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Cardiovascular Research 2005 65(3):743-750; doi:10.1016/j.cardiores.2004.10.020
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Copyright © 2004, European Society of Cardiology

Effects of all-trans retinoic acid on orphan receptor APJ signaling in spontaneously hypertensive rats

Jiu-Chang Zhonga,*, Dong-Yang Huanga,*, Ge-Fei Liua, Hai-Yan Jinb, Yan-Mei Yanga, Yi-Fan Lia, Xu-Hong Songa and Kun Dua

aCenter for Molecular Biology, Shantou University Medical College, 22 Xin Ling Rd., Shantou, Guangdong 515041, China
bInstitute of Cardiovascular Research, Guangdong Provincial People's Hospital, Guangzhou, Guangdong 510080, China

* Corresponding authors. Tel.: +86 754 8900400; fax: +86 754 8557562. Email address: g_jczhong{at}stu.edu.cn huangdy{at}stu.edu.cn

Received 22 June 2004; revised 25 September 2004; accepted 13 October 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Studies show general agreement that all-trans retinoic acid (atRA) has been linked to the regulation of G protein-coupled receptor (GPCRs) signaling. To further validate effects of atRA on the cardiovascular GPCRs, the present study was designed to assess whether atRA will modulate orphan receptor APJ, a homologue of angiotensin II type 1 (AT1) receptor.

Methods: Real-time polymerase chain reaction and Western blot methods were performed to examine the expression of APJ and its endogenous ligand apelin in spontaneously hypertensive rats (SHR) and Wistar–Kyoto (WKY) rats after chronic atRA treatment.

Results: APJ and apelin expression were markedly depressed in placebo-treated SHR, compared with WKY rats (p<0.01). However, in atRA-treated SHR, a significant upregulation of APJ and apelin expression was observed in both heart and aorta (p<0.05), accompanied by a reduction of AT1 expression, an elevation of serum nitric oxide levels and a subsequent decrease of blood pressure.

Conclusions: Chronic atRA treatment activates gene and protein expression of APJ and apelin and reduces blood pressure in SHR, suggesting that atRA may regulate the balance between apelin-APJ and angiotensin II-AT1 signaling and have potential clinical value in the prevention and treatment of human hypertension.

KEYWORDS All-trans retinoic acid; Hypertension; APJ; Apelin; Nitric oxide


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
The cardiovascular system is richly endowed with G protein-coupled receptor (GPCRs), which represent the largest group of transmembrane proteins responsible for transduction of a diverse array of extracellular signals [1,2]. Dysfunctional GPCRs signaling transduction in the cardiovascular system may be an important contributor to the initiation, establishment and/or maintenance of hypertension [1,3]. In fact, increased risk of essential hypertension in certain human populations has been associated with genetic polymorphisms in genes encoding components of GPCR signaling pathways, among which is angiotensin II type 1 (AT1) receptor gene [1,4].

The APJ (putative receptor protein related to AT1) gene, originally identified from a human genomic library, is among the earliest cloned orphan GPCRs that bind an unknown ligand and encodes a seven-transmembrane domain GPCR of 377 amino acids [5,6]. Although "orphan" for many years, its natural ligand was recently isolated and named apelin (for APJ endogenous ligand) [7]. Apelin and APJ are widely expressed in the cardiovascular system in a pattern shared with angiotensin II and the angiotensin receptor AT1, respectively [8–10]. However, angiotensin II does not bind to APJ. The apelin-APJ signaling pathway emerges as an important novel mediator of cardiovascular control and blood pressure homeostasis [8,11]. In addition, abnormalities in apelin-APJ signaling may be involved in the pathogenesis of hypertension and its complications such as heart failure [2,8,11,12]. Drugs that specifically influence the activities of APJ receptor and its ligand apelin may therefore have potential clinical value in the prevention and treatment of hypertension and its relevant complications [1,8,10,11].

