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Adenosine A2A receptor activation prevents DOCA-salt induced hypertensive cardiac remodeling via iBAT
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Adenosine A2A receptor activation prevents DOCA-salt induced hypertensive cardiac remodeling via iBAT Yan-Ping Zhou, Cheng-Chao Ruan, Ling-Ran Kong*, Ping-Jin Gao State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Hypertension, Shanghai Institute of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 January 2020 Accepted 6 February 2020 Available online xxx
Hypertensive cardiac remodeling is a constellation of abnormalities that includes cardiomyocyte hypertrophy and death and tissue fibrosis. Adenosine is a long-known vasodilator, through interacting with its four cell surface receptor subtypes in cardiovascular system. However, it is unclear that whether adenosine A2A receptor (A2AR) activation is involved in the cardiac remodeling in hypertension. WT mice were utilized to induce DOCA-salt sensitive hypertension and received A2AR agonist CGS21680 or antagonist KW6002 treatment. Cardiac functional phenotyping measurement by echocardiography showed that CGS21680 improved cardiac dysfunction in DOCA-salt mice. Moreover, CGS21680 reduced cardiomyocyte hypertrophy, cardiac inflammation and fibrosis. However, iBAT depletion surgery induces dramatic cardiac remodeling in DOCA-salt mice, and the protective function of CGS21680 was blocked without intact iBAT. Mechanistically, A2AR agonist CGS21680 increased iBAT-derived fibroblast growth factor 21 (FGF21). Our data suggest that activation of A2AR could be a potential therapeutic strategy in preventing heart damage in hypertension. © 2020 Elsevier Inc. All rights reserved.
Keywords: Hypertension Cardiac remodeling Adenosine A2AR Brown adipose tissue
1. Introduction Hypertension is a major clinical condition that induces cardiac remodeling, through acting as a contributing factor in both systolic and diastolic dysfunction, arrhythmia and symptomatic heart failure [1]. Long-term cardiac load or injury results in cardiac remodeling, defined as left ventricular hypertrophy and interstitial fibrosis. Therefore, it is necessary to develop effective approach to intervene the process of cardiac remodeling caused by hypertension. Adenosine is a ubiquitous endogenous molecule, which can be released from intracellular pools by membrane transporters or directly produced extracellularly following ATP catabolism by the concerted action of the ecto-enzymes [2,3]. The adenosine signal can be transmitted by its four G protein coupled receptors: adenosine A1 and A3 receptors through Gi or A2A and A2B receptors via Gs. Adenosine A2AR subtype is high sensitive to adenosine and participate in cardiovascular and metabolic regulation because it is widely expressed both in cardiovascular system and metabolismregulating organs [4,5]. Animal studies showed that A2AR
* Corresponding author. E-mail address: [email protected] (L.-R. Kong).
knockout rats are sensitive to the prohypertensive effect in highsalt diet [6]. Furthermore, A2AR agonist has been reported to ameliorate cardiac remodeling following myocardial infarction in spontaneously hypertensive rats [7,8]. However, experiments in vitro showed that pharmacological stimulation of A2AR did not directly affect cardiac fibroblast function [9]. In addition, recent study showed that A2AR activation promotes lipolysis, thermogenesis in adipose tissue, which protected against high-fat diet induced obesity [10]. Therefore, we hypothesized that the protective effects of A2AR in hypertensive cardiac remodeling may through regulating adipose tissue activity. Brown adipose tissue (BAT) is well recognized for its nonshivering thermogenesis mediated by uncoupling protein 1 (UCP1), mainly distributed in interscapular, axillary and cervical regions in rodent [11]. Recent data suggested that the beneficial effects of BAT could involve a previously unrecognized endocrine role through the release of endocrine factors [12]. The BAT hormones are named “batokines including Thriiodothyronine (T3), Fibroblast growth factor-21 (FGF21), Interleukin-6 (IL-6) and so on [13]. Study demonstrated that mice with BAT ablation express an elevation of blood pressure, LVH and increased interstitial tissue in addition to the development of obesity and insulin resistance [14]. However, the precise mechanism underlying the interaction
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Please cite this article as: Y.-P. Zhou et al., Adenosine A2A receptor activation prevents DOCA-salt induced hypertensive cardiac remodeling via iBAT, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.035
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Abbreviations A2AR BAT iBAT DOCA Col1a1 TGFb aSMA UCP1 PPARg PRDM16 Cidea FGF21
Adenosine A2A receptor brown adipose tissue interscapular brown adipose tissue deoxycorticosterone acetate collagen type 1 alpha chain transforming growth factor b a smooth muscle actin uncoupling protein 1 peroxisome proliferator-activated receptor g PR domain containing 16 cell death inducing DNA fragmentation factoralpha-like A fibroblast growth factor 21
between BAT and cardiac remodeling is rarely investigated and remains elusive. In this study, we treated mice with a specific A2AR agonist, CGS21680, to determine whether A2AR activation could prevent hypertension-induced cardiac damage. Our results showed that administration of CGS21680 effectively suppressed cardiac fibrosis and improved cardiac function in deoxycorticosterone acetate (DOCA)-salt hypertensive mice. 2. Methods 2.1. Mice and treatment C57BJ/6J wild type mice aged 8e12 weeks were randomly assigned to SHAM group or DOCA-salt group which underwent uninephrectomy and DOCA pellet (50mg/pellet) subcutaneously together with 1% NaCl in the drinking water for 2 weeks [15]. For pharmacological investigation, DOCA-salt mice received intraperitoneally injection of A2AR agonist CGS21680 (1 mg/kg/day), A2AR antagonist KW6002 (1 mg/kg/day) or PBS. IBAT removal surgery was performed as previously described [16]. Mice belonging to SHAM group were administrated the same procedure, the kidney or iBAT pad was located, exposed, and then been closed. 2.2. Echocardiography Transthoracic echocardiography was performed with a Vevo 2100 instrument (Fuji Film Visual Sonics) equipped with an MS-400 imaging transducer (18e38 MHz) as previously described [17]. Briefly, mice were preconditioned by chest hair removal, exposed to 1%e2% isoflurane to be anaesthetized, and maintained in a supine position on handing platform with limbs attached for electrocardiogram gating during imaging. Body temperature was kept constant by a heating pad, in the meantime, heart and respiratory rates were continuously monitored. M-mode recording was performed at the midventricular level. All images were analyzed using dedicated software (Vero 2100 version 1.4). Percent LV ejection fraction (EF) and fractional shortening (FS) was calculated from M-mode measurements. All procedures were performed under double-blind conditions with regard to treatment. 2.3. In vivo iBAT imaging The iBAT imaging was performed as previously described [18]. Briefly, mice were intravenously injected with micellar SRFluor680 (0.35 mg in 100 mL PBS), after 6e8 h, mice were anaesthetized with
