Aging-Associated Liver Sinusoidal Endothelial Cells Dysfunction Aggravates the Progression of Metabolic Dysfunction-Associated Steatotic Liver Disease

Abstract

Aging increases the susceptibility to metabolic dysfunction-associated steatotic liver disease (MASLD). Liver sinusoidal endothelial cells (LSECs) help in maintaining hepatic homeostasis, but the contribution of age-associated LSECs dysfunction to MASLD is not clear. The aim of this study was to investigate the effect of aging-associated LSECs dysfunction on MASLD. Free fatty acid-treated AML12 cells were co-cultured with young and etoposide-induced senescent TSEC cells to evaluate the senescence-associated endothelial effects on the lipid accumulation in hepatocytes. In addition, young and aged rats were subjected to methionine-choline-deficient diet-induced metabolic dysfunction-associated steatohepatitis (MASH). Hepatic hemodynamics and endothelial dysfunction were evaluated by in situ liver perfusion. Liver tissue samples from young and aged healthy controls and MASH patients were also analyzed. Steatotic AML12 cells co-cultured with young TSEC cells showed less lipid accumulation, and such effect was abolished by eNOS inhibitor or with senescent TSEC cells. However, co-culture with resveratrol-treated senescent TSEC cells could partially resume the NO-mediated protective effects of endothelial cells. Furthermore, aged MASH rats showed more severe liver injury, steatosis, fibrosis, and endothelial and microcirculatory dysfunction. In addition, aged MASH patients showed more pronounced liver injury and fibrosis with lower hepatic eNOS, p-eNOS, and SIRT1 protein levels than in young patients. Senescence compromises the protective effects of LSECs against hepatocyte steatosis. In addition, aging aggravates not only liver steatosis and fibrosis but also intensifies LSECs dysfunction in MASH rats. Accordingly aged MASH patients also showed endothelial dysfunction with more severe liver injury and fibrosis.

Keywords: aging; fibrosis; liver sinusoidal endothelial cells; metabolic dysfunction–associated steatotic liver disease.

FIGURE 1

Endothelial dysfunction and decreased SIRT1 expression in senescent TSEC cells. (A) Western blot and (B) quantitative analysis of eNOS, p‐eNOS, and SIRT1 protein levels in senescent TSEC cells at day 5 (N = 6). (C) Quantitative analysis of senescent TSEC mRNA expression levels of eNOS and SIRT1 at day 5 (N = 6). (D) Nitrite concentration in control and etoposide‐treated TSEC cells at day 5 (N = 6). (E) Western blot and (F) quantitative analysis of iNOS protein levels after treatment with different concentration of etoposide in the presence or absence of iNOS inhibitor 1400 W (day 5) (N = 3). (G) Nitrite concentration in TSEC cells at day 5 after treatment with etoposide with/without 1400 W (N = 6). (H) Quantitative analysis of eNOS and SIRT1 mRNA levels in TSEC cells at day 5 after etoposide treatment in the absence or presence of 1400 W (N = 5). Data expressed as mean ± SEM, one‐way ANOVA followed by Tukey's post hoc test. In B–D, F–H, *, **, ***, ns represent comparison with control group; in F–H, #, ##, ###, ns show intergroup comparison. */#p < 0.05, **/##p < 0.01, ***/###p < 0.001, and ns/ns = not significant.

