Urokinase receptor expression on human microvascular endothelial cells is increased by hypoxia: implications for capillary-like tube formation in a fibrin matrix

Hypoxia stimulates angiogenesis, the formation of new blood vessels. This study evaluates the direct effect of hypoxia (1% oxygen) on the angiogenic response of human microvascular endothelial cells (hMVECs) seeded on top of a 3-dimensional fibrin matrix, hMVECs stimulated with fibroblast growth factor-2 (FGF-2) or vascular endothelial growth factor (VEGF) together with tumor necrosis factor-α (TNF-α) formed 2- to 3-fold more tubular structures under hypoxic conditions than in normoxic (20% oxygen) conditions. In both conditions the in-growth of capillary-like tubular structures into fibrin required cell-bound urokinase-type plasminogen activator (uPA) and plasmin activities. The hypoxia-induced increase in tube formation was accompanied by a decrease in uPA accumulation in the conditioned medium. This decrease in uPA level was completely abolished by uPA receptor-blocking antibodies. During hypoxic culturing uPA receptor activity and messenger RNA (mRNA) were indeed increased. This increase and, as a consequence, an increase in plasmin formation contribute to the hypoxia-induced stimulation of tube formation. A possible contribution of VEGF-A to the increased formation under hypoxic conditions is unlikely because there was no increased VEGF-A expression detected under hypoxic conditions, and the hypoxia-induced tube formation by FGF-2 and TNF-α was not inhibited by soluble VEGFR-1 (sVEGFR-1), or by antibodies blocking VEGFR-2. Furthermore, although the α(v)-integrin subunit was enhanced by hypoxia, blocking antibodies against α(v)β3 and α(v)β5-integrins had no effect on hypoxia-induced tube formation. Hypoxia increases uPA association and the angiogenic response of human endothelial cells in a fibrin matrix; the increase in the uPA receptor is an important determinant in this process. (C) 2000 by The American Society of Hematology. Chemicals/CAS: fibrin, 9001-31-4; phorbol 13 acetate 12 myristate, 16561-29-8; plasmin, 9001-90-5, 9004-09-5; plasminogen activator, 9039-53-6; tissue plasminogen activator, 105913-11-9; urokinase, 139639-24-0; vasculotropin, 127464-60-2; Antigens, CD; Culture Media; Culture Media, Conditioned; DNA, Complementary; Endothelial Growth Factors; Fibrin, 9001-31-4; Fibroblast Growth Factor 2, 103107-01-3; Integrin alphaV; integrin alphaVbeta5; Integrins; Lymphokines; Oxygen, 7782-44-7; Plasmin, EC 3.4.21.7; plasminogen activator, urokinase receptors; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases, EC 2.7.1.112; Receptors, Cell Surface; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor, EC 2.7.1.112; Receptors, Vitronectin; RNA, Messenger; Tumor Necrosis Factor; Urinary Plasminogen Activator, EC 3.4.21.73; Vascular Endothelial Growth Factor Receptor-1, EC 2.7.1.112; vascular endothelial growth factor