Nitric oxide and hypoxia stimulate erythropoietin receptor via MAPK kinase in endothelial cells

Erythropoietin receptor (EPOR) expression level determines the extent of erythropoietin (EPO) response. Previously we showed that EPOR expression in endothelial cells is increased at low oxygen tension and that EPO stimulation of endothelial cells during hypoxia can increase endothelial nitric oxide (NO) synthase (eNOS) expression and activation as well as NO production. We now observe that while EPO can stimulate NO production, NO in turn can regulate EPOR expression. Human umbilical vein endothelial cells (HUVEC) treated with 10-50 pM of NO donor diethylenetriamine NONOate (DETANO) for 24 h showed significant induction of EPOR gene expression at 5% and 2% of oxygen. Also human bone marrow microvascular endothelial cell line (TrHBMEC) cultured at 21 and 2% oxygen with 50 uM DETANO demonstrated a time and oxygen dependent induction of EPOR mRNA expression after 24 and 48 h, particularly at low oxygen tension. EPOR protein was also induced by DETANO at 2% oxygen in TrHBMEC and HUVEC. The activation of signaling pathways by NO donor stimulation appeared to be distinct from EPO stimulation. In reporter gene assays, DETANO treatment of HeLa cells at 2% oxygen increased EPOR promoter activity indicated by a 48% increase in luciferase activity with a 2 kb EPOR promoter fragment and a 71% increase in activity with a minimal EPOR promoter fragment containing 0.2 kb 5'. We found that DETANO activated MAPK kinase in TrHBMEC both in normoxia and hypoxia, while MAPK kinase inhibition showed significant reduction of EPOR mRNA gene expression at low oxygen tension, suggesting MAPK involvement in NO mediated induction of EPOR Furthermore, DETANO stimulated Ala anti-apoptotic activity after 30 min in normoxia, whereas it inhibited Akt phosphorylation in hypoxia. In contrast, EPO did not significantly increase MAPK activity while EPO stimulated Akt phosphorylation in TrHBMEC in normoxia and hypoxia. These observations provide a new effect of NO on EPOR expression to enhance EPO response in endothelial cells, particularly at low oxygen tensions

Low oxygen tension primes aortic endothelial cells to the reparative effect of tissue-protective cytokines

Erythropoietin (EPO) has both erythropoietic and tissue-protective properties. The EPO analogues carbamylated EPO (CEPO) and pyroglutamate helix B surface peptide (pHBSP) lack the erythropoietic activity of EPO but retain the tissue-protective properties that are mediated by a heterocomplex of EPO receptor (EPOR) and the β common receptor (βCR). We studied the action of EPO and its analogues in a model of wound healing where a bovine aortic endothelial cells (BAECs) monolayer was scratched and the scratch closure was assessed over 24 h under different oxygen concentrations. We related the effects of EPO and its analogues on repair to their effect on BAECs proliferation and migration (evaluated using a micro-Boyden chamber). EPO, CEPO and pHBSP enhanced scratch closure only at lower oxygen (5%), while their effect at atmospheric oxygen (21%) was not significant. The mRNA expression of EPOR was doubled in 5% compared to 21% oxygen, and this was associated with increased EPOR assessed by immunofluorescence and Western blot. By contrast βCR mRNA levels were similar in 5% and 21% oxygen. EPO and its analogues increased both BAECs proliferation and migration, suggesting that both may be involved in the reparative process. The priming effect of low oxygen tension on the action of tissue-protective cytokines may be of relevance to vascular disease, including atherogenesis and restenosis

JAK-STAT and AKT pathway-coupled genes in erythroid progenitor cells through ontogeny

Background: It has been reported that the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway regulates erythropoietin (EPO)-induced survival, proliferation, and maturation of early erythroid progenitors. Erythroid cell proliferation and survival have also been related to activation of the JAK-STAT pathway. The goal of this study was to observe the function of EPO activation of JAK-STAT and PI3K/AKT pathways in the development of erythroid progenitors from hematopoietic CD34(+) progenitor cells, as well as to distinguish early EPO target genes in human erythroid progenitors during ontogeny. Methods: Hematopoietic CD34(+) progenitor cells, isolated from fetal and adult hematopoietic tissues, were differentiated into erythroid progenitor cells. We have used microarray analysis to examine JAK-STAT and PI3K/AKT related genes, as well as broad gene expression modulation in these human erythroid progenitor cells. Results: In microarray studies, a total of 1755 genes were expressed in fetal liver, 3844 in cord blood, 1770 in adult bone marrow, and 1325 genes in peripheral blood-derived erythroid progenitor cells. The erythroid progenitor cells shared 1011 common genes. Using the Ingenuity Pathways Analysis software, we evaluated the network pathways of genes linked to hematological system development, cellular growth and proliferation. The KITLG, EPO, GATA1, PIM1 and STAT3 genes represent the major connection points in the hematological system development linked genes. Some JAK-STAT signaling pathway-linked genes were steadily upregulated throughout ontogeny (PIM1, SOCS2, MYC, PTPN11), while others were downregulated (PTPN6, PIAS, SPRED2). In addition, some JAK-STAT pathway related genes are differentially expressed only in some stages of ontogeny (STATs, GRB2, CREBB). Beside the continuously upregulated (AKT1, PPP2CA, CHUK, NFKB1) and downregulated (FOXO1, PDPK1, PIK3CG) genes in the PI3K-AKT signaling pathway, we also observed intermittently regulated gene expression (NFKBIA, YWHAH). Conclusions: This broad overview of gene expression in erythropoiesis revealed transcription factors differentially expressed in some stages of ontogenesis. Finally, our results show that EPO-mediated proliferation and survival of erythroid progenitors occurs mainly through modulation of JAK-STAT pathway associated STATs, GRB2 and PIK3 genes, as well as AKT pathway-coupled NFKBIA and YWHAH genes

