The P2Y12 Receptor Antagonist Ticagrelor Ameliorates Pulmonary Hypertension

Background: Pulmonary arterial hypertension (PAH) is a disease that the pulmonary artery is abnormally elevated. P2Y12 is an adenosine diphosphate (ADP) receptor and it act as the target of thienopyridine antiplatelet drugs by controlling vascular remodeling. Inhibition of P2Y12 receptor in the process of PAH was explored in this study. Methods: The PAH model was established in Sprague-Dawley rats by single subcutaneous injection of 60 mg/kg monocrotaline (MCT). The ticagrelor solution (a selective P2Y12R inhibitor) was intraperitoneally injected into rats at a dose of 14 mg/kg from the time of MCT injection to day 28. Results: In the lung tissues of PAH rats, the marked P2Y12R was detected. Treatment with ticagrelor greatly decreased P2Y12R level and eciently abolished the upregulation of α-SMA as demonstrated by Western blot and RT-PCR. The wall thickness and occlusion score of the pulmonary arterioles showed that blockade of P2Y12R could relieve lung remodeling caused by PAH. The haemodynamic changes at 4 weeks determined that P2Y12R inhibition affected RV pressure and right heart hypertrophy. Conclusions: P2Y12R might be involved in the pathogenesis of PAH. Blockade of P2Y12R has potential in treating PAH.


Background
Pulmonary arterial hypertension (PAH) is a disease that the pulmonary artery is abnormally elevated and ultimately leads to pulmonary vascular remodeling. The proliferation of pulmonary arterial smooth muscle cell (PASMC) and the dysfunction of pulmonary arterial endothelial cell are determining factors involved in PAH pathogenesis. And it has been con rmed that, the two processes have signi cant roles in pulmonary vascular resistance, right heart failure, and death [1][2][3] . Besides that, various pathologic conditions have been revealed to be risk factors of PAH, such as hypoxia, oxidative, and infections.
P2Y12 receptor is one of the members of P2 receptor family. The P2Y12 receptor consist of ion-channel P2X and G-protein-coupled P2Y receptors. P2Y12 receptor was originally found to be expressed in platelet. Recent studies demonstrated that, P2Y12 receptor also expressed in vascular smooth muscle cells (VSMCs) 5 . In platelets, endothelial cells, or immune cells, the adenosine triphosphate (ATP) and adenosine diphosphate (ADP) generated from cell are able to active P2 receptors, including P2Y12 4 . The elevated P2Y12 suppresses adenylyl cyclase level and then participates in regulating the activation of platelet and thrombosis. Thus, the P2Y12 receptor has been clinically used as a target for thromboembolism treatment.
Antiplatelet drugs are widely used clinically, especially for cardiovascular events with thrombotic involvement. But recent clinical studies suggest that antiplatelet drugs may also be useful as agents for primary cardiovascular prevention 2,6 . VSMCs are one of the main cell types involved in most stages of PAH. Inhibition the migration and proliferation of VSMCs are critical in the treatment of PAH. Ticagrelor is a relatively novel antiplatelet agent that has been shown to reversibly inhibit P2Y12 receptors on platelets and smooth muscle cells (SMCs). Here, the role of ticagrelor on the pathogenesis of PAH was tested for the rst time, as well as the therapeutic role of ticagrelor on the treatment of PAH.