Research advances on the key transcription factors and nuclear hormone receptors provide insight into the development of novel transcription-modulating antihypertensive drugs, among which is all-trans retinoic acid (atRA), a biologically active metabolite of vitamin A [13]. Studies show general agreement that atRA influences the activities of many GPCRs and has been linked to the regulation of GPCRs signal transduction pathways [14–18]. However, whether atRA participates in apelin-APJ signaling regulation awaits further investigation. Recent experimental reports have demonstrated that atRA influences gene expression of AT1 and its ligand angiotensin II [15,18,19]. In view of the sequence and distribution similarities between APJ and AT1 (as well as apelin and angiotensin II) [5,7,9], we predicted that atRA might affect the expression of orphan GPCR APJ and its endogenous ligand apelin. Most data have substantiated that the apelin-APJ signaling pathway lowers blood pressure via a nitric oxide (NO)-dependent mechanism [11,12]. Thus, it is of considerable interest to determine the effects of atRA on serum NO levels. In the present work, changes of messenger RNA (mRNA) and protein levels for APJ, apelin and AT1 receptor have been examined in spontaneously hypertensive rats (SHR) and Wistar–Kyoto (WKY) rats after chronic atRA treatment.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Animal and tissue processing
Twelve-week-old male SHR and WKY rats (Shanghai Institute of Hypertension, China) were randomly assigned to five treatment groups: WKY-C (WKY control treated with vehicle 1 ml.kg–1.day–1), WKY-R (WKY treated with atRA 10 mg.kg–1.day–1), SHR-C (SHR control treated with vehicle 1 ml.kg–1.day–1), SHR-L (SHR treated with low-dose atRA 10 mg.kg–1.day–1) and SHR-H (SHR treated with high-dose atRA 20 mg.kg–1.day–1). Under minimal light, fresh suspensions of atRA (Sixth Pharmaceutical, Shanghai, China) in vehicle were prepared each day. Rats were injected intraperitoneally with atRA or vehicle (50% intralipid, 45% saline and 5% ethanol) once a day for a month. Systolic blood pressure (SBP) was measured by the tail-cuff method once a week. Conscious rats were prewarmed for 10 min at 37 °C in a thermostatically controlled heating cabinet. With the rat kept gently in a special cage, the tail was passed through a miniaturized cuff and a tail-cuff sensor that was connected to an amplifier. Tail-cuff SBP is defined as the inflation pressure at which the waveform becomes indistinguishable from baseline noise. Final SBP value was obtained by averaging three successful readings. Rats were housed in a room with a 12:12-h light–dark cycle and fed with a standard animal diet and water ad libitum. At the end of the 1-month treatment period, blood was collected for NO determination. The heart and aorta were carefully excised after the rat was decapitated. Serum and tissue samples were immediately frozen in liquid nitrogen and stored at –70 °C until assay. All procedures used in this study were approved and performed in accordance with the National Animal Protection Law of China, conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2. Isolation of total RNA and synthesis of complementary DNA
Total RNA was extracted from heart and aorta tissues using Trizol Reagent (Invitrogen) according to the manufacturer's instruction. Complementary DNAs (cDNA) were synthesized by standard techniques (Superscript® First Strand Synthesis System for RT-PCR, Invitrogen). An aliquot of the resulting single-strand cDNA was used in the real-time polymerase chain reaction (PCR) experiments as described below.

2.3. Quantitative real-time PCR
Based on the sequences reported in the GenBank database (see Table 1), primers and probes were designed for APJ, apelin, AT1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with the Primer Express Software. Probes were labeled with a 5'-reporter dye (6-carboxy-fluorescein) and a 3'-quencher dye (6-carboxy-tetramethylrodamine). To determine the quantity of each mRNA in real-time PCR reactions, a calibration curve was constructed with a standard. In the present study, we successfully amplified the known amount of plasmids (pGEM-T easy vector, Promega) encompassing rat APJ, apelin, AT1 and GAPDH cDNA to serve as gene standards after they were identified by PCR amplification (Fig. 1) and sequence (ABI Prism 377 DNA Sequencer). Serial dilutions of the positive recombinant plasmids were carried out in duplicate from 1 x 109 to 103 copies and used as standard templates to make the calibration curves for sample array. Real-time PCR was carried out in a 25-µl reaction mixture prepared with a TaqMan PCR core reagent kit (PE-ABI) containing an appropriately diluted cDNA solution, 0.2 µM each primer, 0.2 µM probe and 0.2 µM ROX Reference Dye (Invitrogen) under the following conditions: at 50 °C for 10 min, at 95 °C for 10 min and 40 cycles at 95 °C for 15 s and at 60 °C for 45 s. Real-time PCR reactions were performed, recorded, and analyzed by using the ABI 7700 Prism Sequence Detection System (PE-ABI). Each tissue sample of rats was run in triplicate. Rat housekeeping gene GAPDH mRNA from samples was measured as an internal control.


