The effects of doxycycline and micronized purified flavonoid fraction on human vein wall remodeling are not hypoxia-inducible factor pathway-dependent

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The effects of doxycycline and micronized purified flavonoid fraction on human vein wall remodeling are not hypoxia-inducible factor pathway-dependent Chung Sim Lim, MRCS, PhD, Serafim Kiriakidis, PhD, Ewa M. Paleolog, PhD, Alun H. Davies, DM, FRCS Journal of Vascular Surgery Volume 56, Issue 4, Pages 1069-1077 (October 2012) DOI: 10.1016/j.jvs.2012.02.067 Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 1 A, Expression of hypoxia-inducible factor (HIF)-1α and HIF-2α messenger RNA (mRNA) measured with quantitative polymerase chain reaction relative to normoxia (value = 1) of nonvaricose vein (NVV) organ cultures (n = 6) exposed to 16 hours of hypoxia with or without treatment with doxycycline (5 μg/mL) or micronized purified flavonoid fraction (MPFF; 10–5 mol/L). MPFF was dissolved in dimethyl sulfoxide (DMSO; 0.02% v/v) and therefore, the effects of DMSO on HIF-1α and HIF-2α mRNA in NVV organ cultures exposed to 16 hours of hypoxia were also assessed for comparison with those treated with MPFF. Data are expressed as mean ± standard error of the mean. One-way analysis of variance (ANOVA) with repeated measures, followed by the Dunnett test for multiple comparisons, was used to calculate for any significant differences. The overall P value of the one-way analysis of variance for each gene expression is presented with the individual graph. None of the individual comparisons between groups were significant. B, Representative Western blot analysis of HIF-1α and HIF-2α of NVV organ cultures of a patient exposed to hypoxia with and without treatment of doxycycline (5 μg/mL) or MPFF (10–5 mol/L). The MPFF was dissolved in DMSO (0.02% v/v), and therefore, the effects of DMSO on HIF-1α mRNA in NVV organ cultures exposed to hypoxia were also assessed for comparison with those treated with MPFF. The loading control was assessed with extracellular signal-regulated kinase 2 (ERK-2). Journal of Vascular Surgery 2012 56, 1069-1077DOI: (10.1016/j.jvs.2012.02.067) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 2 A, Expression of hypoxia-inducible factor (HIF)-1α and HIF-2α messenger RNA (mRNA) measured with quantitative polymerase chain reaction relative to normoxia (value = 1) of varicose vein (VV) organ cultures (n = 6) exposed to 16 hours of hypoxia with or without treatment with doxycycline (5 μg/mL) or micronized purified flavonoid fraction (MPFF; 10–5 mol/L). MPFF was dissolved in dimethyl sulfoxide (DMSO; 0.02% v/v), and therefore, the effects of DMSO on HIF-1α and HIF-2α mRNA in VV organ cultures exposed to 16 hours of hypoxia were also assessed for comparison with those treated with MPFF. Data are expressed as mean ± standard error of the mean. §Significantly decreased compared with VV organ cultures exposed to 16 hours of hypoxia and treated with DMSO 0.02% (v/v) only (P < .05; paired t-test). One-way analysis of variance (ANOVA) with repeated measures, followed by the Dunnett test for multiple comparisons, was used to calculate for any significant differences. The overall P value of the one-way analysis of variance for each gene expression is presented with the individual graph. Unless stated, none of the individual comparisons between groups were significant. B, Representative Western blot analysis of HIF-1α and HIF-2α of VV organ cultures exposed to hypoxia with and without treatment of doxycycline (5 μg/mL) or MPFF (10–5 mol/L). The MPFF was dissolved in DMSO (0.02% v/v), and therefore, the effects of DMSO on HIF-1α mRNA in VV organ cultures exposed to hypoxia were also assessed for comparison with VVs treated with MPFF. The loading control was assessed with extracellular signal-regulated kinase 2 (ERK-2). Journal of Vascular Surgery 2012 56, 1069-1077DOI: (10.1016/j.jvs.2012.02.067) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 3 Expression of six hypoxia-inducible factor (HIF) target genes (carbonic anhydrase-9 [CA-9], B-cell lymphoma 2/adenovirus E1B 19-kDa protein-interacting protein 3 [BNIP-3], prolyl hydroxylase domain-2 [PHD-2], and domain-3 [PHD-3], glucose transporter-1 [GLUT-1] and vascular endothelial growth factor [VEGF]) measured with quantitative polymerase chain reaction relative to normoxia (value = 1) of six nonvaricose vein organ cultures exposed to 16 hours of hypoxia with or without treatment with doxycycline (5 μg/mL) or micronized purified flavonoid fraction (MPFF; 10–5 mol/L). The MPFF was dissolved in dimethyl sulfoxide (DMSO; 0.02% v/v), and therefore, the effects of dimethyl sulfoxide on hypoxia-inducible factor target genes in nonvaricose vein organ cultures exposed to hypoxia were also assessed for comparison with those treated with MPFF. Data are expressed as mean ± standard error of the mean. One-way analysis of variance (ANOVA) with repeated measures, followed by the Dunnett test for multiple comparisons, was used to calculate for any significant differences. The overall P value of one-way analysis of variance for each gene expression was presented with the individual graph. All individual comparisons between groups were not significant. Journal of Vascular Surgery 2012 56, 1069-1077DOI: (10.1016/j.jvs.2012.02.067) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 4 Expression of six hypoxia-inducible factor (HIF) target genes (carbonic anhydrase-9 [CA-9], B-cell lymphoma 2/adenovirus E1B 19-kDa protein-interacting protein 3 [BNIP-3], prolyl hydroxylase domain-2 [PHD-2], and domain-3 [PHD-3], glucose transporter-1 [GLUT-1] and vascular endothelial growth factor [VEGF]) measured with quantitative polymerase chain reactions relative to normoxia (value equal to 1) of six varicose vein organ cultures exposed to 16 hours of hypoxia with or without treatment with doxycycline (5 μg/mL) or micronized purified flavonoid fraction (MPFF; 10–5 mol/L). The MPFF was dissolved in dimethyl sulfoxide (DMSO; 0.02% v/v), and therefore, the effects of dimethyl sulfoxide on hypoxia-inducible factor target genes in varicose vein organ cultures exposed to hypoxia were also assessed for comparison with those treated with MPFF. Data are expressed as mean ± standard error of the mean. One-way analysis of variance (ANOVA) with repeated measures, followed by the Dunnett test for multiple comparisons, was used to calculate for any significant differences. The overall P value of one-way analysis of variance for each gene expression is presented with the individual graph. None of the individual comparisons between groups were significant. Journal of Vascular Surgery 2012 56, 1069-1077DOI: (10.1016/j.jvs.2012.02.067) Copyright © 2012 Society for Vascular Surgery Terms and Conditions