Hyaluronic acid has been implicated in cell-cell interaction, cell-matrix adhesion, cell motility, and the ordering of the extracellular matrix (1, 2). In many developing or remodeling tissues a hyaluronate-rich stroma accompanies cell migration (1). Subsequent differentiation and vascular ingrowth are associated with an increase in tissue hyaluronidase activity and a decrease in hyaluronic acid concentration 3). Hyaluronate inhibits vascularization of chick embryo limb buds (4) and delays or reduces development of granulation tissue and newly formed capillaries around subcutaneous implants (5). In this report we describe a further regulatory role for certain products of hyaluronate degradation.
Preparations of both human umbilical (Miles Scientific 36-242-1; Sigma H1751) and bovine vitreous (Sigma H7630) hyaluronic acid were used to minimize the possibility of artifacts produced by contaminating species. Because the major contaminants in such preparations are normally chondroitin 4- and 6-sulfate, a mixed preparation of these (Sigma C3219) was also tested. Each glycosaminoglycan preparation was subjected to enzymatic digestion. Testicular hyaluronidase (Miles Scientific 32-042-1) digests were carried out in 0.1M acetate buffer and 0.15M NaCl (pH 5.4) at an enzyme-to-substrate ratio of 1 to 2 by weight (125 to 250 turbidity-reducing units per milligram of substrate). These were incubated at 37 deg.C for the periods indicated in Table 1, and the reaction was stopped by inactivating the enzyme at 100 deg.C for 5 minutes. The digests were then centrifuged at 2000g for 10 minutes, filtered through a glass fiber filter (pore size, 1 mu m), and freeze-dried.
We determined angiogenic activity by the chick chorioallantoic membrane (CAM) assay (Fig. 1) (6). Hyaluronic acid preparations digested with testicular hyaluronidase for 1 to 10 hours consistently gave a significantly higher proportion of positive responses than undigested control preparations (P...
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