Hypoxia enhances the tissue protective effect of erythropoietin and its analogues in an endothelial cell injury model

PO has tissue protective activities in ischemic disease but also has prothrombotic, erythropoietic effects. Carbamylated EPO (CEPO) retains the protective actions without the erythropoietic effects. To assess the potential of these molecules in atherosclerosis (an ischemic heart disease), we investigated EPO and CEPO in an in vitro model of injury using bovine aortic endothelial cells (BAEC) in hypoxia and normoxia.. BAECs were grown to confluence in 10% FBS in 12 well culture plates. They were then cultured under normoxia (21% oxygen) or hypoxia (5% oxygen) 24 h prior to their use in an injury model using the ‘scratch assay.’ The effects of EPO and CEPO on endothelial closure were assessed using a range of concentrations (0-10 ng/mL). In separate experiments, the effects of EPO and CEPO on BAEC proliferation and chemotaxis were also assessed under similar hypoxic conditions. Gene expression of the receptors that may be involved in their protective pathway [EPOR and the β common chain receptor (βCR)] were assessed using quantitative PCR. The effects of both EPO and CEPO were enhanced under hypoxic conditions (13 ± 2.6 %, and 10 ± 1.69 %, p0.05). Whilst, the expression of EPOR gene increased by 2.1 ± 0.8 folds (p<0.05) In hypoxia, βCR expression was not affected by the change in oxygen tension. The effects of EPO and CEPO in the scratch assay appeared to be mediated by enhancing cell proliferation and migration of BAECs (p<0.05). In conclusion, the enhanced effects of EPO and CEPO on endothelial cells under hypoxia requires further investigation in processes in which hypoxia may play a role, e.gfor example. in atherogenesis and re-stensosis following angioplasty

Definition of a family of tissue-protective cytokines using functional cluster analysis: a proof-of-concept study

The discovery of the tissue-protective activities of erythropoietin (EPO) has underlined the importance of some cytokines in tissue-protection, repair, and remodeling. As such activities have been reported for other cytokines, we asked whether we could define a class of tissue-protective cytokines. We therefore explored a novel approach based on functional clustering. In this pilot study, we started by analyzing a small number of cytokines (30). We functionally classified the 30 cytokines according to their interactions by using the bioinformatics tool STRING (Search Tool for the Retrieval of Interacting Genes), followed by hierarchical cluster analysis. The results of this functional clustering were different from those obtained by clustering cytokines simply according to their sequence. We previously reported that the protective activity of EPO in a model of cerebral ischemia was paralleled by an upregulation of synaptic plasticity genes, particularly early growth response 2 (EGR2). To assess the predictivity of functional clustering, we tested some of the cytokines clustering close to EPO (interleukin-11, IL-11; kit ligand, KITLG; leukemia inhibitory factor, LIF; thrombopoietin, THPO) in an in vitro model of human neuronal cells for their ability to induce EGR2. Two of these, LIF and IL-11, induced EGR2 expression. Although these data would need to be extended to a larger number of cytokines and the biological validation should be done using more robust in vivo models, rather then just one cell line, this study shows the feasibility of this approach. This type of functional cluster analysis could be extended to other fields of cytokine research and help design biological experiments

The role of novel biomarkers of inflammation in arterial stiffness, and in predicting further vascular events after TIA and lacunar stroke

