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. 2024 Nov 1;14:26266. doi: 10.1038/s41598-024-78321-6

Effect of esaxerenone on the onset of aortic endothelial dysfunction and circulating microparticles in type 1 diabetic male mice

Kumiko Taguchi 1,#, Hiroyuki Kondo 1,#, Takayuki Matsumoto 1,2, Tsuneo Kobayashi 1,✉
PMCID: PMC11530539  PMID: 39487333

Abstract

Endothelial dysfunction exacerbates hypertension and other vascular complications in diabetes mellitus (DM). Circulating microparticles (MPs) and extracellular vesicles released in patients with DM have emerged as novel regulators of endothelial dysfunction. The obstruction of mineralocorticoid receptors (MRs) is a potential therapeutic approach to reduce cardiovascular complications. Their impact on the obstruction of MRs on circulating MPs and endothelial dysfunction in DM remains unclear. DM was induced in mice through a single intravenous dose of streptozotocin (STZ; 200 mg/kg). Esaxerenone (ESAX; 3 mg/kg/day), a MR blocker was administered via diet for 8 weeks. In this study, the aortas of the DM group showed the endothelial dysfunction and the administration of ESAX ameliorated the endothelial-dependent responses. Moreover, ESAX influences the impaired endothelial-dependent responses of DM-derived MPs. Interestingly, MP levels increased in DM whereas decreased after ESAX administration. In the aorta, the DM-derived MPs increased the expression of intercellular adhesion molecule-1 (ICAM-1). ESAX inhibited the adhesion of DM-derived MPs. Moreover, the ICAM-1 inhibitor A205804 shows similar effects as ESAX. These results indicate that the release and adhesion properties of MPs can be partially obstructed by ESAX via the ICAM-1 signaling pathway, which clarifies the other functions beyond the anti-hypertensive effects of ESAX.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-78321-6.

Keywords: Diabetes, Endothelial dysfunction, Microparticles, ICAM-1, Mineralocorticoid receptor, Esaxerenone

Subject terms: Cardiovascular biology, Circulation, Cardiovascular diseases

Diabetic cardiovascular disease is a major cause of mortality and morbidity in patients with diabetes mellitus (DM). Vascular endothelial dysfunction is considered a pathophysiological hallmark characterized by DM1. Endothelial dysfunction refers to the condition where the endothelium loses its physiological properties but shows a tendency toward vasoconstrictive, pro-thrombotic, and pro-inflammatory states1. In addition, the reduction in the bioavailability of nitric oxide (NO) subsequent to the reduction of endothelial NO synthase (eNOS) is characteristic of vascular endothelial dysfunction, and eNOS is involved in the regulation of vascular function2. In the circulatory system, NO derived from eNOS is a critical signaling molecule that functions as a crucial vasoactive factor related to endothelial-dependent relaxation2.

Recent preclinical and clinical studies have shown that DM impairs vascular endothelial-dependent relaxation, and mineralocorticoid receptors (MRs) play a crucial role in the development of vascular dysfunction in DM3,4. Moreover, MR antagonists increase eNOS activation and reverse vascular dysfunction5–7. Esaxerenone (ESAX) is a new nonsteroidal MR blocker with higher potency and MR selectivity8. However, the effect of ESAX on the vascular endothelial-dependent function in DM has not been fully investigated. Therefore, we investigated whether ESAX ameliorates diabetes-induced endothelial dysfunction in diabetic mice.

Circulating microparticles (MPs) are submicron vesicular fragments of cells that are released by various cells, such as activated platelets and endothelial cells under stress or injury conditions9,10. A number of studies have revealed that circulating MPs in pathological conditions can lead to the dysfunction or impairment of vascular endothelial cells9,11,12. Our previous work demonstrated that the amount of platelet-derived MPs increases in DM and that the rising MP levels impair vascular endothelial-dependent relaxation by releasing endothelium-derived MPs containing eNOS13–15.

Thus, MPs are considered markers of endothelial dysfunction in DM. Whether MPs participate in the influence of the DM condition and vascular endothelial dysfunction remains unclear. Furthermore, as a MR blocker, ESAX has not been widely used for DM. It was reported that MR stimulation could increase the concentration of MPs and impair endothelial dysfunction16. However, whether ESAX, as a new selective MR blocker, would affect the function of MPs and its mechanism in DM remains unclear. This study aimed to evaluate the effect of ESAX on the function of MPs in DM.

Results

Body weight and non-fasting plasma glucose and insulin levels

Table 1 shows no significant differences between the Control and Control + ESAX groups or between the DM and DM + ESAX groups in body weight and non-fasting glucose and insulin levels. However, in the DM and DM + ESAX groups the body weight and plasma insulin levels were significantly lower than the Control group, and the plasma glucose levels were higher than the Control group.

Table 1.

Body weight and non-fasting plasma glucose and insulin changes in DM mice.

