Abstract Body: Background: Venous thrombosis (VT) can cause vein wall inflammation and fibrotic injury. Fibrinolysis and neovascularization processes are driven by venous endothelial cells (VECs) and proinflammatory (M1) or pro-resolution (M2) monocytes/macrophages (Mo/MΦ). We explore the effect of modulating macrophage receptor with collagenous structure (MARCO) in vein wall inflammation using Poly-lactide-co-glycolide/PLGA acid nanoparticles (NP), which are designed for myeloid cell uptake through MARCO.
Methods: We characterized in vitro and in vivo the effect of PLGA-NP on murine BMDMs (derived from WT C57BL/6, pro-M1 NR4a1-/- or pro-M2 CCR2-/- mice) and human (THP-1, or CD14+ Mo/MΦ) cells. qRT-PCR of Mo/MΦ cultures stimulated with PLGA-NP quantified surface receptors (ccr7), thrombolytic mediators (serpine1, mmp2), and inflammatory markers (tnfa, kmt2a). Mo/MΦ MARCO expression was inhibited through PolyG (1.6ug/ml), followed by PLGA-NP administration. We measured PLGA-NP thrombolytic properties using High whole blood thrombolysis plate assay (HALO). BMDMs cultured with fluorophore-tagged PLGA-NP were imaged to demonstrate uptake. Thrombi weight/length and vein wall thickness (Trichrome staining) measures were obtained from IVC stasis models at 8- and 14-days in C57BL/6 WT mice. Mice were given intravenous PLGA-NP once daily (xx mg/kg) on days 2 through 5 (8d group) and 2 through 11 (14d group).
Results: PLGA-NP increased mmp2 and decreased PAI-1 transcripts in a MARCO-dependent manner. Clot lysis activity was noted with PLGA-NP ex vivo (HALO assay). Fluorescent-tagged PLGA-NP were visualized intracellularly in cultured BMDMs. PLGA-NPs significantly decreased vein wall thickness (VWT) in 8- and 14-day animals treated with PLGA-NPs in the stasis model compared to vehicle controls (Figure 1). There was no difference in thrombus size at day 8 in controls vs PLGA-NP groups (0.012 vs 0.016 gm/cm, n = 4-6; P = 0.41).
Conclusion: PLGA-NPs suppress fibrinolysis inhibitor PAI-1 and upregulate MMP2 in vitro. In vivo, PLGA-based targeting o MARCO in Mo/MΦ ameliorate vein wall injury in thrombosis.