Chondroitin sulfate on leukocytes mediates thrombus resolution and organization: an in vivo imaging study

Accumulating evidence suggests that deep vein thrombosis (DVT) is an inflammatory disease and that chondroitin sulfate glycosaminoglycans (CS-GAGs), a long unbranched polysaccharide, can modulate inflammation. Thus, we aimed to clarify CS-GAGs role in DVT pathophysiology by using mice with target deletion of chondroitin sulfate N-acetylgalactosaminyltransferase-2 [ChGn-2 knockout (KO) mice], in which CS-GAGs chain length is reduced. We found an impaired DVT resolution and organization in both stasis- and stenosis-induced DVT model, with a lower level of inflammation and neutrophil infiltration within DVT of ChGn-2 KO mice. Using serial in vivo imaging, we observed a reduced Ly6G/Ly6C-positive cells migration into DVT in ChGn-2 KO mice. After confirming expression of CS-GAGs on both leukocytes and venous endothelial cells, we performed leukocyte transfer experiments and demonstrated that rhodamine 6 G-stained cells from ChGn-2 KO mice failed to invade into the thrombus in wild-type (WT) mice, whereas rhodamine 6 G-stained cells from WT mice were capable to infiltrate the thrombus in ChGn-2 KO mice. Simultaneous imaging of rhodamine 6 G-stained cells from WT and Hoechst 33342-stained cells from ChGn-2 KO mice confirmed that loss of ChGn-2 attenuated myeloid cell migration into the thrombus. Finally, CS disaccharide composition analysis revealed a shorter CS-GAGs on the leukocytes' cell membrane from ChGn-2 KO mice. Collectively, CS-GAGs on the cell surface of leukocytes and its regulation by ChGn-2 mediate their capability to infiltrate the thrombus, highlighting a potential innovative therapy by modulating CS-GAGs to enhance DVT resolution and organization.NEW & NOTEWORTHY Chondroitin sulfate glycosaminoglycans (CS-GAGs), one of the many factors that regulates inflammation, are found to be crucial in enhancing thrombus resolution and organization during the natural course of DVT. Alteration in CS-GAGs on the cell surface of leukocytes due to ChGn-2, an enzyme that mainly participates in the elongation of CS-GAGs, impacts their capacity to migrate into the thrombus and drive the resolution process.