1%e2% isoflurane with oxygen flow rate of 2 L/min and denuded by depilatory cream to reduce scattering and absorption by the hair. Fluorescence imaging was obtained from IVIS Dpectrum/CT system (PerkinElmer) following the manufacture introductions. The fluorescence acquisition parameters were: Excitation Filter: 615e665 nm, Excitation Filter: 695e770 nm, Acquisition time: 10 s, Subject height: 1.5 cm, Field od view: A (2 cm*2 cm). Then the CT imaging and 3D Reconstruction was obtained through X-Rays Acquire mode. Data analysis and the photon flux data were obtained by using Region of Interest (ROI) tools. 2.4. Histological analysis Heart and iBAT tissues were fixed in 4% formaldehyde for 48 h, dehydrated, embedded in paraffin and sectioned at 5 mm thickness. Then sections were stained with hematoxylin and eosin (HE), Masson’s trichrome to evaluate the degree of fibrosis or wheat germ agglutinin (WGA). Images were captured by a Carl Zeiss Axio Imager M2 microscope (Carl Zeiss Corporation, Germany) by software Imager-Pro Plus (Media Cybernetics, United States). For immunohistochemical staining, heart sections were incubated with primary antibodies for Col1a1 (1:100) or a-SMA (1:1000), iBAT section were incubated with primary antibody for UCP1 (1:100). 2.5. Quantitative RT-PCR analysis Total RNA was extracted from heart and iBAT samples using TRIzol reagent (Sigma-Aldrich, TS424), according to the manufacturer’s specifications. Then, total RNA was reverse-transcribed to cDNA by using Reverse Transcription Master Mix (A0010GQ, EZBioscience). Real-time qRT-PCR was performed with SYBR Premix Ex Taq kits with ROX (Takara, RR420B) according to manufacturer’s instruction. Reactions were run on an ABI PRISM 7900 machine (Applied Biosystems). The housekeeping gene b-Actin was used as internal control: the expression of the genes studied was expressed as a ratio to that b-Actin. The primers used are listed as followed: Col1a1, forward 50 -GAGCGGAGAGTACTGGATCG-30 and reverse 50 -TACTCGAACGGGAATCCATC-3’; TGFb, forward 50 -CAACAATTCCTGG CGTTACCTTGG-30 and reverse 50 -GAAAGCCCTGTATTCCGTCTCCTT-3’; aSMA, forward 50 -CTGACAGAGGCACCATGAA-30 and reverse 50 AGAGGCATAGAGGGACAGCA-3’; UCP1, forward 50 -AGGCTTCCAGTACCATTAGGT-30 and reverse 50 -CTGAGTGAGGCAAAGCTGATTT-3’; PPARg, forward 50 -TTAGATGACAGTGACTTGGC-30 and reserve 50 TCTTCTGGAGCACCTTGG-3’; PRDM16, forward 50 -CAGCACGGTGAAGCCATTC-30 and reverse 50 - GCGTGCATCCGCTTGTG-3’; CIDEA, forward 50 -TGCTCTTCTGTATCGCCCAGT-30 and reverse 50 GCCGTGTTAAGGAATCTGCTG-3’; FGF21, forward 50 -CAGGGAGGATGGAACAGT-30 and reverse 50 -TTTGGGGAGTCCTTCTG-3’; b-Actin, forward 50 - CGTGGGCCGCCCTAGGCACCA-30 and reverse 50 TTGGCCTTAGGGTTCAGGGGGG-3’. 2.6. Western blot analysis The frozen heart, iBAT and liver tissues were lysed in Radio Immunoprecipitation Assay (RIPA) buffer (Merck Millipore, 20e188) containing 1% protease inhibitor cocktail (Biotool, B14002). Protein was run on a 12% SDS page gel and blotted onto PVDF membrane (Millipore) by wet transfer, blots were blocked and blotted according to the antibody manufacturers’ recommendations. The primary antibodies were as follows: anti-Col1a1 (1:1000 dilution) (Santa Cruz, SC-8787-R), anti-TGFb (1:1000 dilution) (CST, 3711S), anti-aSMA (1:1000 dilution) (Sigma, A5228), anti-UCP1 (1:1000 dilution) (Abcam, ab23841), anti-PPARg (1:1000 dilution) (Santa Cruz, sc-7273), anti-PRDM16 (1:1000 dilution) (Abcam, ab106410), anti-CIDEA (1:1000 dilution) (Abcam, ab8402),
Please cite this article as: Y.-P. Zhou et al., Adenosine A2A receptor activation prevents DOCA-salt induced hypertensive cardiac remodeling via iBAT, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.035
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anti-FGF21 (1:1000 dilution) (Abcam, ab171941) and GAPDH monoclonal antibody (1:4000) (proteintech, HRP-60004). Results were quantified by using imaging software Quantity One (Bio-Rad Laboratory, Spain). 2.7. FGF21 measurement Serum was obtained from blood collected from mice. FGF21 levels in blood was determined by using Mouse FGF21 ELISA-Kit (R&D, MF2100) according to the manufacturer’s instruction. 2.8. Statistical analysis All values are presented as means ± SEM. Statistical analysis was performed using one-tailed or two-tailed Student’s t-test. For experiments in which more than two groups were compared, twoway analysis of variant (ANOVA) was used and followed by the post hoc Dunnett’s test for data with more than two groups (Levene’s tests for equalvariance). Dunnett’sT3 test was used for post hoc test comparison for the analysis of unequal variances (Welch’s and BrowneForsythe’s test). The significance level was set at P < 0.05 in all statistics. 3. Results
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3.3. IBAT depletion blockade the cardioprotective effects of CGS21680 To provide more convincing proof that the endocrine of BAT is an indispensable channel for A2AR to improve hypertensive cardiac remolding, mice were administrated iBAT removal surgery (iBAT()) in PBS-treated and CGS21680-treated group. Interestingly, iBAT depletion exacerbated the DOCA-salt-induced cardiac remolding demonstrated by decreased EF and FS between PBSiBAT() group and PBS-iBAT(þ) group. Furtherly, iBAT depletion blocked the protection of CGS21680 in DOCA-salt mice. (Fig. 3A and B). WGA and Masson staining also demonstrated that iBAT() increased the cardiomyocyte size and cardiac interstitial fibrosis (Fig. 3C and D), as well as the increasing expression of Col1a1 and aSMA (Fig. 3E and E). More importantly, there was no statistically significant difference in the WGA and Col1a1 and aSMA staining analysis when compared PBS-iBAT() with CGS21680-iBAT() (Fig. 3D and F). These studies validated the iBAT depletion blockaded the effect of CGS21680 to mitigate DOCA-salt-induced cardiac remodeling. 3.4. A2AR activation increases BAT-derived FGF21 in hypertension
In order to explore the role of A2AR in hypertensive cardiac remodeling, Wild type (WT) DOCA-salt mice were treated with A2AR agonist CGS21680 (1 mg/kg/day) or its antagonist KW6002 (1 mg/kg/day). DOCA-salt treatment induced a reduced ejection fraction (EF) and fractional shortening (FS) in WT mice, but CGS21680 protected against impaired heart function compared with KW6002 group (Fig. 1A and B). Histological analysis revealed that DOCA-salt induced cardiac remodeling was attenuated in CGS21680 group. A2AR activation resulted in decreased cardiomyocyte size and cardiac interstitial fibrosis (Fig. 1C and D), as well as reduced positive staining for Col1a1 and aSMA (Fig. 1E and Fig.S1A). These were further demonstrated by detecting fibrotic makers (Col1a1, TGFb and aSMA) in mRNA and protein levels (Fig. 1F, Fig.S1B and Fig.S1C). These data indicated that A2AR activation ameliorated hypertension-induced cardiac remodeling.
