ROLE OF HEPARIN IN THE ACTIVATION AND MIGRATION OF PRIMARY HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVECS) FOLLOWING SIGNAL TRANSDUCTION BY CD44 AND ITS LIGAND HYALURONIC ACID

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 10μg/ml, 100μg/ml, 250μg/ml and 500μg/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 10μg/ml, 100μg/ml, 250μg/ml and 500μg/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 100μg/ml heparinised endothelial cells showed greater degree of migration with HAF than HA added at the same 100μg/ml. In heparin-free medium, the primary HUVECs migrated more at 10μg/ml for cells seeded with HA while they migrated more with HAF at a concentration of 250μg/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.

[1]  V. Lokeshwar.  Clinical and biologic relevance of hyaluronic acid and its fragments , 2004 .

[2]  I. Fidler,et al.  Regulation of neoplastic angiogenesis. , 2000, Journal of the National Cancer Institute. Monographs.

[3]  V. Lokeshwar,et al.  Differences in hyaluronic acid-mediated functions and signaling in arterial, microvessel, and vein-derived human endothelial cells. , 2000, The Journal of biological chemistry.

[4]  I. Fidler.  Angiogenesis and cancer metastasis. , 2000, Cancer journal.

[5]  D. Mangham,et al.  The normal structure and function of CD44 and its role in neoplasia. , 1998, Molecular pathology : MP.

[6]  R. Klemke,et al.  Integrin αvβ3 Requirement for Sustained Mitogen-activated Protein Kinase Activity during Angiogenesis , 1998, The Journal of cell biology.

[7]  Ronit Vogt Sionov,et al.  CD44: structure, function, and association with the malignant process. , 1997, Advances in cancer research.

[8]  N Iida,et al.  The Cell Adhesion Molecule, GP116, Is a New CD44 Variant (ex14/v10) Involved in Hyaluronic Acid Binding and Endothelial Cell Proliferation* , 1996, The Journal of Biological Chemistry.

[9]  E. Turley,et al.  HA receptors: Regulators of signalling to the cytoskeleton , 1996, Journal of cellular biochemistry.

[10]  W. Stetler-Stevenson,et al.  Localization of Matrix Metalloproteinase MMP-2 to the Surface of Invasive Cells by Interaction with Integrin αvβ3 , 1996, Cell.

[11]  C. Soria,et al.  Evidence of involvement of CD44 in endothelial cell proliferation, migration and angiogenesis in vitro , 1996, International journal of cancer.

[12]  W. Stetler-Stevenson,et al.  Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3. , 1996, Cell.

[13]  David A. Cheresh,et al.  Definition of Two Angiogenic Pathways by Distinct αv Integrins , 1995, Science.

[14]  K. Oritani,et al.  Glycosylation of CD44 negatively regulates its recognition of hyaluronan , 1995, The Journal of experimental medicine.

[15]  D. Cheresh,et al.  Definition of two angiogenic pathways by distinct alpha v integrins. , 1995, Science.

[16]  R. Savani,et al.  Identification of a novel heparin binding domain in RHAMM and evidence that it modifies HA mediated locomotion of ras‐transformed cells , 1994, Journal of cellular biochemistry.

[17]  L. Ellis,et al.  The implications of angiogenesis for the biology and therapy of cancer metastasis , 1994, Cell.

[18]  J. Sleeman,et al.  Hyaluronate receptors: key players in growth, differentiation, migration and tumor progression. , 1994, Current opinion in cell biology.

[19]  R. Auerbach,et al.  Angiogenesis inhibition: a review. , 1994, Pharmacology & therapeutics.

[20]  C. Underhill,et al.  CD44: the hyaluronan receptor. , 1992, Journal of cell science.

[21]  R. Hyman,et al.  CD44 can be activated to function as an hyaluronic acid receptor in normal murine T cells , 1992, European journal of immunology.

[22]  K. Miyake,et al.  Monoclonal antibodies to Pgp-1/CD44 block lympho-hemopoiesis in long- term bone marrow cultures , 1990, The Journal of experimental medicine.

[23]  W. Carter,et al.  Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits , 1987, The Journal of cell biology.

[24]  J. Folkman,et al.  How is blood vessel growth regulated in normal and neoplastic tissue? G.H.A. Clowes memorial Award lecture. , 1986, Cancer research.

[25]  V. Terranova,et al.  Human endothelial cells are chemotactic to endothelial cell growth factor and heparin , 1985, The Journal of cell biology.

[26]  E. Turley.  Purification of a hyaluronate-binding protein fraction that modifies cell social behavior. , 1982, Biochemical and biophysical research communications.