Cytochrome P450 Family <i>4F2</i> and <i>4F11</i> Haplotype Mapping and Association with Hepatic Gene Expression and Vitamin K Hydroxylation Activity

This study evaluated the underlying mechanistic links between genetic variability in vitamin K metabolic pathway genes (CYP4F2 and CYP4F11) and phylloquinone hydroxylation activity using genotype- and haplotype-based approaches. Specifically, we characterized genetic variability in the CYP4F2/CYP4F11 locus and compared common single allele genotypes and common haplotypes as predictors of hepatic gene expression, enzyme abundance, and phylloquinone (VK1) ω-hydroxylation kinetics. We measured CYP4F2 and CYP4F11 mRNA levels, CYP4F2 and CYP4F11 protein abundances, and the VK1 concentration-dependent ω-hydroxylation rate in matched human liver nucleic acid and microsome samples, utilizing a novel in vitro population modeling approach. Results indicate that accounting for the CYP4F2*3 allele alone is sufficient to capture most of the genetic-derived variability in the observed phenotypes. Additionally, our findings highlight the important contribution that CYP4F11 makes toward vitamin K metabolism in the human liver

Effect of BBR derivatives on tubulogenesis of HUVECs.

<p>(A) Representative phase-contrast images of HUVECs plated on growth-factor reduced Matrigel in the presence of the indicated doses of test compounds, photographed 16 hours after plating. (B) Quantitation of the number of tube-like structures formed by HUVECs in the presence of test compounds. The number of tubes was measured using NeuronJ. Data are mean and standard error of two independent experiments. <i>p-values</i> from a one-way ANOVA are shown; ns is not significant, * P<0.05, ** P<0.01, *** P<0.001. (C) Time course of HUVEC tubulogenesis in the presence of either vehicle control (DMSO) or 6OH-BBR, photographed at 1 hour, 2 hours, 6 hours and 20 hours post-seeding.</p

Effect of test compounds on aortic ring sprouting.

<p>(A) Representative images of isolectin-stained aortic rings treated with either vehicle controls or the indicated concentration of various test compounds. (B) Quantitation of the number of sprouts per ring with each bar representing at least 4 rings. *** P<0.001, ** P<0.01, * P<0.05, ns not significant).</p

Proliferation of HUVECs in the presence of 7.5 μM test compounds.

<p>Cell viability was measured at 72 hours post-seeding. Asterisk represents significant differences between groups (*** P<0.001, * P<0.05).</p

Chemical structures of compounds tested as EYA3 inhibitors and anti-angiogenic agents.

<p>(A) Metabolites of BBR. (B) Alternate halogenation of the phenol ring. (C) Alternate substituents at position 2 of the benzofuran.</p

Lateral migration of HUVECs in the presence of test compounds.

<p>(A) HUVECs were scratch wounded and then cultured in the presence of either vehicle or 7.5 μM test compound. Representative phase-contrast photographs were taken 22 hours after wounding. White dotted lines indicate location of edges of the original wound. (B) Number of cells that migrate into the wounded area was counted using the Cell Counter functionality of ImageJ. Data represent three independent experiments and asterisk represent significant differences between groups (*** P<0.001, ** P<0.01, * P<0.05, ns not significant).</p

Effect of EYA3 knock-down on the migration and proliferation of HUVECs.

<p>(A) Western blot showing knockdown of EYA3 in HUVECs by infection with shEYA3 lentivirus relative to scramble (scr) non-specific RNA and control HUVEC cells. (B) EYA3 knock-down leads to a significant reduction in cell migration in a scratch wound healing assay. (C) EYA3 knock-down in HUVECs attenuates cell proliferation 72 hours post-seeding.</p

Protein absorbance (A₂₈₀) and HCTLase activity of column fractions from the chromatographic purification of human liver HCTLase (A–E) and recombinant HCTLase (F, G).

<p>A₂₈₀ nm, solid lines; HCTL activity, solid lines with open circles. (A) Carboxymethyl (CM) column 1, (B) ceramic hydroxyapatite (HA) column, (C) Superdex 200 column 1, (D) CM column 2, (E) Superdex 200 column 2; (F) HA column, (G) Superdex 200 column. Ticks represent fraction changes.</p

Inhibition of human liver HCTL activity.

<p>* HCTLase activity was measured at 10 mM concentration of substrate.</p><p>† Iodoacetamide inhibited rBPHL to the same extent.</p><p>Inhibition of human liver HCTL activity.</p

IEF activity and Coomassie stain of liver BPHL and rBPHL.

<p>(A) HCTLase activity stain of rBHPL (lane 2) and human liver BPHL (lane 3). Hemoglobin shows a faint band as well (lane 1). (B) Coomassie Blue stain of hemoglobin, rBPHL and human liver BPHL (lanes 1–3, respectively).</p