Maladaptive lymphangiogenesis is associated with synovial iron accumulation and delayed clearance in factor VIII-deficient mice after induced hemarthrosis

Background: Mechanisms of iron clearance from hemophilic joints are unknown.

Objectives: To better understand mechanisms of iron clearance following joint bleeding in a mouse model of hemophilia.

Methods: Hemarthrosis was induced by subpatellar puncture in factor VIII (FVIII)-deficient (FVII^-/-) mice, +/- periprocedural recombinant human FVIII, and hypocoagulable (^HypoBALB/c) mice. ^HypoBALB/c mice experienced transient FVIII deficiency (anti-FVIII antibody) at the time of injury combined with warfarin-induced hypocoagulability. Synovial tissue was harvested weekly up to 6 weeks after injury for histological analysis, ferric iron and macrophage accumulation (CD68), blood and lymphatic vessel remodeling (αSMA; LYVE1). Synovial RNA sequencing was performed for FVIII^-/- mice at days 0, 3, and 14 after injury to quantify expression changes of iron regulators and lymphatic markers.

Results: Bleed volumes were similar in FVIII^-/- and ^HypoBALB/c mice. However, pronounced and prolonged synovial iron accumulation colocalizing with macrophages and impaired lymphangiogenesis were detected only in FVIII^-/- mice and were prevented by periprocedural FVIII. Gene expression changes involved in iron handling (some genes with dual roles in inflammation) and lymphatic markers supported proinflammatory milieu with iron retention and disturbed lymphangiogenesis.

Conclusion: Accumulation and delayed clearance of iron-laden macrophages were associated with defective lymphangiogenesis after hemarthrosis in FVIII^-/- mice. The absence of such findings in ^HypoBALB/c mice suggests that intact lymphatics are required for removal of iron-laden macrophages and that these processes depend on FVIII availability. Studies to elucidate the biological mechanisms of disturbed lymphangiogenesis in hemophilia appear critical to develop new therapeutic targets.