Fibroblast growth factor 21 (FGF21), which produced predominantly in liver [19], is a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity [20]. Previous study shows that FGF21 exert a protective action against both antihypertrophic and cardioprotective actions on the heart [21]. And thermogenic activation induces FGF21 gene expression in BAT and triggers the release of FGF21 by BAT [22,23]. In order to explain why BAT thermogenic activity by A2AR activation could alleviate hypertensive cardiac remodeling partly, we examined the protein levels of FGF21 level in serum by ELIAS kit firstly. In DOCA-salt treated mice, FGF21 increased in serum in the CGS21680-treated group (Fig. 4A). Next, we surprisingly found the mRNA expression level and protein level of FGF21 in the iBAT were significantly upregulated due to the activation of A2AR, whereas KW6002 inhibited the expression of FGF21 (Fig. 4B, C and D). As for the expression of FGF21 in heart and liver, there were no significant changes after the stimulation or blockade of A2AR (Fig. 4B). Taken together, these results indicated that A2AR-mediated iBAT thermogenic activation increased the level of serum FGF21, which could act on the target of the heart through blood transport.
3.2. A2AR regulates BAT function in hypertension
4. Discussion
BAT has been always considered a site of metabolic energy consumption to produce heat, however, it has been showed to be as a secretory organ recently [12]. To further understand whether A2AR could activate BAT, we detected BAT activation in DOCA-salt mice treated with CGS21680 or KW6002 by a deep-red fluorescent micellar probe, SRFluor680, which has been showed to be accumulated in iBAT regardless mouse species [18]. As expected, fluorescence imaging combined with microCT showed a dramatic increased fluorescent density in the interscapular region in the CGS21680 group compared with PBS or KW6002 group (Fig. 2A and B). IHC analysis suggested that A2AR agonist significantly promoted iBAT activation demonstrated by smaller-sized adipocyte and increased uncoupling protein 1 (UCP1) staining (Fig. 2C and D). Consistently, the increase of mRNA and protein expression of thermogenic genes including UCP1, PPARg, PRDM16, and CIDEA induced by DOCA-salt treatment became still more in the CGS21680 group. However, KW6002 had the opposite effects on BAT in DOCA-salt mice (Fig. 2E, Fig. S2A and Fig. S2B). These results demonstrated that A2AR regulated BAT function in hypertension.
Overall, our studies illuminated that A2AR agonist could alleviate cardiac remodeling and regulate the BAT function in hypertension. IBAT removal surgery in DOCA-salt mice showed that iBAT depletion blocked the protection of CGS21680 in hypertensive cardiac remodeling. In addition, our data showed that CGS21680 increased FGF21 expression in BAT of DOCA-salt mice. Therefore, our study showed that A2AR activation mitigated the damage induced by hypertension mainly depending on the FGF21 secreted by BAT. It is known that A2AR is highly expressed on the surface of brain, immune system and adipose tissue [24,25]. The A2AR expression in different organs will change under the condition of changeable human disease and animal models [25]. Chronic heart failure exhibits a significance of up-regulation of A2AR in heart and peripheral blood mononuclear cells [26]. Cardiac remodeling involves the production of extracellular matrix (ECM) by cardiac fibroblast and inflammatory process, both of which are sensitive to adenosine [27]. Recent evidence has been presented that A2AR participate in the signaling for inhibition of cardiac fibroblast growth and fibrosis, and A2AR stimulation prevents neutrophil accumulation and matrix
3.1. A2AR activation protects against hypertensive cardiac remodeling
Please cite this article as: Y.-P. Zhou et al., Adenosine A2A receptor activation prevents DOCA-salt induced hypertensive cardiac remodeling via iBAT, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.035
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Fig. 1. A2AR agonist alleviates Hypertensive cardiac remodeling. (A) Representative echocardiography images of SHAM and DOCA-salt mice, injected with PBS, CGS21680 or KW6002 for 2 weeks. (B) Quantitative analysis of ejection fraction (EF), fractional shortening (FS) in diastolic phase obtained from echocardiography. (CeE) Representative cross-sections of heart straining for Masson, WGA, Col1a1 and aSMA in SHAM and DOCA-salt mice, injected with PBS, CGS21680 or KW6002 for 2 weeks and quantitative analysis of cardiomyocyte size (WGA staining) and fibrotic area (blue area in Masson staining). (F) Representative immunoblots of Col1a1, TGFb and aSMA protein expression in heart of SHAM and DOCA-salt mice, injected with PBS, CGS21680 or KW6002 for 2 weeks. Data are presented as means ± SEM; n ¼ 5e6/group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2. A2AR regulates BAT function in hypertension. (A) Representative fluorescence imaging combined with micro CT of SHAM and DOCA-salt mice, treated with PBS, CGS21680 or KW6002 after intravenous injection of SRFluor680 for 6 h. (B) Quantitative analysis of normalized mean intensity of fluorescence. (C) Representative of HE and UCP1 staining images of iBAT from SHAM and DOCA-salt mice, treated with PBS, CGS21680 or KW6002 for 2 weeks. (D) Quantitative analysis of UCP1 signal. (E) Representative immunoblots of UCP1, PPARg, PRDM16, Cidea protein expression in iBAT of SHAM and DOCA-salt mice, treated with PBS, CGS21680 or KW6002 for 2 weeks. Data are presented as means ± SEM; n ¼ 5e6/group.
