Chondroitin Sulfate Enrichment in the Extracellular Matrix Inhibits NETosis in a 3D Calcific Aortic Valve Disease Model

Calcific aortic valve disease (CAVD) is the most common valvular disorder in high-income countries, and its prevalence is projected to rise with an aging population. No pharmacological therapies currently exist to halt or reverse progression, reflecting the complex and poorly understood pathophysiology. A hallmark of CAVD is extracellular matrix (ECM) remodeling and accumulation of proteoglycans such as chondroitin sulfate (CS). CAVD is also recognized as an inflammatory disease, with emerging evidence implicating neutrophils and their extracellular traps (NETs) as active contributors to disease progression. However, the impact of CS enrichment on neutrophil behavior remains unclear. Here, we developed a disease-inspired 3D ECM hydrogel model to investigate how ECM composition regulates neutrophil function and NET formation. Human neutrophils were encapsulated in type I collagen hydrogels enriched with CS, mimicking healthy and diseased valve tissue. CS enrichment impaired neutrophil effector functions, including actin cytoskeleton remodeling and NETosis. Neutrophils in CS-rich matrices showed disrupted actin dynamics, reduced chromatin decondensation, and diminished histone H3 citrullination, resulting in suppressed NET release in response to NADPH oxidase-dependent and calcium-mediated stimuli. These findings identify CS enrichment as a regulator of neutrophil function through disruption of actin cytoskeleton remodeling. Beyond CAVD, this study highlights the importance of ECM composition in modulating immune responses.