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Adenosine A2a Receptors Improve Hypoxic Pulmonary Arterial Hypertension Via Mitochondrial ATP-sensitive Potassium Channels

Material and methods Using wild-type (WT) and A2aR-deficient (A2aR-/-) mice; hypoxic pulmonary artery smooth muscle cells (PASMCs) were induced by a 24-hours hypoxia exposure. Mice and PASMCs were treated with the A2aR agonist CGS21680, MitoKATP blocker 5-hydroxydecanoic acid sodium salt (5HD), or MitoKATP agonist diazoxide. Mitochondrial morphology was observed by electron microscopy. The mitochondrial membrane potential (Δψm); invasive hemodynamic parameters; right ventricular (RV) hypertrophy index; pulmonary arterial remodeling index; proliferative and apoptotic indexes; protein expression levels of A2aR, Bax, Bcl-2, and Caspase-9; and release of cytochrome C from the mitochondria to the cytoplasm were measured.


Introduction 32
Pulmonary hypertension (PH) is a potentially fatal disease characterized by excessive pulmonary 33 P r e p r i n t vasoconstriction, vascular remodeling and pulmonary arteriole occlusion. These pathological 34 symptoms cause a continuous increase in pulmonary artery pressure, which aggravates the right 35 ventricular (RV) afterload, leading to RV failure and even death [1]. The median survival rate of 36 patients treated with traditional methods is only 2.8 years, and the 1-, 3-, and 5-year survival rates 37 are 68%, 48%, and 34%, respectively; therefore, the prognosis remains very poor [2,3,4]. 38 Multiple cell and molecular signaling pathways are involved in the pathological process of 39 pulmonary vascular remodeling [5,6]. At present, the complex pathogenesis of PH is not fully 40 understood; however, an increasing number of studies have confirmed that pulmonary vascular 41 remodeling plays an important role in the development of PH [7][8][9][10]. 42 Adenosine is an endogenous mediator that is often used as a cytoprotective modulator for stress 43 responses and has strong vasodilator and anti-inflammatory effects. Adenosine signaling has an 44 important regulatory role in various physiological and pathological conditions via the four 45 subtypes of G protein-coupled receptors (A1, A2a, A2b, and A3), which are expressed in the lung 46 [11]. Adenosine A2a receptor (A2aR) is a highly expressed receptor with complex functions [12]. 47 metabolism of cells and thereby affect function [16]. The mitochondrial apoptotic pathway is an 55 important signal transduction pathway in apoptosis. Mitochondrial membrane potential is 56 associated with the integrity of mitochondrial function; the mitochondrial ATP-sensitive potassium 57 channel (MitoKATP), a KATP channel in the mitochondrial membrane of PASMCs, is closely 58 associated with the maintenance of mitochondrial membrane potential [17]. Hu [18] found that 59 hypoxia can activate the activity of MitoKATP in PASMCs, promoting their opening and partial 60 depolarization of the mitochondrial membrane potential (Δψm), subsequent inhibition of the 61 release of cytochrome C (Cyt C), and ultimately promoting proliferation and inhibition of 62 apoptosis in smooth muscle cells. The opening of MitoKATP channel can promote 63 hypoxia-induced proliferation of human PASMCs [19]. These findings suggest that MitoKATP is 64 associated with the development of PH. 65 Additionally, adenosine can stimulate the activity of KATP and calcium-activated potassium 66 channels by activating the A2a receptor, thereby expanding the coronary vessels [20]. We 67 speculated that A2aR could also affect pulmonary circulation via MitoKATP, ultimately reducing 68 pulmonary vascular remodeling and preventing PH. To evaluate this hypothesis, the effects of 69 A2aR on HPH via MitoKATP were determined in vivo and in vitro. 70