metalloproteinases (MMPs) secretion to degrade the ECM [28]. In our study, we demonstrated that A2AR agonist improved the cardiac function and decreased the interstitial fibrosis caused by hypertension. And the fibrosis-related genes expression in mRNA and
protein level on heart decreases because of the A2AR stimulation. In the last decade, data generated in both rodents and humans reveal the therapeutic potential of BAT in cardiovascular disease [13]. BAT transplantation improved cardiomyocyte injury in mice
Please cite this article as: Y.-P. Zhou et al., Adenosine A2A receptor activation prevents DOCA-salt induced hypertensive cardiac remodeling via iBAT, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.035
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Fig. 3. iBAT depletion blockade the cardioprotective effects of CGS21680. (A) Representative echocardiography images of PBS-treated or CGS21680-treated DOCA-salt mice with or without iBAT. (B) Quantitative analysis of ejection fraction (EF), fractional shortening (FS) in diastolic phase obtained from echocardiography. (CeE) Representative cross sections of heart staining for Masson, WGA, Col1a1 and a-SMA of PBS-treated or CGS21680-treated DOCA-salt mice with or without iBAT and quantitative analysis of cardiomyocyte size and fibrotic area. (F) Quantitative analysis of Col1a1 and aSMA signals in heart of PBS-treated or CGS21680-treated DOCA-salt mice with or without iBAT. Data are presented as means ± SEM; n ¼ 5e6/group; NS indicates no statistical difference between PBS-treated and CGS21680-treated without iBAT.s.
Fig. 4. A2AR agonist increases iBAT-derived FGF21 in hypertension. (A) Serum FGF21 concentration in SHAM and DOCA-salt mice, treated with PBS, CGS21680 or KW6002 for 2 weeks. (B) Relative FGF21 mRNA expression in iBAT, heart and liver of SHAM and DOCA-salt mice, treated with PBS, CGS21680 or KW6002 for 2 weeks. (C) Representative immunoblots of UCP1 and GAPDH protein expression in iBAT of SHAM and DOCA-salt mice, treated with PBS, CGS21680 or KW6002 for 2 weeks. (D) Quantitative analysis of immunoblots of FGF21. Data are presented as means ± SEM; n ¼ 5e6/group.
[16]. In previous study, we detected that DOCA-salt treatment induced a significant increase of A2AR in BAT, but an unconspicuous change in heart [17]. This study provides the primary data that iBAT
depletion aggravates cardiac damage and blocked A2AR protection in hypertensive mice. Furtherly, data shown that A2AR agonist promoted BAT thermogenesis and secretion of FGF21. Previous
Please cite this article as: Y.-P. Zhou et al., Adenosine A2A receptor activation prevents DOCA-salt induced hypertensive cardiac remodeling via iBAT, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.035
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study has proved that A2AR mediates FGF21 expression via AMPK/ PGC1a signaling pathway [29]. Heart is the key target of FGF21, which is involved in inducing beneficial effects in cardiac remodeling [30,31]. Investigators have showed that FGF21 knockout mice exhibit an increased heart weight and develop enhanced signs of dilatation and cardiac dysfunction in response to isoproterenol infusion [21]. These could be explained by that FGF21 induced the expression of PGC1a, an established repressor of the NF-kB proinflammatory pathway [32]. Our study suggested that A2AR mediates BAT activation leading to an elevated FGF21 level in BAT without affecting cardiac FGF21. The current study demonstrated that iBAT is a vital target against the hypertensive cardiac remodeling. Although A2AR mediates BAT thermogenic activity and elevates the expression of FGF21, the detailed mechanism and signaling pathways of this A2AR-dependent response remain to be investigated in the future. In summary, we demonstrated that A2AR-mediated FGF21 secreted by BAT improved hypertensive cardiac remodeling in DOCA-salt mice. Our data suggested that regulation of adenosine/ A2AR pathway could be a direction for preventing or treating organ damage in hypertension. Sources of funding This work was supported by grants from the National Natural Science Foundation of China (81922004, 91739303 and 81800225). Compliance with ethical standard Ethical approval of animal studies All animal procedures were approved in accordance with institutional guidelines established by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine. Human subjects/informed consent statement No human studies were carried out by the authors for this article. Author contributions L.-R.K., P.-J. G. and Y.-P.Z. designed the experiments, analyzed data and wrote the manuscript. L.-R.K. and Y.-P.Z. performed the animal experiments and analytical methods. Y.-P.Z. and C.-C.R. performed the in vivo images. L.-R.K., C.-C.R. and Y.-P.Z. analyzed the data. Declaration of competing interest The authors declare that they have no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.02.035. References [1] J. Tomek, G. Bub, Hypertension-induced remodelling: on the interactions of cardiac risk factors, J. Physiol. 595 (12) (2017) 4027e4036, https://doi.org/ 10.1113/jp273043. [2] B.B. Fredholm, I.J. Ap, K.A. Jacobson, J. Linden, C.E. Muller, International union of basic and clinical pharmacology. LXXXI. Nomenclature and classification of adenosine receptors–an update, Pharmacol. Rev. 63 (1) (2011) 1e34, https://
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Mampel, et al., Thermogenic activation induces FGF21 expression and release in brown adipose tissue, J. Biol. Chem. 286 (15) (2011) 12983e12990, https://doi.org/ 10.1074/jbc.M110.215889. [23] F.M. Fisher, S. Kleiner, N. Douris, E.C. Fox, R.J. Mepani, F. Verdeguer, et al., FGF21 regulates PGC-1alpha and browning of white adipose tissues in adaptive thermogenesis, Genes Dev. 26 (3) (2012) 271e281, https://doi.org/ 10.1101/gad.177857.111. [24] A. Guerrero, A2A adenosine receptor agonists and their potential therapeutic applications. An update, Curr. Med. Chem. 25 (30) (2018) 3597e3612, doi: 10.2174/0929867325666180313110254. [25] A. van Waarde, R. Dierckx, X. Zhou, S. Khanapur, H. Tsukada, K. Ishiwata, et al., Potential therapeutic applications of adenosine A2A receptor ligands and opportunities for A2A receptor imaging, Med. Res. Rev. 38 (1) (2018) 5e56, https://doi.org/10.1002/med.21432. [26] K. Varani, F. Laghi-Pasini, A. Camurri, P.L. Capecchi, M. Maccherini, F. Diciolla,
