatory process. IL-10, an endogenous angioinhibitor, has been identified as an important anti-inflammatory cytokine [8]. Furthermore, IL-10 has also decreased VEGF and matrix metalloproteinase-9 (MMP-9) synthesis to prevent angiogenesis [9].A diverse group of reports suggested neovascularization contributes to the atherosclerotic lesions growth and is a major factor in plaque destabilization leading to rupture [1, 10, 11]. Ticagrelor, a P2Y12 inhibitor, was shown to lead to a lower incidence of cardiovascular mortality, myocardial infarction, or stroke compared with clopidogrel [12]. In addition, P2Y12 inhibitors significantly inhibited the aggregation of platelet-monocyte and reduced the expression of major proinflammatory cytokines, including tumor necrosis factor- (TNF-) α, IL-6, and chemokine (C–C motif) ligand 2 and conversely increased the expression of IL-10 [13]. These studies indicated that ticagrelor may regulate angiogenesis by inhibiting inflammation to play a plaque-stabilizing role. However, whether P2Y12 inhibitors can inhibit angiogenesis by regulating inflammation is still unclear.In our study, we aimed to determine the effects of ticagrelor on angiogenesis. We demonstrate that the ischemic hindlimb angiogenesis was sharply decreased in IL-10+/+ mice than IL-10-/- mice. Notably, ticagrelor treatment inhibited angiogenesis and blood reperfusion recovery in IL-10+/+ mice. Moreover, ticagrelor treatment also significantly increased the IL-10 protein level and decreased VEGF protein level in ischemic tissues. However, the ticagrelor antiangiogenesis and anti-inflammation effects were abolished in IL-10-deficient mice, suggesting ticagrelor played a vital role in the ischemia-induced angiogenesis.


## 2. Materials and Methods

### 2.1. Animal Model and Groups

C57BL/6J male IL-10+/+ and IL-10-/- mice were obtained from the Laboratory Animal Center of Nanjing Medical University. IL-10+/+ mice were randomly divided into three groups: (1) IL-10+/+ control group (IL-10+/+ group), treatment with saline; (2) sham group, underwent open skin procedure but without femoral artery ligation, then treatment with saline; (3) IL-10+/++ticagrelor group (IL-10+/++tica), treatment with ticagrelor (150 mg•kg-1•d-1). C57BL/6 J IL-10-/- mice were randomly divided into two groups: (1) IL-10-/- control group (IL-10-/-), treatment with saline; (2) IL-10-/- + ticagrelor group (IL-10-/- + tica), treatment with ticagrelor (150 mg•kg-1•d-1). Intragastric treatment of ticagrelor (AstraZeneca) was initiated 3 days before operationally induced unilateral limb ischemia and continued for 2 weeks postoperatively. Animal care and protocols for the experiments were complied with the Guidelines for the Care and Use of Laboratory Animals of Jiangsu University.


### 2.2. Hindlimb Ischemia Model

The mice were underwent unilateral hindlimb ischemia surgery as described previously [14]. Simply, mice were first intraperitoneally anesthetized with pentobarbital sodium (50 mg/kg), and then, the right femoral artery was dissected along its full length. Finally, all branches were ligated and resected, and the left hindlimb remained intact and was used as the nonischemic limb.


### 2.3. Laser Doppler Perfusion Imaging

Two weeks after surgery, laser Doppler perfusion imaging (Perimed, Sweden) was used to detect superficial blood flow in both feet. Then, the ratio of the ischemic (right) to normal (left) limb blood flow relative perfusion data was recorded.


### 2.4. Immunofluorescence Staining

Two weeks after surgery, the ischemic limb muscle tissue sections were fixed in 4% paraformaldehyde and permeabilized with xylene. After blocking (5% BSA in PBS), the sections were immunolabeled with primary antibody: CD31 (1 : 100; Abcam) and then incubated with Alexa Fluor ®488 donkey anti-mouse secondary antibody (1 : 250; Invitrogen) after washing. The nuclei were counterstained using DAPI (Invitrogen), and the cells were observed under a fluorescence microscope (Olympus, Japan).


