Urokinase receptor expression on human microvascular endothelial cells is increased by hypoxia: implications for capillary-like tube formation in a fibrin matrix by Marielle E. Kroon, Pieter Koolwijk, Bea van der Vecht, and Victor W. M. van Hinsbergh Blood Volume 96(8):2775-2783 October 15, 2000 ©2000 by American Society of Hematology

Capillary-like tube formation is increased under hypoxic conditions Capillary-like tube formation is increased under hypoxic conditions.hMVECs were cultured on top of a 3-dimensional fibrin matrix in M199 supplemented with 10% HS and 10% NBCS under (A,C) normoxic or (B,D) hypoxic culture conditions and were not stimulated (... Capillary-like tube formation is increased under hypoxic conditions.hMVECs were cultured on top of a 3-dimensional fibrin matrix in M199 supplemented with 10% HS and 10% NBCS under (A,C) normoxic or (B,D) hypoxic culture conditions and were not stimulated (panels A,B) or stimulated (panels C,D) with 10 ng/mL each FGF-2+TNF-α. After 3 days of culturing, nonphase photomicrographs were taken.The bar indicates 300 μm. Marielle E. Kroon et al. Blood 2000;96:2775-2783 ©2000 by American Society of Hematology

Capillary-like tube formation is increased under hypoxic conditions Capillary-like tube formation is increased under hypoxic conditions.hMVECs were cultured on top of a 3-dimensional fibrin matrix in M199 supplemented with 10% HS and 10% NBCS under normoxic or hypoxic culture conditions and were not stimulated (control) or ... Capillary-like tube formation is increased under hypoxic conditions.hMVECs were cultured on top of a 3-dimensional fibrin matrix in M199 supplemented with 10% HS and 10% NBCS under normoxic or hypoxic culture conditions and were not stimulated (control) or stimulated with 10 ng/mL each FGF-2+TNF-α or with 25 ng/mL VEGF165 and 10 ng/mL TNF-α (VEGF165+TNF-α). After 3 days of culturing, the mean tube length (mm/cm2) was measured as described. The data represent the mean plus or minus SD of 5 independent experiments performed in duplicate wells. Marielle E. Kroon et al. Blood 2000;96:2775-2783 ©2000 by American Society of Hematology

Inhibition of capillary-like tube formation by uPAR antibodies Inhibition of capillary-like tube formation by uPAR antibodies.hMVECs were cultured on top of a 3-dimensional fibrin matrix in M199 supplemented with 10% HS and 10% NBCS under normoxic or hypoxic culture conditions and were stimulated with 10 ng/mL each FGF... Inhibition of capillary-like tube formation by uPAR antibodies.hMVECs were cultured on top of a 3-dimensional fibrin matrix in M199 supplemented with 10% HS and 10% NBCS under normoxic or hypoxic culture conditions and were stimulated with 10 ng/mL each FGF-2+TNF-α in the presence of 100 μg/mL pAb anti-tPA, 100 μg/mL pAb anti-uPA, 5 μg/mL mAb H-2 (anti-uPAR), or 100 U/mL aprotinin. After 3 days of culturing, the mean tube length (mm/cm2) was measured as described and expressed as a percentage of FGF-2+TNF-α. The data represent the mean plus or minus SD of 3 independent experiments performed in duplicate wells. Marielle E. Kroon et al. Blood 2000;96:2775-2783 ©2000 by American Society of Hematology

Effect of hypoxia on uPA and uPAR mRNA expression Effect of hypoxia on uPA and uPAR mRNA expression.(A) hMVECs were cultured for 72 hours in normoxic and hypoxic conditions in M199 supplemented with 10% HS and not stimulated (control) or stimulated with 10 ng/mL TNF-α or 10 ng/mL each FGF-2+TNF-α. Effect of hypoxia on uPA and uPAR mRNA expression.(A) hMVECs were cultured for 72 hours in normoxic and hypoxic conditions in M199 supplemented with 10% HS and not stimulated (control) or stimulated with 10 ng/mL TNF-α or 10 ng/mL each FGF-2+TNF-α. After 72 hours, total RNA was isolated and analyzed by Northern blotting using α-32P CTP-labeled probes for uPA, uPAR, and actin. (B) The signals for uPA and uPAR mRNA were quantified by Phosphorimager analysis and adjusted for the corresponding actin mRNA. The data are expressed as a percentage of normoxic control cells. Similar results were obtained in 3 independent experiments. Marielle E. Kroon et al. Blood 2000;96:2775-2783 ©2000 by American Society of Hematology

Hypoxia increases uPAR antigen levels Hypoxia increases uPAR antigen levels.hMVECs were cultured for 72 hours in normoxic and hypoxic conditions in M199 supplemented with 10% HS and not stimulated (control) or stimulated with 10 ng/mL each FGF-2+TNF-α or with 10−8mol/L phorbol myristate acetate... Hypoxia increases uPAR antigen levels.hMVECs were cultured for 72 hours in normoxic and hypoxic conditions in M199 supplemented with 10% HS and not stimulated (control) or stimulated with 10 ng/mL each FGF-2+TNF-α or with 10−8mol/L phorbol myristate acetate (PMA). Subsequently, the cells were cooled on ice, and the specific binding of 125I-labeled DIP-uPA to hMVECs was determined and expressed as fmol125I-uPA/105 cells. The data represent the mean plus or minus SEM of 4 independent experiments performed in duplicate wells. The asterisk indicates P < .05, which is significantly different from the normoxic counterpart. Marielle E. Kroon et al. Blood 2000;96:2775-2783 ©2000 by American Society of Hematology

