Objective:
Repeated joint bleeding in patients with hemophilia leads to hemophilic arthropathy (HA), which cannot be entirely prevented by clotting factor replacement. Vascular remodeling and permeability are associated with hemarthrosis and may contribute to HA progression; however, the underlying mechanisms and effects of FVIII replacement are poorly understood. Here, we explored vascular changes and synovial gene expression profiles in FVIII-deficient mice after induced hemarthrosis +/- FVIII replacement.
Methods:
Hemarthrosis was induced in FVIII-deficient mice by sub-patellar needle puncture +/- 100-200 IU/kg recombinant human FVIII (rhFVIII) intravenously 2 hours before and 6 hours after injury. Vascularity and gene expression were analyzed at baseline and 2 weeks post-injury. Vascularity was assessed by histology with Safranin-O-Fast Green and α-smooth muscle actin (αSMA) staining, and by musculoskeletal ultrasound with Power Doppler to detect microvascular flow. The permeability of synovial vessels was determined by quantification of extravasated albumin-bound Evans blue dye in knee joints at near-infrared fluorescence. For gene expression studies, RNA was extracted from synovial tissue and cDNA libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit and sequenced on an Illumina NextSeq500 platform (single-end; 75bp reads). The R BioConductor packages tximport, edgeR and limma were used to read Salmon transcript files and implement the limma-voom method for differential expression analyses. Functional enrichment was performed using Signaling Pathway Impact Analysis.
Results:
Knee injury caused profound hemarthrosis in vehicle-treated mice that was largely prevented with rhFVIII prophylaxis (day 2 hematocrit: 26.4% and 46.3%, respectively). Soft tissue proliferation increased to a similar extent in both groups, as did various vascular parameters: microvascular flow (vehicle: 1.8-fold; rhFVIII: 1.5-fold), vessel number (vehicle: 2.2-fold, p=0.0005; rhFVIII: 2.0-fold, p=0.004), vessels with diameter ≥ 20 µm (vehicle: 2.9-fold, p=0.02; rhFVIII: 2.7-fold, p=0.02), and αSMA area per vessel (vehicle: 2.3-fold, p>0.05; rhFVIII: 3.6-fold, p=0.0006). Vascular permeability also increased significantly after joint bleeding (1.7-fold, p=0.0007) and was only partially rescued by rhFVIII prophylaxis (1.3-fold, p>0.05). RNA sequencing revealed a strong transcriptional response (1527 differentially expressed genes (DEG), 13 perturbed pathways), that was not substantially dampened in rhFVIII-treated mice (891 DEG, 20 perturbed pathways). Notably, perturbation of extracellular matrix (ECM)-receptor interactions was highly significant with vehicle (pGFWER=1.7x10-11) and rhFVIII prophylaxis (pGFWER=1.4x10-10). STRING analysis of the top 20 DEG revealed enrichment of ECM components and organization, and numerous genes encoding ECM constituents, including collagens and MMPs, were significantly upregulated. Changes in ECM gene expression may facilitate the observed synovial tissue and vascular remodeling after hemarthrosis.
Conclusions:
Hemarthrosis in FVIII-deficient mice triggers profound changes in synovial gene expression that may drive associated tissue and vascular remodeling processes. These changes are incompletely mitigated by rhFVIII prophylaxis. Further exploration will enable identification of disease markers and targeted drug discovery to intercept the progression of HA.