Animal models 85
Fifty 12-to 14-week-old male BALB/c mice weighing approximately 20-25 g were purchased 86 from Slac Experimental Animal Technology (Shanghai, CHN), and forty 12-to 14-week-old, male 87 A2aR -/-BALB/c mice weighing approximately 20-25 g were purchased from Jackson Laboratory 88 (Bar Harbor, ME, USA). All animals were fed in a specific pathogen-free animal laboratory, and 89 the laboratory and experimental protocols were reviewed and approved by Wenzhou Medical 90 University Animal Experiment Center. In the laboratory, the mice were maintained under a 91 12-hour day-night cycle at 20-24 °C and were allowed free access to sufficient food and water. 92 Fifty WT mice were randomly divided into the following five groups (10 mice per group): a WT 93 normal control group (N, saline-treated), a WT hypoxia group (H, saline-treated), a WT hypoxia 94 plus 5HD group (H5HD, 10 mg/kg), a WT hypoxia plus diazoxide group (HDia, 7 mg/kg), and a 95 WT hypoxia plus CGS21680 group (HCGS, 0.2 mg/kg). Forty A2aR -/mice were randomly 96 P r e p r i n t divided into the following four groups (10 mice per group): an A2aR -/normoxia group (K, 97 saline-treated), A2aR -/hypoxia group (HK, saline-treated), A2aR -/hypoxia plus 5HD group 98 (HK5HD, 10 mg/kg), and A2aR -/hypoxia plus diazoxide group (HKDia, 7 mg/kg). Control mice 99 in groups N and K were exposed to room air while the hypoxia groups were exposed to 9%-11% 100 O2. HPH mouse models were established over a 4-week period by placing mice in the hypoxia 101 groups in a closed chamber (8 hours per day) and by monitoring and automatically controlling the 102 O2 concentration using a detector as previously described [15]. 103

Measurements of hemodynamic parameters and RV hypertrophy 118
At the end of the hypoxia exposure period, the RV systolic pressure (RVSP) and the mean carotid 119 arterial pressure (mCAP) were measured using the method described by Huang et al. [15]. The mice 120 were anesthetized with 20% urethane (1 mL/100 g) and supine fixed, and the right external jugular 121 vein and left carotid artery were separated. Two home-made polyethylene catheters (outer 122 diameter: 0.9 mm, inner diameter: 0.5 mm) connected to pressure transducers and prefilled with 123 heparin were inserted into the RV and left carotid artery, and RVSP and mCAP were measured and 124 analyzed using a PowerLab 8/35 Multi-channel Biological Signal Recording System (AD 125 Instruments, Colorado Springs, Australia). After sacrificing mice by exsanguination, their hearts 126 were removed and divided into the RV, left ventricle (LV), and septum (S), and each section was 127 weighed. The weight ratios RV/(LV+S) and RV/body weight (BW) were calculated as indexes of 128 RV hypertrophy. Additionally, a portion of the lung tissue was homogenized to detect the 129 expression of A2aR in each group by western blotting. 130

Measurement of pulmonary arterial remodeling 131
The lung tissue was dissected and cut into several parts. The upper lobe of the right lung was 132 promptly fixed in 4% paraformaldehyde, conventionally dehydrated, paraffin-embedded, serially 133 sectioned at a thickness of 4 μm, and then stained with hematoxylin-eosin (HE). After HE staining, 134 the structural remodeling of the pulmonary arteries was observed by microscopy. Pulmonary 135 arteries (external diameters, 25-100 μm) were randomly selected and analyzed using Image-Pro 136 Plus 6.0 (Media Cybernetics, Rockville, MD, USA). The ratios (expressed as percentages) of the 137 pulmonary artery wall area to the total area (WA/TA%) and the wall thickness to the total 138 P r e p r i n t thickness (WT/TT%) were calculated to evaluate pulmonary arterial remodeling as previously 139 described [15]. 140

Ultrastructural examination of pulmonary arteries 141
Tissue samples of approximately 1 × 1 × 3 mm 3 were taken from the left lung near the hilum. The 142 tissues were fixed with 2.5% glutaraldehyde and 1% osmic acid, stained with 1% uranium acetate, 143 dehydrated with acetone, and embedded in epoxy resin. Subsequently, the fixed tissues were 144 sectioned (semithin and ultrathin sections), and the ultrathin sections were examined using an 145 H-7500 transmission electron microscope (Hitachi, Tokyo, Japan). 146

Detection of mitochondrial membrane potential 147
Rhodamine-123 can cross the mitochondrial membrane in living cells, and its fluorescence 148 intensity is linearly correlated with Δψm. PASMCs were incubated with rhodamine-123 (10 mg/L) 149 for 30 min at 37 °C. The fluorescence intensity was detected by laser confocal microscopy 150 (Olympus FV1000, Tokyo, Japan) and analyzed using Image-Pro Plus 6.0. The optical densities of 151 the respective images were measured and expressed as the corrected average optical density. 152

