2022 Volume 45 Issue 12 Pages 1825-1831
Endothelial dysfunction contributes to cardiometabolic disorders, including hypertension, obesity, and type 2 diabetes. Esaxerenone is a selective, nonsteroidal, high-affinity mineralocorticoid receptor blocker recently approved in Japan for the treatment of hypertension. Although imbalanced signaling between vasorelaxant and vasocontractile factors induced by endothelial stimulation is often observed in type 2 diabetic vessels, the effects of esaxerenone on endothelium-dependent responses in type 2 diabetes remain unclear. The aim of this study was to investigate the effect of esaxerenone on endothelium-dependent responses in superior mesenteric arteries isolated from type 2 diabetic Goto-Kakizaki (GK) rats. It was found that esaxerenone (3 mg/kg/d for 4 weeks, per os (p.o.)) partially ameliorated acetylcholine (ACh)-induced endothelium-derived hyperpolarizing factor (EDHF)-type relaxation and NS309, a potent activator of small- and intermediate-conductance Ca2+-activated K+ channels, -induced relaxation, and reduced ACh-induced endothelium-derived contracting factor (EDCF)-mediated contraction. These results suggest that esaxerenone ameliorates endothelial function through increased EDHF signaling and suppressed EDCF signaling.
The rising burden of type 2 diabetes (T2D) is a major concern in health care worldwide.1) Endothelial dysfunction is directly responsible for T2D.2) Accumulating evidences suggest that endothelium-dependent responses are altered, for example, impaired relaxation and enhanced contraction in various blood vessels in T2D animals and humans.3,4) These dysfunctions may be attributed to reduced signaling of endothelium-derived relaxing factors [EDRFs; mainly nitric oxide (NO), prostaglandin I2 (PGI2), and endothelium-derived hyperpolarizing factor (EDHF)] and increased signaling of endothelium-derived contracting factors [EDCFs; e.g., vasoconstrictor prostanoids, endothelin-1, and uridine adenosine tetraphosphate].2,4–6) The balance between EDRFs and EDCFs regulates vascular tone in both physiological and pathophysiological states. However, little information is available on the effect of these factors to normalize vasculopathy in T2D.
Aldosterone is a physiological ligand of mineralocorticoid receptor (MR) and is traditionally known as a steroid hormone that controls electrolyte homeostasis, body fluid, and blood pressure.7) Activation of MR in cardiovascular tissues reportedly induces oxidative stress, sustained inflammation, and excess accumulation of extracellular matrix.8) The MR is expressed in fibroblasts, cardiomyocytes, inflammatory cells, vascular smooth muscle cells, and endothelial cells (ECs).8) Spironolactone and eplerenone are clinically available steroidal MR antagonists. Diverse clinical studies have demonstrated that the blockade of MR is an effective therapeutic approach against heart failure, chronic kidney disease, and hypertension.8–10) The association between MR activation and vascular dysfunction in diabetic arteries is reported by various studies.10,11) For example, T2D db/db mice exhibited increased arterial expression of interleukin (IL)-1β and caspase-1, increased levels of IL-1β in plasma, active caspase-1 in macrophages, and impaired acetylcholine (ACh)-induced vasorelaxation as compared to control db/m mice.11) Treatment with MR antagonist spironolactone and an NLRP3 selective inhibitor MCC950 decreased plasma IL-1β and partly improved ACh-induced vasorelaxation in db/db mice.11) Spironolactone decreased active caspase-1-positive macrophages, which contribute to diabetes-associated vascular changes, suggesting that MR and NLRP3 activation contribute to diabetes-associated vascular dysfunction and proinflammatory phenotype.11) EC-MR activation reportedly accounts for diabetes-induced endothelial dysfunction in experimental diabetes [low-dose streptozotocin (STZ)-injected mice] using EC-specific MR knockout mice.12) These evidences indicate that the manipulation of MR activation may be one of the important strategies to prevent and/or treat diabetes-associated vascular dysfunction.
Esaxerenone, has higher MR-binding specificity and nonsteroidal structure13) and is recently approved as an MR blocker in Japan.14) Esaxerenone has greater potency than spironolactone or eplerenone for treating hypertension.15,16) Esaxerenone (3 mg/kg/d for 4 weeks) clearly ameliorated existing renal injury without lowering systolic blood pressure (SBP) in rats treated with deoxycorticosterone acetate (DOCA), which suggested that esaxerenone regressed renal injury independent of its antihypertensive action.16) Extrarenal effects of esaxerenone have recently gained attention. The effect of esaxerenone on vascular function of T2D remains unclear. The Goto-Kakizaki (GK) rat is a highly inbred strain of Wistar rat that spontaneously develop T2D.17) The GK rat is a nonobese, nonhypertensive model of T2D.17) GK rats are reported to display impaired relaxation and increased contraction in various blood vessels.18,19) However, no study focuses on the effect of esaxerenone on endothelium-dependent responses, especially EDHF- and EDCF-mediated responses in T2D GK rats.