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Adenosine A2A receptor activation prevents DOCA-salt induced hypertensive cardiac remodeling via iBAT Yan-Ping Zhou, Cheng-Chao Ruan, Ling-Ran Kong*, Ping-Jin Gao State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Hypertension, Shanghai Institute of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 January 2020 Accepted 6 February 2020 Available online xxx
Hypertensive cardiac remodeling is a constellation of abnormalities that includes cardiomyocyte hypertrophy and death and tissue fibrosis. Adenosine is a long-known vasodilator, through interacting with its four cell surface receptor subtypes in cardiovascular system. However, it is unclear that whether adenosine A2A receptor (A2AR) activation is involved in the cardiac remodeling in hypertension. WT mice were utilized to induce DOCA-salt sensitive hypertension and received A2AR agonist CGS21680 or antagonist KW6002 treatment. Cardiac functional phenotyping measurement by echocardiography showed that CGS21680 improved cardiac dysfunction in DOCA-salt mice. Moreover, CGS21680 reduced cardiomyocyte hypertrophy, cardiac inflammation and fibrosis. However, iBAT depletion surgery induces dramatic cardiac remodeling in DOCA-salt mice, and the protective function of CGS21680 was blocked without intact iBAT. Mechanistically, A2AR agonist CGS21680 increased iBAT-derived fibroblast growth factor 21 (FGF21). Our data suggest that activation of A2AR could be a potential therapeutic strategy in preventing heart damage in hypertension. © 2020 Elsevier Inc. All rights reserved.
Keywords: Hypertension Cardiac remodeling Adenosine A2AR Brown adipose tissue
1. Introduction Hypertension is a major clinical condition that induces cardiac remodeling, through acting as a contributing factor in both systolic and diastolic dysfunction, arrhythmia and symptomatic heart failure [1]. Long-term cardiac load or injury results in cardiac remodeling, defined as left ventricular hypertrophy and interstitial fibrosis. Therefore, it is necessary to develop effective approach to intervene the process of cardiac remodeling caused by hypertension. Adenosine is a ubiquitous endogenous molecule, which can be released from intracellular pools by membrane transporters or directly produced extracellularly following ATP catabolism by the concerted action of the ecto-enzymes [2,3]. The adenosine signal can be transmitted by its four G protein coupled receptors: adenosine A1 and A3 receptors through Gi or A2A and A2B receptors via Gs. Adenosine A2AR subtype is high sensitive to adenosine and participate in cardiovascular and metabolic regulation because it is widely expressed both in cardiovascular system and metabolismregulating organs [4,5]. Animal studies showed that A2AR
* Corresponding author. E-mail address: [email protected] (L.-R. Kong).
knockout rats are sensitive to the prohypertensive effect in highsalt diet [6]. Furthermore, A2AR agonist has been reported to ameliorate cardiac remodeling following myocardial infarction in spontaneously hypertensive rats [7,8]. However, experiments in vitro showed that pharmacological stimulation of A2AR did not directly affect cardiac fibroblast function [9]. In addition, recent study showed that A2AR activation promotes lipolysis, thermogenesis in adipose tissue, which protected against high-fat diet induced obesity [10]. Therefore, we hypothesized that the protective effects of A2AR in hypertensive cardiac remodeling may through regulating adipose tissue activity. Brown adipose tissue (BAT) is well recognized for its nonshivering thermogenesis mediated by uncoupling protein 1 (UCP1), mainly distributed in interscapular, axillary and cervical regions in rodent [11]. Recent data suggested that the beneficial effects of BAT could involve a previously unrecognized endocrine role through the release of endocrine factors [12]. The BAT hormones are named “batokines including Thriiodothyronine (T3), Fibroblast growth factor-21 (FGF21), Interleukin-6 (IL-6) and so on [13]. Study demonstrated that mice with BAT ablation express an elevation of blood pressure, LVH and increased interstitial tissue in addition to the development of obesity and insulin resistance [14]. However, the precise mechanism underlying the interaction
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Please cite this article as: Y.-P. Zhou et al., Adenosine A2A receptor activation prevents DOCA-salt induced hypertensive cardiac remodeling via iBAT, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.035
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Abbreviations A2AR BAT iBAT DOCA Col1a1 TGFb aSMA UCP1 PPARg PRDM16 Cidea FGF21
Adenosine A2A receptor brown adipose tissue interscapular brown adipose tissue deoxycorticosterone acetate collagen type 1 alpha chain transforming growth factor b a smooth muscle actin uncoupling protein 1 peroxisome proliferator-activated receptor g PR domain containing 16 cell death inducing DNA fragmentation factoralpha-like A fibroblast growth factor 21
between BAT and cardiac remodeling is rarely investigated and remains elusive. In this study, we treated mice with a specific A2AR agonist, CGS21680, to determine whether A2AR activation could prevent hypertension-induced cardiac damage. Our results showed that administration of CGS21680 effectively suppressed cardiac fibrosis and improved cardiac function in deoxycorticosterone acetate (DOCA)-salt hypertensive mice. 2. Methods 2.1. Mice and treatment C57BJ/6J wild type mice aged 8e12 weeks were randomly assigned to SHAM group or DOCA-salt group which underwent uninephrectomy and DOCA pellet (50mg/pellet) subcutaneously together with 1% NaCl in the drinking water for 2 weeks [15]. For pharmacological investigation, DOCA-salt mice received intraperitoneally injection of A2AR agonist CGS21680 (1 mg/kg/day), A2AR antagonist KW6002 (1 mg/kg/day) or PBS. IBAT removal surgery was performed as previously described [16]. Mice belonging to SHAM group were administrated the same procedure, the kidney or iBAT pad was located, exposed, and then been closed. 2.2. Echocardiography Transthoracic echocardiography was performed with a Vevo 2100 instrument (Fuji Film Visual Sonics) equipped with an MS-400 imaging transducer (18e38 MHz) as previously described [17]. Briefly, mice were preconditioned by chest hair removal, exposed to 1%e2% isoflurane to be anaesthetized, and maintained in a supine position on handing platform with limbs attached for electrocardiogram gating during imaging. Body temperature was kept constant by a heating pad, in the meantime, heart and respiratory rates were continuously monitored. M-mode recording was performed at the midventricular level. All images were analyzed using dedicated software (Vero 2100 version 1.4). Percent LV ejection fraction (EF) and fractional shortening (FS) was calculated from M-mode measurements. All procedures were performed under double-blind conditions with regard to treatment. 2.3. In vivo iBAT imaging The iBAT imaging was performed as previously described [18]. Briefly, mice were intravenously injected with micellar SRFluor680 (0.35 mg in 100 mL PBS), after 6e8 h, mice were anaesthetized with