Hypoxia does not decrease uPA production Hypoxia does not decrease uPA production.hMVECs were cultured for 72 hours in normoxic and hypoxic conditions in M199 supplemented with 10% HS and not stimulated (control) or stimulated with 10 ng/mL each FGF-2+TNF-α in the presence of 0 or 5 μg/mL mAb H-2. Hypoxia does not decrease uPA production.hMVECs were cultured for 72 hours in normoxic and hypoxic conditions in M199 supplemented with 10% HS and not stimulated (control) or stimulated with 10 ng/mL each FGF-2+TNF-α in the presence of 0 or 5 μg/mL mAb H-2. After the incubation period, the uPA antigen levels were determined in the conditioned media by ELISA, as previously described, and expressed as ng uPA/105 cells. The data represent the mean plus or minus SD of 3 independent experiments performed in duplicate wells. The asterisk indicates P < .05, which is significantly different from the normoxic counterpart. Marielle E. Kroon et al. Blood 2000;96:2775-2783 ©2000 by American Society of Hematology

Plasmin formation is increased in hypoxia Plasmin formation is increased in hypoxia.hMVECs were cultured in M199 supplemented with 10% HS and 10% NBCS and not stimulated (ns) or stimulated with 10 ng/mL each FGF-2+TNF-α in normoxia (bT/normoxia) or hypoxia (bT/hypoxia). Plasmin formation is increased in hypoxia.hMVECs were cultured in M199 supplemented with 10% HS and 10% NBCS and not stimulated (ns) or stimulated with 10 ng/mL each FGF-2+TNF-α in normoxia (bT/normoxia) or hypoxia (bT/hypoxia). Normoxic hMVECs were cultured in the presence of 5 μg/mL mAb H-2 or 200 U aprotinin. After 72 hours the plasmin formation was measured as described in “Materials and methods.” H-2 and aprotinin were also present during the plasmin formation assay. The data are expressed as the mean plus or minus SD. Each condition was performed 8-fold. The asterisk indicatesP < .002, which is significantly different from normoxic FGF-2/TNF-α–stimulated cells. Marielle E. Kroon et al. Blood 2000;96:2775-2783 ©2000 by American Society of Hematology

sVEGFR-1 does not inhibit FGF-2+TNF-α–induced tube formation in hypoxia.hMVECs were cultured on top of a 3-dimensional fibrin matrix in M199 supplemented with 10% HS and 10% NBCS under normoxic or hypoxic culture conditions and were not stimulated (control)... sVEGFR-1 does not inhibit FGF-2+TNF-α–induced tube formation in hypoxia.hMVECs were cultured on top of a 3-dimensional fibrin matrix in M199 supplemented with 10% HS and 10% NBCS under normoxic or hypoxic culture conditions and were not stimulated (control) or stimulated with 10 ng/mL each FGF-2+TNF-α or with 25 ng/mL VEGF165 and 10 ng/mL TNF-α (VEGF165+TNF-α) in the presence of 0 or 2 g nmol/L sVEGFR-1. After 3 days of culturing, the mean tube length (mm/cm2) was measured as described. The data represent the mean plus or minus SD of 3 independent experiments performed in duplicate wells. Marielle E. Kroon et al. Blood 2000;96:2775-2783 ©2000 by American Society of Hematology

Hypoxia increases αv-integrin mRNA expression Hypoxia increases αv-integrin mRNA expression.(A) hMVECs were cultured for 16 hours in normoxic (N) and hypoxic (H) conditions in M199 supplemented with 10% HS and not stimulated (control) or stimulated with 10 ng/mL each FGF-2+TNF-α. Hypoxia increases αv-integrin mRNA expression.(A) hMVECs were cultured for 16 hours in normoxic (N) and hypoxic (H) conditions in M199 supplemented with 10% HS and not stimulated (control) or stimulated with 10 ng/mL each FGF-2+TNF-α. After the incubation period, total RNA was isolated and analyzed by Northern blotting using α-32P CTP-labeled probes for αv-integrin, β5-integrin, and actin. (B) The cell adhesion assay was performed as described in “Materials and methods.” hMVECs were allowed to adhere to vitronectin for 90 minutes in the presence of control antibody 10 μg/mL anti-FITC (fluorescein isothiocyanate), 10 μg/mL LM609, 10 μg/mL P1F6, or a combination of 10 μg/mL each LM609 and P1F6. The data are expressed as the mean percentage of the control plus or minus SD. Each condition was performed 4-fold. The different groups were compared by an analysis of variance (ANOVA). The asterisk indicates P < .001, which is significantly different from control, and the number sign indicatesP = .005, which is significantly different from P1F6. (C) hMVECs were cultured on top of a 3-dimensional fibrin matrix in M199 supplemented with 10% HS and 10% NBCS under normoxic or hypoxic culture conditions and were stimulated with 10 ng/mL each FGF-2+TNF-α (control) alone or in the presence of 10 μg/mL of the αvβ3-blocking mAb LM609, the αvβ5-blocking mAb P1F6, or a combination of these antibodies (LM609/P1F6). After 3 days of culture, the mean tube length (mm/cm2) was measured as described. The effect of LM609 and P1F6 is expressed as the mean percentage of FGF-2+TNF-α–stimulated cells plus or minus the range of 2 independent experiments performed in duplicate wells. Marielle E. Kroon et al. Blood 2000;96:2775-2783 ©2000 by American Society of Hematology