This study investigated the effect of esaxerenone treatment on vasorelaxant responses induced by endothelial stimulators in superior mesenteric arteries (SMAs) isolated from GK rat at the chronic stage of T2D.
Male GK and its control Wistar rats (4 weeks old) were obtained from Clea (Tokyo, Japan) and were housed under standard laboratory conditions with free access to water and food until the rats were 31 or 32 weeks old. Some GK rats were given esaxerenone (3 mg/kg/d, per os (p.o.)) for 4 weeks starting at 31 or 32 weeks. Therefore, this study comprised three groups: vehicle (0.5% methylcellulose solution)-treated Wistar and GK group and esaxerenone-treated GK groups (hereafter termed Wistar, GK, and Esax-GK, respectively). This study was approved by the Hoshi University Animal Care and Use Committee, which was accredited by the Ministry of Education, Culture, Sports, Science, and Technology, Japan (Approval No. P21-035). Esaxerenone was provided by Daiichi Sankyo Co., Ltd. (Tokyo, Japan)
Measurement of Blood Pressure and Blood ParametersSBP was measured using the tail cuff method 24-d after treatment with vehicle or esaxerenone.4)
The nonfasting blood glucose levels were assessed by a glucose meter sampling from the anesthetized rat tail vein. The serum insulin level was measured by enzyme immunoassay (Shibayagi Co., Ltd., Gunma, Japan).
Measurement of Isometric Force in SMARats were anesthetized with isoflurane and euthanized by thoracotomy and exsanguination. After euthanasia, the SMA was isolated and placed in an ice-cold, oxygenated, modified Krebs–Henseleit Solution (KHS). The 2-mm-long SMA segments were suspended by a pair of stainless-steel pins in a well-oxygenated bath containing KHS at 37 °C. The rings were stretched until an optimal resting tension of 9.8 mN was loaded, and allowed to equilibrate for at least 30 min. An isometric transducer (model TB-611T; Nihon Kohden, Tokyo, Japan) was used to monitor force generation as previous studies were using SMAs of rats.4,18) Arterial integrity was assessed by high K+ (80 mM)-induced contraction and subsequently with phenylephrine (PE; 10−6 M), followed by ACh (10−6 M)-induced relaxation.
Once PE (10−6 M)-mediated contraction had stabilized, relaxations were induced by a cumulative manner using one of the following: ACh or sodium nitroprusside (SNP). Concentration–relaxation curves for ACh, NS309, an activator of small- and intermediate-conductance calcium-activated K+ channels (SKCa and IKCa), or GSK1016790A, an agonist of transient receptor potential vanilloid type 4 (TRPV4), were generated in the combined presence of an inhibitor of nitric oxide synthase (NOS) L-NNA (10−4 M) and an inhibitor of cyclo-oxygenase (COX) indomethacin (10−5 M) before precontraction with PE (10−7 M).
SMA rings were treated with L-NNA (10−4 M) for 30 min, and subsequently, cumulative applications of ACh, ATP, or uridine 5′-triphosphate (UTP) were conducted for the contraction studies.
Statistical AnalysisData were expressed as the mean ± standard error (S.E.), and n represents the number of rats. The vasorelaxant responses were expressed as a percent reduction of the precontraction level; the vasocontractile responses were expressed as an absolute force (mN) per mg ring tissue. Graph Pad Prism software (San Diego, CA, U.S.A.) was used for data analysis. Significance was analyzed by using repeated-measures two-way ANOVA with the Tukey multiple comparison tests for concentration–response curves. One-way ANOVA followed by Tukey multiple comparison testing was performed to determine the significance level of other parameters. A p-value < 0.05 was considered significant.