PAH model
SPF grade of Sprague-Dawley rats (all male, weighed 280-330g) were purchased from the Laboratory Animal Center, Chinese Academy of Science (Beijing, China). The rats were housed in a standard animal room at 21±1˚C temperature and 55±5% humidity. The animal room were under a 12-h light/dark cycle and the rats were free access to water and food. After feeding in the animal room for 7 days, he experiments were began. The animal studies performed were all approved by the Shandong University Institutional Animal Care and Use Committee and were conducted according to the standard protocols and guidelines. The rats were randomly divided into 4 groups: Sham group in which rats received water alone (n = 15); Sham + T group in which the rats were intraperi SPF grade of Sprague-Dawley rats (all male, weighed 280-330g) were purchased from the Laboratory Animal Center, Chinese Academy of Science (Beijing, China). The rats were housed in a standard animal room at 21±1˚C temperature and 55±5% humidity. The animal room were under a 12-h light/dark cycle and the rats were free access to water and food. After feeding in the animal room for 7 days, he experiments were began. The animal studies performed were all approved by the Shandong University Institutional Animal Care and Use Committee and were conducted according to the standard protocols and guidelines. The rats were randomly divided into 4 groups: Sham group in which rats received water alone (n = 15); Sham + T group in which the rats were intraperitoneally injected with 14 mg/kg ticagrelor solution (AstraZeneca) every day (n = 15); PAH group in which PAH was induced by left pneumonectomy plus MCT injection 7 (n = 30); and PH + T group in which PAH rats were injected with ticagrelor solution (n = 20). Ticagrelor solution (a selective P2Y12R inhibitor) was made using a 360 mg tablet diluted with 25.5 ml saline water and injected from the time MCT injection to day 28 9 .
oneally injected with 14 mg/kg ticagrelor solution (AstraZeneca) every day (n = 15); PAH group in which PAH was induced by left pneumonectomy plus MCT injection 7 (n = 30); and PH + T group in which PAH rats were injected with ticagrelor solution (n = 20). Ticagrelor solution (a selective P2Y12R inhibitor) was made using a 360 mg tablet diluted with 25.5 ml saline water and injected from the time MCT injection to day 28 9 .
The animals were anaesthetized using 2% xylazine (4 mg/kg)/ketamine (100 mg/kg). The rats received an adjusted rate of 60 breaths/min. Respiratory support was given to the rats using a small animal ventilator (HX-300S; Chengdu TME Technology Co., Ltd.) at a tidal volume of 1.1-1.3 ml/100 g, followed by a left unilateral pneumonectomy 8 . One week following surgery, the rats were subcutaneously injected with 60 mg/kg MCT. All rats were under monitored every day until the PAH symptoms were developed, such as body weight loss and tachypnea.

Echocardiography and haemodynamic measurements
Cardiac function was evaluated using a 14 MHz linear transducer equipped with an echocardiographic machine (Visual Sonics, Toronto, Canada). According to Simpson's method, cardiac output (CO) and Bmode long axis was used to detect stroke volume, and pulmonary artery diameter and M-mode were used to measured RV wall thickness. The acceleration time of the pulmonary artery was obtained by applying ultrasonic Doppler to the pulmonary artery 10 . According to the tail-cuff method, a blood pressure recorder (BP-98A; Softron, Tokyo, Japan), was used to measure the blood pressure of the rats 11 . Pulmonary artery pressure transduction was conducted with correct jugular vein by a 1.4F Millar Mikro-Tip catheter transducer (Millar Instruments Inc., Houston, TX) directed to the main pulmonary artery after insertion into the right ventricular out ow duct, although RV systolic pressure (RVSP) was detected with a power laboratory monitoring device (Miller Instruments). Hemodynamic values were accurately computed by LabChart 7.0 physiological data acquisition system (AD Instruments, Sydney, Australia). The rats were anaesthetised during this process.

Tissue processing and histology
Following the test of echocardiography and haemodynamic measurements, the animals were sacri ced by inducing cardiac arrest by injection of 2 mmol KCl through the catheter. The lungs were isolated. The left one was weighed and the right one was in ated with 0.5% low melting agarose at a constant pressure of 25 cm H 2 O, and xed in 10% formalin for 24 h. Subsequently, the heart was excised.

Western blot
The lysis buffer used for the extraction of proteins from tissues was a mixture of RIPA (Beyotime Institute of Biotechnology) and PMSF at a ratio of 100:1 11 . The extracted proteins were detected using a BCA protein assay reagent kit (Pierce). The proteins were then subjected to a 5-12% SDS-PAGE gel and transferred onto polyvinylidene di uoride (PVDF) membrane. After blocking in the TBST for 1 h at 4˚C, the target proteins were probed by incubation with following antibodies: 1:2000 for P2Y12R (Abcam, USA) and 1:1500 for α-SMA (Abcam, USA). Primary antibodies were detected using horseradish peroxidaseconjugated antibodies: 1:5000 for anti-mouse (ZSJQ-BIO, Beijing, China) and 1:5000 for anti-rabbit (ZSJQ-BIO, Beijing, China), at room temperature for 2 h. The enhanced chemiluminescence (ECL) detection kit (Millipore) was used for blot development. The blots were visualized by the FluroChem E Imager (Protein-Simple, Santa Clara, CA, USA) and semi-quanti ed using ImageJ software (National Institutes of Health).