Objective: To explore the role of biomarkers (hsCRP, sRANKL, PRDX1 and EPO) in arterial stiffness and in predicting further vascular events. Methods: Patients from the ongoing ASIST study each attended a laboratory visit within fourteen days of their diagnosed TIA or lacunar stroke. Arterial stiffness was calculated using cfPWV (carotid-femoral pulse wave velocity) measured with Complior ®Artech, France, and with the CAVI ®Fukuda, Japan (cardio-ankle vascular index) method. Blood samples were taken for ELISA assays. Analysis was completed with SPSS software. Results: Forty patients were evaluated in this preliminary project (29 male/11 female, mean age 70.7 ± 11.99), with four experiencing a further event during the six month follow up (10%). All biomarkers and both measurements for arterial stiffness had a higher mean value in patients with a further event (hsCRP 3.89 vs 1.42 ug/ml, P=0.08; EPO 9.06 vs 9.01 mU/ml, P=0.85; sRANKL 0.05 vs 0.03 pmol/L, P=0.31; PRDX1 6.27 vs 6.21 ng/ml, P=0.95; CAVI 11.13 vs 9.69, P=0.15; cfPWV 10.82 vs 10.2 m/s, P=0.55), however none were statistically significant. Levels of PRDX1 were elevated acutely post-event before falling significantly (R=-0.475, P=0.002), while hsCRP and EPO continued to be elevated at >10 days post-event. In addition, CAVI correlated closely with hsCRP (R=0.28, P=0.09) and EPO (R=0.29, P=0.08), but cfPWV was not closely related to any of the biomarkers. Conclusions: This preliminary data suggests that biomarkers, particularly EPO and hsCRP, are more closely related to CAVI than cfPWV. hsCRP was the most relevant as an independent predictive factor for further vascular events

Glutathione fine-tunes the innate immune response toward antiviral pathways in a macrophage cell line independently of its antioxidant properties

Glutathione (GSH), a major cellular antioxidant, is considered an inhibitor of the inflammatory response involving reactive oxygen species (ROS). However, evidence is largely based on experiments with exogenously added antioxidants/reducing agents or pro-oxidants. We show that depleting macrophages of 99% of GSH does not exacerbate the inflammatory gene expression profile in the RAW264 macrophage cell line or increase expression of inflammatory cytokines in response to the toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS); only two small patterns of LPS-induced genes were sensitive to GSH depletion. One group, mapping to innate immunity and antiviral responses (Oas2, Oas3, Mx2, Irf7, Irf9, STAT1, il1b), required GSH for optimal induction. Consequently, GSH depletion prevented the LPS-induced activation of antiviral response and its inhibition of influenza virus infection. LPS induction of a second group of genes (Prdx1, Srxn1, Hmox1, GSH synthase, cysteine transporters), mapping to nrf2 and the oxidative stress response, was increased by GSH depletion. We conclude that the main function of endogenous GSH is not to limit inflammation but to fine-tune the innate immune response to infection

Hypoxia enhances the regenerative effects of erythropoietin and its non-erythropoietic peptide analogue in models of endothelial cell injury

Background: Hypoxia is invariably associated with wound repair, inflammation, and vascular disease. The induction of Hypoxia Inducible Factor-1 (HIF-1) is a characteristic feature of hypoxia, and orchestrates the profound changes in transcription that accompany hypoxia. HIF-1 expression is localized to several cell types, and regulates several genes that are important to vascular function including vascular endothelial growth factor (VEGF), nitric oxide synthase (NOS), endothelin-1 and erythropoietin (EPO). In fact, EPO derived from vascular endothelial cells appears to be important in protecting the endothelium against ischemic injury. The non-erythropietic analogue of EPO; pyroglutamate helix B surface peptide (pHBSP) retains the protective actions of EPO without its erythropoietic effects. The aim of our study was to assess the reparative effects of these molecules when used in combination with HIF inducers. Method: The reparative effects of EPO and pHBSP were assessed under hypoxia (1% O2) and normoxia (21% O2) as well as in the presence or absence of DMOG; a HIF-1 inducer. An in vitro model of wound healing (the scratch assay) was used: a monolayer of rat aortic endothelial cells (RAECs) was scraped to produce a reproducible injury, and the scratch closure was assessed over 24 h. An in vivo model of vascular injury using a 2F fogarty balloon catheter was introduced into the common carotid artery causing complete removal of the vascular endothelium. Drugs were applied locally onto the injured arteries using a hydrogel (30% w/v) and re-endothelialisation assessed using Evans blue staining injected 30 min intravenously before culling the rat. The effects of EPO and pHBSP on cell proliferation, chemotaxis and apoptosis were assessed in both the in vitro and in vivo models. The potential molecular mechanisms of these effects were also explored. Results: In vitro, EPO and its analogues only exhibited a reparative effect under hypoxic conditions (13 ± 2.6 %, and 10 ± 1.69 %, p0.05). These effects appeared to be mediated by promoting RAEC proliferation and migration of (p<0.05). The priming effect of hypoxia was associated with stabilization of HIF-1α. Hypoxia was associated with a reduction in nitric oxide (NO) production as assessed by its oxidation products nitrite and nitrate, and this was consistent with the oxygen requirement for the endogenous production of NO by NO synthase (NOS).The HIF-1 inducer; DMOG also exhibited reparative effects in a concentration dependent manner. Similar results were observed in vivo where DMOG and EPO accelerated the repair of injured arteries (35 ± 9.8 % recovery compared to untreated injured arteries respectively). This mode of application also caused site-specific increase in VEGF expression on treated arteries compared to untreated ones within the same animal. Conclusion and implication: The tissue-protective effects of EPO-related cytokines in pathophysiological settings are enhanced by hypoxia. These findings may be particularly relevant to atherogenesis and post-angioplasty restenosi

