Differential modulation of angiogenesis by erythropoiesis-stimulating agents in a mouse model of ischaemic retinopathy

Background: Erythropoiesis stimulating agents (ESAs) are widely used to treat anaemia but concerns exist about their potential to promote pathological angiogenesis in some clinical scenarios. In the current study we have assessed the angiogenic potential of three ESAs; epoetin delta, darbepoetin alfa and epoetin beta using in vitro and in vivo models.

Methodology/Principal Findings: The epoetins induced angiogenesis in human microvascular endothelial cells at high doses, although darbepoetin alfa was pro-angiogenic at low-doses (1-20 IU/ml). ESA-induced angiogenesis was VEGF-mediated. In a mouse model of ischaemia-induced retinopathy, all ESAs induced generation of reticulocytes but only epoetin beta exacerbated pathological (pre-retinal) neovascularisation in comparison to controls (p<0.05). Only epoetin delta induced a significant revascularisation response which enhanced normality of the vasculature (p<0.05). This was associated with mobilisation of haematopoietic stem cells and their localisation to the retinal vasculature. Darbepoetin alfa also increased the number of active microglia in the ischaemic retina relative to other ESAs (p<0.05). Darbepoetin alfa induced retinal TNF alpha and VEGF mRNA expression which were up to 4 fold higher than with epoetin delta (p<0.001).

Conclusions: This study has implications for treatment of patients as there are clear differences in the angiogenic potential of the different ESAs.

The use of erythropoiesis-stimulating agents with ruxolitinib in patients with myelofibrosis in COMFORT-II: an open-label, phase 3 study assessing efficacy and safety of ruxolitinib versus best available therapy in the treatment of myelofibrosis

BACKGROUND: Anemia is considered a negative prognostic risk factor for survival in patients with myelofibrosis. Most patients with myelofibrosis are anemic, and 35-54 % present with anemia at diagnosis. Ruxolitinib, a potent inhibitor of Janus kinase (JAK) 1 and JAK2, was associated with an overall survival benefit and improvements in splenomegaly and patient-reported outcomes in patients with myelofibrosis in the two phase 3 COMFORT studies. Consistent with the ruxolitinib mechanism of action, anemia was a frequently reported adverse event. In clinical practice, anemia is sometimes managed with erythropoiesis-stimulating agents (ESAs). This post hoc analysis evaluated the safety and efficacy of concomitant ruxolitinib and ESA administration in patients enrolled in COMFORT-II, an open-label, phase 3 study comparing the efficacy and safety of ruxolitinib with best available therapy for treatment of myelofibrosis. Patients were randomized (2:1) to receive ruxolitinib 15 or 20 mg twice daily or best available therapy. Spleen volume was assessed by magnetic resonance imaging or computed tomography scan.

RESULTS: Thirteen of 146 ruxolitinib-treated patients had concomitant ESA administration (+ESA). The median exposure to ruxolitinib was 114 weeks in the +ESA group and 111 weeks in the overall ruxolitinib arm; the median ruxolitinib dose intensity was 33 mg/day for each group. Six weeks before the first ESA administration, 10 of the 13 patients had grade 3/4 hemoglobin abnormalities. These had improved to grade 2 in 7 of the 13 patients by 6 weeks after the first ESA administration. The rate of packed red blood cell transfusions per month within 12 weeks before and after first ESA administration remained the same in 1 patient, decreased in 2 patients, and increased in 3 patients; 7 patients remained transfusion independent. Reductions in splenomegaly were observed in 69 % of evaluable patients (9/13) following first ESA administration.

CONCLUSIONS: Concomitant use of an ESA with ruxolitinib was well tolerated and did not affect the efficacy of ruxolitinib. Further investigations evaluating the effects of ESAs to alleviate anemia in ruxolitinib-treated patients are warranted (ClinicalTrials.gov identifier, NCT00934544; July 6, 2009).

A common polymorphism in the oxygen-dependent degradation (ODD) domain of hypoxia inducible factor-1alpha (HIF-1alpha) does not impair Pro-564 hydroxylation

The hypoxia-inducible factor (HIF) transcription complex, which is activated by low oxygen tension, controls a diverse range of cellular processes including angiogenesis and erythropoiesis. Under normoxic conditions, the alpha subunit of HIF is rapidly degraded in a manner dependent on hydroxylation of two conserved proline residues at positions 402 and 564 in HIF-1alpha in the oxygen-dependent degradation (ODD) domain. This allows subsequent recognition by the von Hippel-Lindau (VHL) tumor suppressor protein, which targets HIF for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, prolyl hydroxylation of HIF is inhibited, allowing it to escape VHL-mediated degradation. The transcriptional regulation of the erythropoietin gene by HIF raises the possibility that HIF may play a role in disorders of erythropoiesis, such as idiopathic erythrocytosis (IE).

