OC 33.2 - Maladaptive Lymphangiogenesis is Associated with Synovial Iron Accumulation and Delayed Clearance in FVIII-/- Mice After Induced Hemarthrosis

Background: Hemophilic hemarthroses lead to toxic synovial iron accumulation. Mechanisms of iron clearance are unknown.

Aims: To study synovial iron clearance and relationships with macrophage infiltration and lymphatic vessel remodeling in Factor VIII deficient (FVIII-/-) mice and hypocoagulable wild-type mice (HypoBALB/c) after induced hemarthrosis.

Methods: Hemarthrosis was induced by subpatellar puncture in FVIII-/- mice [+/- peri-procedural recombinant human FVIII (rhFVIII)] and in HypoBALB/c mice [transient FVIII-deficiency (anti-FVIII antibody and warfarin)]. Knee joints were harvested up to 6 weeks after injury for histological, iron and macrophage (CD68) analysis, blood (αSMA) and lymphatic vessel (LYVE1) remodeling, and gene expression changes of iron and lymphatic regulators (RNAseq).

Results: Knee injury caused similar bleed volumes in FVIII-/- and HypoBALB/c mice. However, pronounced, and prolonged iron accumulation in macrophages was only detected in FVIII-/- mice and was prevented by peri-procedural FVIII. Gene expression changes were present for iron regulators, some with dual roles in inflammation and suggested pro-inflammatory M1-polarization of macrophages with iron retention.

Vascular remodeling was comparable between FVIII-/- and HypoBALB/c mice, but lymphangiogenesis was only impaired in FVIII-/- mice. Synovial LYVE1 protein expression in FVIII-/- mice at baseline was significantly lower compared to HypoBALB/c mice, and was strongly suppressed after hemarthrosis, accompanied by lymphatic vessel rarefication, and marked gene expression changes of lymphatic regulators. Those included impressive down-regulation of Lyve1, paralleling LYVE1 protein expression, but also Vegfc and Vegfd, genes encoding growth factors critical for lymphangiogenesis.

Conclusion(s): Pronounced accumulation and delayed clearance of iron-laden pro-inflammatory macrophages was associated with defective lymphangiogenesis after hemarthrosis in FVIII-/- mice. The absence of such findings in HypoBALB/c mice with transient FVIII deficiency but similar joint bleed volumes suggests that processes involving adaptive lymphangiogenesis are critical for the removal of synovial iron-laden macrophages, and are dependent on FVIII availability during the healing phase.