Anti-CCR2 Nanotherapeutics to Reduce Vein Wall Fibrosis Following Deep Vein Thrombosis

The post-thrombotic syndrome (PTS) occurs in part due to valvular reflux caused by macrophage- and fibroblast-induced vein wall fibrosis. C-C chemokine receptor type 2 (CCR2) mediates monocyte-macrophage recruitment during thrombus formation and mediates vein wall injury in murine chronic DVT settings. Here, we used a multifunctional anti-CCR2 nanoparticle to investigate the effects of anti-CCR2 (IncB) therapy in experimental murine DVT resolution and vein wall fibrosis. Human fresh frozen plasma (FFP) clots were incubated with 0.5 U thrombin and 0.4 M CaCl2 to evaluate targeting of various poly(lactic-co-glycolic acid (PLGA) fluorescent nanoparticles, with the full nanoparticle as PLGA-IncB (Anti-CCR2)-GPRP (peptide targeting fibrin)-CyAl5.5 (NIR fluorophore). Clots were washed, centrifuged, and imaged by fluorescence reflectance imaging (LICOR Odyssey, ex/em 680/780nm). Next, C57/BL6 male mice (n = 38) underwent inferior vena cava (IVC) complete ligation to develop full-stasis DVT (Fig 1, B). On day 4, mice randomly received intravenous phosphate buffered saline (PBS), PLGA-IncB-GPRP-CyAl5.5, or PLGA-IncB-CyAl5.5 (20mg/kg PLGA for nanoparticles). IVC DVT were resected on D8 to measure thrombus burden, or for Masson's trichrome (MT) and picrosirius red assessment (PSR) of thrombus and vein wall collagen, respectively. Human FFP clots showed ∼6× and 3× higher binding of PLGA compounds compared with PBS control and FTP11-CyAl5, a positive control fibrin-binding peptide (Fig 1, A) (P < .0001). In vivo, both PLGA-GPRP-IncB and PLGA-IncB did not alter d8 thrombus burden (Fig 1, C). However, histological assessment showed reduced fibrin and erythrocytes in PLGA groups compared with PBS control (Fig 1, E). Further, d8 vein wall PSR analyses showed significantly reduced vein wall collagen in both PLGA-IncB groups (Fig 1, D; PBS vs PLGA-IncB; P < .0001; PBS vs PLGA-GPRP-IncB; P = .007), demonstrating PLGA-IncB salutary reductions on vein wall fibrosis, regardless of additional fibrin/GPRP targeting. Anti-CCR2 nanotherapy significantly improves DVT resolution by reducing vein wall collagen thickness in murine DVT, without impairing thrombus resolution. This data provides a foundation for targeting CCR2 to reduce PTS.