Fig 1.
Physiologic APN doses facilitate ACh-induced vasorelaxation in an endothelium-dependent and NO-mediated manner.
(A) Different physiologic APN levels (5, 10, 15μg/ml) significantly enhanced ACh-induced vasorelaxation in a concentration-dependent fashion. (B) APN-induced aortic vascular ring vasorelaxation is blocked after pre-treatment with L-NAME (NO synthesis inhibitor, 100 μmol/L). n = 5–7 mice/group. #,P<0.05; ##, P<0.01.
Fig 2.
Knockout of adiponectin receptor 1 (AdipoR1), but not receptor 2 (AdipoR2), abolishes APN-enhanced ACh-induced vasorelaxation.
(A) Enhanced vasorelaxation to ACh by APN (5μg/ml) was significantly decreased in AdipoR1-KO mouse aorta. (B) Enhanced vasorelaxation to ACh by APN (5μg/ml) was not affected in AdipoR2-KO mouse aorta. n = 5–7 mice/group. #, P<0.05; ##, P<0.01.
Fig 3.
Physiologic APN levels induced eNOS phosphorylation in an AdipoR1-dependent fashion.
HUVECs were incubated with different doses of APN (5, 10, and 15μg/ml) (A) and eNOS phosphorylation (Ser1177) was determined at different time periods (0, 15, 30, and 60 min) (B). (C) Western blot analysis confirmed successful knockdown of AdipoR1 and AdipoR2 by siRNA. (D) AdipoR1, but not AdipoR2, knockdown significantly attenuated APN-induced eNOS phosphorylation. n = 6–8 dishes/group. #, P<0.05; ##, P<0.01 vs respective control. R1, R2 represent AdipoR1 and AdipoR2, respectively.
Fig 4.
Cav-1 is required for AdipoR1-dependent APN-enhanced vasorelaxation.
(A) Immunoblot demonstrating interaction of Cav1 with AdipoR1 in the presence or absence of APN (5μg/ml). (B) Western blot analysis confirmed successful knockdown of Cav-1 by siRNA. n = 6–8 dishes/group. (C) Knockdown of Cav-1 significantly attenuated APN-induced eNOS phosphorylation. (D) Cav-1 KO mouse aorta exhibited significantly decreased APN enhancement to ACh-induced vasorelaxation. Ir, irrelevant antibody. n = 5–7 mice/group. #, P<0.05; ##, P<0.01 vs respective control.
Fig 5.
Physiologic APN concentrations increased NO production in an AdipoR1 and Cav-1 dependent manner.
NO production in HUVEC medium was determined after incubation with vehicle or APN (5μg/ml) for 15 minutes. Knockdown of AdipoR1 (but not AdipoR2), knockdown of Cav-1, and treatment with L-NAME (100 μmol/L) abolished APN-induced NO production enhancement. n = 6–8 dishes/group. ##, P<0.01 vs respective control. R1, R2 represent AdipoR1 and AdipoR2, respectively.
Fig 6.
Schematic of the mechanisms underlying APN-induced vasodilation.