Control Control
+ESAX		 DM DM
+ESAX
Body weight (g) 56.3 ± 1.5 56.3 ± 1.6 37.6 ± 1.3* 37.5 ± 0.9*
Plasma glucose (mg/dL) 221.2 ± 11.1 230.7 ± 11.2 975.6 ± 52.0* 1105.0 ± 49.6*
Plasma insulin (ng/mL) 2.0 ± 0.4 2.2 ± 0.6 0.1 ± 0.1* 0.1 ± 0.1*
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All values are presented as the mean ± SE (n = 6–19).

ESAX esaxerenone.

*P < 0.05 vs. the Control.

ESAX alleviated the endothelial dysfunction associated with DM

The aortas of the Control, Control + ESAX, DM, and DM + ESAX groups were dissected for vascular functional assays. Compared with the Control group, the acetylcholine (ACh)-induced endothelial-dependent relaxations were markedly reduced in the DM group (Fig. 1

Fig. 1.

Fig. 1

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Effect of ESAX on vascular functional responses and NO production in the aortas of Control and DM groups. (A, B) Effect of ESAX on acetylcholine (ACh)-induced (A) and sodium nitroprusside (SNP)-induced (B) relaxations and of prostaglandin F2a (PGF2a)-contracted aortic rings from the control and DM groups. (C) Effect of ESAX on NO production in the aortic rings from the control and DM groups. Total vascular nitrate/nitrite (NOx) measurement. The aortic rings of the Control, Control + ESAX, DM, or DM + ESAX were determined according to the levels of ACh (1 × 10− 6 M, for 20 min)-stimulated NO production. (D, E) Effect of ESAX on the phenylephrine (PE)-induced contraction of aortic rings from the Control and DM groups in the absence (D) or presence (E) of NG-nitro-L-arginine (L-NNA; 1 × 10− 4 M, for 30 min). The percentage was calculated from the highest peaks of the potassium-induced contraction. Data are expressed as the mean ± SE (n = 7–15). *P < 0.05 vs. Control. †P < 0.05 vs. DM.

1A; Table 2). Treatment with ESAX at 3 mg/kg/day for 8 weeks significantly reversed these impairments in the aortas. On the other hand, DM or DM + ESAX treatment groups did not affect the sodium nitroprusside (SNP)-induced endothelial-independent relaxation (Fig. 1B; Table 2), which indicates that the vascular smooth muscle function response to NO was unaltered. Furthermore, impairment of the functional response was supported by a decline in ACh-stimulated NOx production in the DM group (Fig. 1C). ESAX did not increase the ACh-stimulated NO production in the Control group, whereas ESAX treatment significantly enhanced the ACh-stimulated NO production level in the DM group.

Table 2.

Maximal responses (Emax) and pD2 values for ACh-, SNP-, and PE-induced vascular response in mice aorta.

Agonist/
Treatment		 Control Control-ESAX DM DM-ESAX
Emax pD2 Emax pD2 Emax pD2 Emax pD2
ACh 89.8 ± 0.9 7.45 ± 0.03 88.7 ± 1.9 7.49 ± 0.06

*

65.6 ± 1.4

*

6.87 ± 0.04

 88.9 ± 1.5

#

7.16 ± 0.03
SNP 100.0 ± 2.6 8.22 ± 0.10 100.0 ± 2.0 8.24 ± 0.08 99.3 ± 1.7 8.65 ± 0.10 96.7 ± 3.93 8.29 ± 0.17
PE 47.7 ± 3.0 6.11 ± 0.08 44.8 ± 3.5 5.97 ± 0.10

*

77.7 ± 2.27

*

6.63 ± 0.05

 48.6 ± 5.6

#

6.08 ± 0.16
PE/L-NNA 114.1 ± 2.9 6.89 ± 0.05 116.3 ± 3.0 6.83 ± 0.05 105.1 ± 2.5 7.12 ± 0.05 107.2 ± 2.7 6.95 ± 0.05
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Data are expressed as the mean ± SE (n = 7). Emax: maximal agonist-induced response expressed as % of precontraction (ACh- and SNP-induced) or 80-mM high K (PE-induced); pD2: −log (corresponding agonist) required to produce 50% of the maximal response.

*P < 0.05 vs. the corresponding control group.

#P < 0.05 vs. the corresponding DM group.

In addition, compared with the Control group, the DM condition increased the phenylephrine (PE)-induced contractile response in the aortas (Fig. 1D; Table 2). Treatment with ESAX decreased the sensitivity to PE in the DM group (Fig. 1D). To investigate the putative role of NO, the aortas were incubated with the NOS inhibitor L-NNA (100 µM). Under NOS inhibition the PE-induced contractile response in the aortas obtained from each experimental group increased and did not modify the PE-induced contractile response in all groups (Fig. 1E; Table 2), which suggests that the endothelial negative modulation to a1-adrenergic receptor agonist-induced contraction was reduced with ESAX treatment.