Detection of proliferation 153
PCNA is usually expressed in the DNA synthesis phase of the cell cycle. To detect PCNA 154 expression levels, 10-μm-thick paraffin sections of lung tissues were dehydrated with ethanol, heat 155 induced for antigen retrieval, blocked with normal goat serum, and incubated with anti-PCNA 156 antibody (1:100) overnight at 4 °C. Subsequently, the sections were incubated with goat 157 anti-rabbit IgG conjugated to horseradish peroxidase (1:100). Diaminobenzidine was used as the 158 chromogen, and hematoxylin was used for counterstaining. The sections were then observed under 159 a microscope (ICC50W0859, Leica, Germany), and the percentages of positive cells were 160 assessed in five randomly selected pulmonary arteries (external diameters of 100 μm). 161

Detection of apoptosis 162
The terminal deoxyribonucleotide transferase mediated dUTP nick-labeling (TUNEL) assay 163 (using the in situ Cell Death Detection Kit, POD) was used to detect apoptosis. After tissue 164 sections were dewaxed and rehydrated with xylene and ethanol, they were treated with a 3% 165 hydrogen peroxide solution to block endogenous peroxidase. After cell drilling with Triton-X100, 166 every tissue section was incubated with the TUNEL reaction mixture and transforming agent 167

Statistical analysis 187
All statistical analyses were performed using SPSS 20.0 (IBM, Somers, NY, USA). All results are 188 expressed as mean ± standard deviation (SD) and were tested for normality. Comparisons between 189 two groups were performed using Student's t-tests, and comparisons among multiple groups were 190 performed using one-way analysis of variance with the LSD test (equal variances assumed). A 191 p-value of < 0.05 was considered statistically significant. 192

A2aR reversed the hypoxia-induced opening of MitoKATP in PASMCs 194
To assess whether A2aR reverses the hypoxia-induced opening of MitoKATP in PASMCs, we first groups. As shown in Fig. 1b and c, compared with that in the N group, the rhodamine-123 200 fluorescence intensity in the H group was significantly higher (p < 0.01), which indicated that 201 P r e p r i n t hypoxia promoted the opening of MitoKATP in PASMCs. Interestingly, the rhodamine-123 202 fluorescence intensity ratio was significantly decreased post CGS21680 treatment (p < 0.05). In 203 contrast, when the A2aR gene was knocked down, the fluorescence intensity ratio was further 204 enhanced in the HK group (p < 0.01). Ultimately, these results indicate that A2aR reversed the 205 hypoxia-induced opening of MitoKATP in PASMCs. 206

A2aR alleviated hypoxia-induced hemodynamic changes by blocking MitoKATP 207
To determine the protective effects of A2aR in HPH, A2aR protein expression in lung homogenates 208 was first examined by western blotting. In vivo, as shown in Fig. 2a, A2aR was not expressed in the 209 A2aR -/groups. A2aR expression increased in hypoxia-exposed WT mice than that in normoxic 210 mice (p < 0.05). Moreover, CGS21680 treatment significantly enhanced A2aR expression 211 compared with hypoxia treatment (p < 0.05). Then, RVSP in each group was recorded to reflect 212 the pulmonary arterial pressure after 4 weeks of treatment. As shown in Fig. 2b and d, RVSP was 213 significantly higher in groups exposed to hypoxia (groups H and HK) than in groups exposed to 214 normoxia (groups N and K) (p < 0.01), and RVSP in the HK group was higher than in the H group 215 (p < 0.05). The hypoxia-induced increase in RVSP was inhibited by CGS21680 treatment (p < 216 0.05). These results indicate that the hypoxia-induced increase in RVSP is alleviated by an 217 increase A2aR and exacerbated by its decrease. The hypoxia-induced increase in RVSP was 218 reduced by treatment with 5HD (p < 0.05), but it was further increased by treatment with 219 diazoxide in both WT and A2aR -/groups (p < 0.05). Accordingly, hypoxia-induced increases in 220 RVSP are alleviated by blocking MitoKATP and exacerbated by promoting its opening. 221 Furthermore, RVSP in the HK5HD group was significantly higher than in the H5HD group (p < 222 0.01), and it was higher in the HKDia group than that in the HDia group (p < 0.05). Combined 223 with previous research that found that A2aR could reverse the hypoxia-induced opening of 224 MitoKATP in PASMCs, these results indicate that A2aR alleviated hypoxia-induced hemodynamic 225 changes partially by blocking MitoKATP. However, as shown in Fig. 2c and e, there were no 226 significant differences in mCAP among these nine groups. 227