1%e2% isoflurane with oxygen flow rate of 2 L/min and denuded by depilatory cream to reduce scattering and absorption by the hair. Fluorescence imaging was obtained from IVIS Dpectrum/CT system (PerkinElmer) following the manufacture introductions. The fluorescence acquisition parameters were: Excitation Filter: 615e665 nm, Excitation Filter: 695e770 nm, Acquisition time: 10 s, Subject height: 1.5 cm, Field od view: A (2 cm*2 cm). Then the CT imaging and 3D Reconstruction was obtained through X-Rays Acquire mode. Data analysis and the photon flux data were obtained by using Region of Interest (ROI) tools. 2.4. Histological analysis Heart and iBAT tissues were fixed in 4% formaldehyde for 48 h, dehydrated, embedded in paraffin and sectioned at 5 mm thickness. Then sections were stained with hematoxylin and eosin (HE), Masson’s trichrome to evaluate the degree of fibrosis or wheat germ agglutinin (WGA). Images were captured by a Carl Zeiss Axio Imager M2 microscope (Carl Zeiss Corporation, Germany) by software Imager-Pro Plus (Media Cybernetics, United States). For immunohistochemical staining, heart sections were incubated with primary antibodies for Col1a1 (1:100) or a-SMA (1:1000), iBAT section were incubated with primary antibody for UCP1 (1:100). 2.5. Quantitative RT-PCR analysis Total RNA was extracted from heart and iBAT samples using TRIzol reagent (Sigma-Aldrich, TS424), according to the manufacturer’s specifications. Then, total RNA was reverse-transcribed to cDNA by using Reverse Transcription Master Mix (A0010GQ, EZBioscience). Real-time qRT-PCR was performed with SYBR Premix Ex Taq kits with ROX (Takara, RR420B) according to manufacturer’s instruction. Reactions were run on an ABI PRISM 7900 machine (Applied Biosystems). The housekeeping gene b-Actin was used as internal control: the expression of the genes studied was expressed as a ratio to that b-Actin. The primers used are listed as followed: Col1a1, forward 50 -GAGCGGAGAGTACTGGATCG-30 and reverse 50 -TACTCGAACGGGAATCCATC-3’; TGFb, forward 50 -CAACAATTCCTGG CGTTACCTTGG-30 and reverse 50 -GAAAGCCCTGTATTCCGTCTCCTT-3’; aSMA, forward 50 -CTGACAGAGGCACCATGAA-30 and reverse 50 AGAGGCATAGAGGGACAGCA-3’; UCP1, forward 50 -AGGCTTCCAGTACCATTAGGT-30 and reverse 50 -CTGAGTGAGGCAAAGCTGATTT-3’; PPARg, forward 50 -TTAGATGACAGTGACTTGGC-30 and reserve 50 TCTTCTGGAGCACCTTGG-3’; PRDM16, forward 50 -CAGCACGGTGAAGCCATTC-30 and reverse 50 - GCGTGCATCCGCTTGTG-3’; CIDEA, forward 50 -TGCTCTTCTGTATCGCCCAGT-30 and reverse 50 GCCGTGTTAAGGAATCTGCTG-3’; FGF21, forward 50 -CAGGGAGGATGGAACAGT-30 and reverse 50 -TTTGGGGAGTCCTTCTG-3’; b-Actin, forward 50 - CGTGGGCCGCCCTAGGCACCA-30 and reverse 50 TTGGCCTTAGGGTTCAGGGGGG-3’. 2.6. Western blot analysis The frozen heart, iBAT and liver tissues were lysed in Radio Immunoprecipitation Assay (RIPA) buffer (Merck Millipore, 20e188) containing 1% protease inhibitor cocktail (Biotool, B14002). Protein was run on a 12% SDS page gel and blotted onto PVDF membrane (Millipore) by wet transfer, blots were blocked and blotted according to the antibody manufacturers’ recommendations. The primary antibodies were as follows: anti-Col1a1 (1:1000 dilution) (Santa Cruz, SC-8787-R), anti-TGFb (1:1000 dilution) (CST, 3711S), anti-aSMA (1:1000 dilution) (Sigma, A5228), anti-UCP1 (1:1000 dilution) (Abcam, ab23841), anti-PPARg (1:1000 dilution) (Santa Cruz, sc-7273), anti-PRDM16 (1:1000 dilution) (Abcam, ab106410), anti-CIDEA (1:1000 dilution) (Abcam, ab8402),
Please cite this article as: Y.-P. Zhou et al., Adenosine A2A receptor activation prevents DOCA-salt induced hypertensive cardiac remodeling via iBAT, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.035
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anti-FGF21 (1:1000 dilution) (Abcam, ab171941) and GAPDH monoclonal antibody (1:4000) (proteintech, HRP-60004). Results were quantified by using imaging software Quantity One (Bio-Rad Laboratory, Spain). 2.7. FGF21 measurement Serum was obtained from blood collected from mice. FGF21 levels in blood was determined by using Mouse FGF21 ELISA-Kit (R&D, MF2100) according to the manufacturer’s instruction. 2.8. Statistical analysis All values are presented as means ± SEM. Statistical analysis was performed using one-tailed or two-tailed Student’s t-test. For experiments in which more than two groups were compared, twoway analysis of variant (ANOVA) was used and followed by the post hoc Dunnett’s test for data with more than two groups (Levene’s tests for equalvariance). Dunnett’sT3 test was used for post hoc test comparison for the analysis of unequal variances (Welch’s and BrowneForsythe’s test). The significance level was set at P < 0.05 in all statistics. 3. Results
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3.3. IBAT depletion blockade the cardioprotective effects of CGS21680 To provide more convincing proof that the endocrine of BAT is an indispensable channel for A2AR to improve hypertensive cardiac remolding, mice were administrated iBAT removal surgery (iBAT()) in PBS-treated and CGS21680-treated group. Interestingly, iBAT depletion exacerbated the DOCA-salt-induced cardiac remolding demonstrated by decreased EF and FS between PBSiBAT() group and PBS-iBAT(þ) group. Furtherly, iBAT depletion blocked the protection of CGS21680 in DOCA-salt mice. (Fig. 3A and B). WGA and Masson staining also demonstrated that iBAT() increased the cardiomyocyte size and cardiac interstitial fibrosis (Fig. 3C and D), as well as the increasing expression of Col1a1 and aSMA (Fig. 3E and E). More importantly, there was no statistically significant difference in the WGA and Col1a1 and aSMA staining analysis when compared PBS-iBAT() with CGS21680-iBAT() (Fig. 3D and F). These studies validated the iBAT depletion blockaded the effect of CGS21680 to mitigate DOCA-salt-induced cardiac remodeling. 3.4. A2AR activation increases BAT-derived FGF21 in hypertension