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  		Table 1 Primers and probes used in real-time PCR reactions

 

Figure 1
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  		Fig. 1 The identification of gene standards by PCR amplification. The purified apelin, APJ, AT1 and GAPDH were cloned into the pGEM-T easy vector. PCR amplification was performed to identify positive recombinant plasmids with 2% agarose gel electrophoresis at 100 V for 35 min. M indicates DNA marker (GeneRulerTM 50 bp DNA Ladder, Fermentas); 1–4, plasmid-apelin selected at random (101 bp); 5–8, plasmid-APJ selected at random (111 bp); 9–12, plasmid-AT1 selected at random (89 bp); 13–16, plasmid-GAPDH selected at random (83 bp).

 
2.4. Western blotting
Heart and aorta were quickly minced, resuspended and homogenized in lysis buffer and then centrifuged. Protein concentrations were determined using a Bio-Rad protein assay kit with bovine serum albumin (BSA) standards. Protein samples (80 µg) were separated on a sodium dodecyl sulfate (SDS)-polyacrylamide denaturing gels, transblotted to nitrocellulose filter (Hybond ECL, Amersham). The membranes were blocked overnight at 4 °C with 1% Blot-Qualified BSA (Promega) in TBST solution [25 mM Tris, pH 7.5, 150 mM NaCl, 0.05% (vol/vol) Tween 20], and then incubated for 2 h at room temperature under gentle agitation with the primary antibody (rabbit anti-APJ and apelin-12, Phoenix Pharmaceuticals, USA; rabbit anti-AT1 and β-actin, Boster Biotechnology, China). After three washings with TBST, the membranes were incubated with AP-labeled secondary antibody (Santa Cruz) in TBST solution for 1 h at room temperature, followed by washing as above. The positive protein bands were developed with the use of the Western Blue® Stabilized Substrate for Alkaline Phosphatase (ProtoBlot® II AP System, Promega) and quantified on an Automated Imaging System (Bio-Rad). Results were expressed as the ratio of the optical density of the protein band of interest to that of rat β-actin.

2.5. Measurement of serum atRA levels
Serum atRA levels were measured by reversed-phase high-performance liquid chromatography (RP-HPLC). Under reduced lighting conditions, blood samples were drawn from the tail vein 2 h after administration of atRA or vehicle on the day of treatment (day 0) and at 15 and 30 days after treatment. Serum (0.2 ml) was mixed with 0.2 ml of 0.25 M ammonium acetate (pH 3.8), 0.6 ml of acetonitrile (Sigma) and 1 ml of hexane. The resulting supernatant was extracted and evaporated to dryness, then reconstituted in 100 µl methanol (Sigma) and injected (50 µl) for analysis. RP-HPLC analysis was performed on a HP 1090 Liquid Chromatograph System by using a Symmetry® C18 analytical column (150 x 4.6 mm) (Ireland) at UV 340 nm with a flow rate of 1 ml/min and column temperature of 25 °C. Mobile phase consisted of 30 mM ammonium acetate/acetonitrile (15:85). A standard solution of atRA (Sigma) was used to obtain the calibration curves, from which serum atRA levels could be quantified. As presented in Fig. 2, a sustained elevation of serum atRA was observed in atRA-treated rats (WKY-R, SHR-L and SHR-H) after atRA administration. In the placebo-treated rats (WKY-C and SHR-C); however, serum samples contained essentially no measurable atRA.


Figure 2
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  		Fig. 2 Serum concentration of atRA over a 30-day time course. Blood was drawn from the tail vein of SHR and WKY rats 2 h after administration of atRA on the day of treatment (day 0) and at 15 and 30 days after treatment. All SHR-H measurements were statistically significantly different from SHR-L and WKY-R measurements (p<0.05). The measurements of serum atRA on day 30 were significantly higher than those of 15 days after atRA treatment (p<0.05). Mean ± S.E.M. values are shown. WKY-R indicates WKY treated with atRA; SHR-L, SHR treated with low-dose atRA; SHR-H, SHR treated with high-dose atRA; *p<0.05 vs. SHR-L; #p<0.05 vs. 15 days post-atRA-treatment.

