Human intervertebral disc aggrecan inhibits endothelial cell adhesion and cell migration in vitro.
METHODS
The effect of human intervertebral disc aggrecan on endothelial cell growth was examined using cell culture assays.


OBJECTIVE
To determine the response of endothelial cells to human intervertebral disc aggrecan, and whether the amount and type of aggrecan present in the intervertebral disc may be implicated in disc vascularization.


BACKGROUND
Intervertebral disc degeneration has been associated with a loss of proteoglycan, and the ingrowth of blood vessels and nerves. Neovascularization is a common feature also of disc herniation. Intervertebral disc aggrecan is inhibitory to sensory nerve growth, but the effects of disc aggrecan on endothelial cell growth are not known.


METHODS
Aggrecan monomers were isolated separately from the anulus fibrosus and nucleus pulposus of human lumbar intervertebral discs, and characterized to determine the amount and type of sulfated glycosaminoglycan side chains present. The effects of these aggrecan isolates on the cellular adhesion and migration of the human endothelial cell lines, HMEC-1 and EAhy-926, were examined in vitro.


RESULTS
Homogenous substrata of disc aggrecan inhibited endothelial cell adhesion and cell spreading in a concentration dependent manner. In substrata choice assays, endothelial cells seeded onto collagen type I migrated over the collagen until they encountered substrata of disc aggrecan, where they either stopped migrating, retreated onto the collagen, or, more commonly, changed direction to align along the collagen-aggrecan border. The inhibitory effect of aggrecan on endothelial cell migration was concentration dependent, and reduced by enzymatic treatment of the aggrecan monomers with a combination of chondroitinase ABC and keratinase/keratinase II. Anulus fibrosus aggrecan was more inhibitory to endothelial cell adhesion than nucleus pulposus aggrecan. However, this difference did not relate to the extent to which the different aggrecan isolates were charged, as determined by colorimetric assay with 1,9-dimethylmethylene blue, or to marked differences in the distribution of chondroitin sulfated and keratan sulfated side chains.


CONCLUSIONS
Human intervertebral disc aggrecan is inhibitory to endothelial cell migration, and this inhibitory effect appears to depend, in part, on the presence of glycosaminoglycan side chains on the aggrecan monomer.
Johnson
William E B
WE
J.P. O'Brien Laboratory, Centre for Spinal Studies, Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, United Kingdom. Eustace.Johnson@rjah.nhs.uk
Caterson
Bruce
B
Eisenstein
Stephen M
SM
Roberts
Sally
S
D053674:Aggrecans; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000473:pathology / Q000503:physiopathology; D016326:Extracellular Matrix Proteins / Q000494:pharmacology*; D006025:Glycosaminoglycans / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007403:Intervertebral Disc / Q000737:chemistry / Q000378:metabolism*; D037181:Lectins, C-Type; D008159:Lumbar Vertebrae; D011509:Proteoglycans / Q000494:pharmacology*
D053674:Aggrecans; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans; D037181:Lectins, C-Type; D011509:Proteoglycans