Renin-angiotensin-aldosterone (RAS) signal plays a role in developing arterial senescence and arteriosclerosis. Autophagy is a system to maintain cell homeostasis. Besides the conventional form of autophagy (CA), the presence of an alternative form of autophagy (AA) has recently been reported. Lipidation of LC3, a hallmark of CA, and Atg7 are not involved in AA. Instead, autophagosomes are associated with Rab9, and Ulk1 is required in AA. The aim of this study is to investigate how autophagy is involved in the effect of RAS-inhibition on arterial senescence and arteriosclerosis.Design and Method:
We used double knockout mice (DKO) of angiotensin II type Ia receptor KO and apolipoprotein E KO (apoE KO).Results:
The degree of aortic senescence and atherosclerotic lesions in DKO were significantly lower than those of apoE KO. Aortic expressions of senescence marker, p21 and PAI-1, in DKO were significantly lower than in apoE KO. Whereas electron microscopic analysis revealed the number of autophagosome are higher in DKO than in apoE KO, there was no difference in the ration of LC3II/LC3I between them, suggesting that CA is not involved in this model. Aortic expressions of the ratio of p-Ulk1 at Ser555/total Ulk1 and Rab9 were significantly higher in DKO than apoE KO, suggesting that AA is induced in DKO. In in vitro experiments using HUVEC and SMC, energy starvation with RAS-inhibition increased the number of Rab9 puncta co-localized with Lamp2 puncta, which indicate autolysosomes, in cells of control and Atg7 knock-down (KD), but not in Ulk1 KD. Degradation of long-lived proteins were significantly attenuated in the presence of Ulk1 KD, but not Atg7 KD in cells with RAS-inhibition.Conclusions:
Alternative autophagy, but not conventional autophagy, plays a crucial role in the effect of RAS-inhibition on arterial senescence and arteriosclerosis.