 
2.6. Measurement of serum NO levels
Serum NO concentrations were determined with the Greiss reagent (Nanjing Jiancheng Bioengineering Institute, China) after reduction with nitrate reductase. The resulting supernatant of sample mixture with medium was measured at 550 nm with a Beckman DU650 spectrophotometer. Conditioned medium from untreated samples with no added Greiss reagent were used as sample blanks. Known concentrations of KNO2 and KNO3 were used as standards. All procedures were carried out according to the manufacturer's protocol.

2.7. Statistical analysis
Values are shown as mean ± S.E.M. Statistical analyses were performed with the use of ANOVA, followed by Bonferroni or Student–Newman–Keuls test, if appropriate. Significance was taken at p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Effects of atRA on blood pressure in SHR
Fig. 3 illustrates that SBP was markedly increased in SHR (SHR-C, SHR-L and SHR-H), compared with WKY-C rats (210 ± 11, 188 ± 8 and 185 ± 7 mm Hg vs.136 ± 9 mm Hg, p<0.01, respectively). Treatment with atRA significantly decreased SBP in both SHR-L and SHR-H, compared with SHR-C (188 ± 8 and 185 ± 7 mm Hg vs. 210 ± 11 mm Hg, p<0.05, respectively). However, no significant differences in SBP changes were shown between WKY-C and WKY-R rats (136 ± 9 vs.132 ± 8 mm Hg, p>0.05), indicating that chronic atRA treatment reduced SBP in SHR but had no effects in WKY rats.


Figure 3
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  		Fig. 3 Effects of atRA on systolic blood pressure. Values represent mean ± S.E.M. of eight or nine rats, with three successful readings taken from each rat. WKY-C indicates WKY rats control; WKY-R, WKY treated with atRA; SHR-C, SHR control; SHR-L, SHR treated with low-dose atRA; SHR-H, SHR treated with high-dose atRA; **p<0.01 vs. WKY-C; #p<0.05 vs. SHR-C.

 
3.2. Effects of atRA on APJ, apelin and AT1 mRNA expression in SHR
Fig. 4 indicates that, in placebo-treated SHR, a significant reduction of APJ and apelin mRNA expression and an obvious rise of AT1 mRNA expression were shown in heart and aorta, compared with WKY-C rats (p<0.01, respectively). In atRA-treated SHR (SHR-L and SHR-H), supplementation with atRA upregulated mRNA expression of APJ and apelin and downregulated mRNA expression of AT1 in heart and aorta, compared with SHR-C (p<0.05, respectively). However, in WKY rats, mRNA expression of APJ, apelin and AT1 were unchanged in heart and aorta after atRA treatment. The housekeeping gene GAPDH mRNA did not differ among the various experimental groups.


Figure 4
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  		Fig. 4 Quantification of APJ, apelin and AT1 mRNA in heart and aorta. Shown are mRNA copies x 10–6/ml. Rat GAPDH mRNA was measured as an internal control. WKY-C indicates WKY rats control; WKY-R, WKY treated with atRA; SHR-C, SHR control; SHR-L, SHR treated with low-dose atRA; SHR-H, SHR treated with high-dose atRA; **p<0.01 vs. WKY-C; #p<0.05 vs. SHR-C. Data are mean ± S.E.M. of eight or nine rats, with each sample assayed in triplicate.

 
3.3. Effects of atRA on APJ, apelin and AT1 protein expression in SHR
As shown in Fig. 5A, B and C, in placebo-treated SHR, both heart and aorta APJ and apelin protein expression were markedly depressed, whereas AT1 protein expression was significantly increased when compared with WKY-C rats (p<0.01, respectively). By contrast, in atRA-treated SHR (SHR-L and SHR-H), both heart and aorta APJ and apelin protein expression were significantly enhanced, whereas AT1 protein expression was significantly decreased compared with SHR-C (p<0.05, respectively). No differences in changes of APJ, apelin and AT1 protein expression were shown between WKY-C and WKY-R rats.


Figure 5
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  		Fig. 5 Quantification of APJ, apelin and AT1 protein in heart and aorta (A, B and C). The relative level of each protein was normalized to the amount of rat β-actin. Bars show mean ± S.E.M. (n=5, for all groups); WKY-C, WKY rats control; WKY-R, WKY treated with atRA; SHR-C, SHR control; SHR-L, SHR treated with low-dose atRA; SHR-H, SHR treated with high-dose atRA; **p<0.01 vs. WKY-C; #p<0.05 vs. SHR-C.

