RECOMBINANT ERYTHROPOIETIN MITIGATES REPERFUSION INJURY IN NEONATAL RAT CARDIOMYOCYTES BY NOVEL MULTIPLE SIGNALLING PATHWAYS

Objective: Recombinant Human Erythropoietin (rhEPO) is strongly inferred to protect the cardiomyocytes from the reperfusion injury and our aim is to elucidate the cardioprotective effect and the exact mechanism behind the cardioprotection.Methods: Neonatal rat cardiomyocytes (NCM) exposed to Hypoxia/Reperfusion (H/R) with or without pretreatment using various concentrations of rhEPO. To determine the cell viability-MTT assay, Acridine orange and Ethidium Bromide (Ao/EtBr) staining was performed. To determine the reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm), Dichlorofluorescein diacetate (DCF-DA) and Rhodamine-123 was used. To determine the signaling pathways Western blot analysis of pAkt, pp38 MAPK, cytochrome-c were performed.Results: rhEPO was found to reduce the cell death by stabilizing ROS significantly, Δψm, cytochrome c release, and caspase-3. rhEPO, increases the phosphorylation of p38 MAPK, Akt and BAD compared to H/R. Further myocytes blocked with Wortmannin (WT), and SB203580 showed increased caspase-3 activity.Conclusion: Hence we conclude from this study that rhEPO regulated the factors involved in reperfusion injury through modulation of Akt and p38 MAPK pathways

Correction: Brugia malayi Asparaginyl-tRNA Synthetase Stimulates Endothelial Cell Proliferation, Vasodilation and Angiogenesis.

[This corrects the article DOI: 10.1371/journal.pone.0146132.]

Brugia malayi Asparaginyl-tRNA Synthetase Stimulates Endothelial Cell Proliferation, Vasodilation and Angiogenesis.

A hallmark of chronic infection with lymphatic filarial parasites is the development of lymphatic disease which often results in permanent vasodilation and lymphedema, but all of the mechanisms by which filarial parasites induce pathology are not known. Prior work showed that the asparaginyl-tRNA synthetase (BmAsnRS) of Brugia malayi, an etiological agent of lymphatic filariasis, acts as a physiocrine that binds specifically to interleukin-8 (IL-8) chemokine receptors. Endothelial cells are one of the many cell types that express IL-8 receptors. IL-8 also has been reported previously to induce angiogenesis and vasodilation, however, the effect of BmAsnRS on endothelial cells has not been reported. Therefore, we tested the hypothesis that BmAsnRS might produce physiological changes in endothelial by studying the in vitro effects of BmAsnRS using a human umbilical vein cell line EA.hy926 and six different endothelial cell assays. Our results demonstrated that BmAsnRS produces consistent and statistically significant effects on endothelial cells that are identical to the effects of VEGF, vascular endothelial growth factor. This study supports the idea that new drugs or immunotherapies that counteract the adverse effects of parasite-derived physiocrines may prevent or ameliorate the vascular pathology observed in patients with lymphatic filariasis

The effect of rBmAsnRS and VEGF on EA.hy926 cell migration was determined in Boyden chamber experiments.

<p>Data are presented as means ± SEM. The number of cells migrated in response to 1 ng/ml rBmAsnRS was 14, and 10 ng/ml rBmAsnRS caused the greatest migration (72.75) similar to 10 ng/ml VEGF. Asterisk (*) denotes significant differences (p<0.0001) for negative control vs 10 ng/ml rBmAsnRS and VEGF.</p

The effect of rBmAsnRS and VEGF <i>in ex vivo</i> angiogenesis.

<p>Fig 6A. Photomicrographs denote areas with angiogenesis induced by 10 ng/ml rBmAsnRS and 10 ng/ml VEGF. Angiogenesis was induced by placing sterile paper discs containing rBmAsnRS, VEGF or negative control (PBS) on the egg yolk vascular bed for 30 min, then incubated for another 24 hours. Images are representative of five different experiments. Fig 6B. Graphical representation of the fold increase in vessel length induced by 10 ng/ml rBmAsnRS and 10 ng/ml VEGF. Data are presented as means ± SEM of five experiments. The fold increase in length of blood vessel in 10 ng/ml rBmAsnRS and 10 ng/ml VEGF were significantly greater than negative controls. Asterisk (*) denotes statistically significant difference (p<0.005).</p

Hexa-histidine tagged rBmAsnRS was purified from the sonicated supernatant of IPTG induced <i>E</i>. <i>coli</i> cultures expressing the full length wild type BmAsnRS cDNA.

<p>Purified rBmAsnRS was subjected to electrophoresis in a 12%SDS-polyacrylamide gel and visualized by staining with Coomassie blue R250. Lane 1: molecular weight markers. Lane 2: purified rBmAsnRS.</p

The effect of rBmAsnRS and VEGF in endothelial ring formation.

<p>Fig 4A. Photomicrographs show the morphology of endothelial rings (arrows) formed by EA.hy926 cells in response to stimulation with 10 ng/ml rBmAsnRS and 10 ng/ml VEGF. Fig 4B. Graphical representation of the induction of endothelial rings in response to rBmAsnRS and VEGF. Data are means ± SEM. The percentage of cells forming endothelial rings in 10 ng/ml rBmAsnRS and 10 ng/ml VEGF were similar and both were significantly greater than the PBS control. Asterisk (*) denotes p<0.0001.</p

The effect of rBmAsnRS and VEGF on tube formation.

<p>Fig 5A. Images indicate with arrows the endothelial tube formation in negative control, 10 ng/ml rBmAsnRS and 10 ng/ml VEGF groups. Photomicrographs were taken with 20X magnification under an inverted bright field microscope. Fig 5B. Graphical representation of the number of tubes formed by EA.hy926 cells exposed to negative control, 10 ng/ml rBmAsnRS or 10 ng/ml VEGF for 24 hours in Matrigel-coated 24-well plates. Data are presented as means ± SEM. Asterisk (*) denotes statistically significant differences compared to negative control.</p

The effect of rBmAsnRS and VEGF on vasodilation of capillary blood vessels.

<p>Fig 7A. Images represent the change in capillary width induced by 10 ng/ml rBmAsnRS and 10 ng/ml VEGF. Images were captured after 20 minutes observation and are representative of 3 sets of experiments. Fig 7B. Graphical representation of changes in vessel width induced by rBmAsnRS and VEGF. Data are presented as means ± SEM. rBmAsnRS and VEGF treated blood vessels were both significantly dilated compared to controls. Asterisk (*) indicates statistically significant difference between control vs rBmAsnRS or VEGF (p < 0.005.).</p