ESAX increased the resilience to MP-induced endothelial dysfunction

To determine the effect of ESAX on the MP-incubated vascular responses, we assessed the ACh-induced endothelial-dependent relaxation, SNP-induced endothelial-independent relaxation, and PE-induced contraction in the aortic rings incubated with the Control-derived MPs, Control + ESAX-derived MPs, DM-derived MPs, and DM + ESAX-derived MPs (Fig. 2; Table 3). DM-derived MPs significantly decreased the ACh-induced relaxations in the aortic rings compared with the vehicle alone and Control-derived MPs, whereas the Control-derived MPs, Control + ESAX-derived MPs, and DM + ESAX-derived MPs had no effect (Fig. 2A; Table 3). Notably, the DM + ESAX-derived MPs significantly enhanced the ACh-induced endothelial-dependent relaxation compared with the DM-derived MPs. On the other hand, MP stimulation did not cause any significant change in the SNP-induced endothelial-independent relaxations (Fig. 2B; Table 3). Furthermore, impairment of the functional response was supported by the decline in ACh-stimulated NOx production in the DM-derived MPs (Fig. 2C). The effect of ESAX did not increase the ACh-stimulated NO production in the Control + ESAX-derived MPs, but the effect of ESAX significantly increased the ACh-stimulated NO production level in the DM + ESAX-derived MPs. In addition, compared with the vehicle alone and Control-derived MPs, PE significantly induced contraction of the aortic rings in the DM-derived MP treatment. However, the tension of the aortic rings in the treatment of the DM + ESAX-derived MPs did not show a significant difference compared to the treatment with the vehicle and Control-derived MPs (Fig. 2D; Table 3). The aortic rings were incubated with L-NNA before PE-induced contractions. Pretreatment with L-NNA significantly augmented the PE-induced contractile response in all groups, and the presence of L-NNA abolished the differences between the DM-derived MPs and the other groups (Fig. 2E; Table 3), which suggests that the endothelial negative modulation caused by DM-derived MPs against PE-induced contraction was reduced in the MPs produced by ESAX administration.

Fig. 2.

Fig. 2

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Effect of ESAX on the vascular effects of MPs assessed via vascular functional response and NO production in the aortas of the Control and DM groups. (A, B) ACh (A) and SNP (B)-induced relaxation responses in the vehicle (PBS)-treated Control aortas, or those incubated with Control-derived MPs, Control + ESAX-derived MPs, DM-derived MPs, or DM + ESAX-derived MPs (1 × 105 events/mL, for 30 min). (C) ACh-stimulated NO production in the Control aortic rings under MP treatment. Total vascular nitrate/nitrite (NOx) measurement. The aortic rings of the Control were used to determine the levels of ACh (1 × 10− 6 M, for 20 min)-stimulated NO production under Control-derived MPs, Control + ESAX-derived MPs, DM-derived MPs, or DM + ESAX-derived MPs (1 × 105 events/mL, 30 min). (D, E) PE-induced contraction response in vehicle-treated control aortas, or those incubated with Control-derived MPs, Control + ESAX-derived MPs, DM-derived MPs, or DM + ESAX-derived MPs (1 × 105 events/mL, for 30 min) in the absence (D) or presence (E) of NG-nitro-L-arginine (L-NNA; 1 × 10− 4 M, 30 min). The percentage was calculated from the highest peaks of the 80 mM high K+-induced contraction. Data are expressed as the mean ± SE (n = 5–9). *P < 0.05 vs. vehicle (+ veh). †P < 0.05 vs. DM-derived MPs (+ DM MPs). #P < 0.05 vs. Control-derived MPs (+ Cont MPs).

Table 3.

Maximal responses (Emax) and pD2 values for ACh-, SNP-, and PE-induced vascular response in mice aorta treated with MPs induced by control, control-ESAX, DM, and DM-ESAX.

Agonist/
Treatment		 +veh +Cont MPs +Cont-ESAX MPs +DM MPs +DM-ESAX MPs
Emax pD2 Emax pD2 Emax pD2 Emax pD2 Emax pD2
ACh 91.2 ± 1.9 7.42 ± 0.05 93.1 ± 1.5 7.35 ± 0.04 90.0 ± 2.0 7.27 ± 0.05

*

76.3 ± 1.6

#

*

6.91 ± 0.04

 88.9 ± 2.5

†

7.14 ± 0.06
SNP 100.0 ± 3.4 8.83 ± 0.26 100.0 ± 5.9 9.12 ± 0.66 98.4 ± 4.9 8.58 ± 0.29 99.4 ± 3.5 8.46 ± 0.18 100.0 ± 1.9 8.84 ± 0.13
PE 52.0 ± 3.0 6.25 ± 0.08 51.7 ± 3.3 6.38 ± 0.09 50.2 ± 5.6 6.12 ± 0.15 75.4 ± 2.0

*

6.40 ± 0.04

 66.1 ± 4.1

*

6.23 ± 0.09
PE/L-NNA 115.6 ± 3.6 6.98 ± 0.06 106.3 ± 3.7 6.91 ± 0.06 105.6 ± 3.5 7.08 ± 0.06 106.0 ± 3.4 6.83 ± 0.06 111.8 ± 2.4 6.93 ± 0.04
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Data are expressed as the mean ± SE (n = 6–9). Emax: maximal agonist-induced response expressed as % of precontraction (ACh- and SNP-induced) or 80-mM high K (PE-induced); pD2: −log (corresponding agonist) required to produce 50% of the maximal response.