In order to explore the role of A2AR in hypertensive cardiac remodeling, Wild type (WT) DOCA-salt mice were treated with A2AR agonist CGS21680 (1 mg/kg/day) or its antagonist KW6002 (1 mg/kg/day). DOCA-salt treatment induced a reduced ejection fraction (EF) and fractional shortening (FS) in WT mice, but CGS21680 protected against impaired heart function compared with KW6002 group (Fig. 1A and B). Histological analysis revealed that DOCA-salt induced cardiac remodeling was attenuated in CGS21680 group. A2AR activation resulted in decreased cardiomyocyte size and cardiac interstitial fibrosis (Fig. 1C and D), as well as reduced positive staining for Col1a1 and aSMA (Fig. 1E and Fig.S1A). These were further demonstrated by detecting fibrotic makers (Col1a1, TGFb and aSMA) in mRNA and protein levels (Fig. 1F, Fig.S1B and Fig.S1C). These data indicated that A2AR activation ameliorated hypertension-induced cardiac remodeling.
Fibroblast growth factor 21 (FGF21), which produced predominantly in liver [19], is a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity [20]. Previous study shows that FGF21 exert a protective action against both antihypertrophic and cardioprotective actions on the heart [21]. And thermogenic activation induces FGF21 gene expression in BAT and triggers the release of FGF21 by BAT [22,23]. In order to explain why BAT thermogenic activity by A2AR activation could alleviate hypertensive cardiac remodeling partly, we examined the protein levels of FGF21 level in serum by ELIAS kit firstly. In DOCA-salt treated mice, FGF21 increased in serum in the CGS21680-treated group (Fig. 4A). Next, we surprisingly found the mRNA expression level and protein level of FGF21 in the iBAT were significantly upregulated due to the activation of A2AR, whereas KW6002 inhibited the expression of FGF21 (Fig. 4B, C and D). As for the expression of FGF21 in heart and liver, there were no significant changes after the stimulation or blockade of A2AR (Fig. 4B). Taken together, these results indicated that A2AR-mediated iBAT thermogenic activation increased the level of serum FGF21, which could act on the target of the heart through blood transport.
3.2. A2AR regulates BAT function in hypertension
4. Discussion
BAT has been always considered a site of metabolic energy consumption to produce heat, however, it has been showed to be as a secretory organ recently [12]. To further understand whether A2AR could activate BAT, we detected BAT activation in DOCA-salt mice treated with CGS21680 or KW6002 by a deep-red fluorescent micellar probe, SRFluor680, which has been showed to be accumulated in iBAT regardless mouse species [18]. As expected, fluorescence imaging combined with microCT showed a dramatic increased fluorescent density in the interscapular region in the CGS21680 group compared with PBS or KW6002 group (Fig. 2A and B). IHC analysis suggested that A2AR agonist significantly promoted iBAT activation demonstrated by smaller-sized adipocyte and increased uncoupling protein 1 (UCP1) staining (Fig. 2C and D). Consistently, the increase of mRNA and protein expression of thermogenic genes including UCP1, PPARg, PRDM16, and CIDEA induced by DOCA-salt treatment became still more in the CGS21680 group. However, KW6002 had the opposite effects on BAT in DOCA-salt mice (Fig. 2E, Fig. S2A and Fig. S2B). These results demonstrated that A2AR regulated BAT function in hypertension.
Overall, our studies illuminated that A2AR agonist could alleviate cardiac remodeling and regulate the BAT function in hypertension. IBAT removal surgery in DOCA-salt mice showed that iBAT depletion blocked the protection of CGS21680 in hypertensive cardiac remodeling. In addition, our data showed that CGS21680 increased FGF21 expression in BAT of DOCA-salt mice. Therefore, our study showed that A2AR activation mitigated the damage induced by hypertension mainly depending on the FGF21 secreted by BAT. It is known that A2AR is highly expressed on the surface of brain, immune system and adipose tissue [24,25]. The A2AR expression in different organs will change under the condition of changeable human disease and animal models [25]. Chronic heart failure exhibits a significance of up-regulation of A2AR in heart and peripheral blood mononuclear cells [26]. Cardiac remodeling involves the production of extracellular matrix (ECM) by cardiac fibroblast and inflammatory process, both of which are sensitive to adenosine [27]. Recent evidence has been presented that A2AR participate in the signaling for inhibition of cardiac fibroblast growth and fibrosis, and A2AR stimulation prevents neutrophil accumulation and matrix
3.1. A2AR activation protects against hypertensive cardiac remodeling
Please cite this article as: Y.-P. Zhou et al., Adenosine A2A receptor activation prevents DOCA-salt induced hypertensive cardiac remodeling via iBAT, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.035
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Fig. 1. A2AR agonist alleviates Hypertensive cardiac remodeling. (A) Representative echocardiography images of SHAM and DOCA-salt mice, injected with PBS, CGS21680 or KW6002 for 2 weeks. (B) Quantitative analysis of ejection fraction (EF), fractional shortening (FS) in diastolic phase obtained from echocardiography. (CeE) Representative cross-sections of heart straining for Masson, WGA, Col1a1 and aSMA in SHAM and DOCA-salt mice, injected with PBS, CGS21680 or KW6002 for 2 weeks and quantitative analysis of cardiomyocyte size (WGA staining) and fibrotic area (blue area in Masson staining). (F) Representative immunoblots of Col1a1, TGFb and aSMA protein expression in heart of SHAM and DOCA-salt mice, injected with PBS, CGS21680 or KW6002 for 2 weeks. Data are presented as means ± SEM; n ¼ 5e6/group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2. A2AR regulates BAT function in hypertension. (A) Representative fluorescence imaging combined with micro CT of SHAM and DOCA-salt mice, treated with PBS, CGS21680 or KW6002 after intravenous injection of SRFluor680 for 6 h. (B) Quantitative analysis of normalized mean intensity of fluorescence. (C) Representative of HE and UCP1 staining images of iBAT from SHAM and DOCA-salt mice, treated with PBS, CGS21680 or KW6002 for 2 weeks. (D) Quantitative analysis of UCP1 signal. (E) Representative immunoblots of UCP1, PPARg, PRDM16, Cidea protein expression in iBAT of SHAM and DOCA-salt mice, treated with PBS, CGS21680 or KW6002 for 2 weeks. Data are presented as means ± SEM; n ¼ 5e6/group.