Immunohistochemistry
The right lung tissues were formalin-xed, para n-embedded and used for HE or regular immunohistochemistry staining 2 . The OCT-embedded tissue was placed into a freezing microtome (CM3050; Leica Microsystems GbmH) and tissue samples were cut into 5 μm sections 8 . In each lung section, 30 small PAs (50-100 μm in diameter) were analyzed at × 40 magni cation in a blinded manner. The medial wall thickness was expressed as the summation of two points of medial thickness/ external diameter × 100 (%). Intraacinar (precapillary) PAs (20-30 μm in diameter, 25 vessels each) were assessed for occlusive lesions, de ned as Grade 0 when there was no evidence of neointimal lesion, Grade 1 when there was less than 50% luminal occlusion, and Grade 2 when there was more than 50% luminal occlusion 13 . There was no evidence of neointimal lesion formation in any PAs from normal rats (all PAs were graded as 0). Anti-α-SMA (1:200; Abcam) antibodies were used as primary antibodies. After xing the frozen sections with cold acetone at 25˚C for 5 min and blocking with QuickBlock™ Blocking Buffer for Immunol Staining (cat. no. P0260; Beyotime Institute of Biotechnology) for 10 min at 4˚C, they were treated overnight at 4˚C with anti-P2Y12R antibody (1:200; Novus) and α-SMA (1:200; Abcam). Following incubation with primary antibody, Alexa 546-conjugated donkey anti-rabbit (1:200; Invitrogen) and FITCconjugated rabbit anti-mouse (1:200; Abcam) secondary antibodies were added, respectively, and the sections were incubated for 2 h at room temperature. The sections were counterstained with DAPI (Life Technologies) to identify nuclei. The sections were then washed and placed under a uorescence microscope for observation and image capture. Nerve density was measured and evaluated using ImageJ software.

Statistics
Data are expressed as the mean ± SEM. The signi cant difference between two groups were analyzed by unpaired t-test. For three or more groups, analysis of variance (ANOVA) followed by a Newman-Keuls test was utilized. Statistical analyses were performed using SPSS 20.0 software (SPSS Inc. Chicago, IL, USA), and p-value < 0.05 was considered statistically signi cant.

PAH rats show signi cant high P2Y12R level in lungs
Co-staining of P2Y12R with α-SMA shown that P2Y12R was largely distributed in PASMCs from the hypertrophied media of pulmonary vessels in PAH lung tissue (Fig 1), indicating P2Y12R as a central risk factor of PAH. To further investigate the role of P2Y12R in PAH, a specific P2Y12R inhibitor, ticagrelor, was applied.

Effects of ticagreloron P2Y12R and α-SMA expression in lung tissues
The effects of ticagrelor on P2Y12R expression were assessed. The expression level of P2Y12R (Fig. 2B, D, F) was upregulated in PAH rats. Treatment with ticagrelor greatly decreased P2Y12R level and e ciently abolished the upregulation of α-SMA as demonstrated by Western blot and RT-PCR ( Fig. 2A, C,  E). Results showed that there was little difference between the two sham groups, which con rmed that interference by ticagrelor to PAH may be related to inhibition of P2Y12R to expression of α-SMA.

P2Y12R inhibition inhibits pulmonary vascular remodeling
PAH leads to pulmonary vascular remodeling 14 , thus we further studied the effects of ticagrelor on remodeling. By measuring the wall thickness and occlusion score of the pulmonary arterioles, we found that wall thickness was remarkably increased from 60.8% ± 4.7% to 81.2% ± 4.4% (p < 0.05) in vessels with diameters ranging from 50 to 100 μm (Fig. 3). Treatment with ticagrelor suppressed the wall thickness to 67.6% ± 3.5% (p < 0.05; Fig.3C). Decreases in Grade I and II occlusion were also demonstrated (15 and 73% in PAH vehicle group vs. 25 and 29% in the ticagrelor administrated PAH group respectively; Fig.3D). Therefore, blockade of P2Y12R could relieve lung remodeling caused by PAH.