*P < 0.05 vs. the corresponding vehicle group.

#P < 0.05 vs. the corresponding DM group.

†P < 0.05 vs. the corresponding DM.

Effect of ESAX on ERK1/2 and eNOS in MPs

Platelet-derived MPs represented the majority of circulating MPs in DM17,18. Levels of platelet-derived MPs have been shown to be correlated with DM complications19. To study the protective effect of ESAX on MPs, we first determined if ESAX could suppress the production and release of MPs by platelets under DM conditions. As shown in Fig. 3A, the DM group released significantly more platelet-derived MPs than the Control group. Although there was no significant difference in the release of platelet-derived MPs in the Control + ESAX group, the increased release of platelet-derived MPs in the DM group was significantly reduced after ESAX administration.

Fig. 3.

Fig. 3

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Characterization of MPs isolated from the Control, Control + ESAX, DM, and DM + ESAX groups. The count of platelet-derived MPs (A), eNOS (B), ERK1/2 (C), and phosphorylated ERK1/2 (D) levels was measured by ELISA. Data are expressed as the mean ± SE (n = 5–6). *P < 0.05 vs. Control-derived MPs or Control. †P < 0.05 vs. DM-derived MPs or DM.

A previous study showed that DM-derived MPs had more activated ERK1/2 and emitted eNOS-containing endothelial-derived MPs from the endothelial cells to the blood15. Therefore, to further assess the impact of ESAX on the MP contents, we examined eNOS and extracellular signal-regulated kinase 1/2 (ERK1/2) in the MPs. As shown in Fig. 3B, DM-derived MPs had significantly more eNOS than the Control-derived MPs. Consistent with the MP release, eNOS-containing MPs were not different in the Control + ESAX group, whereas increased eNOS-containing DM-derived MPs were significantly reduced when ESAX was administered. Moreover, the total ERK1/2 and phosphorylated ERK1/2 levels were upregulated in the DM-derived MPs (Fig. 3C,D). Although there was no significant difference in the ERK1/2-containing MPs and the phosphorylation of ERK1/2 in the Control + ESAX-derived MPs, the increased total ERK1/2 and phosphorylated ERK1/2 levels in the DM-derived MPs were not significantly reduced (tendency to be reduced) when ESAX was administered in the DM group. These results suggest that ESAX could protect endothelial cells from released eNOS-containing MPs by inhibiting the production of ERK1/2-containing platelet-derived MPs in DM.

Effect of ESAX on ERK1/2, eNOS, and ICAM-1 in MPs-treated aortas

Since ERK1/2-containing DM-derived MPs attached strongly to the aortas and induced eNOS-containing MP release from the endothelial cells of the aortas14,15, the role of the MP attachment factor in the ERK1/2-containing DM-derived MPs-induced expression of ERK1/2, eNOS, and intercellular adhesion molecule-1 (ICAM-1), an attachment factor, was evaluated. The higher expression of ERK1/2 and ICAM-1 was observed in the control aortas in response to DM-derived MPs (Fig. 4A–C). The DM + ESAX-derived MPs abolished the stimulatory effect of the DM-derived MPs on the ERK1/2 protein expression level, and the stimulatory effect of the DM-derived MPs on the ICAM-1 protein levels was not observed following treatment with DM + ESAX-derived MPs. The DM-derived MPs caused the downregulation of the eNOS protein expression levels in the aortas associated with the reduced ACh-induced NO production (Figs. 2C, 4A,D). The DM-derived MPs-induced eNOS downregulation was prevented by DM + ESAX-derived MPs. These results suggest that ICAM-1 has a determining role in the ERK1/2-containing DM-derived MPs-induced eNOS-containing endothelial-derived MPs production signal. Moreover, it is suggested that ESAX may produce MPs that do not release eNOS-containing endothelial-derived MPs without inducing ICAM-1.

Fig. 4.

Fig. 4

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Effect of ESAX on the expression of ERK1/2, eNOS, and ICAM-1 in the MPs-treated Control aorta. Mice aortas were incubated with either vehicle, Control-derived MPs, Control + ESAX-derived MPs, DM-derived MPs, or DM + ESAX-derived MPs for 30 min, and the subsequent expression levels of ERK1/2; eNOS; and ICAM-1 were assessed by western blot analysis. Results are shown as representative immunoblots (A) and the corresponding cumulative data (B; ERK1/2, C; ICAM-1, D; eNOS). Immunoblots were quantified by densitometric analysis and normalized with β-actin (n = 6). *P < 0.05 vs. Control-derived MPs; †P < 0.05 vs. DM-derived MPs.

Effects of ESAX and ICAM-1 on the NO production in aortas

An increased ICAM-1 expression level was observed in the aorta of the DM group compared to that of the Control group, and the DM-induced upregulation of the ICAM-1 protein was prevented by ESAX (Fig. 5A). Therefore, these findings indicate the involvement of ICAM-1 in DM.

Fig. 5.