### 2.5. Detection of IL-10 by ELSIA

Two weeks after surgery, the concentration of IL-10 (ab108870; Abcam) in the muscle was assessed using commercially available ELISA kits (USCN Business Co., Ltd.) according to the manufacturer’s protocol.


### 2.6. Western Blot

At 2 weeks postsurgery, ischemic limb muscle tissue homogenate lysate was harvested for western blot analysis as described study [15]. The lysates were electrophoresed and separated on 10% SDS-PAGE and transblotted onto nitrocellulose membranes (Bio-Rad, Hercules, USA). After blocked with milk, the membranes were incubated against primary antibodies: VEGF (1 : 1000) and GAPDH (1 : 4000; Cell Signaling Technology) followed by secondary antibody for 30 min at room temperature.


### 2.7. Statistical Analysis

SPSS version 13.0 (IBM Corp.) was used to analyze all the data. Differences were statistically analyzed using a one-way ANOVA or two-tailed Studentst-test. The Newman-Keuls test was used to show post hoc differences. P value of <0.05 shows the significant difference.


## 2.1. Animal Model and Groups

C57BL/6J male IL-10+/+ and IL-10-/- mice were obtained from the Laboratory Animal Center of Nanjing Medical University. IL-10+/+ mice were randomly divided into three groups: (1) IL-10+/+ control group (IL-10+/+ group), treatment with saline; (2) sham group, underwent open skin procedure but without femoral artery ligation, then treatment with saline; (3) IL-10+/++ticagrelor group (IL-10+/++tica), treatment with ticagrelor (150 mg•kg-1•d-1). C57BL/6 J IL-10-/- mice were randomly divided into two groups: (1) IL-10-/- control group (IL-10-/-), treatment with saline; (2) IL-10-/- + ticagrelor group (IL-10-/- + tica), treatment with ticagrelor (150 mg•kg-1•d-1). Intragastric treatment of ticagrelor (AstraZeneca) was initiated 3 days before operationally induced unilateral limb ischemia and continued for 2 weeks postoperatively. Animal care and protocols for the experiments were complied with the Guidelines for the Care and Use of Laboratory Animals of Jiangsu University.


## 2.2. Hindlimb Ischemia Model

The mice were underwent unilateral hindlimb ischemia surgery as described previously [14]. Simply, mice were first intraperitoneally anesthetized with pentobarbital sodium (50 mg/kg), and then, the right femoral artery was dissected along its full length. Finally, all branches were ligated and resected, and the left hindlimb remained intact and was used as the nonischemic limb.


## 2.3. Laser Doppler Perfusion Imaging

Two weeks after surgery, laser Doppler perfusion imaging (Perimed, Sweden) was used to detect superficial blood flow in both feet. Then, the ratio of the ischemic (right) to normal (left) limb blood flow relative perfusion data was recorded.


## 2.4. Immunofluorescence Staining

Two weeks after surgery, the ischemic limb muscle tissue sections were fixed in 4% paraformaldehyde and permeabilized with xylene. After blocking (5% BSA in PBS), the sections were immunolabeled with primary antibody: CD31 (1 : 100; Abcam) and then incubated with Alexa Fluor ®488 donkey anti-mouse secondary antibody (1 : 250; Invitrogen) after washing. The nuclei were counterstained using DAPI (Invitrogen), and the cells were observed under a fluorescence microscope (Olympus, Japan).


## 2.5. Detection of IL-10 by ELSIA

Two weeks after surgery, the concentration of IL-10 (ab108870; Abcam) in the muscle was assessed using commercially available ELISA kits (USCN Business Co., Ltd.) according to the manufacturer’s protocol.


## 2.6. Western Blot

At 2 weeks postsurgery, ischemic limb muscle tissue homogenate lysate was harvested for western blot analysis as described study [15]. The lysates were electrophoresed and separated on 10% SDS-PAGE and transblotted onto nitrocellulose membranes (Bio-Rad, Hercules, USA). After blocked with milk, the membranes were incubated against primary antibodies: VEGF (1 : 1000) and GAPDH (1 : 4000; Cell Signaling Technology) followed by secondary antibody for 30 min at room temperature.


