LARP7 overexpression alleviates aortic senescence and atherosclerosis

Abstract

Atherosclerosis, characterized by the accumulation of lipid plaques on the inner walls of arteries, is the leading cause of heart attack, stroke and severe ischemic injuries. Senescent cells have been found to accumulate within atherosclerotic lesions and contribute to the progression of atherosclerosis. In our previous study, we discovered that suppressing Larp7 accelerates senescence by inhibiting Sirt1 activity, resulting in increased atherosclerosis in high-fat diet (HFD) fed and ApoE deficient (ApoE^KO) mice. However, there has been no direct evidence demonstrating Larp7 per se could attenuate atherosclerosis. To this end, we generated a tetO-controlled and Cre-activated Larp7 gain-of-function mouse. Through RT-PCR and western blotting, we confirmed Larp7 overexpression in the aortas of HFD-fed ApoE^KO; Larp7^tetO mice. Larp7 overexpression led to increased Sirt1 activity and decreased cellular senescence signals mediated by p53/p65 in the aortas. Additionally, Larp7 overexpression reduced the presence of p16-positive senescent cells in the aortic lesions. Furthermore, Larp7 overexpression resulted in a decrease in pro-inflammatory macrophages and SASP factors. Consequently, Larp7 overexpression led to a reduction in the area of atherosclerotic lesions in HFD-fed ApoE^KO; Larp7^tetO mice. In summary, our study provides evidence that Larp7 overexpression holds promise as an approach to inhibit cellular senescence and prevent atherosclerosis.

Keywords: Larp7; Sirt1; atherosclerosis; cellular senescence.

FIGURE 1

Strategy for generating inducible Larp7 ^tetO mouse. (A) Strategy for generating an inducible overexpression mouse by combining the Tet‐On system with the Cre‐loxp system. SA‐pA means splice acceptor and polyA signal. P1 and P2 are specific primers for Larp7 ^tetO mice genotyping. (B) Representative gel image of genotyping results. Larp7 ^tetO mice had a 413 bp product band.

FIGURE 2

Larp7 overexpressed in ApoE ^KO; Larp7 ^tetO mice and activated Sirt1 in the arotas. (A) Experimental schedule. Tamoxifen (TAM) was consecutively injected at 5‐day intervals, followed by a 12‐week high‐fat diet (HFD) to expedite the progression of atherosclerosis. During the HFD period, oral administration of doxycycline was utilized to induce Larp7 overexpression in ApoE ^KO; Larp7 ^tetO mice. ApoE ^KO; WT mice treated with a vehicle were used as controls. (B) Larp7 mRNA overexpressed in aortas of ApoE ^KO; Larp7 ^tetO mice compared with ApoE ^KO; WT mice. n = 5 mice per group. Data = Mean ± SD, two‐tailed student's t‐test. p < 0.05 indicated significance. (C) Larp7 protein increased in the aortas of ApoE ^KO; Larp7 ^tetO mice compared with ApoE ^KO; WT mice. (D) Sirt1 activity assays illustrating nuclear Sirt1 activity significantly increased in aortas of ApoE ^KO; Larp7 ^tetO mice compared with ApoE ^KO; WT mice. n = 3 mice per group. Data = Mean ± SD, two‐tailed student's t‐test. p < 0.05 indicated significance.

FIGURE 3

Larp7 overexpression reduced p53/p65 mediated senescent signals in the aortas. (A) Aortas of ApoE ^KO; Larp7 ^tetO mice showing elevated acetylation of p65 and p53 after overexpression of Larp7 for 12 weeks. (B) Cdkn1a (p21) mRNA was decreased in ApoE ^KO; Larp7 ^tetO aortas 12 weeks after HFD. n = 5 mice per group. Data = Mean ± SD, two‐tailed student's t‐test. p < 0.05 indicated significance. (C) Immunoblotting showed that p16 protein decreased in ApoE ^KO; Larp7 ^tetO aortas compared with ApoE ^KO; WT aortas. (D) p16 immunostaining showed Larp7 overexpression decreased the senescent cells in the atherosclerotic lesion of ApoE ^KO; Larp7 ^tetO mice compared with ApoE ^KO; WT mice. Scale bar: 500 μm. (a) indicated ApoE ^KO; WT group, (b) indicated ApoE ^KO; Larp7 ^tetO group. (E) p16 positive fluorescent intensity was calculated. n = 11 mice per group. Data = Mean ± SD, two‐tailed student's t‐test. p < 0.05 indicated significance.

FIGURE 4

Larp7 overexpression alleviates inflammatory phenotypes in the aortas. (A) Immunostaining of Mac3 in the aortic roots showed Larp7 overexpression decreased the inflammatory macrophage infiltration in the ApoE ^KO; Larp7 ^tetO aortas compared with ApoE ^KO; WT aortas. Scale bar: 500 μm. (a) indicated ApoE ^KO; WT group, (b) indicated ApoE ^KO; Larp7 ^tetO group. (B) Fluorescent intensity of Mac3 was quantified by imageJ. n = 11 mice per group. Data = Mean ± SD, two‐tailed student's t‐test. p < 0.05 indicated significance. (C) RT‐PCR results showed SASP factors were decreased in the ApoE ^KO; Larp7 ^tetO aortas compared with ApoE ^KO; WT aortas. n = 5 mice per group. Data = Mean ± SD, two‐tailed student's t‐test. p < 0.05 indicated significance.

FIGURE 5

Larp7 overexpression reduced aortic lesion area. (A, B) Larp7 overexpression decreased the atherogenesis. The representative images of atherosclerosis lesions of aorta en face stained by oil red O (A). The calculation of percentage of lesion area relative to total region (B). (a) indicated ApoE ^KO; WT group, (b) indicated ApoE ^KO; Larp7 ^tetO group. Scale bar: 1 mm. n = 11 mice per group. (C, D) Oil red staining of aortic root. The aortic roots of ApoE ^KO;WT and ApoE ^KO; Larp7 ^tetO mice were cryosectioned and subjected to oil red staining (C), and the relative lesion area (%) was calculated and statistically analysed (D). (a) indicated ApoE ^KO; WT group, (b) indicated ApoE ^KO; Larp7 ^tetO group. Scale bar: 500 μm. n = 11 mice per group. Data = Mean ± SD, two‐tailed student's t‐test. p < 0.05 indicated significance.

FIGURE 6

Research summary. (A) HFD intake in ApoE ^KO; WT mice reduces LARP7 protein, lowering SIRT1 activity and increasing p53 and p65 acetylation. This enhances transcriptional activity of Cdkn1a, p16 and SASP factors, leading to cellular senescence and inflammation, promoting atherosclerosis. (B) LARP7 overexpression in ApoE ^KO; Larp7 ^tetO mice restores SIRT1 activity and decreases p53 and p65 acetylation. This inhibits expression of Cdkn1a, p16 and SASP factors, leading to reduced cellular senescence and inflammation, promoting atherosclerosis alleviation.