 
3.4. Effects of atRA on serum NO levels in SHR
Fig. 6 shows that serum NO was greatly reduced in placebo-treated SHR compared with WKY-C rats (p<0.01), but significantly elevated in atRA-treated SHR (SHR-L and SHR-H, p<0.05). No significant difference in serum NO change was shown between WKY-C and WKY-R rats, suggesting that atRA accelerated the release of NO in SHR but had no effect in WKY rats.


Figure 6
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  		Fig. 6 atRA-mediated changes of serum nitric oxide (NO) levels. Blood was drawn from SHR and WKY rats a month after treatment. Values are mean ± S.E.M. of eight or nine rats, with each sample assayed in triplicate. WKY-C indicates WKY control; WKY-R, WKY treated with atRA; SHR-C, SHR control; SHR-L, SHR treated with low-dose atRA; SHR-H, SHR treated with high-dose atRA; **p<0.01 vs. WKY-C; #p<0.05 vs. SHR-C.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The cardiovascular GPCR system plays an important role in the cardiovascular physiology modulation and blood pressure homeostasis via various signal transduction pathways [1,20]. In the GPCR family, AT1 is a vasoconstrictor whereas APJ acts as a vasodilator despite sharing an identity of 54% in the transmembrance regions with AT1 [9,11]. It has been increasingly validated that there are close relations between hypertension, AT1 and APJ receptor [9,11,18,21]. In the present study, both APJ and apelin mRNA and protein expressions were markedly depressed in placebo-treated SHR when compared with WKY rats, suggesting that there is an abnormal apelin-APJ signaling pathway in SHR. In atRA-treated SHR, there was a significant upregulation of APJ and apelin mRNA and protein expression in heart and aorta, accompanied by a reduction of AT1 expression, an elevation of serum NO levels and a subsequent decrease of blood pressure. However, in WKY rats, chronic atRA treatment had no effect on APJ, apelin and AT1 expression, serum NO levels and blood pressure. These results have confirmed and extended the effects of atRA on cardiovascular GPCR signaling and revealed the intriguing possibility that atRA might regulate the balance between apelin-APJ and angiotensin II-AT1 signaling and therefore elicit a beneficial effect on blood pressure in hypertension.

In this report, we have shown for the first time depressed gene and protein expression of APJ and apelin in SHR. The APJ cognate ligand apelin is secreted as a 77-amino acid preproprotein, and processed to 12-, 13- and 36-amino acid moieties, which are variably produced in different tissues and show varying activity levels [7–9]. Signaling of the apelin and angiotensin II peptide is mediated by GPCR related through structure and similarities of physiological function [22]. Both sequence and mRNA expression distribution analyses revealed similarities between apelin and angiotensin II, suggesting that they share related physiological roles such as the thirst-promoting and blood pressure-regulating actions [9 10]. However, the systemic administration in rats or mice revealed apelin to have hypotensive properties, eliciting a brief drop in blood pressure, in contrast to the well-known vasopressor effects of angiotensin II [9,11,12]. Very recent research has demonstrated that apelin-APJ signaling may function as a member of the vasorelaxation system in blood pressure regulation as apposed to the vasoconstriction system including angiotensin II-induced AT1 signaling [11]. An increased vasopressor response to the potent vasoconstrictor angiotensin II shown in APJ deficient mice indicates that the apelin-APJ system exerts the hypotensive effect in vivo and plays a counter-regulatory role against the pressor action of angiotensin II-AT1 [11]. Here, in placebo-treated SHR, the relative absence of apelin-APJ expression may be implicated in elevated levels of AT1 receptor and high blood pressure, whereas in atRA-treated SHR, the rise of apelin-APJ expression may result in the reduction of AT1 levels and the decrease of blood pressure. Given the fact that hypertension can be caused by deletion of genes encoding vasodilating GPCRs or by overproduction of vasoconstrictive GPCRs [1,3,11], it is reasonable to surmise that the vasoactive GPCR pair (AT1/APJ) may be a pivotal regulator in hypertension.