A2aR alleviated hypoxia-induced RV hypertrophy and pulmonary arterial remodeling by 228 blocking MitoKATP 229
To evaluate the degree of RV hypertrophy, we measured the RV/(LV+S) and RV/BW ratios of 230 mice after hypoxia exposure (4 weeks, 8hours per day). As shown in Fig. 3a and b, both these 231 ratios were significantly higher in WT and A2aR -/mice exposed to hypoxia than in those exposed 232 to normoxia (p < 0.01). Treatment with CGS21680 reduced the RV/(LV+S) (p < 0.05) and 233 RV/BW ratios (p < 0.05); the ratios in the HK group were higher than those in the H group (p < 234 0.05). These results demonstrate that hypoxia-induced RV hypertrophy was alleviated by an 235 increase in A2aR and exacerbated by its decrease. Repeated administration of 5HD also reduced 236 the RV/(LV+S) (p < 0.05) and RV/BW ratios (p < 0.05) in WT mice while repeated 237 administration of diazoxide led to the opposite effects (p < 0.05). These results show that 238 hypoxia-induced RV hypertrophy is alleviated by blocking MitoKATP and aggravated by 239 promoting its opening. Furthermore, the RV/(LV+S) (p < 0.05) and RV/BW (p < 0.01) ratios were 240 significantly higher in the HK5HD group than in the H5HD group, and they were also higher in 241 the HKDia group than in the HDia group (p < 0.05). These results indicate that A2aR alleviated 242 hypoxia-induced RV hypertrophy partly by blocking the opening of MitoKATP. 243 To investigate pulmonary arterial remodeling, we estimated the pulmonary artery wall area 244 P r e p r i n t relative to the total area (WA/TA%) and the wall thickness relative to total thickness (WT/TT%) 245 by HE staining. As shown in Fig. 3c-e, exposure to hypoxia for 4 weeks caused a significant 246 increase in the wall area and thickness of the pulmonary artery in WT and A2aR -/mice compared 247 with normoxia exposure (p < 0.01). As expected, CGS21680 exerted the strongest inhibitory effect 248 and dramatically decreased WA/TA and WT/TT ratios (p < 0.05). These results indicate that an 249 increase in A2aR alleviates hypoxia-induced pulmonary arterial remodeling. Moreover, the 250 hypoxia-induced increase in WA/TA and WT/TT ratios was aggravated by diazoxide (p < 0.05), 251 which was reversed by treatment with 5HD in both WT and A2aR -/groups. These findings suggest 252 that hypoxia-induced pulmonary arterial remodeling is alleviated by blocking the opening of 253 MitoKATP and aggravated by promotion of its opening. Additionally, WA/TA and WT/TT 254 percentages were higher in the HK5HD (p < 0.05) and HKDia (p < 0.05) groups than in the H5HD 255 and HDia groups. These results demonstrate that A2aR alleviates hypoxia-induced pulmonary 256 arterial remodeling partially through blocking the opening of MitoKATP. 257

A2aR alleviated hypoxia-induced excessive proliferation in PASMCs by blocking MitoKATP 258
To evaluate the proliferation of PASMCs in vivo, proliferating cell nuclear antigen (PCNA) 259 expression was measured by immunohistochemistry. As shown in Fig. 4a and c, PCNA expression 260 was significantly higher in the hypoxia groups than in the control groups (p < 0.01). CGS21680 261 treatment significantly decreased the expression of PCNA in the HCGS group compared with that 262 in the H group (p < 0.01). Meanwhile, the expression of PCNA in the HK group was higher than 263 in the H group (p < 0.05). The groups treated with 5HD had observably reduced percentages of 264 positive cells compared with that in the hypoxia groups (p < 0.01). In contrast, repeated 265 administration of diazoxide further increased the percentage of PCNA-positive cells (p < 0.05). 266 These results suggest hypoxia-induced excessive proliferation of PASMCs is alleviated by 267 blocking MitoKATP and promoting the opening. Additionally, the percentage of PCNA-positive 268 cells was significantly higher in the HK5HD group than in the H5HD group (p < 0.01), and it was 269 also higher in the HKDia group than in the HDia group (p < 0.05). 270

