Antiangiogenic antithrombin down-regulates the expression of the proangiogenic heparan sulfate proteoglycan, perlecan, in endothelial cells by Weiqing Zhang, Yung-Jen Chuang, Richard Swanson, Juan Li, Kyunga Seo, Lawrence Leung, Lester F. Lau, and Steven T. Olson Blood Volume 103(4):1185-1191 February 15, 2004 ©2004 by American Society of Hematology

Inhibition of bFGF-induced endothelial cell proliferation by cleaved and latent antithrombins. Inhibition of bFGF-induced endothelial cell proliferation by cleaved and latent antithrombins. Resting HUVEC cells were cultured in the absence or presence of 10 μg/mL each of native, cleaved, or latent forms of antithrombin both in the absence and presence of 10 ng/mL bFGF as indicated. The number of viable cells was determined after 72 hours of incubation as described in “Materials and methods.” Mean values ± SD (bars) derived from 5 independent determinations are shown. *A statistically significant difference from the control (P < .001) based on a Student t test. Weiqing Zhang et al. Blood 2004;103:1185-1191 ©2004 by American Society of Hematology

Effects of antithrombin forms on endothelial cell cycle transitions. Effects of antithrombin forms on endothelial cell cycle transitions. Synchronized HUVEC cells (2 × 106) were cultured with different antithrombin forms (20 μg/mL) in the presence or absence of bFGF (10 ng/mL) for 48 hours. Cells were fixed and stained with propidium iodide for detection of DNA and then analyzed by flow cytometry as detailed in “Materials and methods.” (A) Plots of the distribution of cells among the G1, S, and G2 phases of the cell cycle as reflected by their DNA content under the indicated culture conditions. (B) The percentage of cells in each cell cycle phase measured from the data in panel A and other experiments as mean values ± SD (bars) from 3 independent determinations. The different bar patterns represent from left to right the same sequence of conditions given in panel A. *A statistically significant difference from the control (P < .001). Weiqing Zhang et al. Blood 2004;103:1185-1191 ©2004 by American Society of Hematology

cDNA microarry analysis of the effects of antithrombin forms on the expression of select heparan sulfate proteoglycans in primary HUVECs. HUVECs were cultured with or without different antithrombin forms (20 μg/mL) in the presence and absence of VEGF (10 ng... cDNA microarry analysis of the effects of antithrombin forms on the expression of select heparan sulfate proteoglycans in primary HUVECs. HUVECs were cultured with or without different antithrombin forms (20 μg/mL) in the presence and absence of VEGF (10 ng/mL) as indicated. Total mRNA was isolated from the cells, amplified, and analyzed by cDNA microarray as detailed in “Materials and methods.” The expression of the indicated heparan sulfate proteoglycan mRNAs relative to a universal RNA reference is shown for the different treatment conditions ± SEM. Weiqing Zhang et al. Blood 2004;103:1185-1191 ©2004 by American Society of Hematology

Semiquantiative RT-PCR analysis of perlecan mRNA expression in antithrombin-treated HUVECs. HUVECs were cultured with or without 10 μg/mL of the different antithrombin forms in the presence and absence of 10 ng/mL bFGF as indicated for 72 hours. Semiquantiative RT-PCR analysis of perlecan mRNA expression in antithrombin-treated HUVECs. HUVECs were cultured with or without 10 μg/mL of the different antithrombin forms in the presence and absence of 10 ng/mL bFGF as indicated for 72 hours. Total mRNA was isolated, and the content of perlecan mRNA relative to β-actin mRNA was analyzed by semiquantitative RT-PCR. The inset shows the relative intensities of 500 bp perlecan and 336 bp β-actin cDNA fragments amplified from reverse-transcribed mRNA with specific primers under the different experimental conditions, as described in “Materials and methods.” Band intensities from the representative experiment shown were quantified and are presented as the ratio of perlecan to β-actin bands in the bar graph. Similar results were obtained in replicate experiments. Weiqing Zhang et al. Blood 2004;103:1185-1191 ©2004 by American Society of Hematology

Northern blotting analysis of perlecan mRNA expression in antithrombin-treated HUVECs. Total mRNA was isolated from HUVECs cultured under the conditions of Figure 4 and perlecan mRNA expression was analyzed by Northern blotting using a perlecan domain III c... Northern blotting analysis of perlecan mRNA expression in antithrombin-treated HUVECs. Total mRNA was isolated from HUVECs cultured under the conditions of Figure 4 and perlecan mRNA expression was analyzed by Northern blotting using a perlecan domain III cDNA probe as described in “Materials and methods.” The same blot was probed with GAPDH cDNA as a loading control. Normalized band intensities are shown. Weiqing Zhang et al. Blood 2004;103:1185-1191 ©2004 by American Society of Hematology

Cleaved and latent forms of antithrombin down-regulate perlecan protein expression. Cleaved and latent forms of antithrombin down-regulate perlecan protein expression. HUVECs were cultured in serum-free media with or without 10 μg/mL of the different forms of antithrombin in the presence and absence of 10 ng/mL bFGF as indicated for 72 hours. The conditioned media were applied to a nitrocellulose membrane in the indicated amounts, and perlecan protein was detected by enhanced chemiluminescence using a primary antibody to domain III of perlecan followed by a secondary enzyme-conjugated antibody as detailed in “Materials and methods.” Weiqing Zhang et al. Blood 2004;103:1185-1191 ©2004 by American Society of Hematology

Reversal of the antiproliferative effect of cleaved antithrombin on bFGF-stimulated HUVECs by TGF-β1–induced overexpression of perlecan. Reversal of the antiproliferative effect of cleaved antithrombin on bFGF-stimulated HUVECs by TGF-β1–induced overexpression of perlecan. HUVECs were cultured with or without 10 μg/mL native (N) or cleaved (C) forms of antithrombin in the absence or presence of 10 ng/mL bFGF and/or 5 ng/mL TGF-β1 as indicated for 48 hours, and then the numbers of viable cells were counted as described in “Materials and methods.” (A) The number of viable cells expressed relative to the control as mean ± SD (bars) from 3 independent determinations. *A statistically significant difference from the control in the absence of TGF-β1 (P < .001). (B) The relative perlecan and β-actin control mRNA levels measured in bFGF-stimulated HUVECs cultured in the absence (lanes 1 to 3) or in the presence of TGF-β1 (lanes 4 to 6) by semiquantitative RT-PCR as in Figure 4. Weiqing Zhang et al. Blood 2004;103:1185-1191 ©2004 by American Society of Hematology