The body weight at the time of the experiment and SBP after 24-d treatment starting at 31- or 32-week were found to be significantly lower in GK than in Wistar. The nonfasting blood glucose levels were found to be significantly higher in GK than in Wistar. Serum insulin levels were similar between Wistar and GK. Treatment with esaxerenone did not change the body weight, glucose, insulin, and SBP (vs. untreated GK rats) (Table 1).
| Wistar | GK | Esax-GK | |
|---|---|---|---|
| Body weight (g) | 516.0 ± 10.6 (10) | 351.6 ± 3.2 (10)a) | 348.1 ± 3.5 (10)b) |
| Glucose (mg/dL) | 100.5 ± 4.9 (10) | 239.9 ± 30.5 (10)a) | 236.2 ± 22.5 (10)b) |
| Insulin (ng/mL) | 1.39 ± 0.53 (10) | 1.22 ± 0.11 (9) | 1.44 ± 0.11 (10) |
| Systolic blood pressure (mmHg) | 131 ± 1 (10) | 118 ± 4 (10)a) | 123 ± 3 (10) |
Values are presented as the mean ± S.E. The number of experiments is shown within parentheses. a) p < 0.05, Wistar vs. GK. b) p < 0.05, Wistar vs. Esax-GK (by one-way ANOVA with Tukey’s post hoc test).
ACh-induced relaxation in SMA was significantly weaker in GK at 10−6 to 10−5 M than in Wistar. In Esax-GK, such relaxation was slightly increased at 3 × 10−6 to 10−5 M compared to GK. However, the relaxation was significantly weaker at 3 × 10−6 to 10−5 M in Esax-GK than in Wistar (Fig. 1A).
The ordinate shows the relaxation response normalized by phenylephrine (PE)-induced precontraction, which contractile forces were not significantly altered among three groups. n = 9 or 10. ap < 0.05, Wistar vs. GK. bp < 0.05, Wistar vs. Esax-GK.
SNP-induced relaxation was significantly weaker in GK at 3 × 10−8 to 10−7 M than in Wistar. The relaxation was similar between GK and Esax-GK. This relaxation was significantly weaker in Esax-GK at 10−8 to 10−7 M than in Wistar (Fig. 1B).
Treatment with Esaxerenone Partly Normalized the EDHF-Type Relaxation in SMA of GK RatsThe cumulative effect of ACh to rings precontracted by PE in the presence of L-NNA (10−4 M) and indomethacin (10−5 M) to block NOS and COX, respectively, was investigated to understand the EDHF-type relaxation. The EDHF-type relaxation was significantly weaker in SMA rings of GK than Wistar. Esaxerenone treatment significantly improved the relaxation (Fig. 2A).
n = 9 or 10. ap < 0.05, Wistar vs. GK. bp < 0.05, Wistar vs. Esax-GK. cp < 0.05, GK vs. Esax-GK.
The activation of SKCa and IKCa initiated EDHF-mediated relaxation.20) NS309-mediated relaxation in the presence of L-NNA (10−4 M) and indomethacin (10−5 M) was assessed to understand the effect of esaxerenone on responses induced by these K+ channels. The NS309-induced relaxation was reported to be slightly weaker (but significantly weaker at 10−6 M) in GK than in Wistar (Fig. 2B). Notably, this relaxation was significantly improved in Esax-GK than in GK.
An increase in the intracellular Ca2+ and the activation of TRPV4 generate EDHF signaling in ECs.20,21) GSK1016790A-mediated relaxation in the presence of L-NNA and indomethacin was observed to investigate the effect of esaxerenone treatment on responses mediated by TRPV4 activation. The GSK1016790A-mediated relaxation was significantly weaker in GK than in Wistar (Fig. 2C). The GSK1016790A-mediated relaxation was slightly increased compared to GK, whereas, the relaxation was still significantly weaker compared to Wistar than in Esax-GK.
Effects of Esaxerenone on Endothelium-Mediated Contraction in GK RatsSince vascular responsiveness to endothelium stimulators including ACh and extracellular nucleotides is regulated by EDRFs and EDCFs,4–6) the contractile component in SMAs in the presence of L-NNA was investigated. ACh-induced contraction was observed in GK (Fig. 3A). This contraction was significantly weaker in Esax-GK than in GK. When ATP was added cumulatively to SMA rings in the presence of L-NNA, a contraction was observed in all groups. The ATP-induced contraction was found to be significantly higher in GK than in Wistar. The ATP-induced contraction was slightly weaker in Esax-GK than in GK. When UTP was added cumulatively to SMA rings in the presence of L-NNA, a contraction was observed in all groups. The UTP-induced contraction was observed to be significantly higher in GK than in Wistar. This was greater in Esax-GK than in Wistar.
n = 9 or 10. ap < 0.05, Wistar vs. GK. bp < 0.05, Wistar vs. Esax-GK. cp < 0.05, GK vs. Esax-GK.
Two major effects of chronic esaxerenone treatment of T2D GK rats were detected in the present study: an improvement of endothelium-dependent responses such as increased EDHF-type relaxation and suppressed EDCF-mediated contraction in isolated SMAs.