A family with erythrocytosis establishes a role for prolyl hydroxylase domain protein 2 in oxygen homeostasis

The number of red blood cells is normally tightly regulated by a classic homeostatic mechanism based on oxygen sensing in the kidney. Decreased oxygen delivery resulting from anemia induces the production of erythropoietin, which increases red cell production and hence oxygen delivery. Investigations of erythropoietin regulation identified the transcription factor hypoxia-inducible factor (HIF). HIF is now recognized as being a key regulator of genes that function in a comprehensive range of processes besides erythropoiesis, including energy metabolism and angiogenesis. HIF itself is regulated through the -subunit, which is hydroxylated in the presence of oxygen by a family of three prolyl hydroxylase domain proteins (PHDs)/HIF prolyl hydroxylases/egg-laying-defective nine enzymes. Hydroxylation allows capture by the von Hippel–Lindau tumor suppressor gene product, ubiquitination, and destruction by the proteasome. Here we describe an inherited mutation in a mammalian PHD enzyme. We show that this mutation in PHD2 results in a marked decrease in enzyme activity and is associated with familial erythrocytosis, identifying a previously unrecognized cause of this condition. Our findings indicate that PHD2 is critical for normal regulation of HIF in humans.

A gain-of-function mutation in the HIF2A gene in familial erythrocytosis

Hypoxia-inducible factor (HIF) a, which has three isoforms, is central to the continuous balancing of the supply and demand of oxygen throughout the body. HIF-a is a transcription factor that modulates a wide range of processes, including erythropoiesis, angiogenesis, and cellular metabolism. We describe a family with erythrocytosis and a mutation in the HIF2A gene, which encodes the HIF-2a protein. Our functional studies indicate that this mutation leads to stabilization of the HIF-2a protein and suggest that wild-type HIF-2a regulates erythropoietin production in adults.

The classification and diagnosis of erythrocytosis

An absolute erythrocytosis is present when the red cell mass is raised and the haematocrit is elevated above prescribed limits. Causes of an absolute erythrocytosis can be primary where there is an intrinsic problem in the bone marrow and secondary where there an event outside the bone marrow driving erythropoiesis. This can further be divided into congenital and acquired causes. There remain an unexplained group idiopathic erythrocytosis. Investigation commencing with thorough history taking and examination and then investigation depending on initial features is required. Clear simple criteria for polycythaemia vera are now defined. Those who do not fulfil these criteria require further investigation depending on the clinical scenario and initial results. The erythropoietin level provides some guidance as to the direction in which to proceed and the order and extent of investigation necessary in an individual patient. It should thus be possible to make an accurate diagnosis in the majority of patients.

Pathophysiology of anemia and erythrocytosis.

An increasing understanding of the process of erythropoiesis raises some interesting questions about the pathophysiology, diagnosis and treatment of anemia and erythrocytosis. The mechanisms underlying the development of many of the erythrocytoses, previously characterised as idiopathic, have been elucidated leading to an increased understanding of oxygen homeostasis. Characterisation of anemia and erythrocytosis in relation to serum erythropoietin levels can be a useful addition to clinical diagnostic criteria and provide a rationale for treatment with erythropoiesis stimulating agents (ESAs). Recombinant human erythropoietin as well as other ESAs are now widely used to treat anemias associated with a range of conditions, including chronic kidney disease, chronic inflammatory disorders and cancer. There is also heightened awareness of the potential abuse of ESAs to boost athletic performance in competitive sport. The discovery of erythropoietin receptors outside of the erythropoietic compartment may herald future applications for ESAs in the management of neurological and cardiac diseases. The current controversy concerning optimal hemoglobin levels in chronic kidney disease patients treated with ESAs and the potential negative clinical outcomes of ESA treatment in cancer reinforces the need for cautious evaluation of the pleiotropic effects of ESAs in non-erythroid tissues.

Prevalence and Management of Anaemia in Renal Transplant Recipients: Data from Ten European Centres.

Background: Although it is a known predictor of mortality, there is a relative lack of recent information about anaemia in kidney transplant recipients. Thus, we now report data about the prevalence and management of post-transplant anaemia (PTA) in Europe 5 years after the TRansplant European Survey on Anemia Management (TRESAM) study. Methods: In a cross-sectional study enrolling the largest number of patients to date, data were obtained from 5,834 patients followed at 10 outpatient transplant clinics in four European countries using the American Society of Transplantation anaemia guideline. Results: More than one third (42%) of the patients were anaemic. The haemoglobin (Hb) concentration was significantly correlated with the estimated glomerular filtration rate (eGFR) (r = 0.4, p < 0.001). In multivariate analysis, eGFR, serum ferritin, age, gender, time since transplantation and centres were independently and significantly associated with Hb. Only 24% of the patients who had a Hb concentration <110 g/l were treated with an erythropoiesis-stimulating agent. The prevalence of anaemia and also the use of erythropoiesis-stimulating agents were significantly different across the different centres, suggesting substantial practice variations. Conclusions: PTA is still common and under-treated. The prevalence and management of PTA have not changed substantially since the TRESAM survey.