Fig. 5

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Effects of ESAX on the expression of ICAM-1 in mouse aortas, and NO production in ACh-stimulated aortas under ICAM-1 inhibitor treatment. (A) The mouse aortas of the Control, Control + ESAX, DM or DM + ESAX groups were used to determine the protein levels of ICAM-1 by western blot analysis. Results are shown as representative immunoblots (upper panels) and the corresponding cumulative data (lower panels). Immunoblots were quantified by densitometric analysis and normalized with β-actin (n = 6). *P < 0.05 vs. Control; †P < 0.05 vs. DM. (B) NO production in the mouse aortas from the Control, Control + ESAX, DM, and DM + ESAX groups under ACh-stimulation (20 min) in the presence of A205804 (for 60 min), an ICAM-1 inhibitor. Data are expressed as mean ± SE (n = 5–6).

There is suspicion that DM-derived MPs-induced endothelial NO production counteracts the expression of ICAM-1. Therefore, we studied whether ICAM-1 was involved in the DM-induced decrease of NO production and the DM + ESAX-induced improvement of NO production. In Fig. 1C, DM decreased the ACh-induced NO production, and in Fig. 5B, A205804, an ICAM-1 inhibitor, prevented the DM-decreased ACh-induced NO production.

We found that ICAM-1 was responsible for the DM-induced decrease in ACh-induced NO production. Therefore, we studied whether ICAM-1 inhibition was improved in the DM-derived MPs-induced impaired endothelial dysfunction. We determined the ACh-induced NO-dependent relaxation response under A205804 treatment. As shown in Fig. 6, A205804 prevented the impaired ACh-induced endothelial-dependent relaxation response with DM-derived MPs, and the ACh-induced endothelial-dependent relaxation responses were not attenuated by the Control-derived MPs, Control + ESAX-derived MPs, and DM + ESAX-derived MPs were further unchanged by A205804 treatment.

Fig. 6.

Fig. 6

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Effects of ESAX and ICAM-1 on the impaired endothelial-dependent relaxation response by the DM-derived MPs. ACh-induced relaxation response in vehicle (PBS)-treated Control aortas (A), or those incubated with Control-derived MPs (B), Control + ESAX-derived MPs (C), DM-derived MPs (D), or DM + ESAX-derived MPs (1 × 105 events/mL, for 30 min) (E) in the absence or presence of A205804 (1 × 10− 6 M, for 60 min). Data are expressed as the mean ± SE (n = 6). †P < 0.05 vs. DM-derived MPs (+ DM MPs).

Discussion

Our study indicated that MR obstruction by ESAX ameliorated the ACh-induced vascular endothelial-dependent relaxation and PE-induced vascular contractile response by enhancing the NO production that was impaired by the induction of DM by STZ. Particularly, the present findings indicated that ESAX administration also blocked the MP-induced endothelial dysfunction through increased ERK1/2, promoted ICAM-1, and decreased eNOS. The effects of ESAX on the promoted ICAM-1 by DM-derived MPs can be repeated by the ICAM-1 inducible inhibitor. These results suggest that ESAX ameliorates the DM-derived MPs-induced endothelial dysfunction by uncoupling the association between ICAM-1 and the endothelial cells and DM-derived MPs.

DM is an established risk factor for cardiovascular disease, and vascular dysfunction is a primary contributor to cardiovascular disease in DM20,21. Moreover, other researchers have reported that the aldosterone or corticosterone levels, which are MR agonists, increase in STZ-induced diabetes models22,23. The pharmacological obstruction of MRs significantly reduced morbidity, improved the survival of patients with heart failure, and decreased postmyocardial infarction hospitalization in several clinical trials24,25. MR is a ligand-activated transcription factor that is functionally expressed in both vascular smooth muscle and endothelial cells24,26. Accumulating evidence implicates vascular MR signaling as underlying receptors for vasoactive ligands27,28, adhesion or inflammatory molecules29,30, and critical pathways for vascular (dys)function and coronary blood flow control31. Importantly, it was recently demonstrated that MR antagonists improved the microvascular dysfunction in patients with DM, independent of blood pressure32,33. Furthermore, MR antagonists ameliorated vascular dysfunction in DM models6,7,34. In this study, we used ESAX, a recently approved MR blocker in Japan. ESAX has a higher MR-binding specificity and a nonsteroidal structure8. Therefore, we focused on the effects of ESAX on vascular function in DM. Our observation of an impaired ACh-induced endothelium-dependent vascular relaxation response in DM mice is consistent with previous reports14,15. Moreover, we found that ACh-induced endothelial-dependent vascular NO production and relaxation response were improved by ESAX administration in DM mice. Enhanced PE-induced constriction has been attributed to the reduced NO production in DM mice; ESAX administration ameliorated the PE-induced contraction, which is attributed to the increased NO production in DM mice. It is well known that SNP acts via the direct stimulation of vascular smooth muscle cells, independent of an intact endothelium. In the current study, the responses to SNP were not different among the Control, Control-ESAX, DM, and DM-ESAX mice aortas. Therefore, in this study, ESAX exhibited protective effects on endothelial function in DM mice.