Another striking observation is that chronic atRA treatment impressively induced expression of APJ and apelin in SHR. In the present study, the dose of atRA (10 or 20 mg.kg–1.day–1) was selected according to the previous experimental research that showed an efficient reduction of blood pressure [15]. A sustained elevation of serum atRA was displayed in atRA-treated rats, which may be responsible for the potentiation of apelin-APJ signaling and the subsequent reduction of AT1 levels in SHR. Our findings provide novel evidence for a possible interaction between atRA and GPCRs signal pathways. Signals of atRA are mediated by binding the retinoic acid receptor (RAR) and retinoid X receptor (RXR) [14,23]. There are many known ways in which atRA can influence gene expression and protein production, but in terms of molecular mechanisms, a predominant, classical pathway has emerged: ligand involvement (atRA) plus receptor dimerization (RAR/RXR), DNA binding, and retinoic acid receptor response element (RARE) regulation [23]. Initially, atRA activates a heterodimer RAR/RXR, which further recognizes and binds to RARE consensus sequence, thereby activating or repressing target gene transcription [14,23]. A degree of variability can be tolerated in the sequence of RARE, which are located in a variety of GPCR genes [13]. We conjecture that atRA augments expression of APJ and its ligand apelin, perhaps by this pathway via the RAR/RXR-mediated signaling. It is hoped that future studies will begin to confirm some of the molecular mechanisms discussed above for the effects of atRA on APJ, AT1 and other GPCRs expression.

It is well recognized that NO, a potent vasodilator produced by endothelial NO synthase (eNOS), has been implicated in the regulation of blood pressure [21,24,25]. eNOS-deficient mice that exhibit a relative lack of the vasodilator effect of NO develop hypertension, whereas, in normotensive mice, higher NO levels could account for the "resistance" to hypertension [21,24,25]. Interestingly, in the present study, serum NO levels was significantly reduced in placebo-treated SHR compared with WKY control, but markedly elevated in atRA-treated SHR, suggesting that the elevation of NO may mediate certain antihypertensive action of atRA in SHR. Studies have substantiated that apelin-APJ signaling causes vasodilation and hypotension via the activation of the NO/L-arginine system [11,12]. Thus, chronic atRA treatment potentiates the activity of apelin-APJ signaling, which may, at least in part, contribute to the increase of serum NO levels and the subsequent beneficial effects on blood pressure in SHR. There are several possible reasons for stimulation of the NO production by apelin-APJ signaling. First, apelin can facilitate the activity of eNOS in endothelial cells and APJ is crucially involved in apelin-induced phosphorylation of eNOS, which promotes the release of NO [11,12]. Second, the apelin-APJ system may activate phospholipase C/protein kinase C pathway via pertussis toxin-sensitive Gi proteins, leading to augmented Ca2+ levels and subsequent elevation of NO production by promotion of eNOS activity [2,21]. Third, inducible NO synthase (iNOS) is also likely to mediate the regulation of apelin-APJ system in NO production in atRA-treated SHR. In vivo, supplementation with atRA enhanced the expression of rat iNOS in both mRNA and protein levels [26]. Finally, the apelin-APJ system plays a counter-regulatory role against angiotensin II-induced AT1 signaling, leading to the fall of AT1 expression and the subsequent reduction of NO inactivation [11,21].

Taken together, chronic atRA treatment augments gene and protein expression of orphan receptor APJ and its endogenous ligand apelin in both heart and aorta, resulting in a reduction of AT1 expression, an elevation of serum NO levels and a subsequent decrease of blood pressure in SHR. Our findings provide a novel insight into the role of atRA as a transcription-modulating drug, which may regulate the balance between apelin-APJ and angiotensin II-AT1 signaling and have potential clinical value in the prevention and treatment of human essential hypertension. The GPCR pair (AT1/APJ) may be a pivotal regulator in hypertension. An understanding of the relations between hypertension, the angiotensin II-AT1 and the apelin-APJ signaling pathway leads one to believe that available therapeutic strategies capable of restoring the homeostatic balance of these vasoactive agents would be effective in preventing or treating hypertension. Therefore, the present study provides the basis for further scientific inquiry relating to the possible effects of atRA on apelin-APJ signaling and other benefits to apply in hypertension.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by National Natural Science Foundation of China Grant 30370340. We gratefully acknowledge Drs Min-Jie Luo, Wen-Hong Luo, Ji-Kai Jiang and Yu-Cai Fu for their helpful advice and collaboration.


   Notes
 
Time for primary review 20 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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