## 2.7. Statistical Analysis

SPSS version 13.0 (IBM Corp.) was used to analyze all the data. Differences were statistically analyzed using a one-way ANOVA or two-tailed Studentst-test. The Newman-Keuls test was used to show post hoc differences. P value of <0.05 shows the significant difference.


## 3. Results

### 3.1. Ticagrelor Therapy Decreases Blood Perfusion in IL-10+/+ Mice

As depicted in Figures1(a) and 1(b), compared in the IL-10+/+ mice, the amount of ischemia (right leg) perfusion in mice treated with saline was significantly lower than that in the sham group (0.55±0.08vs.1.01±0.05, P<0.01). After treatment with ticagrelor, the ischemic tissue blood flow restoration was decreased in the ticagrelor-treated IL-10+/+ mice than the saline-treated controls (0.39±0.12vs.0.55±0.08, P<0.05).Figure 1
Effect of ticagrelor therapy on blood perfusion in IL-10+/+ and IL-10-/- mice. (a) Representative figures of ischemic limb blood flow after the right femoral artery ligation for 14 days; arrows represent ischemic hindlimbs. (b) Quantitative analysis of superficial blood flow in ischemic and normal limbs (n=6‐10). #P<0.01 indicated compared with the sham group; $P<0.05 indicated compared with the IL-10+/+ group; ∗P<0.01 indicated compared with the IL-10+/+ group; ∗∗P<0.01 indicated compared with the IL-10+/+ + tica group.
(a)(b)


### 3.2. Ticagrelor Therapy Decreases the Capillary Density in IL-10+/+ Mice

Measurement of capillary density corresponded to reduced ischemic hindlimb perfusion in mice. Indeed, the IL-10+/+ mice showed the capillary number ratio was decreased in the ischemic leg in the saline-treated groups compared with the sham group (0.45±0.06vs.0.91±0.03, P<0.01). After ticagrelor treatment, lower capillary density was seen in the ticagrelor-treated groups as comparison to the saline-treated groups (0.22±0.08vs.0.45±0.06, P<0.05, Figures 2(a) and 2(b)).Figure 2
Effect of ticagrelor therapy on capillary density in IL-10+/+ and IL-10-/- mice: (a) immunofluorescence staining of CD31; (b) quantification analysis of the CD31-stained vessel numbers in the sections of gastrocnemius muscle (n=6‐10). #P<0.01 indicated compared with the sham group; $P<0.05 indicated compared with the IL-10+/+ group; ∗P<0.01 indicated compared with the IL-10+/+ group; ∗∗P<0.01 indicated compared with the IL-10+/+ +tica group.
(a)(b)


### 3.3. Determination the IL-10 and VEGF Level in IL-10+/+ Mice

As presented in Figure3, the IL-10 expression was evidently elevated in the IL-10+/++tica group than the IL-10+/+ group (489.3±170.9vs.305.9±127.5 pg/100 mg protein, respectively, P<0.05). We also checked whether treatment with ticagrelor changes the VEGF protein level of ischemic gastrocnemius muscle tissue. As expected, in Figures 4(a) and 4(b), the results showed that the VEGF protein expression was decreased in the IL-10+/++tica group than the IL-10+/+ group.Figure 3
Ticagrelor therapy increases IL-10 protein levels in IL-10+/+ mice. ELISA was conducted to detect the IL-10 expression in indicated groups (n=6‐10). #P<0.01 indicated compared with the sham group; $P<0.05 indicated compared with the IL-10+/+ group.Figure 4
The detection of VEGF protein levels in the calf muscles of mice. (a) The VEGF protein levels in indicated groups were determined by western blot. (b) Densitometry of the VEGF expression normalized to GAPDH (n=6‐10). #P<0.01 indicated compared with the sham group; $P<0.05 indicated compared with the IL-10+/+ group; ∗P<0.01 indicated compared with the IL-10+/+ group; ∗∗P<0.01 indicated compared with the IL-10+/+ +tica group.
(a)(b)


