![Figure 1. MCP-1 up-regulates hypoxia-inducible factor 1α and induces VEGF production by vascular endothelial cells. (A) Aortic rings prepared from thoracic and abdominal aortas of healthy male rats were embedded into growth factor–deficient Matrigel in 24-well plates and incubated for 24 hours in the presence of MCP-1 (10 or 100 ng/mL). In some experiments, Clostridium difficile (C difficile) toxin (Rho inhibitor; 7 pM; Sigma, St Louis, MO) or C3 transferase (RhoA inhibitor; 100 μg/mL; Cytoskeleton, Denver, CO) was added to the culture 24 hours prior to simulation with MCP-1. After 48 hours, the outgrowth of microvessels was photographed under the microscope. (B) HAEC monolayers were treated for 24 hours with 1 to 100 ng/mL MCP-1. The amounts of HIF-1α protein (120 kDa; A, top row) and B actin (A, middle row) in the cell lysate were determined by immunoblotting assay. The amount of HIF-1α protein bound to HRE was also determined by EMSA (a bottom figure). (C) Aortic rings were prepared and treated with MCP-1 as described in panel A. After 48 hours, RNA was isolated and reverse transcribed, and RT-PCR or real-time PCR was performed as described in “Study design.” The results of agarose gel electrophoresis shown in the figures are representative of 5 independent experiments. The Ct value (the cycle number at which emitted fluorescence exceeded an automatically determined threshold) for VEGF-A165 gene expression was corrected by the Ct value for the GAPDH housekeeping gene, and the relative amount of VEGF-A165 mRNA was calculated and expressed as fold changes (mean ± SE). *P < .05, as determined by Mann-Whitney U test. (D) Cultured HAEC monolayers were stimulated with MCP-1 (10 ng/mL) for 24 hours in the presence or absence of 100 μg/mL C3 transferase (RhoA inhibitor), 10 μM SB203580 (p38 MAPK inhibitor), 10 μM PD98059 (p42/44 MAPK inhibitor), or 2 μM GF109203x (protein kinase C [PKC] inhibitor), and the concentration of VEGF-A in the supernatant was measured by ELISA. The results shown are the means ± SE values of 4 independent experiments. *P < .05, as determined by Mann-Whitney U test.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/105/4/10.1182_blood-2004-08-3178/6/zh80040574340001.jpeg?Expires=1724253918&Signature=Nn1n80~Y6QSOwQeFuPX3-m8NzrxPX4GQzgebb0VT3eg~-YG65bRRt4Q0AzZwsjGPm3ycp2~u9ZulYQf9tY5cR6wq4slLeyUZyn-0p2zsde0hfoQpQ07xj7yOnLeLRWrRPbEdrGR3DmpIU1-vnf~U68vzeXiZ5HlKmmpY8sCA1Z-7k7IFtBCKvX5TMjOZdWAPqs0URG2IKPtqXwuZ6NhXeGqFIi-R8y98UIbdnLniMvYAQfOL0TQRyf45W1kEJw6af5r4CutEBbxZsmeIx-mqwsegn8xHhzoAA6tgGswwar4EkRwMIH2QoserVCIL6sdJQd7xo6~bDNajSiBApoNl4Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
MCP-1 up-regulates hypoxia-inducible factor 1α and induces VEGF production by vascular endothelial cells. (A) Aortic rings prepared from thoracic and abdominal aortas of healthy male rats were embedded into growth factor–deficient Matrigel in 24-well plates and incubated for 24 hours in the presence of MCP-1 (10 or 100 ng/mL). In some experiments, Clostridium difficile (C difficile) toxin (Rho inhibitor; 7 pM; Sigma, St Louis, MO) or C3 transferase (RhoA inhibitor; 100 μg/mL; Cytoskeleton, Denver, CO) was added to the culture 24 hours prior to simulation with MCP-1. After 48 hours, the outgrowth of microvessels was photographed under the microscope. (B) HAEC monolayers were treated for 24 hours with 1 to 100 ng/mL MCP-1. The amounts of HIF-1α protein (120 kDa; A, top row) and B actin (A, middle row) in the cell lysate were determined by immunoblotting assay. The amount of HIF-1α protein bound to HRE was also determined by EMSA (a bottom figure). (C) Aortic rings were prepared and treated with MCP-1 as described in panel A. After 48 hours, RNA was isolated and reverse transcribed, and RT-PCR or real-time PCR was performed as described in “Study design.” The results of agarose gel electrophoresis shown in the figures are representative of 5 independent experiments. The Ct value (the cycle number at which emitted fluorescence exceeded an automatically determined threshold) for VEGF-A165 gene expression was corrected by the Ct value for the GAPDH housekeeping gene, and the relative amount of VEGF-A165 mRNA was calculated and expressed as fold changes (mean ± SE). *P < .05, as determined by Mann-Whitney U test. (D) Cultured HAEC monolayers were stimulated with MCP-1 (10 ng/mL) for 24 hours in the presence or absence of 100 μg/mL C3 transferase (RhoA inhibitor), 10 μM SB203580 (p38 MAPK inhibitor), 10 μM PD98059 (p42/44 MAPK inhibitor), or 2 μM GF109203x (protein kinase C [PKC] inhibitor), and the concentration of VEGF-A in the supernatant was measured by ELISA. The results shown are the means ± SE values of 4 independent experiments. *P < .05, as determined by Mann-Whitney U test.
