Rivaroxaban, a specific FXa inhibitor, improved endothelium-dependent relaxation of aortic segments in diabetic mice

Figure 1. Induction of diabetes increased expression of PAR2 in aorta. (A) qPCR analysis using thoracic aorta demonstrated that induction of diabetes by STZ increased the expression of PAR2 (n = 6). (B) Western blot data demonstrated that induction of diabetes increased the expression of PAR2 in the aorta (n = 8–10). *P < 0.05. All values are mean ± SEM.

Figure 2. Rivaroxaban attenuated endothelial dysfunction in diabetic mice. (A,B) Induction of diabetes by STZ impaired endothelium-dependent vasodilation in response to Ach compared with the non-diabetes group. Rivaroxaban administration to diabetic mice for 3 weeks ameliorated endothelial function compared with the non-treated group (A). Vasorelaxation in response to SNP did not differ among the three groups (B) (n = 8–12). ***P < 0.001 vs. NT, and ††P < 0.01 vs. STZ. (C) Induction of diabetes attenuated eNOS phosphorylation at Ser1177; however, rivaroxaban treatment ameliorated this response (n = 10–13). There was no significant difference in total eNOS expression among 3 groups. *P < 0.05. All values are mean ± SEM.

Figure 3. JNK inhibitor attenuated endothelial dysfunction induced by FXa. (A) FXa promoted the phosphorylation of JNK in HCAEC (n = 9). **P < 0.01. (B) FXa attenuated the phosphorylation of eNOS at Ser1177 in HCAEC. Pretreatment with SP600125, a JNK inhibitor, ameliorated this response (n = 9). *P < 0.05. (C,D) Vascular reactivity in response to Ach (C) or SNP (D) was examined using aortic segments obtained from WT mice (n = 5–12). FXa impaired endothelium-dependent vasodilation in response to Ach. SP600125 attenuated endothelial dysfunction induced by FXa. H2O2 was used for showing the impaired endothelial dysfunction. Vasorelaxation in response to SNP did not differ in the presence of FXa or SP600125. ***P < 0.001 vs. NT, and †††P < 0.001 vs. FXa. All values are mean ± SEM.

Figure 4. Genetic deletion of PAR2 prevented endothelial dysfunction caused by diabetes. (A,B) Vascular reactivity in response to Ach (A) or SNP (B) was examined using aortic segments obtained from diabetic or non-diabetic mice (n = 8–14). Induction of diabetes impaired endothelial function determined by the response to Ach in WT mice. Induction of diabetes to PAR2−/− mice did not affect vascular response to Ach or SNP. ***P < 0.001 vs. non-diabetic WT mice. (C,D) Induction of diabetes in PAR2−/− mice did not affect the phosphorylation of eNOSSer1177 (C) and JNK (D) (n = 6). There was no significant difference in total eNOS and JNK expression between 2 groups. All values are mean ± SEM.

Figure 5. Activation of PAR2 impaired endothelial function. (A,B) Vascular reactivity in response to Ach (A) or SNP (B) was examined using aortic segments obtained from WT mice or PAR2−/− mice (n = 6–11). FXa did not impair endothelium-dependent (A) and -independent (B) vascular response in aortic segments obtained from PAR2−/− mice. H2O2 was used for showing the impaired endothelial dysfunction in WT mice. ***P < 0.001 vs. WT + NT. (C,D) Vascular reactivity to AP-II, a specific agonist for PAR2, was determined using aortic segments isolated from WT and PAR2−/− mice (n = 5–7). AP-II impaired endothelium-dependent vasodilation in response to Ach in aortic segments obtained from WT mice. However, AP-II did not affect endothelial function in aortic segments obtained from PAR2−/− mice (C). AP-II did not affect vasorelaxation in response to SNP in aortic segments obtained from both strains of mouse (D). ***P < 0.001 vs. WT + NT. All values are mean ± SEM.

Figure 6. Rivaroxaban ameliorated FXa-induced endothelial dysfunction. Rivaroxaban ameliorated FXa-induced endothelial dysfunction (A), although it did not have effects on endothelial dysfunction induced by AP-II (C) or H2O2 (E). Rivaroxaban did not affect endothelial-independent vascular response induced by FXa (B), AP-II (D), or H2O2 (F) (n = 4–7). †††P < 0.001 vs. FXa. All values are mean ± SEM.