metalloproteinases (MMPs) secretion to degrade the ECM [28]. In our study, we demonstrated that A2AR agonist improved the cardiac function and decreased the interstitial fibrosis caused by hypertension. And the fibrosis-related genes expression in mRNA and
protein level on heart decreases because of the A2AR stimulation. In the last decade, data generated in both rodents and humans reveal the therapeutic potential of BAT in cardiovascular disease [13]. BAT transplantation improved cardiomyocyte injury in mice
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Fig. 3. iBAT depletion blockade the cardioprotective effects of CGS21680. (A) Representative echocardiography images of PBS-treated or CGS21680-treated DOCA-salt mice with or without iBAT. (B) Quantitative analysis of ejection fraction (EF), fractional shortening (FS) in diastolic phase obtained from echocardiography. (CeE) Representative cross sections of heart staining for Masson, WGA, Col1a1 and a-SMA of PBS-treated or CGS21680-treated DOCA-salt mice with or without iBAT and quantitative analysis of cardiomyocyte size and fibrotic area. (F) Quantitative analysis of Col1a1 and aSMA signals in heart of PBS-treated or CGS21680-treated DOCA-salt mice with or without iBAT. Data are presented as means ± SEM; n ¼ 5e6/group; NS indicates no statistical difference between PBS-treated and CGS21680-treated without iBAT.s.
Fig. 4. A2AR agonist increases iBAT-derived FGF21 in hypertension. (A) Serum FGF21 concentration in SHAM and DOCA-salt mice, treated with PBS, CGS21680 or KW6002 for 2 weeks. (B) Relative FGF21 mRNA expression in iBAT, heart and liver of SHAM and DOCA-salt mice, treated with PBS, CGS21680 or KW6002 for 2 weeks. (C) Representative immunoblots of UCP1 and GAPDH protein expression in iBAT of SHAM and DOCA-salt mice, treated with PBS, CGS21680 or KW6002 for 2 weeks. (D) Quantitative analysis of immunoblots of FGF21. Data are presented as means ± SEM; n ¼ 5e6/group.
[16]. In previous study, we detected that DOCA-salt treatment induced a significant increase of A2AR in BAT, but an unconspicuous change in heart [17]. This study provides the primary data that iBAT
depletion aggravates cardiac damage and blocked A2AR protection in hypertensive mice. Furtherly, data shown that A2AR agonist promoted BAT thermogenesis and secretion of FGF21. Previous
Please cite this article as: Y.-P. Zhou et al., Adenosine A2A receptor activation prevents DOCA-salt induced hypertensive cardiac remodeling via iBAT, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.035
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study has proved that A2AR mediates FGF21 expression via AMPK/ PGC1a signaling pathway [29]. Heart is the key target of FGF21, which is involved in inducing beneficial effects in cardiac remodeling [30,31]. Investigators have showed that FGF21 knockout mice exhibit an increased heart weight and develop enhanced signs of dilatation and cardiac dysfunction in response to isoproterenol infusion [21]. These could be explained by that FGF21 induced the expression of PGC1a, an established repressor of the NF-kB proinflammatory pathway [32]. Our study suggested that A2AR mediates BAT activation leading to an elevated FGF21 level in BAT without affecting cardiac FGF21. The current study demonstrated that iBAT is a vital target against the hypertensive cardiac remodeling. Although A2AR mediates BAT thermogenic activity and elevates the expression of FGF21, the detailed mechanism and signaling pathways of this A2AR-dependent response remain to be investigated in the future. In summary, we demonstrated that A2AR-mediated FGF21 secreted by BAT improved hypertensive cardiac remodeling in DOCA-salt mice. Our data suggested that regulation of adenosine/ A2AR pathway could be a direction for preventing or treating organ damage in hypertension. Sources of funding This work was supported by grants from the National Natural Science Foundation of China (81922004, 91739303 and 81800225). Compliance with ethical standard Ethical approval of animal studies All animal procedures were approved in accordance with institutional guidelines established by the Committee of Ethics on Animal Experiments at the Shanghai Jiao Tong University School of Medicine. Human subjects/informed consent statement No human studies were carried out by the authors for this article. Author contributions L.-R.K., P.-J. G. and Y.-P.Z. designed the experiments, analyzed data and wrote the manuscript. L.-R.K. and Y.-P.Z. performed the animal experiments and analytical methods. Y.-P.Z. and C.-C.R. performed the in vivo images. L.-R.K., C.-C.R. and Y.-P.Z. analyzed the data. Declaration of competing interest The authors declare that they have no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.02.035. References [1] J. Tomek, G. Bub, Hypertension-induced remodelling: on the interactions of cardiac risk factors, J. Physiol. 595 (12) (2017) 4027e4036, https://doi.org/ 10.1113/jp273043. [2] B.B. Fredholm, I.J. Ap, K.A. Jacobson, J. Linden, C.E. Muller, International union of basic and clinical pharmacology. LXXXI. Nomenclature and classification of adenosine receptors–an update, Pharmacol. Rev. 63 (1) (2011) 1e34, https://
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Please cite this article as: Y.-P. Zhou et al., Adenosine A2A receptor activation prevents DOCA-salt induced hypertensive cardiac remodeling via iBAT, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.035