A2aR decreased hypoxia-induced apoptosis resistance in PASMCs by blocking MitoKATP 271
We further investigated the effects of A2aR on cell apoptosis. In vivo, as shown in Fig. 4b and d,  272 the percentage of apoptotic cells was significantly lower in the hypoxia groups than in the control 273 groups (p < 0.01). However, the decreased apoptosis index was significantly increased by Bcl-2, and calculated the ratio of Bax to Bcl-2 expression. As shown in Fig. 5a, Bax expression  282 was significantly down-regulated in the lung tissue homogenate of the hypoxia groups compared 283 with that in the control groups (p < 0.01). However, treatment with CGS21680 reversed this effect 284 (p < 0.05) (Fig. 5a). Additionally, the expression of Bax in the HK group was lower than in the H 285 group (p < 0.05) (Fig. 5b). These results indicate that an increase in A2aR up-regulates the 286 expression of Bax. As shown in Fig. 5b, decreases in Bax expression induced by hypoxia were 287 reversed by treatment with 5HD in the WT group (p < 0.05). Compared with expression levels in 288 the hypoxia groups, diazoxide further down-regulated the expression of Bax (p < 0.05) (Fig. 5c). 289 According to these results, the expression of Bax is up-regulated by blocking MitoKATP and 290 down-regulated by promoting its opening. As shown in Fig. 5b, the effects of 5HD up-regulation 291 of Bax was reversed in the A2aR -/group, and the expression of Bax in the HK5HD group was 292 lower than in the H5HD group (p < 0.05). 293 The expression levels of Bcl-2 were opposite to those of Bax in each group (Fig. 5d-f). We also 294 computed the ratio of Bax to Bcl-2 expression and found that the ratio was the same as Bax. As 295 shown in Fig. 5g, the ratio was significantly down-regulated in the hypoxia groups compared with 296 that in the control groups (p < 0.01) and treatment with CGS21680 reversed this effect (p < 0.01) . 297 Additionally, the ratio in the HK group was lower than in the H group (p < 0.05) (Fig. 5h-i). As 298 shown in Fig. 5h, decreases in the ratio of Bax to Bcl-2 expression induced by hypoxia were 299 reversed by treatment with 5HD in the WT group (p < 0.01), and the effects of 5HD were reversed 300 in the A2aR -/group, and the ratio in the HK5HD group was lower than that in the H5HD group (p 301 < 0.01). Compared with expression levels in the hypoxia groups, diazoxide further down-regulated 302 the ratio (p < 0.01) (Fig. 5i). 303

A2aR modulated cell apoptosis via the mitochondrial-dependent apoptosis pathway 304
To investigate whether A2aR modulates cell apoptosis via the mitochondrial-dependent apoptosis 305 pathway, electron microscopy was used to observe the ultrastructure of mitochondria. In vivo, as 306 shown in Fig. 6a, in the N and K groups, the mitochondrial bilayer membrane structure was 307 complete, and the cristae of the mitochondria were compact. Compared with the N and K groups, 308 abnormal mitochondria membrane swelling was observed in the H and HK groups (i.e., the 309 structure of mitochondrial cristae was disordered, vacuoles appeared in few mitochondria, and 310 some were broken). However, after treatment with CGS21680, the mitochondrial cristae became 311 dense, the membrane was intact, and the structure tended to be normal. The destructive effects of 312 hypoxia were reversed with 5HD treatment but aggravated by diazoxide treatment. The 313 mitochondrial damage was more severe in the HK5HD and HKDia groups than in the H5HD and 314 HDia groups. In groups HK5HD and HKDia, mitochondrial swelling was obvious, most of the 315 cristae were broken and appeared blurry, and some mitochondria exhibited pyknosis. 316 To further clarify the relationship between A2aR and MitoKATP in the mitochondrial-dependent 317 apoptotic pathway, the expression levels of Cyt C and Caspase-9 were detected by western 318 blotting. As shown in Fig. 6b and d, the release of Cyt C from the mitochondria to the cytoplasm 319 was significantly inhibited in the hypoxia groups compared with that in the normoxia groups (p < 320 0.01), which was reversed after treatment with CGS21680(P<0.01). Meanwhile, inhibition of the 321 pathway was reversed by treatment with 5HD in both the WT and A2aR -/groups (p < 0.01) (Fig.  322 6f-g). Additionally, the mice treated with diazoxide exhibited (HDia) further reductions in the 323 release of Cyt C from the mitochondria to the cytoplasm than those exposed to hypoxia(H) (p < 324 0.05) (Fig. 6h-j). These results suggest that the mitochondrial-dependent apoptotic pathway is 325 activated by blocking MitoKATP and inhibited by promoting its opening. In addition, the effects of 326 5HD up-regulation of the release of Cyt C from the mitochondria to the cytoplasm was reversed in 327 A2aR -/mice, and there was a significant difference in the release of Cyt C from the mitochondria 328 to the cytoplasm between the H5HD and HK5HD groups (p < 0.05) (Fig. 6f-g); a similarly 329 significant difference between the HDia and HKDia groups was observed (p < 0.05) (Fig. 6h-j). 330 Thus, these results indicate that A2aR activates the mitochondrial-dependent apoptotic pathway 331 partially by blocking the opening of MitoKATP. 332 As shown in Fig. 6k, the expression of Caspase-9 was decreased in the lung tissues of 333 hypoxia-exposed mice (p < 0.01), which was ameliorated by CGS21680 treatment (p < 0.05). 334 Repeated administration of 5HD reversed chronic hypoxia-induced down-regulation of Caspase-9 335 expression (p < 0.01) (Fig. 6l), and the expression of Caspase-9 was lower in the HK5HD group 336 than in the H5HD group (p < 0.05), indicating that A2aR activates the mitochondrial-dependent 337 apoptotic pathway by blocking MitoKATP. The decrease in Caspase-9 expression induced by 338 hypoxia was further inhibited by diazoxide treatment (p < 0.01) (Fig. 6m). 339