FIGURE 2

The effects of endothelial cells and resveratrol‐treated senescent endothelial cells co‐culture on hepatocyte fat accumulation. (A) Western blot and (B) quantitative analysis of eNOS, p‐eNOS, and SIRT1 protein levels of TSEC cells after etoposide and/or resveratrol treatment along with 1400 W. (C) TSEC nitrite levels after treatment with etoposide, resveratrol and 1400 W. (D) Oil Red O staining and (E) evaluation of triglyceride concentration in F‐AML12 mono‐culture or co‐cultured with YTSEC, mono‐culture with addition of nitric oxide donor SNAP, or inhibitor L‐NAME, or co‐cultured with Y‐TSEC in the presence of L‐NAME. (F) Oil Red O staining and (G) triglyceride concentration estimation in F‐AML12 cells cultured alone or co‐cultured with the indicated TSEC cells. In (D, F), scale bar = 5 μm. Data expressed as mean ± SEM (N = 6), one‐way ANOVA followed by Tukey's post hoc test. In (E) and (G), *, ** and ns show comparison vs. control group; in (B) and (C), #, ##, ###, ns show intergroup comparisons. */#p < 0.05, **/##p < 0.01, ***/###p < 0.001, and ns/ns = not significant. F‐AML12, high free fatty acid–treated AML12 cells; L‐NAME, NG‐Nitroarginine methyl ester hydrochloride; R‐STSEC, resveratrol‐treated senescent TSEC cells; R‐YTSEC, resveratrol‐treated young TSEC cells; SNAP, S‐Nitroso‐N‐acetyl‐DL‐penicillamine; STSEC, senescent TSEC cells; YTSEC, young TSEC cells.

FIGURE 3

Age‐associated effects on liver injury and fibrosis of diet‐induced MASH model. (A) Liver/body weight ratio. (B) Spleen/body weight ratio. (C) Serum ALT and AST levels. (D) Hepatic triglyceride concentration. (E) Representative images of liver morphology, H & E staining, and Sirius Red staining (scale bar = 100 μm). (F) Western blot and (G) quantitative analysis of hepatic eNOS, p‐eNOS, SIRT1, and α‐SMA protein expression levels. Data expressed as mean ± SEM (N = 9–12 for A and B, N = 6 for C–G), one‐way ANOVA followed by Tukey's post hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, and ns = not significant.

FIGURE 4

Effect of aging on liver injury, fibrosis, and LSECs‐related protein expression in MASH patients. (A) Serum ALT and AST levels. (B) Representative images of H & E staining and Sirius Red staining (scale bar = 50 μm). (C) Western blot and (D) quantitative analysis of hepatic eNOS, p‐eNOS, SIRT1, and α‐SMA protein expression levels. Data expressed as mean ± SEM (N = 9), one‐way ANOVA followed by Tukey's post hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, and ns = not significant.

FIGURE 5

Effects of aging and MASH on the portal vascular bed reactivity. The PVR in response to increasing concentrations of (A–D) ACH and (E–H) SNAP of young (8 weeks) and aged (78 weeks) rats after 12 weeks of MCD diet or standard chow diet feeding. Data expressed as mean ± SEM (N = 9–12), general linear model for repeated measurements. ACH, acetylcholine; MTX, methoxamine; PVR, portal venous resistance; SNAP, S‐Nitroso‐N‐acetyl‐DL‐penicillamine.

FIGURE 6

Influence of aging and MASH on the hepatic artery vascular bed reactivity. The HAR in response to increasing concentrations of MTX in the (A, C, E, and G) absence and (B, D, F, and H) presence of L‐NMMA of young (8 weeks) and aged (78 weeks) rats fed with standard chow diet or MCD diet. Data expressed as mean ± SEM (N = 9–12), general linear model for repeated measurements. HAR, hepatic arterial resistance; L‐NMMA, NG‐Methyl‐L‐arginine acetate salt; MTX, methoxamine.

FIGURE 7

Combined effects of aging and MASH on the sinusoidal vascular bed reactivity in response to vasoactive drugs. The SVR in response to increasing concentrations of (A, C, E, and G) ACH and (B, D, F, and H) SNAP administration in the portal vein of young (8 weeks) and aged (78 weeks) control and MASH rats. Data expressed as mean ± SEM (N = 9–12), general linear model for repeated measurements. ACH, acetylcholine; MTX, methoxamine; SNAP, S‐Nitroso‐N‐acetyl‐DL‐penicillamine; SVR, sinusoidal vascular resistance.