Deficiency of S100 calcium binding protein A9 attenuates vascular dysfunction in aged mice

Abstract

Background: S100 calcium-binding protein A9 (S100A9) is a danger-associated molecular pattern molecule that mediates the inflammatory response. Inflammation is essential in aging-related cardiovascular diseases. However, less is known regarding the role of S100A9 in vascular aging.

Methods: S100A9 null mice were used to investigate the role of S100A9 in aging-related pathologies. Artery rings were used to measure the functional characteristics of vascular with a pressurized myograph. Telomere length, Sirtuin activity, oxidative stress, and endothelial nitric oxide synthetase (eNOS) activity were used to elevate vascular senescence. Intraperitoneal glucose tolerance (IPGTT) and insulin sensitivity test (IST) were employed to investigate the effects of S100A9 on insulin resistance. Inflammation response was reflected by the concentration of inflammatory cytokines. The Toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE) inhibitors were used to identify the downstream molecular mechanisms of S100A9 in aging-induced senescence in endothelial cells.

Results: S100A9 expression in vascular increased with aging in mice and humans. Deficiency of S100A9 alleviated vascular senescence in aged mice, as evidenced by increased telomere length, Sirtuin activity, and eNOS activity. Meanwhile, S100A9 knockout improved endothelium-dependent vasodilatation and endothelial continuity in aged mice. Moreover, the increased insulin resistance, oxidative stress, and inflammation were mitigated by S100A9 deletion in aged mice. In vitro, S100A9 induced senescence in endothelial cells, and that effect was blunted by TLR4 but not RAGE inhibitors.

Conclusion: The present study suggested that S100A9 may contribute to aging-related pathologies and endothelial dysfunction via the TLR4 pathway. Therefore, targeting S100A9/TLR4 signaling pathway may represent a crucial therapeutic strategy to prevent age-related cardiovascular diseases.

Keywords: Aging; Insulin resistance; Oxidative stress; S100A9; Vascular senescence.

Fig. 1

S100A9 expression increased in the aged aortas of mice and humans. (A) S100A9 expression in the aortas of mice at 3-, 12- and 24-month was measured using Western blots. The statistical results of S100A9 were presented in (B) (n = 6). (C) S100A9 expression in mice aortas was measured using IHC at 3- and 24-month and the related statistical results were displayed in (D) (n = 4). (E) S100A9 expression in young and aged human aortas was measured using Western blots. (F) The statistical results of blots in E (young group, <40 years, n = 3; aged group, >60 years, n = 5). (G) Representative IHC images of S100A9 expression in the aorta of young and aged humans and the related statistical results were depicted in (H) (n = 3). ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2

S100A9 knockout alleviates vascular senescence in aged mice. (A) S100A9 knockout increased as evidenced by the increased ratio of telomeric repeats with single-copy gene (T/S ratio) (n = 6). (B) Representative Western blots of Phospho-γH2AX expression in nuclear. The statistical results were illustrated in (C) (n = 6). (D) Representative Western blots of p21 and p53 in mice aortas of different ages. The related statistical results were exhibited in (E) and (F) (n = 6). (G) S100A9 knockout increased the Sirtuin activity in aged mice aorta (n = 6). ns, not significant. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Fig. 3

S100A9 knockout improves vascular function in aged mice. (A) S100A9 knockout improved endothelium-dependent vasodilatation induced by Ach in aged mice (n = 6). (B) The area under curve of different groups (n = 6). (C) Representative Western blots of Phospho-eNOS in mice aortas of different ages. The related statistical results were shown in (D) (n = 6). (E) S100A9 knockout increased the NO content in the aged mice aorta (n = 6). (F) Representative IHC images of CD31 in the aorta of wild-type and S100A9 null-aged mice and the related statistical results were presented in (G). ns, not significant. *P < 0.05, ***P < 0.001, ****P < 0.0001. ^{^ ^}P < 0.01 Aged vs. Aged-S100A9^−/−. ^##P < 0.01 Aged + l-NAME vs. Aged. ^&&P < 0.01 Aged-S100A9−/− + L-NAME vs. Aged-S100A9^−/−.

Fig. 4

S100A9 knockout improves insulin sensitivity in aged mice. (A–C) The body weight (A) fasting blood glucose (B) and serum insulin level (C) in mice from different groups (n = 6). (D) The results of the intraperitoneal glucose tolerance test (IPGTT) in mice from different groups. (E) The area under curve of IPGTT results from different groups (n = 6). (F) The results of insulin sensitivity test (IST) in mice from different groups. (G) The area under curve of IST results from different groups (n = 6). ns, not significant. *P < 0.05, ***P < 0.001, ****P < 0.0001. ^&&P < 0.01 Aged vs. Young-control. ^#P < 0.05, ^##P < 0.01 Aged-S100A9^−/− vs. Aged.

Fig. 5

S100A9 knockout mitigates oxidative stress in the aorta of aged mice. (A) Representative Western blots of Nox1 in the aortas from mice in different groups. The statistical results were displayed in (B) (n = 6). (C) The results of NADPH oxidase activity in mice from different groups (n = 6). (D) The MDA content in aortas from different groups (n = 6). (E) Representative images of dihydroethidium (DHE) staining in aortas from different groups and the statistical results were exposed in (F), n = 4. ns, not significant. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Fig. 6

S100A9 knockout attenuates inflammatory response in aged mice. (A–C) The interleukin 1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α) levels in the serum of mice in different groups (n = 6). (D) Representative Western blots of Phospho-P38 and P65 in the aortas from mice in different groups. The related statistical results were exhibited in (E) and (F) (n = 6). ns, not significant. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Fig. 7

S100A9 induces cellular senescence in human aortic endothelial cells via TLR4. (A) Representative Western blots of Nox1 in HAECs treated with rhS100A9 with TLR4 or RAGE inhibitors. The statistical results were presented in (B) (n = 6). (C–E) The results of NADPH oxidase and Sirtuin activities and NO content in HAECs from different groups (n = 6). (F–G) Representative pictures of SA-β-Gal-stained HAECs and quantification of SA-β-Gal-staining positive cells (n = 5). ns, not significant. *P < 0.05, **P < 0.001, ***P < 0.001, ****P < 0.0001.