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Proteolysis of the urokinase-type plasminogen activator receptor by metalloproteinase-12: Implication for angiogenesis in fibrin matrices

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Author: Koolwijk, P. · Sidenius, N. · Peters, E. · Sier, C.F.M. · Hanemaaijer, R. · Blasi, F. · Hinsbergh, V.W.M. van
Institution: Gaubius Instituut TNO
Source:Blood, 10, 97, 3123-3131
Identifier: 236067
doi: doi:10.1182/blood.V97.10.3123
Keywords: Binding Sites · Blotting, Western · Cells, Cultured · Endothelium, Vascular · Fibrin · Fibroblast Growth Factor 2 · Gene Expression · Humans · Metalloendopeptidases · Microcirculation · Neovascularization, Physiologic · Phenylalanine · Protease Inhibitors · Receptors, Cell Surface · Recombinant Proteins · Reverse Transcriptase Polymerase Chain Reaction · RNA, Messenger · Thiophenes · Transfection · Tumor Necrosis Factor-alpha · Urinary Plasminogen Activator


Pericellular proteolysis plays an important role in cell migration and the formation of new capillary structures. The plasminogen activator/plasmin and matrix degrading metalloproteinase (MMP) cascades act together in the remodeling of matrix and cell-matrix contacts. Previously we have shown that the formation of capillary structures by human foreskin microvascular endothelial cells (hMVECs) in a 3-dimensional fibrin matrix requires a functional urokinase-type plasminogen activator receptor (u-PAR). Here we report on the unexpected finding that inhibition of hMVEC-derived MMP activity by BB94 (batimastat) increased the outgrowth of capillary structures in a fibrin matrix. BB94 prevented the release of the u-PA binding domain D1 of u-PAR and thereby increased the number of functional u-PARs on hMVECs without affecting the u-PAR messenger RNA levels. Comparison of various types of protease inhibitors pointed to the prime involvement of MMP activity. Using recombinant MMPs it was shown that MMP-12 activity was able to release the D1 domain of cellularly expressed u-PAR. In addition, the expression of MMP-12 in control and basic fibroblast growth factor/tumor necrosis factor-α-stimulated hMVECs was shown by reverse transcriptase-polymerase chain reaction, suggesting that endothelial cell-derived MMP-12 may be involved in angiogenesis-related u-PAR shedding. This new mechanism of u-PAR cleavage provides new insights into the mutual interactions between the MMP and u-PA/plasmin systems. Moreover, it may be helpful in the interpretation of recent data on the use of specific MMP inhibitors in the treatment of several types of cancer. © 2001 by The American Society of Hematology. Chemicals/CAS: batimastat, 130370-60-4; Fibrin, 9001-31-4; Fibroblast Growth Factor 2, 103107-01-3; Metalloendopeptidases, EC 3.4.24.-; Phenylalanine, 63-91-2; plasminogen activator, urokinase receptors; Protease Inhibitors; Receptors, Cell Surface; Recombinant Proteins; RNA, Messenger; Thiophenes; Tumor Necrosis Factor-alpha; Urinary Plasminogen Activator, EC