Intrahepatic crystals from elevated dietary cholesterol are sufficient to stiffen the liver

Chronic lipid accumulation is a hallmark of metabolic dysfunction-associated steatotic liver disease (MASLD), and dyslipidemia is associated with disease progression and poorer patient outcomes. Tissue stiffening is an established fibrogenic cue, but how changes in lipid accumulation affect tissue mechanics is not fully understood. Here we show that cholesterol-containing lipid crystals stiffen steatotic liver tissue. We show that rats fed elevated dietary cholesterol develop both liquid and solid cholesterol-containing lipid crystals in the liver. While steatotic livers without crystals are softer than control livers, livers with lipid crystals show increased baseline stiffness and compression stiffening as well as increased progression to fibrosis. Lipid extracts from livers containing crystals stiffen fibrous tissue mimics, while depletion of cholesterol using methyl-ꞵ-cyclodextrin reduces both crystal abundance and tissue stiffness. Our results demonstrate in a rat model that a high cholesterol diet leads to formation of liquid and solid crystals and that cholesterol-containing crystals stiffen tissues. This work implicates lipid crystals arising from dyslipidemia as a key driver of MASLD progression. The presence of cholesterol crystals could lead to new diagnostic tools for progressive MASLD and could be a therapeutic target.