Discussion 340
In this study, we verified that A2aR and MitoKATP both play an important role in the treatment of 341 hypoxic pulmonary arterial hypertension, and that A2aR can improve HPH via mitochondrial 342 ATP-sensitive potassium channels. 343 The vasoconstriction and pulmonary vascular remodeling is the primary pathophysiological 344 features of PH [21]. Humbert et al. found that excessive vasoconstriction is associated with the 345 abnormal expression of potassium channels and dysfunction of endothelial cells [22]. In addition, 346 the primary factor contributing to pulmonary vascular remodeling is the imbalance between 347 proliferation and apoptosis [23]. The purpose of this study was to explore whether MitoKATP is 348 involved in the mechanism underlying improvement of PH and whether it mediates the function of 349

A2aR in regulating PASMC apoptosis. 350
Adenosine is an endogenous purine nucleoside that regulates a series of physiological and 351 pathological processes by binding to adenosine receptors [24]. Recently, the relationship between 352 adenosine and PH has been a focus of research. In other types of PH models, A2aR has been 353 confirmed to play an important role in the pathogenesis of PH. Alencar et al. reported that an 354 A2aR agonist can reverse pulmonary vascular remodeling and endothelial dysfunction in rats with 355 monocrotaline-induced PH [25]. Shang et al. established the A2aR knockout mice model and found 356 that A2aR -/mice are more likely to exhibit the pathological features of PH [14,26]. Like previous 357 studies, A2aR showed the same effects in an HPH animal model. Consistent with the results of this 358 study, these studies all indicate that A2aR plays an important role in the treatment of HPH. 359 An imbalance between PASMC proliferation and apoptosis is a primary cause of pulmonary 360 vascular remodeling. It has been found that, in HPH, some drugs can regulate cell apoptosis 361 through A2aR [27]. Youle et al. reported that members of the Bcl-2 protein family play important 362 roles in regulating apoptosis: Bax promotes the opening of the mitochondrial permeability 363 transition pore to induce apoptosis and Bcl-2 inhibits its opening and prevents apoptosis [28]. In 364 this study, we found that the Bax/Bcl-2 ratio in the lung homogenates with CGS21680 treatment 365 was significantly increased than that of the hypoxia group, objectively proving that the increase in 366 A2aR expression promotes PASMC apoptosis in the treatment of HPH. Although the Bax/Bcl-2 367 ratio was significantly decreased in A2aR -/mice, a certain level was still maintained. These 368 findings indicate that A2aR partially influences PASMC apoptosis induced by hypoxia. 369