Orally administration of esaxerenone could reportedly produce strong MR blockers because of sustained exposure and have greater antihypertensive effects than spironolactone or eplerenone in a DOCA-salt hypertensive rat model.13) Esaxerenone reportedly lowers blood pressure in several hypertensive populations such as essential hypertension and those with chronic kidney disease and/or diabetes.22) Esaxerenone could regress renal injury without affecting blood pressure in the model of established hypertension.13,23) These evidences suggest that esaxerenone may have beneficial effects on various functions independent of blood pressure regulation. In the present study, esaxerenone did not affect glucose and insulin levels and SBP in GK rats, which suggested that this could improve endothelial function without changing metabolic rate and blood pressure.
NO is a major EDRF. Several studies suggest the role of MR activation on NO signaling. EC-MR contributes NO bioavailability and oxidative stress through rapid nongenomic signaling in addition to its traditional gene-transcription role.24) Moreover, treatment with esaxerenone (3 mg/kg/d, for three weeks) in STZ-induced diabetic C57BL/6 mice, starting at three days after STZ injection, ameliorated ACh-induced aortic relaxation through increased endothelial NOS (eNOS) phosphorylation at Ser1177 without affecting metabolic parameters, renal function and blood pressure.25) In the present study, treatment with esaxerenone did not alter SNP-induced relaxation in SMAs of GK rats. This finding suggested that sensitivity of NO in vascular smooth muscle cells was not affected by esaxerenone. However, it was not clear whether treatment with esaxerenone could change in the NO generation, eNOS status (i.e., phosphorylation, dimer/monomer, and cofactors) in SMAs of T2D GK rats.
EDHF is another EDRF and plays a pivotal role in smaller arteries.6) In SMA of rats, the ACh-induced relaxation is mediated by both NO and EDHF.4) The ACh-induced EDHF-type relaxation was impaired in T2D arteries,4,26) although the mechanisms and causal factors responsible for this remain unexplored. The signaling of EDHF is initiated by endothelial intracellular calcium levels induced by blood flow and by several ligands following activation of SKCa and IKCa channels.6,20) Activations of SKCa and IKCa reportedly contribute to EDHF-type relaxation in arteries and impaired these channel-mediated relaxations in diabetic arteries.26) In a previous study, we discovered that ACh-induced EDHF-type relaxation was impaired in SMAs of GK rats compared with age-matched control Wistar rats.27) This impairment was caused by a decrease in SKCa and IKCa activities as each specific inhibitor reduced the ACh-induced EDHF-type relaxation in Wistar arteries but was ineffective in GK arteries.27) TRPV4 channel activation has been linked to increased intracellular calcium levels in endothelium and mediating EDHF-type relaxation.21) In the current study, ACh-induced EDHF-type relaxation was impaired in GK rats, which was partly alleviated by esaxerenone treatment. Furthermore, the SKCa/IKCa activator NS309-induced relaxation in GK rats was reduced, which was partially alleviated by esaxerenone treatment. The agonist of TRPV4 GSK1016790A-induced relaxation, in contrast, was impaired in GK rats and was slightly but not significantly increased by esaxerenone treatment. These findings and supporting evidence suggest that esaxerenone may improve ACh-induced EDHF-type relaxation, which can be attributed to increased SKCa/IKCa-mediated signaling. Numerous studies have suggested that ACh-induced EDHF-type relaxation was independent of the TRPV4-mediated pathway in some arteries, including SMAs of rats.28) It is currently unknown how esaxerenone affects endothelial intracellular calcium levels in SMA of GK rats by altering TRPV4-dependent and -independent events in response to ACh stimulation. TRPV4 and SKCa/IKCa channel gene expression changes are reportedly prevented by MR antagonists in isolated cerebral arteries of angiotensin II-infused mice.29) As a result, the gene expressions of putative EDHF generators in ECs, such as TRPV4 and SKCa/IKCa channels, may be affected by sustained MR blockade by esaxerenone in GK rats’ SMA; further research is needed. Furthermore, the putative mediator of EDHF-type relaxation could be several factors, as evidenced by the heterogeneity among species, vessel types, and (patho)physiological states.6,30) Different factors, such as hydrogen peroxide, potassium ion, and nondiffusible phenomena through myoendothelial gap junctions, could all contribute to EDHF-type relaxation.6,26,30) For example, in the case of the MR-gap junction relationship, aldosterone activation disrupted the gap junction.31) Furthernore, MR blockade inhibits the development of gap junction remodeling associated with pathological cardiac hypertrophy at both at the molecular and functional levels.32) Because ACh-induced EDHF-type relaxation is partly mediated by gap junction in rat SMA,33) chronic MR blockade by esaxerenone may alter not only gap junction activity but also gene/protein expressions of connexins, which are gap junction components. Therefore, more research will be needed to determine the extent to which esaxerenone can control the generation/release of EDHF and/or function in the gap junction in diabetic arteries.