P2Y12R inhibition ameliorates pulmonary hypertension
As shown in Fig.4, RVSP was signi cantly inhibited by ticagrelor treatment in rats (39.3 ± 4.5 mm Hg, vs. 53.9±4.8 mm Hg the P/ MCT group, p < 0.05). Also, ticagrelor treatment prior to or after MCT administration signi cantly reduced the thickness of RV wall, RV area, and pulmonary artery diameter (table 1). It was also observed that, ticagrelor treatment increased the mean acceleration time of the pulmonary artery as compared with PAH group.

Discussion:
Since platelet P2Y12 ADP receptor has been considered as one important target of thienopyridine-type antiplatelet drugs, herein we investigated the impacts of ticagrelor (a selective P2Y12R inhibitor) in the pathogenesis of PAH. We for the rst time described a functionally active P2Y12 in SMC proliferation post pulmonary hypertension. Firstly, it was demonstrated that P2Y12R expression was updated in SMC in PH rats. Secondly, this upregulation positively enhanced vascular proliferation. Therefore, the application of antiplatelet drugs could be important for the treatment of PH.
VSMCs are the major cell type in vessel walls and they play central roles in the most stages of pulmonary hypertension. Initially, P2Y12 receptors were found to be expressed in platelets and microglia in the brain sub region. Recently, studies shown that, it was also expressed in a variety of cells, such as VSMCs 15 . This is consistent with the results presented here which show signi cant P2Y12 upregulation on VSMC in pulmonary hypertension rats. In the current study, MCT-challenged left pneumonectomised rats showed a marked increase in the P2Y12R expression into peri-vascular and peri-alveolar areas of pulmonary tissues and bronchoalveolar lavage samples. It seems that inhibition of P2Y12 may have additional therapeutic bene ts on pulmonary hypertension beyond anti-thrombotic effect, like anti-PAH.
Under the stimulations such as hypoxia or shear stress, and mediate vasodilatatory, in ammatory, and thrombotic responses, extracellular nucleotides including the purines ATP, ADP, and adenosine monophosphate (AMP) as well as pyrimidines uridine-5′-triphosphate (UTP) and uridine-diphosphate (UDP) are released within the pulmonary vascular bed and then involved in the pathogenesis of PH 16 . It has been reported that ADP induces VSMC contraction via P2Y12, and promotes proliferation. ADP elicits pulmonary vasoconstriction through P2Y1 and P2Y12 receptor activation 17 . It was shown here that P2Y12R was upregulated and co-stained with α-SMA in PAH rats. Furthermore, the P2Y12R level was positively related with α-SMA expression. The P2Y12 inhibitor ticagrelor reversed pulmonary hypertension, as well as α-SMA downregulation, indicating that activation of P2Y12 is required for proliferation of PASMCs.
The mechanism underlying P2Y12R mediated pulmonary remodeling may include cAMP/PKA signaling, which has been shown to be the key link in PASMCs proliferation 18 , and is the downstream pathway under the stimulation of ADP 19 . Besides, the P2 receptor mediated Ca 2+ signalosome of the human pulmonary endothelium may be implicated in pulmonary arterial hypertension 20 . The exact mechanism requires for further investigation.

Conclusion And Perspectives:
The vessel wall P2Y12 receptor promotes vascular remodeling in the PAH pathological process. Therefore, antiplatelet agents such as ticagrelor may be used as a therapeutic target for pulmonary hypertension.
It remains to determine whether P2Y12 receptor has potentials in regulating other cell types of PAH pathogenesis, like pulmonary arterial endothelial cell. Besides, a mass of clinical trials are required before     Ticagrelor administration prevented the pulmonary hypertension and improves RV function of PAH rats. (A) and (B) RVSP changes of PAH rats which were treated with ticagrelor. (C) The RV/LV+ S ratio of PAH rats. The representative visual shape of the RV is shown (D). * *p<0.05 and *p< 0.05 mean that results had signi cant difference countered to sham and PAH group, respectively. RVSP= right ventricle systolic pressure.

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