Synovial Fluid-Derived Extracellular Vesicles: Critical Modulators of Endothelial Cell Function

Introduction Inflammation plays a crucial role in the pathophysiology of several musculoskeletal disorders, including Femoroacetabular Impingement (FAI) and Osteoarthritis (OA). The progression of inflammation in OA is influenced by its microenvironment, which includes endothelial cells (ECs). ECs play a key role in the abnormal angiogenesis seen in OA, increasing vascularization and contributing to inflammation. Extracellular vesicles (EVs) are nanoscale, lipid-bound particles found in the synovial fluid (SF) important in both pathological and physiological processes. They are involved in intercellular communication, mediating the transfer of numerous bioactive molecules that can impact angiogenesis and ECs activation. Even though EVs studies have been exponentially expanding over the last years, the specific effect of the SF derived EVs on ECs functions remain poorly understood. Methods SFs were collected from patients with FAI, categorized as having either “low” or “high” inflammation levels based on cartilage damage scores, as well from patients with OA. The levels of interleukin-6 (IL-6) in SFs were measured using an ELISA immunoassay. Ultracentrifugation (UC) was used to isolate EVs, and nanoparticle tracking analysis (NTA) to quantify them. BCA assay was used to quantify the protein concentration in the EVs population. Dot Blot for CD63 and NanoFCM for both CD9 and CD63 were used to characterize the EVs. CryoTEM was used to evaluate the isolated EVs size and morphology. The experiments in this study are limited to small extracellular vesicles (sEVs), as these were the primary focus of subsequent analysis and experimental approaches. Confocal microscopy was used to assess the uptake of SF- sEVs labelled with PKH67 by human umbilical vein endothelial cells (HUVECs). Cell viability was evaluated by Calcein AM Assay when treated with 2 and 4 µg/mL of SF-sEVs. HUVECs migratory potential when treated was investigated using Wound healing and transwell migration assays. Tube formation assay was performed on HUVECs to study the impact of SF-sEVs on angiogenesis. The expression and localization of β-catenin and VE-cadherin were analyzed using immunofluorescence. The presence of Weibel-Palade bodies (WPBs) was investigated through transmission electron microscopy (TEM). MCP-1, ICAM-1 and VCAM-1 mRNA expressions were evaluated by RT-PCR. Results and Conclusion Our results show increased IL-6 levels in the SF of patients, progressing from low inflammation (LI) to OA. The successful separation of EVs was confirmed through NTA, enrichment in CD63 and CD9 markers as well by the morphology and size within the typical range of sEVs. Protein concentration within the sEVs under the three experimental conditions correlated with the inflammatory state. SF-sEVs were successfully delivered into HUVECs without showing any significant toxicity. Cells showed a significant increase in their migration capacity when treated with OA-SF-sEVs. The Pro-angiogenic effects of sEVs on ECs were confirmed by the formation of capillary-like structures, with distinct variations in the density and complexity of the networks depending on the treatment. Fluorescence intensity of β-catenin and VE-cadherin were significantly reduced in cells treated with LI and OA-sEVs. TEM analysis revealed the presence of WPBs in HUVECs treated with LI-sEVs. MCP1, VCAM1 and ICAM1 mRNA levels were overexpressed. Our preliminary data suggests that SF-sEVs influence ECs functions by modulating endothelial permeability and the inflammatory response.