Abstract 4141101: Loss of Chondroitin Sulfate N-acetylgalactosaminyltransferase-2 (ChGn-2) Impairs Thrombus Resolution in Mice Model

Background: Deep vein thrombosis (DVT) remains a significant health problem where current therapy management still possesses risks. Thrombus resolution during natural DVT progression involves various leukocyte influx and pro-inflammatory responses. Chondroitin sulfate glycosaminoglycans (CS-GAGs), linear polysaccharides found on blood vessel endothelium and leukocyte cell surfaces, have been reported to regulate leukocyte trafficking in several inflammatory diseases. Furthermore, GAG chain function depends on the alteration in GAG chain length or sulfation. One of the involved enzymes is Chondroitin Sulfate N-acetylgalactosaminyltransferase-2 (ChGn-2), an enzyme which is able to elongate GAG chain. In humans, the ChGn-2 gene is expressed widely, implying that ChGn-2 might play roles in various mechanisms throughout the body. Nonetheless, ChGn-2 role in the pathology of DVT remains unclear. Methods and results: We investigated ChGn-2 role in DVT by completely ligating the inferior vena cava in 10 – 12-week-old male C57BL/6J mice (n = 25) and mice with global deletion of ChGn-2 (n = 25). We found a higher thrombus burden (p<0.0001) in day 4 and day 7 after surgery with a slower thrombus organization in the transgenic mice by Carstair staining in day 2 (p<0.05) and day 4 (p<0.01). Interestingly, loss of this gene also reduced MMP activity (p<0.001) in day 7 along with pro-inflammatory cytokines and leukocyte markers. Moreover, immunohistochemistry staining also showed that leukocytes were mostly found at the edge of the thrombus in the transgenic mice. Further examination by in vivo imaging of femoral vein ligation revealed an impaired leukocyte influx in the transgenic mice. Transferring leukocytes from WT mice into transgenic mice rescued the phenotype, while the reverse experiment led to an impaired leukocyte influx. Finally, simultaneous imaging of WT and ChGn-2 KO leukocytes revealed that WT leukocytes could infiltrate inside the thrombus but not ChGn-2 KO leukocytes, confirming that ChGn-2 in leukocytes is important in our study.. Conclusion: Insufficient CS-GAGs length caused by the loss of ChGn-2 impaired thrombus resolution due to reduced leukocyte infiltration.