Vascular Permeability and Remodelling Coincide with Inflammatory and Reparative Processes after Joint Bleeding in Factor VIII-Deficient Mice

Abstract

Vascular remodelling is a prominent feature of haemophilic arthropathy (HA) that may underlie re-bleeding, yet the nature of vascular changes and underlying mechanisms remain largely unknown. Here, we aimed to characterize synovial vascular remodelling and vessel integrity after haemarthrosis, as well as temporal changes in inflammatory and tissue-reparative pathways. Thirty acutely painful joints in patients with haemophilia (PWH) were imaged by musculoskeletal ultrasound with Power Doppler (MSKUS/PD) to detect vascular abnormalities and bloody effusions. Nineteen out of 30 painful joint episodes in PWH were associated with haemarthrosis, and abnormal vascular perfusion was unique to bleeding joints. A model of induced haemarthrosis in factor VIII (FVIII)-deficient mice was used for histological assessment of vascular remodelling (α-smooth muscle actin [αSMA] expression), and monitoring of in vivo vascular perfusion and permeability by MSKUS/PD and albumin extravasation, respectively. Inflammatory (M1) and reparative (M2) macrophage markers were quantified in murine synovium over a 10-week time course by real-time polymerase chain reaction. The abnormal vascular perfusion observed in PWH was recapitulated in FVIII-deficient mice after induced haemarthrosis. Neovascularization and increased vessel permeability were apparent 2 weeks post-bleed in FVIII-deficient mice, after a transient elevation of inflammatory macrophage M1 markers. These vascular changes subsided by week 4, while vascular remodelling, evidenced by architectural changes and pronounced αSMA expression, persisted alongside a reparative macrophage M2 response. In conclusion, haemarthrosis leads to transient inflammation coupled with neovascularization and associated vascular permeability, while subsequent tissue repair mechanisms coincide with vascular remodelling. Together, these vascular changes may promote re-bleeding and HA progression.

Mechanisms of vascular permeability and remodeling associated with hemarthrosis in factor VIII‐deficient mice

“…Hemarthrosis in FVIII‐deficient mice leads to synovial neovascularization and associated vascular permeability, followed by formation of large, irregular vessels with increased α‐smooth muscle actin (αSMA) expression . The findings in mice align with patient case studies .…”

“…Clinically noticeable hemarthrosis (swelling, bruising, and immobility) was induced by several subpatellar punctures of the right knee with a 30‐G needle. The extent of intra‐/peri‐articular bleeding was determined by hematocrit measurement on day 2 after injury . The hematocrit was monitored every 3‐4 days for 2 weeks in some mice to check for rebleeding events.…”

“…Right knee joints were harvested at baseline and 2 or 4 weeks post injury. Joints were fixed, decalcified, embedded, and sectioned as described previously . Vascular changes were studied histologically by Safranin‐O‐Fast Green staining.…”

“…Expression of αSMA, collagen IV (Col4), and MMP‐9 was detected using rabbit polyclonal antibodies purchased from abcam (αSMA: ab5964, Col4: ab6586, MMP‐9: ab38898). Stained areas corresponding to αSMA were quantified using ImageJ …”

“…Synovial vascular changes may be fueled by upregulation of pro‐angiogenic factors, such as vascular endothelial growth factor, matrix metalloproteinase (MMP)‐9, and stromal‐derived factor‐1, and inflammatory responses to hemarthrosis, including macrophage infiltration and upregulation of proinflammatory cytokines (interleukin (IL)‐1β, IL‐6, and tumor necrosis factor‐α) . We recently demonstrated that a transient proinflammatory macrophage M1 response is superseded by a prolonged tissue‐reparative macrophage M2 response . The timing of these responses coincided with early soft tissue inflammation, vascular permeability, and the time course of vascular remodeling.…”