Role of heparin in the activation and migration of primary human umbilical ligand hyaluronic acid

Abstract

• BACKGROUND: CD44 is an extracellular matrix molecule that has hyaluronan as one of its principal ligands. The interaction between CD44 and its ligand initiates activation and migration of endothelial cells. Migration of endothelial cells holds importance in neovascularization during cancer metastasis where new blood vessels are formed in order to nurture metastasized cancerous tissue. Studies on bovine endothelial cells showed that greater degree of migrations occurred with addition of lower molecular weight hyaluronic acid fragments (HAF) than the whole molecule of hyaluronan (HA). Our study included addition of heparin to the seedings of HA and HAF at concentrations of 105g/ml, 1005g/ml, 2505g/ml and 5005g/ml to endothelial cells and measuring the degrees of migrations obtained on migration assay.
• METHODS: HUVECs were isolated from umbilical cords obtained from Kasturba Medical College, Manipal, India. The cells were standardized to 2000 cells/ml and cultured in co-star well plates and were seeded with HA and HAF at concentrations of 105g/ml, 1005g/ml, 2505g/ml and 5005g/ml. Basal level of control were resting cells and PMA treated cells were positive control. Migration assay was done on which the endothelial cells were embedded. After treatments, the migrations were measured and compared. Expression of CD44 was done using SDS-PAGE and Western Blot analysis.
• RESULTS: Heparin seems to play a role in the activation and migration of endothelial cells and their sustenance and viability in the medium. In Heparin M199 medium, the endothelial cells showed greater migrations when seeded with HAF than with HA. At 1005g/ml heparinised endothelial cells showed greater degree of migration with HAF than HA added at the same 1005g/ml. In heparin-free medium, the primary HUVECs migrated more at 105g/ml for cells seeded with HA while they migrated more with HAF at a concentration of 2505g/ml. SDS–PAGE and western blot analysis showed CD44 molecules on the primary HUVECs.
• CONCLUSION: The assays were set up and studied in duplicate. Further analysis should be done in order to substantiate the role of heparin in the migration of primary human umbilical vein endothelial cells following signaling with CD44 and HA. This might hold promise for therapeutic research into cancer metastasis and neovascularisation.
• KEY WORDS: CD44, hylauronic acid, cancer metastasis, signal transduction, heparin, activation, migration, human umbilical vein endothelial cells HUVECs, neovascularisation.