An Autocrine Loop Mediates Expression of Vascular Endothelial Growth Factor in Human Dermal Microvascular Endothelial Cells  Barbara Vega-Diaz, Serge Michel  Journal of Investigative Dermatology  Volume 116, Issue 4, Pages 525-530 (April 2001) DOI: 10.1046/j.1523-1747.2001.01294.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Time course of VEGF mRNA induction by VEGF in cultured HDMEC. Total RNA was isolated at the indicated time points from HDMEC following incubation in EBM-2 in the absence or presence of 10 ng per ml VEGF (a) or 50 units per ml of TNFα (b). The expression level of VEGF mRNA was determined relative to that of GAPDH mRNA by semiquantitative RT-PCR analysis as described in Materials and Methods. Results were normalized to the values obtained with untreated cells. Journal of Investigative Dermatology 2001 116, 525-530DOI: (10.1046/j.1523-1747.2001.01294.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Induction of VEGF mRNA level in cultured HDMEC treated with different concentrations of VEGF. Total RNA was isolated at the indicated time points from HDMEC following incubation in EBM-2 in the absence or presence of the indicated concentrations of VEGF. The expression level of VEGF mRNA was determined relative to that of GAPDH mRNA by semiquantitative RT-PCR analysis as described in Materials and Methods. Results were normalized to the values obtained with untreated cells. Journal of Investigative Dermatology 2001 116, 525-530DOI: (10.1046/j.1523-1747.2001.01294.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effect of anti-VEGF antibody on VEGF mRNA expression in cultured HDMEC treated with VEGF. HDMEC were preincubated for 1 h with a polyclonal antibody raised against VEGF (2 µg per ml) before the addition of VEGF (20 ng per ml). After 4 h, total RNA was isolated and the expression level of VEGF mRNA was determined relative to that of GAPDH mRNA by semiquantitative RT-PCR analysis as described in Materials and Methods. Results were normalized to the values obtained with untreated cells. Journal of Investigative Dermatology 2001 116, 525-530DOI: (10.1046/j.1523-1747.2001.01294.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Induction of VEGF polypeptide production by cultured HDMEC treated with VEGF. (a) HDMEC were labeled with [35S]-cysteine in EBM-2 for 24 h and then treated until the indicated time points with or without VEGF at 10 ng per ml. The cell lysates were collected and VEGF was quantitated using a radioimmunoassay with a specific anti-VEGF antibody. (b) HDMEC were labeled with [35S]-cysteine in EBM-2 for 24 h and then treated for 4 h without (lanes 1 and 2) or with (lanes 3 and 4) VEGF at 10 ng per ml. The cell lysate was then immunoprecipitated with a control (lanes 1 and 3) or an anti-VEGF antibody (lanes 2 and 4). Immunoprecipitated VEGF was revealed by polyacrylamide gel electrophoresis and autoradiography. Journal of Investigative Dermatology 2001 116, 525-530DOI: (10.1046/j.1523-1747.2001.01294.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Effect of anti-KDR or anti-Flt-1 antibody on VEGF mRNA expression in cultured HDMEC treated with VEGF. HDMEC were preincubated for 1 h with an antibody raised against KDR (1 µg per ml) or Flt-1 (100 µg per ml) before the addition of VEGF (20 ng per ml). After 4 h, total RNA was isolated and the expression level of VEGF mRNA was determined relative to that of GAPDH mRNA by semiquantitative RT-PCR analysis as described in Materials and Methods. Results were normalized to the values obtained with untreated cells. Journal of Investigative Dermatology 2001 116, 525-530DOI: (10.1046/j.1523-1747.2001.01294.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Effect of actinomycin D on VEGF mRNA expression in cultured HDMEC treated with VEGF. Semiquantitative RT-PCR analysis was performed on total RNA from cells grown in EBM-2 and treated for 4 h either with or without VEGF 20 ng per ml in the presence of actinomycin D 10 µg per ml. The expression level of VEGF mRNA was determined relative to that of GAPDH mRNA as described in Materials and Methods. Results were normalized to the values obtained with untreated cells. Journal of Investigative Dermatology 2001 116, 525-530DOI: (10.1046/j.1523-1747.2001.01294.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Effect of VEGF on the transactivation of the human VEGF promoter in HDMEC. HDMEC were transiently transfected with a plasmid containing a 3.2 kb human VEGF promoter fragment cloned upstream of a luciferase reporter gene. Transfected cells grown in EBM-2 were treated with or without VEGF 20 ng per ml or 100 nM TPA for 4 h. Luciferase activity was determined as described in Materials and Methods. Results were normalized to the values obtained with untreated cells. Journal of Investigative Dermatology 2001 116, 525-530DOI: (10.1046/j.1523-1747.2001.01294.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Effect of the MAP kinase inhibitor PD 098059 on VEGF mRNA expression in cultured HDMEC treated with VEGF. Semiquantitative RT-PCR analysis was performed on total RNA from cells grown in EBM-2 and treated for 4 h either with or without VEGF 20 ng per ml in the presence of PD 098059 20 µM. The expression level of VEGF mRNA was determined relative to that of GAPDH mRNA as described in Materials and Methods. Results were normalized to the values obtained with untreated cells. Journal of Investigative Dermatology 2001 116, 525-530DOI: (10.1046/j.1523-1747.2001.01294.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Effect of VEGF treatment of cultured HDMEC on JNK and ERK activation. HDMEC were incubated in EBM-2 for 24 h and then treated with or without VEGF at 10 ng per ml. Immunoprecipitated ERK and JNK were incubated in the presence of [32P]-ATP with their respective substrate MBP or GST-c-jun. 32P incorporation into the substrates was revealed by polyacrylamide gel electrophoresis and autoradiography. Journal of Investigative Dermatology 2001 116, 525-530DOI: (10.1046/j.1523-1747.2001.01294.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions