The SIRT3–GPX4 Axis Is Associated with Ferroptosis-Related Endothelial Senescence and Vascular Aging

Technical Summary: The SIRT3–GPX4 Axis in Ferroptosis-Related Endothelial Senescence and Vascular Aging

Problem Statement
Vascular aging is a fundamental pathological process characterized by structural remodeling (e.g., intimal thickening, elastic fiber fragmentation) and functional deterioration, serving as a precursor to hypertension and atherosclerosis. While endothelial senescence is recognized as an early driver of these changes, the molecular mechanisms linking iron dyshomeostasis to vascular aging remain incompletely understood. Recent concepts of "ferro-aging" suggest that progressive iron accumulation leads to iron-dependent lipid peroxidation and ferroptosis, a regulated cell death form. Although the Sirtuin 3 (SIRT3)–Glutathione Peroxidase 4 (GPX4) axis has been implicated in ferroptosis in other tissues, its specific role in endothelial senescence and the regulation of GPX4 via acetylation during vascular aging has not been established.

Methodology
The study employed complementary in vivo and in vitro models to investigate the SIRT3–GPX4 axis:

• In Vivo Model: Middle-aged (15-month-old) C57BL/6J mice were subjected to iron overload via intraperitoneal iron dextran injections to simulate age-associated iron accumulation. A subset of these mice received Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, to assess the contribution of ferroptosis to vascular pathology. Young (2-month-old) mice served as controls.
  • Assessments: Histological analysis (EVG, Masson's trichrome), immunohistochemistry (p21, γ-H2AX, ACSL4, GPX4, SIRT3), and serum biochemical assays (MDA, GSH, Fe²⁺) were performed. RNA sequencing (RNA-seq) was conducted on aortic tissues to analyze transcriptomic changes.
• In Vitro Model: Human endothelial EA.hy926 cells were induced into senescence using D-galactose (D-gal).
  • Interventions: Cells were treated with Fer-1 to inhibit ferroptosis or transduced with a lentiviral vector to overexpress SIRT3 (SIRT3-OE).
  • Assessments: Senescence was evaluated via SA-β-gal staining and Western blotting for p21/p53. Ferroptosis markers included ACSL4, GPX4, lipid ROS (C11-BODIPY), intracellular Fe²⁺ (FerroOrange), MDA, and GSH levels. Mitochondrial function was assessed via JC-1 (membrane potential) and MitoSOX (superoxide) staining.
  • Molecular Mechanism: Co-immunoprecipitation (Co-IP) and acetyl-lysine immunoblotting were used to examine the physical interaction between SIRT3 and GPX4 and to quantify GPX4 acetylation levels.

Key Results

1. Iron Overload Exacerbates Vascular Aging and Ferroptosis:

   • In middle-aged mice, iron overload significantly worsened vascular remodeling, evidenced by increased elastic fiber disruption and collagen deposition.
   • Iron overload upregulated senescence markers (p21, γ-H2AX) and ferroptosis-related markers (increased ACSL4, decreased GPX4 and SIRT3).
   • Fer-1 treatment partially attenuated these structural and molecular alterations, confirming the involvement of ferroptosis in iron-induced vascular aging.

2. Transcriptomic Insights:

   • RNA-seq revealed that iron overload altered metabolic pathways related to fatty acid metabolism, oxidative phosphorylation, and glutathione metabolism.
   • Crucially, while Gpx4 mRNA levels remained unchanged, GPX4 protein abundance was significantly reduced in iron-overloaded tissues. This discrepancy suggests post-transcriptional or post-translational regulation of GPX4.
   • Sirt3 mRNA expression was significantly downregulated in the iron-overload group.

3. Ferroptosis Inhibition vs. SIRT3 Restoration:

   • In D-gal-treated endothelial cells, Fer-1 successfully reduced lipid peroxidation, restored mitochondrial membrane potential, and decreased senescence markers. However, Fer-1 did not restore SIRT3 protein levels, suggesting SIRT3 downregulation is not merely a downstream consequence of ferroptosis.
   • Conversely, SIRT3 overexpression in senescent cells significantly reduced senescence markers (p21, p53, SA-β-gal), restored GPX4 protein abundance, decreased ACSL4, and improved mitochondrial function (reduced superoxide, restored membrane potential).

4. SIRT3–GPX4 Interaction and Acetylation:

   • Co-immunoprecipitation confirmed an endogenous physical association between SIRT3 and GPX4 in endothelial cells.
   • Immunofluorescence showed colocalization of SIRT3 and GPX4.
   • D-gal treatment increased GPX4 acetylation levels. SIRT3 overexpression significantly reduced GPX4 acetylation and concurrently increased GPX4 protein abundance.

Key Contributions

• Mechanistic Link: The study establishes a functional link between the SIRT3–GPX4 axis and ferroptosis-related endothelial senescence, proposing that SIRT3 downregulation contributes to vascular aging by promoting GPX4 acetylation and subsequent loss of GPX4 protein.
• Post-Translational Regulation: It identifies GPX4 acetylation as a critical regulatory mechanism in vascular aging, explaining the discrepancy between stable Gpx4 mRNA and reduced GPX4 protein levels observed in iron-overload models.
• Dissociation of Mechanisms: The findings demonstrate that while inhibiting ferroptosis (via Fer-1) alleviates mitochondrial dysfunction and senescence, it does not reverse SIRT3 downregulation. This suggests SIRT3 acts upstream or in parallel to ferroptotic stress, rather than solely as a downstream effector.

Significance and Claims
The authors claim that their findings support a working model where aging or iron overload leads to SIRT3 downregulation. This reduction impairs the deacetylation of GPX4, leading to increased GPX4 acetylation, reduced GPX4 protein stability/abundance, and compromised antioxidant defense. The resulting accumulation of lipid peroxides drives mitochondrial dysfunction, ferroptotic stress, and endothelial senescence, ultimately contributing to vascular aging.

The study concludes that the SIRT3–GPX4 axis represents a potential regulatory pathway linking iron dyshomeostasis to vascular aging. However, the authors maintain a modest tone, acknowledging limitations such as the reliance on pharmacological interventions rather than genetic knockouts, the exploratory nature of the transcriptomic analysis due to sample size, and the need for future studies to identify specific acetylation sites and validate these mechanisms in other vascular cell types (e.g., vascular smooth muscle cells). They do not claim to have fully resolved the mechanism of ferro-aging but provide preliminary experimental evidence supporting the SIRT3–GPX4 axis as a candidate target for delaying vascular aging.