Intermittent supplementation with fisetin improves arterial function in old mice by decreasing cellular senescence

Abstract

Cellular senescence and the senescence-associated secretory phenotype (SASP) contribute to age-related arterial dysfunction, in part, by promoting oxidative stress and inflammation, which reduce the bioavailability of the vasodilatory molecule nitric oxide (NO). In the present study, we assessed the efficacy of fisetin, a natural compound, as a senolytic to reduce vascular cell senescence and SASP factors and improve arterial function in old mice. We found that fisetin decreased cellular senescence in human endothelial cell culture. In old mice, vascular cell senescence and SASP-related inflammation were lower 1 week after the final dose of oral intermittent (1 week on-2 weeks off-1 weeks on dosing) fisetin supplementation. Old fisetin-supplemented mice had higher endothelial function. Leveraging old p16-3MR mice, a transgenic model allowing genetic clearance of p16^INK4A -positive senescent cells, we found that ex vivo removal of senescent cells from arteries isolated from vehicle- but not fisetin-treated mice increased endothelium-dependent dilation, demonstrating that fisetin improved endothelial function through senolysis. Enhanced endothelial function with fisetin was mediated by increased NO bioavailability and reduced cellular- and mitochondrial-related oxidative stress. Arterial stiffness was lower in fisetin-treated mice. Ex vivo genetic senolysis in aorta rings from p16-3MR mice did not further reduce mechanical wall stiffness in fisetin-treated mice, demonstrating lower arterial stiffness after fisetin was due to senolysis. Lower arterial stiffness with fisetin was accompanied by favorable arterial wall remodeling. The findings from this study identify fisetin as promising therapy for clinical translation to target excess cell senescence to treat age-related arterial dysfunction.

Keywords: aging; arterial stiffness; cellular senescence; endothelial function; nutraceutical; vascular dysfunction.

FIGURE 1

Fisetin suppresses cellular senescence in endothelial cells. Cell viability in replicating (control) and senescent human umbilical vein endothelial cells (HUVECs) to increasing doses of fisetin (dashed line represents the IC₅₀). IC₅₀ values of control and senescent HUVECs with 95% confidence intervals. Viability of control and senescent HUVECs at 1 μM fisetin (n = 4–8) (a). Senescence‐associate beta galactosidase (SA‐β‐Gal) signal in control and senescent HUVECs to increasing doses of fisetin (n = 3) and representative images, right (b). mRNA gene expression of cellular senescence markers Cdkn2a and Cdkn1a in control, senescent, or 1 μM fisetin‐treated senescent human aortic endothelial cells (HAECs) (n = 3) (c). Values represent mean ± SEM. *p < 0.05 senescent versus senescent + treatment; ^p < 0.05 versus control.

FIGURE 2

Fisetin reduces cellular senescence in the vasculature of old mice. Study design used in the animal model (a). Aortic mRNA gene expression of cellular senescence markers in old p16‐3MR (3MR) mice (n = 12–15) (b). Aortic protein abundance of p16^INK4A in wild‐type mice with representative virtual blot bands (n = 8) (c). Aortic mRNA gene expression of senescence‐associated secretory phenotypes factors (n = 14–15) (d). Color intensities represent log₂‐fold changes. Values represent mean ± SEM. *p < 0.05 vehicle versus fisetin.

FIGURE 3

Fisetin improves endothelial function in old mice by suppressing cellular senescence and increasing nitric oxide (NO) bioavailability. Endothelium‐dependent dilation (EDD) in isolated carotid arteries in response to acetylcholine (ACh; 1 × 10⁻⁹ to 1 × 10⁻⁴ M; n = 12–18) (a,b). EDD in response to ACh in the presence or absence of ex vivo genetic elimination of cellular senescence via ganciclovir (GCV; 5 μm; 180 min preincubation; n = 9–11) (c). NO‐mediated EDD in response to ACh in the presence or absence of the NO‐synthase inhibitor, L‐NAME (0.1 mM; 30 min preincubation; n = 15) (d,e). Endothelium‐independent dilation to increasing doses of the NO donor, sodium nitroprusside (SNP; 1 × 10⁻¹⁰ to 1 × 10⁻⁴ M; n = 9) (f). Values represent mean ± SEM. *p < 0.05 old vehicle versus old fisetin; ^p < 0.05 versus ACh alone.

FIGURE 4

Fisetin improves endothelial function by ameliorating whole‐cell and mitochondrial oxidative stress. Whole‐cell aortic reactive oxygen species (ROS) levels (n = 6–8) (a). Aortic protein abundance of NADPH oxidase (n = 8) (b) and CuZn superoxide dismutase (SOD; n = 11–12) (c) with representative virtual blot bands, right. Endothelium‐dependent dilation (EDD) in isolated carotid arteries in response to acetylcholine (ACh) in the presence or absence of the SOD mimetic, TEMPOL (1 mM, 60 min preincubation; n = 7–8) (d). Aortic mitochondrial ROS levels (n = 6–10) (e). Aortic protein abundance of phosphorylated p66^SHC (p‐p66^SHC; n = 11–12) (f) and MnSOD (n = 11–12) (g) with representative virtual blot bands, right. EDD in carotid arteries in response to ACh in the presence or absence of the mitochondrial‐specific antioxidant, MitoQ (1 μM; 60 min preincubation; n = 5–7) (h). Values represent mean ± SEM. *p < 0.05 old vehicle versus old fisetin; ^p < 0.05 versus ACh alone.

FIGURE 5

Fisetin reduces arterial stiffness in old mice by improving aortic intrinsic mechanical wall stiffness. Aortic pulse wave velocity (PWV) was measured pre‐ and post‐intervention (n = 9–10) (a). Representative stress–strain curve for determination of ex vivo intrinsic mechanical wall stiffness which was assessed by elastic modulus in aortic rings (calculated as the slope of the final four points in the stress–strain curve) (n = 7) (b). Elastic modulus was assessed after ex vivo genetic (GCV) (n = 8–10) (c) and pharmacological (ABT‐263) (n = 9–10) (d) suppression of cellular senescence in aortas obtained from old p16‐3MR (3MR) and wild‐type mice, respectively. Values represent mean ± SEM. *p < 0.05 old vehicle versus old fisetin; ^p < 0.05 versus media only.

FIGURE 6

Fisetin reduces aortic intrinsic mechanical wall stiffness by favorably remodeling components of the arterial wall. Aortic protein abundance of advanced glycation end products (AGEs) (n = 9) (a), collagen‐1 (n = 9) (b), and α‐elastin (n = 9) (c) with representative virtual blot bands. Representative immunohistochemical staining of aortic AGEs (d) and collagen‐1 (e). Arrows denote protein accumulation in the medial‐adventitial layer. Values represent mean ± SEM. *p < 0.05 old vehicle versus old fisetin.