Homocysteinylated Albumin Promotes Increased Monocyte-Endothelial Cell Adhesion and Up-Regulation of MCP1, Hsp60 and ADAM17

RATIONALE:The cardiovascular risk factor homocysteine is mainly bound to proteins in human plasma, and it has been hypothesized that homocysteinylated proteins are important mediators of the toxic effects of hyperhomocysteinemia. It has been recently demonstrated that homocysteinylated proteins are elevated in hemodialysis patients, a high cardiovascular risk population, and that homocysteinylated albumin shows altered properties. OBJECTIVE:Aim of this work was to investigate the effects of homocysteinylated albumin - the circulating form of this amino acid, utilized at the concentration present in uremia - on monocyte adhesion to a human endothelial cell culture monolayer and the relevant molecular changes induced at both cell levels. METHODS AND RESULTS:Treated endothelial cells showed a significant increase in monocyte adhesion. Endothelial cells showed after treatment a significant, specific and time-dependent increase in ICAM1 and VCAM1. Expression profiling and real time PCR, as well as protein analysis, showed an increase in the expression of genes encoding for chemokines/cytokines regulating the adhesion process and mediators of vascular remodeling (ADAM17, MCP1, and Hsp60). The mature form of ADAM17 was also increased as well as Tnf-α released in the cell medium. At monocyte level, treatment induced up-regulation of ICAM1, MCP1 and its receptor CCR2. CONCLUSIONS:Treatment with homocysteinylated albumin specifically increases monocyte adhesion to endothelial cells through up-regulation of effectors involved in vascular remodeling

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

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

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

Validation by Q-PCR of transcriptome results relevant to upregulated gene involved in endothelial dysfunction.

<p>A: unmodified albumin; AH: homocysteinylated albumin. Gene expression in the AH sample group was significantly increased with respect to the corresponding genes in the A sample group (p<0.001).</p

Characterization of homocysteinylated albumin by mass spectrometry.

<p>Panel A: ESI-MS of human serum albumin. Panel B: ESI-MS of homocysteinylated albumin. Inset: magnification on expanded scale of the signal at Da = 67805. The family of molecular ions is compatible with the structures shown in the panel.</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

De novo transcriptome assembly from inflorescence of Orchis italica: analysis of coding and non-coding transcripts

The floral transcriptome of Orchis italica, a wild orchid species, was obtained using Illumina RNA-seq technology and specific de novo assembly and analysis tools. More than 100 million raw reads were processed resulting in 132,565 assembled transcripts and 86,079 unigenes with an average length of 606 bp and N50 of 956 bp. Functional annotation assigned 38,984 of the unigenes to records present in the NCBI non-redundant protein database, 32,161 of them to Gene Ontology terms, 15,775 of them to Eukaryotic Orthologous Groups (KOG) and 7,143 of them to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The in silico expression analysis based on the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) was confirmed by real-time RT-PCR experiments on 10 selected unigenes, which showed high and statistically significant positive correlation with the RNA-seq based expression data. The prediction of putative long noncoding RNAs was assessed using two different software packages, CPC and Portrait, resulting in 7,779 unannotated unigenes that matched the threshold values for both of the analyses. Among the predicted long non-coding RNAs, one is the homologue of TAS3, a long non-coding RNA precursor of trans-acting small interfering RNAs (ta-siRNAs). The differential expression pattern observed for the selected putative long non-coding RNAs suggests their possible functional role in different floral tissues