Relatively low levels of circulating MPs exist in healthy individuals. However, numerous studies have reported that increased MPs are found in a number of disease states associated with the vascular system35,36. Our previous study15 revealed that circulating MPs were increased in DM mice; this decreases NO production and impairs vascular endothelial-dependent relaxation responses by strongly attaching to the endothelial cells, prompting the release of eNOS-containing endothelial cell-derived MPs. Considering that endothelial cells are in direct contact with the blood and is vulnerable to circulating MPs, we studied the effect of MPs and ESAX on the endothelial cells of the aorta. First, we demonstrated that normal aortas treated with DM-derived MPs were more responsive to PE, while the relaxation response to ACh was significantly decreased compared to those of the Control-derived MPs. Similar results showing increased vascular PE responsiveness and decreased ACh-induced relaxation in DM-derived MPs have been reported in previous studies14,15. There is a possible mechanism for the reduction of ACh-induced relaxation and enhancement in PE-induced constriction, i.e., a decrease in NO production from endothelial cells. DM + ESAX-derived MPs tended to reduce the PE-enhanced contractility, and ACh-induced relaxation was restored. In the aortas from the DM + ESAX-derived MPs there was an increase in the ACh-induced relaxation, which may be due to the involvement of the NO pathway because NO production increased under ACh stimulation and the presence of L-NNA, a NOS inhibitor, did not cause changes in PE contraction curves in the aortas of MPs derived from all treatment groups.

MPs are found in the blood circulation of healthy individuals, and their number increases in cardiovascular disease and conditions that predispose one to cardiovascular disease37. The number of MPs is suggested as a marker of endothelial damage and platelet activation37. We previously reported that higher platelet-derived MP levels were observed in DM mice13–15. Whether ESAX can affect the concentration or function of the MPs in DM remains unknown. In the present study, we found that increased platelet-derived MPs in DM mice were reduced by ESAX administration. ELISA analysis further indicated that ESAX obviously decreased eNOS and slightly reduced ERK1/2 contained in the DM-derived MPs.

ERK1/2 plays an important role in several cellular processes. Our study has recently identified that ERK1/2-containing DM-derived MPs contributed to the impaired ACh-induced endothelial-dependent relaxation response14. ERK1/2 activation is predominantly associated with eNOS activation. ERK1/2 upregulation and consequent eNOS inhibition have been considered a major event in vascular complications in some cardiovascular-related pathologies, including DM. Conversely, ERK1/2 is directly involved in vascular smooth muscle cell contraction. However, Fig. 2D,E illustrate that the increased contractile response induced by DM-derived MPs treatment was abolished in all groups after pretreatment with L-NNA, which is a NO synthase inhibitor. Hence, the ERK1/2 may play a role in the influence of MPs on endothelial cells. In the present study, we found that the DM-derived MPs increased the expression of ERK1/2. However, ESAX administration blocked the effects of MPs on ERK1/2 expression in DM. Consistent with these results, ERK1/2 contained within the MPs has recently been shown to be involved in ERK1/2 phosphorylation in endothelial cells and induces the production or release of endothelium-derived MPs14,15. These results demonstrate that ESAX may partially depend on the blocking of the production or release of endothelial-derived MPs through the ERK pathway.

An imbalance between the production of vasorelaxing (mainly NO) and vasoconstricting factors plays a key role in the development of DM. eNOS-derived NO regulates the vessel’s inflammatory status and tone38. eNOS regulation is complex and is determined by a cascade of events that include changes in eNOS protein levels39. In line with this concept, we previously reported that ERK1/2-containing platelet-derived MPs from DM mice showed reduced eNOS expression in the aortas and increased eNOS expression in the endothelial MPs without phosphorylating eNOS, which indicates that these MPs are able to regulate the eNOS protein levels in the aortas and induce endothelial dysfunction14,15. Moreover, previously, we reported no change in the phosphorylation of eNOS when DM-derived MPs are treated in normal blood vessels, but the total amount of eNOS decreases and both the total amount and the phosphorylation of ERK1/2 increase15. Therefore, in the present study, we only measured the total amount of eNOS and ERK1/2. Additionally, eNOS is only present in the endothelium in blood vessels, and separating the endothelial layer from the smooth muscle layer is quite difficult. Therefore, in this study, we used the whole vessel to examine eNOS. We demonstrated that ESAX can block the reduction of eNOS in the aorta and the enhanced eNOS in the MPs from DM mice. Similar effects of PD98059, an ERK1/2 inhibitor, and ESAX administration on eNOS expression and NO production may result from inhibition of ERK in MPs, or the reduced adhesion of MPs to aortas (reducing the endothelial dysfunction effects of MPs), or both14.

