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 DM 20,21. Moreover, the pharmacological obstruction of MRs significantly reduced morbidity, improved the survival of patients with heart failure, and decreased postmyocardial infarction hospitalization in several clinical trials 22,23. MR is a ligand-activated transcription factor that is functionally expressed in both vascular smooth muscle and endothelial cells 24. Accumulating evidence implicates vascular MR signaling as underlying receptors for vasoactive ligands 25,26, adhesion or inflammatory molecules 27,28, and critical pathways for vascular (dys)function and coronary blood flow control 29. Importantly, it was recently demonstrated that MR antagonists improved the microvascular dysfunction in patients with DM, independent of blood pressure 30,31. Furthermore, MR antagonists ameliorated vascular dysfunction in DM models 6,7,32. In this study, we used ESAX, a recently approved MR blocker in Japan. ESAX has a higher MR-binding specificity and a nonsteroidal structure 8. 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 reports 14,15. 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 system 33,34. Our previous study 15 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 studies 14,15. 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 disease 35. The number of MPs is suggested as a marker of endothelial damage and platelet activation 35. We previously reported that higher platelet-derived MP levels were observed in DM mice 13–15. 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. 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 MPs 14,15. 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 tone 36. eNOS regulation is complex and is determined by a cascade of events that include changes in eNOS protein levels 37. 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 the endothelial MPs, which indicates that these MPs are able to regulate the eNOS protein levels in the aortas and induce endothelial dysfunction 14,15. In the present study, 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 both 14.
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 responses 38,39. ICAM-1 is generally expressed at low basal levels in endothelial cells, but its expression is upregulated by inflammatory cytokines 40. Earlier studies have demonstrated elevated ICAM-1 levels in the DM complications, thus indicating endothelial dysfunction 41,42. Rautou et al. reported that MPs were shown to transfer ICAM-1 to endothelial cells and promote monocyte adhesion and transendothelial migration 43. We previously demonstrated that DM-derived MPs induced the expression of ICAM-1, and PD98059-treated DM-derived MPs did not change 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. 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.