Leukemia inhibitory factor inhibits erythropoietin-induced myelin gene expression in oligodendrocytes

Background: The pro-myelinating effects of leukemia inhibitory factor (LIF) and other cytokines of the gp130 family, including oncostatin M (OSM) and ciliary neurotrophic factor (CNTF), have long been known, but controversial results have also been reported. We recently overexpressed erythropoietin receptor (EPOR) in rat central glia-4 (CG4) oligodendrocyte progenitor cells (OPCs) to study the mechanisms mediating the pro-myelinating effects of erythropoietin (EPO). In this study, we investigated the effect of co-treatment with EPO and LIF. Methods: Gene expression in undifferentiated and differentiating CG4 cells in response to EPO and LIF was analysed by DNA microarrays and by RT-qPCR. Experiments were performed in biological replicates of N ≥ 4. Functional annotation and biological term enrichment was performed using DAVID (Database for Annotation, Visualization and Integrated Discovery). The gene-gene interaction network was visualised using STRING (Search Tool for the Retrieval of Interacting Genes). Results: In CG4 cells treated with 10 ng/ml of EPO and 10 ng/ml of LIF, EPO-induced myelin oligodendrocyte glycoprotein (MOG) expression, measured at day 3 of differentiation, was inhibited ≥ 4-fold (N=5, P < 0.001). Inhibition of EPO-induced MOG was also observed with OSM and CNTF. Analysis of the gene expression profile of CG4 differentiating cells treated for 20 h with EPO and LIF revealed LIF inhibition of EPO-induced genes involved in lipid transport and metabolism, previously identified as positive regulators of myelination in this system. In addition, among the genes induced by LIF, and not by differentiation or by EPO, the role of suppressor of cytokine signaling 3 (SOCS3) and toll like receptor 2 (TLR2) as negative regulators of myelination was further explored. LIF-induced SOCS3 was associated with MOG inhibition; Pam3, an agonist of TLR2, inhibited EPO-induced MOG expression, suggesting that TLR2 is functional and its activation decreases myelination. Conclusions: Cytokines of the gp130 family may have negative effects on myelination, depending on the cytokine environment

Vitamins D3 and D2 have marked but different global effects on gene expression in a rat oligodendrocyte precursor cell line

Background: Vitamin D deficiency increases the risk of developing multiple sclerosis (MS) but it is unclear whether vitamin D supplementation improves the clinical course of MS, and there is uncertainty about the dose and form of vitamin D (D2 or D3) to be used. The mechanisms underlying the effects of vitamin D in MS are not clear. Vitamin D3 increases the rate of differentiation of primary oligodendrocyte precursor cells (OPCs), suggesting that it might help remyelination in addition to modulating the immune response. Here we analyzed the transcriptome of differentiating rat CG4 OPCs treated with vitamin D2 or with vitamin D3 at 24 h and 72 h following onset of differentiation. Methods: Gene expression in differentiating CG4 cells in response to vitamin D2 or D3 was quantified using Agilent DNA microarrays (n=4 replicates), and the transcriptome data were processed and analysed using the R software environment. Differential expression between the experimental conditions was determined using LIMMA, applying the Benjamini and Hochberg multiple testing correction to p-values, and significant genes were grouped into co-expression clusters by hierarchical clustering. The functional significance of gene groups was explored by pathway enrichment analysis using the clusterProfiler package. Results: Differentiation alone changed the expression of about 10% of the genes at 72 h compared to 24 h. Vitamin D2 and D3 exerted different effects on gene expression, with D3 influencing 1,272 genes and D2 574 at 24 h. The expression of the vast majority of these genes was either not changed in differentiating cells not exposed to vitamin D or followed the same trajectory as the latter. D3-repressed genes were enriched for Gene Ontology (GO) categories including transcription factors and the Notch pathway, while D3-induced genes were enriched for the Ras pathway. Conclusions: This study shows that vitamin D3, compared with D2, changes the expression of a larger number of genes in OLs. Identification of genes affected by D3 in OLs should help to identify mechanisms mediating its action in MS