It is well known that ICAM-1 is a key endothelial receptor contributing to blood cell-endothelial cell interactions and plays a critical role in inflammatory responses40,41. ICAM-1 is generally expressed at low basal levels in endothelial cells, but its expression is upregulated by inflammatory cytokines42. Earlier studies have demonstrated elevated ICAM-1 levels in the DM complications, thus indicating endothelial dysfunction43,44. Rautou et al. reported that MPs were shown to transfer ICAM-1 to endothelial cells and promote monocyte adhesion and transendothelial migration45. We previously demonstrated that DM-derived MPs induced the expression of ICAM-1, and PD98059, which is an ERK1/2 inhibitor, -treated DM-derived MPs did not induce the expression of ICAM-1 14. Our findings indicate that DM mice and DM-derived MPs upregulated the ICAM-1 levels, while ESAX partially blocked these effects. While the pre-incubation with A205804 showed uniform levels as ESAX for the ACh-induced NO production, the ACh-induced endothelial-dependent relaxation response in DM-derived MPs-treated aortas was enhanced in the presence of A205804, but was not influenced in the DM + ESAX-derived MPs-treated aortas. This provides evidence that ICAM-1 induction partially and negatively regulates endothelial function, and ESAX administration blocked these effects. This study revealed that ESAX demonstrated similar results to an ICAM-1 inhibitor and an ERK1/2 inhibitor. These data indicated that the protective benefits of ESAX may be partly caused by preventing endothelial dysfunction via ERK1/2 and ICAM-1 interaction. These results further indicate that the vasculoprotective benefits of ESAX may partially occur due to preventing the MPs-endothelial cell interaction by promoting ICAM-1.

In summary, our data demonstrated that ESAX administration ameliorates the DM-induced impaired endothelial function in DM mice. Moreover, ESAX influences the vascular impaired effects of DM-derived MPs by inhibiting the adhesion between MPs and endothelial cells by downregulating the levels of platelet-derived MPs and ICAM-1 and upregulating the NO content by maintaining the eNOS levels. These results confirm that ESAX may be a useful drug to treat endothelial dysfunction and the potential of MPs as a clinically useful marker for endothelial dysfunction in DM.

Methods

Experimental animals and protocol

Male ICR mice (4 weeks old) were obtained from the Tokyo Animal Laboratories (Tokyo, Japan) and randomly divided into cages (n = 5 per cage). All mice were allowed to acclimate to the laboratory environment for 7 days before the start of the study. During the experimental period, the mice were housed under standard laboratory conditions with free access to food and water. All mice used in this study were treated in accordance with ARRIVE guidelines and relevant animal welfare guidelines and regulations and the principles and guidelines of animal care of the Hoshi University Animal Care and Use Committee, and the study protocols were reviewed and approved by an ethics committee (approval ID: #P23-046) that is accredited by the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Popularly, ICR mice are stably generated when diabetes is induced using streptozotocin (STZ; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Several research studies have used ICR mice, and we have published many reports on them14,15; hence, a wealth of basic data is available. Additionally, we utilized animals that are consistent with previous STZ mice model data in this study14,15. The mice were assigned to four groups: non-diabetic Control, STZ-induced type 1 DM, Control + ESAX (Daiichi Sankyo Co., Ltd., Tokyo, Japan), and STZ-induced DM + ESAX groups. Mice in the DM and DM + ESAX groups received a single injection via the tail vein of 200 mg/kg STZ freshly dissolved in a citrate acid buffer, while the non-diabetic mice (Control group) were injected with the buffer only. Plasma glucose levels in the mouse groups were measured 12 − 16 weeks after STZ injection. This is because severe aortic endothelial dysfunction induced by STZ mice is relatively more stable after 12 weeks of STZ injection14,15. Hyperglycemic mice that exhibited a plasma glucose level higher than 400 mg/dL were used as the DM and DM + ESAX groups. The DM + ESAX group and Control + ESAX group received a dose of 3 mg/kg ESAX via their diet (ESAX was mixed into the regular chow at 0.003%) for 8 weeks46–48, and the other groups were fed regular chow. The mice were anesthetized, and the samples, including the plasma and thoracic aortas, were collected and used for further analyses. The plasma samples were collected, centrifuged at 3,500 rpm for 10 min, and kept at − 20℃ until the measurement of glucose and insulin levels.

Plasma glucose and insulin levels

Plasma glucose was assessed using a commercial assay kit (FUJIFILM Wako Oure Chemical Corporation). Plasma insulin levels were determined using an ELISA kit (FUJIFILM Wako Pure Chemical Corporation).

Microparticle isolation and characterization

The protocol used for MP isolation was adapted from those previously published by our laboratory13–15. In brief, whole blood was collected from the abdominal aorta with heparin as the anticoagulant. The blood was centrifuged at 5000×g for 10 min to remove the platelets, and the collected supernatant was centrifuged at 30,000×g for 2 h. The pellet containing the MPs was resuspended in phosphate-buffered saline (PBS) to be used for the subsequent experiments.

