Hypothesis

In aged MASLD livers, hypoxia‑inducible factor‑1α (HIF‑1α) is stabilized in zone 3 hepatocytes by mitochondrial ROS generated from excess fatty acid oxidation, triggering a glycolytic shift that depletes NADPH, elevates labile iron, and sensitizes cells to lipid‑peroxidation‑driven ferroptosis. This HIF‑1α‑dependent metabolic rewiring creates the Aging Hepatocyte Gene Signature (AHGS) and couples ferroptosis to senescence‑associated secretory phenotype (SASP) expansion, thereby linking zonated steatosis to progressive inflammation and fibrosis. Pharmacologic or genetic inhibition of HIF‑1α in zone 3 should reduce AHGS burden, restore DNA repair, and attenuate fibrosis even after steatosis is established.

Mechanistic Rationale

• Zone 3 hepatocytes already exhibit high SREBP‑1c‑driven lipogenesis and limited oxidative capacity, making them prone to mitochondrial overload when fatty acid influx rises (3).
• Mitochondrial NADH accumulation increases superoxide production, which inhibits prolyl‑hydroxylase domain (PHD) enzymes, preventing HIF‑1α degradation and allowing its nuclear translocation.
• HIF‑1α transcriptionally upregulates GLUT1, LDHA, and PDK1, shifting pyruvate away from the TCA cycle toward lactate, thereby lowering mitochondrial NADH oxidation and NADPH generation via the pentose‑phosphate pathway.
• Reduced NADPH compromises glutathione peroxidase 4 (GPX4) activity, while HIF‑1α‑induced transferrin receptor (TFRC) upregulation increases intracellular labile iron, together creating a permissive environment for ferroptosis (1).
• Ferroptotic lipid peroxidation triggers DNA damage responses that activate p21‑mediated senescence and SASP, amplifying macrophage recruitment and fibrotic signaling in the pericentral niche ([synthetic_research_questions_feedback_2.md]).

Testable Predictions

1. In liver sections from aged MASLD patients, HIF‑1α nuclear positivity will colocalize with AHGS markers (e.g., p16, 4‑HNE) specifically in zone 3, and the degree of colocalization will correlate with fibrosis stage (F0‑F4).
2. Genetic deletion of Hif1a in albumin‑Cre hepatocytes (or AAV8‑mediated CRISPRi targeting Hif1a) in aged, high‑fat diet‑fed mice will:
   • Decrease the proportion of AHGS+ hepatocytes by >40% compared with littermate controls.
   • Reduce labile iron pool (measured by Calcein‑AM quenching) and lipid‑ROS (C11‑BODIPY) in isolated zone 3 hepatocytes.
   • Improve GPX4 activity and restore NADPH/NADP+ ratio.
   • Lower serum ALT, hepatic hydroxyproline, and Sirius Red area despite continued steatosis.
3. Pharmacologic inhibition of HIF‑1α (e.g., with PT2385) administered after establishment of F1‑F2 fibrosis will still attenuate further fibrosis progression, whereas treatment initiated after F3 fibrosis will show minimal benefit, defining a therapeutic window.
4. Combined HIF‑1α inhibition with ferrostatin‑1 will produce synergistic reduction of AHGS burden and SASP cytokines (IL‑6, CCL2) beyond either monotherapy, as measured by multiplex ELISA of liver homogenates.

Experimental Approaches

• Spatial transcriptomics (10x Visium) on human liver biopsies to map HIF‑1α target genes alongside AHGS signatures across zones.
• Zone‑specific isolation via perfusion‑based collagenase digestion followed by laser‑capture microdissection to assess iron, NADPH, and GPX4 activity.
• In vivo lineage tracing using ROS‑reporter mice (MitoSOX‑GFP) to link mitochondrial ROS spikes to HIF‑1α activation.
• Intervention trials in aged MASH mice with inducible Hif1a deletion or PT2385 dosing, longitudinal MRI‑PDFF and elastography, endpoint histology.

Falsifiability

If HIF‑1α activation is not enriched in zone 3 AHGS+ hepatocytes, or if its inhibition fails to reduce AHGS burden, iron load, or fibrosis despite adequate target engagement, the hypothesis would be refuted. Conversely, confirmation would support a zonated metabolic‑oxidative axis linking aging, ferroptosis, and fibrosis, suggesting HIF‑1α as a druggable node for early MASLD.