Erythropoietin-Induced Activation of the JAK2/STAT5, PI3K/Akt, and Ras/ERK Pathways Promotes Malignant Cell Behavior in a Modified Breast Cancer Cell Line

Abstract Erythropoietin (Epo), the major regulator of erythropoiesis, and its cognate receptor (EpoR) are also expressed in nonerythroid tissues, including tumors. Clinical studies have highlighted the potential adverse effects of erythropoiesis-stimulating agents when used to treat cancer-related anemia. We assessed the ability of EpoR to enhance tumor growth and invasiveness following Epo stimulation. A benign noninvasive rat mammary cell line, Rama 37, was used as a model system. Cell signaling and malignant cell behavior were compared between parental Rama 37 cells, which express few or no endogenous EpoRs, and a modified cell line stably transfected with human EpoR (Rama 37-28). The incubation of Rama 37-28 cells with pharmacologic levels of Epo led to the rapid and sustained increases in phosphorylation of signal transducers and activators of transcription 5, Akt, and extracellular signal-regulated kinase. The activation of these signaling pathways significantly increased invasion, migration, adhesion, and colony formation. The Epo-induced invasion capacity of Rama 37-28 cells was reduced by the small interfering RNA-mediated knockdown of EpoR mRNA levels and by inhibitors of the phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways with adhesion also reduced by Janus-activated kinase 2/signal transducers and activators of transcription 5 inhibition. These data show that Epo induces phenotypic changes in the behavior of breast cancer cell lines and establishes links between individual cell signaling pathways and the potential for cancer spread.

Philadelphia-Negative Classical Myeloproliferative Neoplasms: Critical Concepts and Management Recommendations From European LeukemiaNet

We present a review of critical concepts and produce recommendations on the management of Philadelphia-negative classical myeloproliferative neoplasms, including monitoring, response definition, first-and second-line therapy, and therapy for special issues. Key questions were selected according the criterion of clinical relevance. Statements were produced using a Delphi process, and two consensus conferences involving a panel of 21 experts appointed by the European LeukemiaNet (ELN) were convened. Patients with polycythemia vera (PV) and essential thrombocythemia (ET) should be defined as high risk if age is greater than 60 years or there is a history of previous thrombosis. Risk stratification in primary myelofibrosis (PMF) should start with the International Prognostic Scoring System (IPSS) for newly diagnosed patients and dynamic IPSS for patients being seen during their disease course, with the addition of cytogenetics evaluation and transfusion status. High-risk patients with PV should be managed with phlebotomy, low-dose aspirin, and cytoreduction, with either hydroxyurea or interferon at any age. High-risk patients with ET should be managed with cytoreduction, using hydroxyurea at any age. Monitoring response in PV and ET should use the ELN clinicohematologic criteria. Corticosteroids, androgens, erythropoiesis-stimulating agents, and immunomodulators are recommended to treat anemia of PMF, whereas hydroxyurea is the first-line treatment of PMF-associated splenomegaly. Indications for splenectomy include symptomatic portal hypertension, drug-refractory painful splenomegaly, and frequent RBC transfusions. The risk of allogeneic stem-cell transplantation-related complications is justified in transplantation-eligible patients whose median survival time is expected to be less than 5 years.

Maintenance treatment of renal anaemia in haemodialysis patients with methoxy polyethylene glycol-epoetin beta versus darbepoetin alfa administered monthly:a randomized comparative trial

Several studies with erythropoiesis-stimulating agents claim that maintenance therapy of renal anaemia may be possible at extended dosing intervals; however, few studies were randomized, results varied, and comparisons between agents were absent. We report results of a multi-national, randomized, prospective trial comparing haemoglobin maintenance with methoxy polyethylene glycol-epoetin beta and darbepoetin alfa administered once monthly.

Localization of erythropoietin gene expression in proximal renal tubular cells detected by digoxigenin-labelled oligonucleotide probes

Erythropoietin (EPO) is the main humoral stimulus of erythropoiesis. In adult mammals, the kidney releases EPO in response to hypoxic stress. Conflicting data have suggested either renal tubular or peritubular cell origins of EPO synthesis in vivo. In situ hybridization studies were performed to define further the kidney cell type(s) capable of increasing EPO gene expression during hypoxic stimulation. EPO gene expression was stimulated in mice exposed to acute hypobaric hypoxia. Kidneys from hypoxic and control normoxic mice were obtained. Six digoxigenin-labelled oligonucleotide probes complementary to EPO exon sequences were utilized for in situ hybridization for EPO messenger RNA. Positive hybridization signals were identified in some proximal tubular cells, confined to the inner third of the renal cortex of hypoxic mouse kidney.