Lymphatic Remodeling Associated with Hemophilic Joint Bleeding in FVIII-Deficient Mice
Authors: Cooke Esther J , Nasamran Chanond A , Fisch Kathleen , von Drygalski Annette 
Published: November 2020

Introduction
Hemophilic joint bleeding leads to toxic synovial iron accumulation, contributing to synovitis. Mechanisms of blood and iron clearance from the joint remain largely unknown. This study aimed to explore synovial lymphatic vessel remodeling in association with hemarthrosis and iron accumulation in Factor VIII (FVIII)-deficient mice as a potential mechanism of iron clearance.
Methods
Hemarthrosis was induced by sub-patellar puncture in FVIII-deficient mice and the hematocrit determined on day 2 as a measure of joint bleeding. Synovial tissue was harvested at baseline and 2 weeks after knee injury for gene expression analyses by RNA sequencing. Briefly, RNA was purified from synovial tissue and sequenced (single-end; 75 bp) on an Illumina NextSeq500 platform. The limma-voom method (R BioConductor) was used for differential expression analyses. Whole knee joints were harvested at baseline and 2 or 4 weeks after knee injury for histological analysis of iron accumulation, as well as blood and lymphatic vessels. Ferric iron (Fe3+) was detected by Prussian blue staining during joint decalcification. Remodeling blood and lymphatic vessels were detected using antibodies for α-smooth muscle actin (αSMA) and lymphatic vascular endothelial hyaluronan receptor 1 (LYVE1), respectively. Total soft tissue and stained areas in histology images were quantified using ImageJ. Co-localization of αSMA and LYVE1 was assessed by laser scanning confocal microscopy with a Zeiss LSM 800 microscope.
Results
Knee injury induced hemarthrosis in FVIII-deficient mice (mean day 2 hematocrit: 27 %) and significant soft tissue proliferation, indicative of synovitis, at 2 weeks (2.9-fold increase in mean tissue area, p=0.007). Gene expression analyses by RNA sequencing revealed differential expression of lymphatic markers, including very high up-regulation of chemokine (C-C motif) ligand 21 (194-fold, p=0.001) and moderate up-regulation of semaphorin 7a (4.9-fold, p=0.02). Up-regulation of these genes may promote homing and adherence of macrophages and dendritic cells to lymphatic endothelial cells (LECs), facilitating lymphangiogenesis. Up-regulation of FMS-like tyrosine kinase 4 (receptor for vascular endothelial growth factor (VEGF)-C and -D) (5.7-fold, p=0.0007) and collagen and calcium-binding EGF domain-1 (5.6-fold, p=0.03) may also favor lymphangiogenesis. However, VEGF-C expression was unchanged and VEGF-D was 2.5-fold down-regulated (p=0.0002). Likewise, expression of LEC marker LYVE1 was down-regulated (2.6-fold, p=0.05). These findings stimulated histological evaluations of lymphatic vessels in relation to iron and blood vessels in synovial tissue. Prussian blue staining showed significant increases in ferric iron accumulation at 2 weeks (38-fold, p=0.005) and 4 weeks relative to baseline (23-fold, p=0.03). This coincided with blood vessel remodeling, evidenced by increased αSMA expression (r=0.6, p=0.0008). Unlike αSMA, an abundance of LYVE1 was detected in baseline synovial tissue. LYVE1-positive vessels appeared as small, flat structures, in contrast to larger, more rounded αSMA-positive vessels. Confocal microscopy confirmed staining of distinct blood and lymphatic vascular structures. When normalized for tissue area, LYVE1 expression was 1.8-fold decreased at 2 weeks (p=0.04) after knee injury compared to baseline and returned to near-baseline levels at 4 weeks. There was a significant negative correlation between iron accumulation (Prussian blue) and lymphatic vessel staining normalized to tissue area (r=-0.6223, p=0.006). The morphology of lymphatic vessels after joint bleeding was different to baseline, with many vessels appearing as larger, more confluent structures with visible lumens, sometimes wrapping around αSMA-positive blood vessels.
Conclusions
Synovial tissue in FVIII-deficient mice contains a rich network of lymphatics. Gene expression changes and altered lymphatic vessel morphology after joint bleeding point toward profound changes in lymphatic homeostasis in inflamed synovium. The reduction in lymphatic vessel density suggests an inadequate lymphangiogenic response, which may hamper iron clearance. The role of the lymphatic network after hemophilic joint bleeding warrants further investigation and may represent a novel target for improving bleed resolution and reducing synovitis.

Disclosures
von Drygalski: Bioverativ/Sanofi: Honoraria, Research Funding; Takeda: Honoraria; Novo-Nordisk: Honoraria; Biomarin: Honoraria; Uniqure: Honoraria; Pfizer: Research Funding; Hematherix LLC: Membership on an entity's Board of Directors or advisory committees, Other: co-founder.