Erythropoietin and a nonerythropoietic peptide analog promote aortic endothelial cell repair under hypoxic conditions: role of nitric oxide

The cytoprotective effects of erythropoietin (EPO) and an EPO-related nonerythropoietic analog, pyroglutamate helix B surface peptide (pHBSP), were investigated in an in vitro model of bovine aortic endothelial cell injury under normoxic (21% O2) and hypoxic (1% O2) conditions. The potential molecular mechanisms of these effects were also explored. Using a model of endothelial injury (the scratch assay), we found that, under hypoxic conditions, EPO and pHBSP enhanced scratch closure by promoting cell migration and proliferation, but did not show any effect under normoxic conditions. Furthermore, EPO protected bovine aortic endothelial cells from staurosporine-induced apoptosis under hypoxic conditions. The priming effect of hypoxia was associated with stabilization of hypoxia inducible factor-1α, EPO receptor upregulation, and decreased Ser-1177 phosphorylation of endothelial nitric oxide synthase (NOS); the effect of hypoxia on the latter was rescued by EPO. Hypoxia was associated with a reduction in nitric oxide (NO) production as assessed by its oxidation products, nitrite and nitrate, consistent with the oxygen requirement for endogenous production of NO by endothelial NOS. However, while EPO did not affect NO formation in normoxia, it markedly increased NO production, in a manner sensitive to NOS inhibition, under hypoxic conditions. These data are consistent with the notion that the tissue-protective actions of EPO-related cytokines in pathophysiological settings associated with poor oxygenation are mediated by NO. These findings may be particularly relevant to atherogenesis and postangioplasty restenosis

Association between inflammatory biomarkers and neointimal response following elective implantation of the ABSORB bioresorbable vascular scaffold

Introduction The ABSORB bioresorbable vascular scaffold (BVS) is associated with greater neointimal proliferation and thrombotic rate than the metal stent. The role of inflammatory biomarkers on neointimal proliferation has not been studied in the setting of BVS implantation. Patients and methods Thirty patients had arterial blood sampling before elective percutaneous coronary intervention with the ABSORB BVS and at 9-months follow-up. Plasma levels of interleukin-6, soluble CD40 ligand, monocyte chemotactic protein-1 and C-reactive protein were measured using enzyme-linked immunosorbent assay. Baseline and follow-up levels were compared for each biomarker. Optical frequency domain imaging was performed at follow-up and the neointimal burden was calculated as the ratio of neointimal area to scaffold area. The levels of inflammatory mediators were correlated with the neointimal burden. Results There was no significant increase in the levels of biomarkers from baseline to follow-up. Median C-reactive protein levels changed from 1.1 [interquartile range (IQR): 0.5–2.5] to 2.2 (IQR: 0.5–3.5) μg/ml, interleukin-6 from 1.0 (IQR: 0.6–1.4) to 1.0 (95% confidence interval: 0.6–1.4) pg/ml, monocyte chemotactic protein-1 from 120.4 (IQR: 86.0–153.4) to 102.0 (IQR: 70.3–148.1) pg/ml and soluble CD40 ligand from 108.3 (IQR: 74.1–173.7) to 112.0 (IQR: 71.0–225.9) pg/ml. The average neointimal burden in the cohort was 18±6%. Baseline, follow-up and change in plasma levels of inflammatory markers between these two time points did not correlate with the neointimal burden. Conclusion Elective percutaneous coronary intervention with the ABSORB BVS does not provoke a chronic inflammatory response. The degree of neointimal proliferation after elective implantation of the ABSORB BVS is independent of the pre-existing inflammatory environment