Microarray and Proteomic Analyses of Myeloproliferative Neoplasms with a Highlight on the mTOR Signaling Pathway

The gene and protein expression profiles in myeloproliferative neoplasms (MPNs) may reveal gene and protein markers of a potential clinical relevance in diagnosis, treatment and prediction of response to therapy. Using cDNA microarray analysis of 25,100 unique genes, we studied the gene expression profile of CD34(+) cells and granulocytes obtained from peripheral blood of subjects with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). The microarray analyses of the CD34(+) cells and granulocytes were performed from 20 de novo MPN subjects: JAK2 positive ET, PV, PMF subjects, and JAK2 negative ET/PMF subjects. The granulocytes for proteomic studies were pooled in 4 groups: PV with JAK2 mutant allele burden above 80%, ET with JAK2 mutation, PMF with JAK2 mutation and ET/PMF with no JAK2 mutation. The number of differentially regulated genes was about two fold larger in CD34(+) cells compared to granulocytes. Thirty-six genes (including RUNX1, TNFRSF19) were persistently highly expressed, while 42 genes (including FOXD4, PDE4A) were underexpressed both in CD34(+) cells and granulocytes. Using proteomic studies, significant up-regulation was observed for MAPK and PI3K/AKT signaling regulators that control myeloid cell apoptosis and proliferation: RAC2, MNDA, S100A8/9, CORO1A, and GNAI2. When the status of the mTOR signaling pathway related genes was analyzed, PI3K/AKT regulators were preferentially up-regulated in CD34(+) cells of MPNs, with down-regulated major components of the protein complex EIF4F. Molecular profiling of CD34(+) cells and granulocytes of MPN determined gene expression patterns beyond their recognized function in disease pathogenesis that included dominant up-regulation of PI3K/AKT signaling

ERYTHROPOIETIN FOR THE TREATMENT OF SUBARACHNOID HEMORRAGE: A FEASIBLE INGREDIENT FOR A SUCCESS MEDICAL RECIPE

Subaracnhoid hemorrage (SAH) following aneurysm bleeding accounts for 6% to 8% of all cerebrovascular accidents. Althoug an aneurysm can be effectively managed by surgery or endovascular therapy, delayed cerebral ischemia is diagnosed in a high percentage of patients resulting in significant morbility and mortality. Cerebral vasospasm occurs in more than half of all patients after aneurysm rupture and is recognized as the leading cause of delayed cerebral ischemia after SAH. Hemodynamic strategies and endovascular procedures may be considered fo the treatment of cerebral vasospasm. In recent years, the mechanism contributing to the development of vasospasm, abnormal reactivity of cerebral arteries and cerebral ischemia following SAH, have been intensively investigated. A number of pathological processes have been identified in the pathogenesis of vasospasm including endothelial injury, smooth muscle cell contraction from spasmogenic substances produced by the subarachnoid blood clots, changes in vascular responsiveness and inflammatory response of the vascular endothelium. to date, the current therapeutic interventions remain ineffective being limited to the manipulation os systemic blood pressure, variation of blood volume and viscosity, and control of arterial carbon dioxide tension. In this scenario, the hormone erythropoietin (EPO), has been found to exert neuroprotective action during experimental SAH when its recombinant form (rHuEPO) is systematically administered. However, recent translation of experimental data into clinical trials has suggested an unclear role of recombinant human EPO in the setting of SAH. In this context, the aim of the recurrent review is to present current evidence on the potential role of EPO in cerebrovascular dysfunction following aneurysmal subarachnoid hemorrage

Erythropoietin in the intensive care unit: beyond treatment of anemia

Erythropoietin (EPO) is the major hormone stimulating the production and differentiation of red blood cells. EPO is used widely for treating anemia of critical illness or anemia induced by chemotherapy. EPO at pharmacological doses is used in this setting to raise hemoglobin levels (by preventing the apoptosis of erythroid progenitor cells) and is designed to reduce patient exposure to allogenic blood through transfusions. Stroke, heart failure, and acute kidney injury are a frequently encountered clinical problem. Unfortunately, in the intensive care unit advances in supportive interventions have done little to reduce the high mortality associated with these conditions. Tissue protection with EPO at high, nonpharmacological doses after injury has been found in the brain, heart, and kidney of several animal models. It is now well known that EPO has anti-apoptotic effects in cells other than erythroid progenitor cells, which is considered to be independent of EPOs erythropoietic activities. This review article summarizes what is known in preclinical models of critical illness and discusses why this does not correlate with randomized, controlled clinical trials

Erythropoietin: a multimodal neuroprotective agent

The tissue protective functions of the hematopoietic growth factor erythropoietin (EPO) are independent of its action on erythropoiesis. EPO and its receptors (EPOR) are expressed in multiple brain cells during brain development and upregulated in the adult brain after injury. Peripherally administered EPO crosses the blood-brain barrier and activates in the brain anti-apoptotic, anti-oxidant and anti-inflammatory signaling in neurons, glial and cerebrovascular endothelial cells and stimulates angiogenesis and neurogenesis. These mechanisms underlie its potent tissue protective effects in experimental models of stroke, cerebral hemorrhage, traumatic brain injury, neuroinflammatory and neurodegenerative disease. The preclinical data in support of the use of EPO in brain disease have already been translated to first clinical pilot studies with encouraging results with the use of EPO as a neuroprotective agent