Vascular experimental protocol

The mice were anesthetized with isoflurane (inhalation). The thoracic aorta was removed and immersed in a modified Krebs-Henseleit solution (KHS) containing 118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO3, 1.8 mM CaCl2, 1.2 mM NaH2PO4, 1.2 mM MgSO4, and 11 mM glucose. The aorta was cleaned of any adhering fat and connective tissue under a dissecting microscope and sliced into rings of 2-mm length. The aortic rings were mounted under an optimal resting tension of 1.5 g in a 10-mL organ bath containing KHS aerated with 95% O2 and 5% CO2 and maintained at 37℃. The vascular isometric force was measured using a force-displacement transducer (TB-611T; Nihon Kohden, Tokyo, Japan) connected to a PowerLab recording system (AD Instruments, Australia). The aortic rings were equilibrated for 45 min prior to the potassium chloride (80 mM)-induced contraction, during which time the KHS was replaced every 15 min with fresh solution. After the equilibration period, the aortic ring was contracted with 1.0 g tension using 80 mM potassium chloride to confirm that the ring generated a normal level of contraction and had fully recovered by washing in fresh solution. The ring was contracted again using prostaglandin F2α (PGF2α; 1 × 10− 6−3 × 10− 6 M) (Fuji Pharma, Tokyo, Japan), which produced approximately 1 g force. When the PGF2α-induced contraction reached a steady-state level, ACh (1 × 10− 9−1 × 10− 5 M) (Daiichi Sankyo Co., Ltd.) or SNP (1 × 10− 10−1 × 10− 5 M) (Wako) were cumulatively applied to the bath solution to determine their concentration-response relationships, which were regarded as the control responses. To examine the effects of the MPs or the ICAM-1 inhibitor A205804 on ACh-induced relaxation, the MPs or inhibitor were applied to the bath solution 30 min before the precontraction of PGF2α14,15.

The aortic rings were prepared as described above and then exposed to cumulatively increasing concentrations of High K+ solution (10 − 80 mM). After three successive washes with KHS to remove the potassium and a 45-min equilibration period, PE (1 × 10− 10−1 × 10− 5 M) concentration-response curve was obtained by measuring the developed tension at various PE concentrations. To examine the effects of NO on PE-induced contraction, the NOS inhibitor NG-nitro-L-arginine (L-NNA; 10− 4 M) (Sigma-Aldrich, St. Louis, MO, USA) was applied to the bath solution 30 min before the administration of PE.

Release of NO

The release of NO was measured using an ENO20 NOx Analyzer (Eicom, Kyoto, Japan), based on liquid chromatography method with post-column derivatization using Griess reagent, as previously described13–15,49. Briefly, the isolated aortas were cleared from the surrounding tissue, and cut into 4-mm lengths, added to tubes containing KHS with or without MPs (1 × 105 events/mL), and incubated for 30 min at 37℃. Furthermore, the other aortic segments were added to tubes containing KHS with A250804 (1 × 10− 6 M) and incubated for 60 min at 37℃. Then, ACh (1 × 10− 6 M) was added to the tube for a further 20 min. Finally, the semi-dried aortas were weighed and frozen in liquid nitrogen. The frozen aortas were used for WB.

MP characterization

Commercial ELISA kits were used to measure the levels of platelet-derived MPs (Protein Purify Co., Ltd., Gunma, Japan), eNOS (RayBiotech, Norcross, GA, USA), ERK1/2, and p-ERK1/2 (Abcam, UK) in accordance with the manufacturer’s instructions.

WB analysis

The frozen aortas were lysed in RIPA buffer supplemented with protease inhibitors. The proteins were quantified using a BCA assay (Thermo Scientific, Rockford, IL, USA). Equal amounts of proteins were loaded onto SDS/PAGE and transferred onto PVDF membranes, followed by immunoblotting with anti-eNOS (cat.no. 610296; BD Biosciensce, NJ, USA), anti-ERK1/2 (cat.no. #4696; Cell Signaling Technology, Danvers, MA, USA), anti-ICAM-1 (cat.no. NBP2-22541; Novus Biologicals, Centennial, CO, USA) (all dilution in 1:1000) and anti-b-actin (cat.no. A5316; Sigma Chemical Co., St. Louis, MO, USA) (dilution in 1:10000). The WB were analyzed using a CS Analyzer system (ATTO, NY, USA).

Statistical analysis

The data were analyzed using one-way or two-way repeated analysis of variance and are presented as the mean ± standard error (SE). Tukey’s test was used for the comparison of multiple means. A pairwise comparison was performed when a significant interaction effect was noted. Differences were considered statistically significant if P < 0.05. All study procedures were performed by an individual that was blinded to the treatment and control groups of the animals.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (966KB, doc)

Acknowledgements

We thank Enago (www. Enago.jp) for the English language review.

Author contributions

K.T. and T. K. conceived, designed and supervised the study. K. T. performed experiments and wrote the main manuscript text. H. K analyzed the final data set and wrote the main manuscript text. T. M. analyzed the final data set. All authors contributed to revising and editing of the manuscript and approved the final version of manuscript.

Funding

We thank Enago (www.Enago.jp) for the English language review. This work was partially supported by the JSPS KAKENHI (Grant Numbers JP21K06878, and JP21K06811), the Science Research Promotion Fund of the Promotion and Mutual Aid Corporation for Private Schools of Japan, and The Research Foundation for Pharmaceutical Sciences.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

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.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Kumiko Taguchi and Hiroyuki Kondo.

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Supplementary Materials

Supplementary Material 1 (966KB, doc)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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