Dromparis et al. have reported that the mitochondrial-dependent apoptotic pathway plays an 370
important role in the occurrence and development of HPH [29]; our study also supports this view. 371 MitoKATP is an important determinant of the mitochondrial membrane potential, which is sensitive 372 to hypoxia. According to a previous study, hypoxia or diazoxide can lead to the depolarization of 373 the mitochondrial membrane in PASMCs, thereby ultimately reducing apoptosis in these cells. The 374 results of this study support this conclusion [18]. Moreover, we found that 5HD ameliorates the 375 effects of hypoxia. The RVSP and RV/(LV+S), RV/BW, WA/TA, and WT/TT ratios in 376 hypoxia-exposed mice were significantly reduced by repeated 5HD treatment, and the 377 ultrastructure of mitochondria in the 5HD group was also relatively intact. Compared with the 378 hypoxia control group, the percentage of PCNA-positive cells in 5HD-treated mice was 379 significantly lower, and the apoptotic index of PASMCs was significantly higher. These findings 380 firmly prove that hypoxia-induced excessive proliferation and apoptosis resistance of PASMCs 381 were inhibited by blocking MitoKATP exacerbated by promoting its opening, which contributes to 382 alleviation of HPH. 383 Our previous study found that A2aR could alleviate HPH via mitochondrial-dependent apoptotic 384 pathway [27], we further explored whether there is an interaction between A2aR and MitoKATP. In 385 our study, the ability of 5HD to improve pathological changes of HPH was significantly inhibited 386 in A2aR -/mice than that in WT mice. The RVSP, RV/(LV+S), RV/BW, WA/TA, and WT/TT values 387 in the A2aR -/group treated with 5HD were significantly higher than those in the WT group. 388 Coincidentally, the initial components of the mitochondrial-dependent apoptotic pathway are 389 apoptosis-regulating proteins of the Bcl-2 protein family. Within this family, Bax is a 390 pro-apoptotic protein and Bcl-2 is an anti-apoptotic protein, and down-regulation of Bax/Bcl-2 in 391 the mitochondrial apoptotic pathway inhibits the release of Cyt C and Caspase-9. Bax promotes 392 the release of mitochondrial Cyt C into the cytoplasm and then activates Caspase-9 to produce a 393 cascade reaction [28]. To explore whether A2aR-induced blocking of MitoKATP could activate the 394 mitochondrial-dependent apoptotic pathway, we detected the expression of Bax, Bcl-2, Cyt C, and 395 Caspase 9. We found that deletion of A2aR diminished the positive effect of the MitoKATP blocker 396 on the mitochondrial apoptotic pathway. This indicates that A2aR up-regulates the expression of 397 Bax partially by blocking MitoKATP and that A2aR thus prevents HPH via blocking MitoKATP. 398 Endogenous nitric oxide (NO) has been reported to promote mitokATP channel opening [30], and 399 its inhibitor can block the cardioprotective effect induced by mitokATP channel inhibitor 400 diazoxide [31]. A recent study found that A2a receptor agonist could suppress the expression of 401 NO synthase protein, thus inhibited the release of NO [32]. In conclusion, A2aR may block the 402 MitoKATP channels by decreasing the expression of NO. But the certain mechanisms between the 403 A2aR and MitoKATP channel remains to be further verified. 404 In this study, we established a hypoxic animal model and verified that A2aR and MitoKATP both 405 play an important role in the treatment of HPH, and we demonstrated an interaction between the 406 two. A2aR can activate the mitochondrial-dependent apoptotic pathway and inhibit PASMC 407 proliferation partially by blocking the opening of MitoKATP, thereby alleviating pulmonary 408 vascular structural remodeling and attenuating HPH. The specific mechanism between A2aR and 409 MitoKATP needs to be further investigated. 410

Conclusions 411
This study showed that an increase in A2aR and blocking of MitoKATP alleviate HPH. Furthermore, 412 A2aR induced the mitochondrial-dependent apoptosis pathway and inhibited PASMC proliferation

Compliance with ethics guidelines 430
All institutional and national guidelines for the care and use of laboratory animals were followed. 431 All experimental protocols conformed to the Guide for the Care and Use of Laboratory Animals, 432 which was published by the US National Institutes of Health, and were approved by the Animal 433 Ethics Committee of Wenzhou Medical University. 434

Competing interests 435
The authors have declared that no competing interest exists. 436

Availability of data and material 437
All relevant data and materials are stored in the Key Laboratory of Heart and Lung of Wenzhou 438 Medical University and can be obtained from the first author and corresponding author.