Reportedly, the abnormal production/release of EDCFs and/or their signaling are seen in diabetic arteries.4–6) One of the endothelium-dependent contractions is mediated by metabolites of COXs and mainly through the thromboxane–prostanoid (TP) receptor activation on smooth muscle.5,6) ACh-induced endothelium-dependent contractions and release of prostanoids were found to be increased in SMAs of T2D Otsuka Long-Evans Tokushima fatty rats.4) In the present study, it was found that esaxerenone could suppress the increased ACh-mediated contraction in the GK artery. Extracellular nucleotides could stimulate endothelium and release various substances from ECs.18,34) In SMAs of GK rats (vs. those from age-matched control Wistar rats), ATP- and UTP-mediated contractions, which were inhibited by the non-selective P2 receptor antagonist, were augmented, and these augmentations were reduced by endothelial denudation and COX inhibition.18) Esaxerenone slightly reduced ATP-mediated contraction but not UTP-mediated contraction. Although the difference of suppressive effect of esaxerenone on contractions induced by ACh, ATP, and UTP remains unclear, the production of prostanoids and/or sensitivities of prostanoid receptor on vascular smooth muscle cells may vary in the esaxerenone treatment.
Vascular tone is regulated by EDRFs and EDCFs. Endothelial dysfunction may occur by decreased EDRFs signaling or by increased EDCF signaling.5,6) The generation/release of EDCFs is tempered by the presence of NO5,6) and EDHFs in healthy ECs.5,6,35) Endothelium-dependent contractions were relatively weak under basal states, and inhibition of NOS augmented them.5,6,18) The production of EDCF may predominate in cases of impaired bioavailability of EDRFs. TP receptor activation suppressed SKCa activity and impaired EDHF-mediated relaxation in the mesenteric artery of rat.36) A defect of EDHF-induced responses facilitated the EDCF-induced responses in the rat renal artery.35) These evidences suggest that these EDRF/EDCF signaling make cross-talk rather than independent pathway in tuning vascular tone. However, it is not known whether increased EDHF-mediated response or suppressed EDCF-mediated response is the prior event or simultaneous event during esaxerenone treatment in GK artery.
This study has certain limitations. Mueller et al.37) demonstrated that the deletion of EC-MR did not change renal sodium handling, basal blood pressure, or blood pressure after various hypertensive stimuli; EC-MR did not contribute to basal endothelium-dependent relaxation in coronary or mesenteric arterioles, and deletion of EC-MR could improve endothelial function in mesenteric arterioles but not in coronary arterioles after exposure to angiotensin II hypertension. Although EC-MR may play a minor role in healthy vessels, it contributes to the development of endothelial dysfunction in response to various risk factors such as hypertension37,38) and obesity.39) In this study, we discovered that blocking MR with esaxerenone could partially normalize endothelial function, including increased EDHF and suppressed EDCF responses in T2D SMA. The differences in vascular responses between the EC-MR deleted mouse and the T2D GK rat can be attributed to species differences, vessel types, and pathophysiological states, as well as EC-specific deletion and blockade of the entire MR. Further research into the role of MR in ECs and the mechanisms involved in the esaxerenone-induced improvement of endothelial dysfunction in various T2D arteries will be required. Furthermore, while the current study indicates that MR blockade is an important strategy against T2D-associated vascular dysfunction, whether these beneficial effects in T2D arteries are caused by esaxerenone or other MR blockers, such as spironolactone and eplerenone, remains unknown. More research will be needed to determine the role of MR blockers in the onset and/or progression of T2D vascular dysfunction.
In conclusion, this study showed esaxerenone could improve endothelial function in SMAs of T2D GK rats, by increasing EDHF-mediated relaxations and reducing EDCF-mediated contractions. These study findings should stimulate further interest in esaxerenone as a potential therapeutic drug for use against diabetes-associated vasculopathy.
The authors would also like to thank Y. Ezaki, M. Ito, H. Yokoyama, Y. Mae, Y. Sato, A. Kurakata, K. Ozawa, and T. Katome for the technical assistance. This study was partly supported by JSPS KAKENHI [JP21K06811 (K.T.) and JP21K06878 (T.K.)].
This study was a collaborative study supported by Daiichi Sankyo Co., Ltd. The Funder had no role in finalizing the study design, execution of experiments, decision to publish, or preparation of the manuscript.
