A protector sequence against the NOR-1 3’-UTR seed region prevents the regulation by miR-17 and -20.

<p>HUVEC were transfected with precursors of either miR-17 or miR-20a (P17 or P20) or a scramble sequence (Scr) in the presence or in the absence of a miScript target protector that blocks the NOR-1 3’-UTR seed sequence recognized by the miR-17 and -20 (NOR1-Prot). Then cells were stimulated with VEGF (100 ng/ml, 2 h). NOR-1 expression was assessed by real-time PCR <b>(A)</b> and Western-blot <b>(B)</b>. Results are expressed as mean ± SD from at least n = 4. (<i>p</i><0.05: *, <i>vs</i>. untransfected cells [Control, CT] or cells transfected with Scr).</p

The NOR-1 protector prevents the down-regulation of VCAM-1 promoted by miR-17 and -20.

<p>HUVEC were transfected with precursors of either miR-17 or miR-20a (P17 or P20) or a scramble sequence (Scr) in the presence or in the absence of a miScript target protector that blocks the NOR-1 3’-UTR seed sequence recognized by the miR-17 and -20a (NOR1-Prot). Then cells were stimulated with VEGF (100 ng/ml, 2 h). VCAM-1 expression was assessed by real-time PCR <b>(A)</b> and Western-blot <b>(B)</b>. Results are expressed as mean ± SD from at least n = 4. (<i>p</i><0.05: *, <i>vs</i>. untransfected cells [Control, CT] or cells transfected with Scr; #, <i>vs</i>. cells transfected with Scr and stimulated with VEGF; †, <i>vs</i>. cells exposed to the same condition but without NOR1-Prot).</p

miR-17 and -20a regulate the expression of NOR-1.

<p>HUVEC were transfected with precursors of either miR-17 or miR-20a (P17 or P20) or a scramble sequence (Scr) in the presence or in the absence of their corresponding specific antagomirs (A17 or A20). Cells were stimulated with VEGF (100 ng/ml, 2 h). NOR-1 expression was assessed by real-time PCR <b>(A)</b>, Western-blot <b>(B)</b> and immunocytochemistry <b>(C)</b>. Results are expressed as mean ± SD from at least n = 4. (<i>p</i><0.05: *, <i>vs</i>. untransfected cells [Control, CT] or cells transfected with Scr).</p

NOR-1 transcript contains a miR-17/-20 putative binding site in the 3’-UTR.

<p>Schematic representation of the human NOR-1 3’-UTR transcript (NM_006981) showing the highly conserved miR-17/-20 binding site among species (positions 2894–2900 in NM_006981 sequence). The seed sequence of miR-17/-20 (nt 2–10) is indicated in bold.</p

Time-dependent, increased expression of ADAM17, MCP1 and Hsp60 in endothelial cells upon homocysteinylated albumin treatment.

<p>Panel A: Real time PCR evaluation during time course of <i>ADAM17</i>, <i>MCP1</i> and <i>Hsp60</i> mRNA. Panel B: ELISA assay of MCP1 released in the culture medium of treated cells. Panel C: Western blotting analysis of intracellular levels of ADAM17, and Hsp60, and analysis of Hsp60 released in the medium by immunoprecipitation and western blotting (Hsp60 IP). A: unmodified albumin control; AH: homocysteinylated albumin treatment. Levels of transcripts or proteins in the homocysteinylated albumin sample group were significantly increased compared to control (p<0.001).</p

VCAM1 expression in endothelial cells treated with N-homocysteinylated albumin.

<p>Panel A: Time course of induction of VCAM1 transcripts, in EAhy926 endothelial cells, by treatment with 1 µmol/L homocysteinylated albumin. Panel B: cytofluorimetric analysis of ICAM1 time course surface expression by EAhy926 endothelial cells treated with homocysteinylated albumin. (C: unmodified albumin negative control; Tnf-α: positive control). Panel C: Time course of ICAM1 release in the culture medium, quantitated by ELISA assay. C: negative control (untreated cells); A: unmodified albumin; AH: 1 µmol/L homocysteinylated albumin. (p<0.001).</p

Amplification conditions and primer pairs for PCR experiments.

<p>All conditions are relevant to real time PCR except for <i>VCAM1</i>, where tradition PCR has been employed. 35 amplification cycles have been performed.</p

Effects of homocysteinylated albumin on monocyte adhesion.

<p>U937 monocytoid cells adhesion onto an endothelial monolayer (EAhy926) expressed as adherent cells (number/field; panel A) and percentage adherent cells compared to positive control (panel B). Counts are the mean of ten independent experiments, each carried out by counting five different fields/sample of triplicate samples. Examples of microscopic fields are shown on the right. C: negative control (untreated cells); A: unmodified albumin; AH: homocysteinylated albumin; AC: carboxymethylated albumin; T: positive control (Tnf-α). 0.3 or 1: homocysteine micromolar concentration present in the assay in the form of <i>N</i>-homocysteinyl groups bound to albumin, as comparable to the in vivo situation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031388#pone.0031388-Perna1" target="_blank">[14]</a>; p<0.0001.</p

Upregulated genes from transcriptional analysis of EAhy926 cells treated with homocysteinylated albumin compared to control.

<p>Homocysteinylated albumin concentration was comparable to that detected <i>in vivo</i> in hyperhomocysteinemic uremic patients compared to control.</p

ICAM1 expression in endothelial cells treated with N-homocysteinylated albumin.

<p>Panel A: expression levels of ICAM1 transcripts quantitated by real time PCR (treated: 1 µmol/L homocysteinylated albumin; control: unmodified albumin); (p<0.001). Panel B: cytofluorimetric analysis of ICAM1 time course surface expression by EAhy926 endothelial cells treated with homocysteinylated albumin (C: unmodified albumin negative control; Tnf-α: positive control). Panel C: Time course of ICAM1 release in the culture medium, quantitated by ELISA assay. C: negative control (untreated cells); A: unmodified albumin; AH: 1 µmol/L homocysteinylated albumin; (p<0.001).</p