P r e p r i n t
Effects of CGS21680 (+CGS, 0.2 mg/kg/day), 5HD (+5HD, 10 mg/kg/day), and Dia (+Dia, 7 mg/kg/day) on PH at the ultrastructure level. Ultrathin sections of lung tissues from WT and A2aR -/mice were observed by a Hitachi H-7500 transmission electron microscopy (a, ×20000), the white arrow indicates the mitochondria. The expression levels of cytochrome C in mitochondrial and cytosol pellets and Caspase-9 in lung tissue were examined by western blotting with antibodies against cytochrome C with COX IV as a mitochondria marker and GAPDH as the internal control. Effects of CGS21680 (+CGS, 0.2 mg/kg/day) on cytochrome C in mitochondrial and cytosol pellets and Caspase-9 expression in lung homogenate of WT mice (b, c, k; n = 3).

P r e p r i n t
Powered by TCPDF (www.tcpdf.org) P r e p r i n t Fig. 4 A2aR alleviated hypoxia-induced excessive proliferation and apoptosis resistance in PASMCs via MitoKATP Representative photomicrographs of PCNA (brown) expression in the pulmonary arteries of WT and A2aR-/-groups (a, ×400), the black arrow indicates the PCNA in the pulmonary arteries. Representative photomicrographs of apoptotic cells (brown) in the pulmonary arteries of WT and A2aR-/-groups (b, ×400), the black arrow indicates the apoptotic cells in the pulmonary arteries. PCNA expression levels in PASMCs were examined by immunohistochemistry, and apoptosis levels of PASMCs were examined by TUNEL assay. Effects of CGS21680 (+CGS, 0.2 mg/kg/day), 5HD (+5HD, 10 mg/kg/day), and Dia (+Dia, 7 mg /kg/day) on PCNA expression in PASMCs (c; n = 5), and the apoptosis of PASMCs (d; n = 5) in WT and A2aR-/-mice. Data are presented as the mean ± standard deviation (SD). # p < 0.05, ## p < 0.01 vs normoxic group; * p < 0.05, ** p < 0.01 vs hypoxic group; + p < 0.05, ++ p < 0.01 between A2aR-/-and WT mice groups. A2aR: A2a receptors; MitoKATP: mitochondrial ATPsensitive potassium channels; PCNA, proliferating cell nuclear antigen; WT, wild-type; PASMCs, pulmonary artery smooth muscle cells; 5HD, 5-hydroxydecanoic Fig. 6 A2aR modulated cell apoptosis via the mitochondrial-dependent apoptosis pathway Effects of CGS21680 (+CGS, 0.2 mg/kg/day), 5HD (+5HD, 10 mg/kg/day), and Dia (+Dia, 7 mg/kg/day) on PH at the ultrastructure level. Ultrathin sections of lung tissues from WT and A2aR-/-mice were observed by a Hitachi H-7500 transmission electron microscopy (a, ×20000), the white arrow indicates the mitochondria. The expression levels of cytochrome C in mitochondrial and cytosol pellets and Caspase-9 in lung tissue were examined by western blotting with antibodies against cytochrome C with COX IV as a mitochondria marker and GAPDH as the internal control. Effects of CGS21680 (+CGS, 0.2 mg/kg/day) on cytochrome C in mitochondrial and cytosol pellets and Caspase-9 expression in lung P r e p r i n t homogenate of WT mice (b, c, k; n = 3). Effects of 5HD (+5HD, 10 mg/kg/day) on cytochrome C in mitochondrial and cytosol pellets and Caspase-9 expression in lung homogenate of WT mice (e, f, l; n = 3). Effects of Dia (+Dia, 7 mg/kg/day) on cytochrome C in mitochondrial and cytosol pellets and Caspase-9 expression in lung homogenate of WT mice (h, i, m; n = 3). Protein expression ratios of cytochrome C in mitochondrial and cytosol pellets were also calculated (d, g, j; n = 3). Data are presented as the mean ± standard deviation (SD). # p < 0.05, ## p < 0.01 vs normoxic group; * p < 0.05, ** p < 0.01 vs hypoxic group; + p < 0.05, ++ p < 0.01 between A2aR-/-and WT mice groups. A2aR: A2a receptors; 5HD, 5-hydroxydecanoic acid sodium salt; Dia, diazoxide; PH, pulmonary hypertension; WT, wild-type.
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