### 3.4. Function of Ticagrelor in IL-10-/- Mice

To verify that ticagrelor treatment of IL-10+/+ mice with decreased ischemia-induced angiogenesis is associated with IL-10 pathway, ticagrelor treatment was tested in IL-10-/- mice. In the two groups treated with ticagrelor, we found that the angiogenesis of ischemic hindlimbs in IL-10-/- mice is significantly higher than that in IL-10+/+ mice (Figure 1(a) and 1(b) and Figures 2(a) and 2(b)). Besides, Figure 3 showed that the anti-inflammation effects of ticagrelor were abolished in IL-10-deficient mice. Moreover, the IL-10-/- mice treated with ticagrelor showed no significant change in VEGF level (Figures 4(a) and 4(b)).


## 3.1. Ticagrelor Therapy Decreases Blood Perfusion in IL-10+/+ Mice

As depicted in Figures1(a) and 1(b), compared in the IL-10+/+ mice, the amount of ischemia (right leg) perfusion in mice treated with saline was significantly lower than that in the sham group (0.55±0.08vs.1.01±0.05, P<0.01). After treatment with ticagrelor, the ischemic tissue blood flow restoration was decreased in the ticagrelor-treated IL-10+/+ mice than the saline-treated controls (0.39±0.12vs.0.55±0.08, P<0.05).Figure 1
Effect of ticagrelor therapy on blood perfusion in IL-10+/+ and IL-10-/- mice. (a) Representative figures of ischemic limb blood flow after the right femoral artery ligation for 14 days; arrows represent ischemic hindlimbs. (b) Quantitative analysis of superficial blood flow in ischemic and normal limbs (n=6‐10). #P<0.01 indicated compared with the sham group; $P<0.05 indicated compared with the IL-10+/+ group; ∗P<0.01 indicated compared with the IL-10+/+ group; ∗∗P<0.01 indicated compared with the IL-10+/+ + tica group.
(a)(b)


## 3.2. Ticagrelor Therapy Decreases the Capillary Density in IL-10+/+ Mice

Measurement of capillary density corresponded to reduced ischemic hindlimb perfusion in mice. Indeed, the IL-10+/+ mice showed the capillary number ratio was decreased in the ischemic leg in the saline-treated groups compared with the sham group (0.45±0.06vs.0.91±0.03, P<0.01). After ticagrelor treatment, lower capillary density was seen in the ticagrelor-treated groups as comparison to the saline-treated groups (0.22±0.08vs.0.45±0.06, P<0.05, Figures 2(a) and 2(b)).Figure 2
Effect of ticagrelor therapy on capillary density in IL-10+/+ and IL-10-/- mice: (a) immunofluorescence staining of CD31; (b) quantification analysis of the CD31-stained vessel numbers in the sections of gastrocnemius muscle (n=6‐10). #P<0.01 indicated compared with the sham group; $P<0.05 indicated compared with the IL-10+/+ group; ∗P<0.01 indicated compared with the IL-10+/+ group; ∗∗P<0.01 indicated compared with the IL-10+/+ +tica group.
(a)(b)


## 3.3. Determination the IL-10 and VEGF Level in IL-10+/+ Mice

As presented in Figure3, the IL-10 expression was evidently elevated in the IL-10+/++tica group than the IL-10+/+ group (489.3±170.9vs.305.9±127.5 pg/100 mg protein, respectively, P<0.05). We also checked whether treatment with ticagrelor changes the VEGF protein level of ischemic gastrocnemius muscle tissue. As expected, in Figures 4(a) and 4(b), the results showed that the VEGF protein expression was decreased in the IL-10+/++tica group than the IL-10+/+ group.Figure 3
Ticagrelor therapy increases IL-10 protein levels in IL-10+/+ mice. ELISA was conducted to detect the IL-10 expression in indicated groups (n=6‐10). #P<0.01 indicated compared with the sham group; $P<0.05 indicated compared with the IL-10+/+ group.Figure 4
The detection of VEGF protein levels in the calf muscles of mice. (a) The VEGF protein levels in indicated groups were determined by western blot. (b) Densitometry of the VEGF expression normalized to GAPDH (n=6‐10). #P<0.01 indicated compared with the sham group; $P<0.05 indicated compared with the IL-10+/+ group; ∗P<0.01 indicated compared with the IL-10+/+ group; ∗∗P<0.01 indicated compared with the IL-10+/+ +tica group.
(a)(b)


