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Chung S. Lim, MRCS, PhD, Serafim Kiriakidis, PhD, Ewa M

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1 Increased activation of the hypoxia-inducible factor pathway in varicose veins 
Chung S. Lim, MRCS, PhD, Serafim Kiriakidis, PhD, Ewa M. Paleolog, PhD, Alun H. Davies, DM, FRCS  Journal of Vascular Surgery  Volume 55, Issue 5, Pages e1 (May 2012) DOI: /j.jvs Copyright © 2012 Society for Vascular Surgery Terms and Conditions

2 Fig 1 Hypoxia-inducible factor (HIF)-1α and HIF-2α expression in varicose and non-varicose veins. A, Relative mRNA expression of HIF-1α and HIF-2α in varicose and non-varicose veins. Gene expression was assayed by quantitative polymerase chain reaction (Q-PCR), and normalized to 18S RNA. Data are median fold change (and interquartile range) of mRNA expression relative to the gene expression of a non-varicose vein specimen, calculated using 2-ΔΔCt method, in 11 varicose and five non-varicose veins, and were compared using unpaired t-test and Mann-Whitney U test, respectively. * P < .05; ** P < .01. B, Expression of HIF-1α and HIF-2α protein in varicose and non-varicose veins assessed using Western blot, with extracellular signal-regulated kinase (ERK)-2 shown as a loading control. Journal of Vascular Surgery  , e1DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

3 Fig 2 Hypoxia-inducible factor (HIF) target gene expression in varicose and non-varicose veins. A, Relative mRNA expression of HIF target genes, namely BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP-3), carbonic anhydrase (CA)-9, glucose transporter-1 (GLUT-1), and vascular endothelial growth factor (VEGF), in varicose and non-varicose veins. The mRNA was assayed by quantitative polymerase chain reaction (Q-PCR), and normalized to 18S RNA. Data are median (and interquartile range) of the fold change of mRNA relative to the gene expression of a non-varicose vein specimen, calculated using 2-ΔΔCt method, in 20 varicose and 10 non-varicose veins. The differences in gene expression between varicose and non-varicose veins were compared using unpaired t-test (BNIP-3) or Mann-Whitney U test (CA9, GLUT-1, and VEGF). ** P < .01; *** P < B, Expression of CA9 protein in varicose and non-varicose veins assessed using Western blot with extracellular signal-regulated kinase (ERK)-2 shown as a loading control. Journal of Vascular Surgery  , e1DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

4 Fig 3 Hypoxia-inducible factor (HIF) regulatory enzymes expression in varicose and non-varicose veins. A, Relative mRNA expression of HIF regulatory enzymes prolyl-hydroxylase domain (PHD)-1, PHD-2, PHD-3, and factor-inhibiting HIF-1 (FIH-1), in varicose and non-varicose veins. Gene expression was assayed by quantitative polymerase chain reaction (Q-PCR), and normalized to 18S RNA. Data are median (and interquartile range) of the fold change of mRNA relative to the gene expression of a non-varicose vein specimen, calculated using 2-ΔΔCt method, in 11 varicose and five non-varicose veins. The differences in gene expression between varicose and non-varicose veins were compared using unpaired t-test (PHD-2 and PHD-3) or Mann-Whitney U test (PHD-1 and FIH-1). * P < .05. B, Western blot analysis of HIF regulatory enzymes, with extracellular signal-regulated kinase (ERK)-2 shown as a loading control. Journal of Vascular Surgery  , e1DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

5 Fig 4 Hypoxia-inducible factor (HIF)-1α and HIF-2α mRNA and protein expression in varicose and non-varicose vein organ culture exposed to normoxia, hypoxia, and dimethyloxallyl glycine (DMOG). A, Relative expression of HIF-1α and HIF-2α mRNA measured by quantitative polymerase chain reaction (Q-PCR) relative to normoxia of non-varicose vein organ cultures of 6 patients (value equal to 1), and varicose vein organ cultures of 6 patients, exposed to 16 hours of normoxia, hypoxia, or DMOG 1 mM. Data are mean ± SEM and were analyzed by one-way analysis of variance (ANOVA) with repeated measures followed by Dunnett's correction for multiple comparisons vs normoxia. * P < .05; *** P < B, Representative Western blot analysis of HIF-1α and HIF-2α protein expression of varicose and non-varicose vein organ cultures exposed to 16 hours of normoxia (N), hypoxia (H), or DMOG 1 mM (D). Extracellular signal-regulated kinase (ERK)-2 is shown as a loading control. Journal of Vascular Surgery  , e1DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

6 Fig 5 Hypoxia-inducible factor (HIF) target gene expression in non-varicose vein organ cultures exposed to normoxia, hypoxia, and dimethyloxallyl glycine (DMOG). A, Expression of six HIF target genes mRNA measured by quantitative polymerase chain reaction (Q-PCR) relative to normoxia (value equal to 1) of non-varicose vein organ cultures of 6 patients exposed to 16 hours of normoxia, hypoxia, or DMOG. The HIF target genes assessed were carbonic anhydrase (CA)-9, BCL2/adenovirus E1B 19-kDa protein-interacting protein-3 (BNIP-3), prolyl-hydroxylase domain (PHD)-2, PHD-3, glucose transporter-1 (GLUT-1), and vascular endothelial growth factor (VEGF). Data are expressed as mean ± SEM and were analyzed by one-way analysis of variance (ANOVA) with repeated measures followed by Dunnett's correction for multiple comparisons. * P < .05; ** P < .01; *** P < B, Representative Western blot analysis of GLUT-1 and PHD-3 protein expression of non-varicose vein organ cultures exposed to 16 hours of normoxia only (N), hypoxia (H), or DMOG 1 mM (D). Extracellular signal-regulated kinase (ERK)-2 is shown as a loading control. Journal of Vascular Surgery  , e1DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

7 Fig 6 Hypoxia-inducible factor (HIF) target gene expression in varicose vein organ cultures exposed to normoxia, hypoxia, and dimethyloxallyl glycine (DMOG). A, Expression of six HIF target genes mRNA measured with quantitative polymerase chain reaction (Q-PCR) relative to normoxia (value equal to 1) of varicose vein organ cultures of 6 patients exposed to 16 hours of normoxia only, hypoxia, or DMOG. The HIF target genes assessed were carbonic anhydrase (CA)-9, BCL2/adenovirus E1B 19-kDa protein-interacting protein-3 (BNIP-3), prolyl-hydroxylase domain (PHD)-2, PHD-3, glucose transporter-1 (GLUT-1), and vascular endothelial growth factor (VEGF). Data are expressed as mean ± SEM and were analyzed by one-way analysis of variance (ANOVA) with repeated measures followed by Dunnett's correction for multiple comparisons. * P < .05; ** P < .01; *** P < B, Representative Western blot analysis of GLUT-1 and PHD-3 protein expression of varicose vein organ cultures of a patient, exposed to 16 hours of normoxia only (N), hypoxia (H), or DMOG 1 mM (D). Extracellular signal-regulated kinase (ERK)-2 is shown as a loading control. Journal of Vascular Surgery  , e1DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

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