## 3.4. Function of Ticagrelor in IL-10-/- Mice

To verify that ticagrelor treatment of IL-10+/+ mice with decreased ischemia-induced angiogenesis is associated with IL-10 pathway, ticagrelor treatment was tested in IL-10-/- mice. In the two groups treated with ticagrelor, we found that the angiogenesis of ischemic hindlimbs in IL-10-/- mice is significantly higher than that in IL-10+/+ mice (Figure 1(a) and 1(b) and Figures 2(a) and 2(b)). Besides, Figure 3 showed that the anti-inflammation effects of ticagrelor were abolished in IL-10-deficient mice. Moreover, the IL-10-/- mice treated with ticagrelor showed no significant change in VEGF level (Figures 4(a) and 4(b)).


## 4. Discussion

The major results of the study showed that IL-10 negatively adjusts angiogenesis induced by ischemia, and ticagrelor inhibits the angiogenesis and blood reperfusion recovery, significantly increases IL-10 level, and reduces the VEGF expression in the IL-10+/+ mouse ischemic hindlimb, which were abolished in IL-10-/- mice.Many experiments showed that the neovascularization contributes to the atherosclerotic lesions growth, and it plays a key role in plaque destabilization leading to rupture [1, 10, 11]. Wallentin et al. showed that the P2Y12 inhibitor, ticagrelor, achieved better clinical outcomes in patients with ACS compared with clopidogrel treatment [12]. Besides, Thomas et al. demonstrated that P2Y12 inhibitors remarkably decreased the expression of major proinflammatory cytokines and conversely increased the expression of anti-inflammatory cytokines such as IL-10 [13]. These studies indicated that ticagrelor may regulate angiogenesis by inhibiting inflammation to play a plaque-stabilizing role. Indeed, our study showed that ticagrelor inhibited angiogenesis and blood reperfusion recovery in IL-10+/+ mice ischemic hindlimb. In addition, it also indicated that ticagrelor also significantly enhanced the IL-10 protein expression level in the IL-10+/+ mice ischemic tissues.Accumulating data have suggested that postischemic proinflammatory cytokines play an important role in inducing angiogenesis [16, 17]. The inflammatory action is simultaneously adjusted by anti-inflammatory cytokine production in major ischemic area. IL-10, as an anti-inflammatory cytokine produced by macrophages, has an antiangiogenic effect [18]. Consistent with these results, we verified that marked IL-10 was secreted in IL-10+/+ mice ischemic tissue. Furthermore, we found that the ischemic hindlimb angiogenesis was sharply decreased in IL-10+/+ mice than IL-10-/- mice.VEGF protein has been shown to play a vital role in the development of hindlimb ischemia angiogenic. It is well known that tissue hypoxia and proinflammatory mediators induce high expression of VEGF [19]. In addition, VEGF can in turn activate the inflammatory pathways by inducing the expression of other proinflammatory cytokines [20]. In a previous study, IL-10 was proved to decrease the VEGF synthesis and prevent angiogenesis [18]. Similarly, the results in our study also showed that the expression of VEGF protein was evidently increased in the hindlimb ischemia IL-10-/-mice, accompanied with increased angiogenesis. We also demonstrated that the inhibitory effect of ticagrelor on angiogenesis and the VEGF expression in ischemic IL-10+/+ mice was eliminated in IL-10-/- mice.These studies, in addition to our data, suggested that the antiangiogenic effect of ticagrelor was associated with the elevated IL-10 expression.


## 5. Conclusion

In summary, ticagrelor, a platelet P2Y12 inhibitor has an antiangiogenic effect in hindlimb ischemia mouse. Furthermore, ticagrelor treatment significantly increased the IL-10 protein expression and inhibited the VEGF protein expression in ischemic tissues of IL-10+/+ mice. However, this antiangiogenic effect was not seen in IL-10-/- mice. The present work demonstrated that ticagrelor may regulate angiogenesis by inhibiting inflammation.



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