Frontiers in Drug Discovery

Hematopoiesis is a dynamic process resulting in continuous production of mature blood cells, from a small population of pluripotent stem cells through several proliferative and differentiative events. Different functions of hematopoietic cells are regulated by growth factors and cytokines, and by the interaction with other cells and extracellular matrix.

Erythropoiesis is mainly orchestrated by erythropoietin and its receptor. Binding of this growth factor to its receptor induces the activation of signal transduction intermediates, especially the JAK2/STAT5 (Janus associated kinase 2/signal transducers and activators of transcription 5), the mitogen activated protein (MAP) kinase and the phosphatidylinositol 3 (PI3) kinase pathways. Several transcription factors are then activated, which will initiate transcription of specific genes. Embryonic and adult erythropoiesis require broad spectrum transcription factors, as well as, specific erythroid transcription factors, which are necessary for the regulation of cell cycle, cell survival, differentiation, proliferation and for the intermediary metabolism of cells and to block apoptosis.

Regulation of erythropoiesis and the maintenance of erythroid homeostasis rely on modulation of erythropoietin (EPO) gene expression in response to tissue oxygen tension. EPO expression is tightly regulated in a tissue-specific manner and by development stage. However, is the hypoxic stimulation that primarily determines the EPO transcription. A 50 bp hypoxia-inducible enhancer, located 120 bp 3′ to the polyadenylation site, mediates the transcriptional response to hypoxia by binding several stimulatory transcription factors, such as hypoxia-inducible transcription factors (HIF-1α and HIF-2α) and hepatocyte nuclear factor-4α (HNF-4 α). Pro-inflammatory cytokines, as interleukin (IL)-1β and tumour necrosis factor (TNF)-α, inhibit EPO expression, apparently by increasing the binding of GATA factors to the EPO promoter and by down-regulation of HNF-4α. Understanding oxygen and tissue-specific regulation of EPO production is of high physiologic relevance. Moreover, this knowledge might be useful for new therapies to treat clinical states associated with aberrant EPO gene expression.

MicroRNAs (miRNAs) are endogenously produced as a family of a small noncoding RNAs encoded by intergenic chromosomal regions that must be processed and matured to target a specific mRNA causing translational repression or mRNA degradation. MicroRNAs play essential functions in many biological processes and their deregulation is associated with pathologies, such as erythropoiesis. Erythropoietin (EPO) promotes the proliferation and differentiation of erythroid precursor cells, and requires the interaction with the EPO receptor and the presence of transcription factors. Erythropoietic stimulating agents are clinically used to improve erythropoietin production and relieve anaemia, which is a hallmark of several diseases. At defined stages of erythropoiesis, the expression of specific miRNAs promotes stem cell proliferation or erythroid cell differentiation. There are currently several approaches to silence, replace or mimetic miRNAs, making them potential tools for gene therapy. In addition, miRNAs may be used as diagnostic and prognostic biomarkers as well as therapeutic targets.

Erythropoietin (EPO) is a peptide hormone that stimulates erythropoiesis. The lack of EPO synthesis is the main cause of anemia associated with chronic disease. Treatment with recombinant human erythropoietin (rhEPO) has proved most useful to increase the quality of life in anemic patients. Furthermore, rhEPO has been associated with cytoprotective effects beyond the erythropoietic action on non-renal tissues. However, several questions deserve better elucidation, which cannot be achieved only with human studies, including the mechanisms underlying resistance to rhEPO therapy, the deleterious effects at high rhEPO doses, as well as the nature of the renoprotective properties. Animal models of kidney diseases/anemia are important tools to study pathophysiological events in kidney diseases that potentially could be translated into improved management of patients with these pathologies. In this chapter some of the main animal models used for studying the effects of rhEPO on kidney disease/anemia will be reviewed.

The introduction of erythropoiesis stimulating agents (ESAs) for the treatment of anemia in chronic kidney disease patients and chemotherapy-induced anemia associated with non-myeloid malignancies, improved the quality of life in these patients. Recently, several pleiotropic effects have been attributed to ESAs, especially under doses higher than the doses used to treat anemia. However, recent studies have raised some concerns about the use of high doses in patients that do not respond properly to the therapy. To use the pleiotropic actions of ESAs new molecules have been developed, that do not present hematopoietic actions. In this book chapter, we present a review of the risks and benefits associated with the use of high ESAs. We also present the recent proposal for new agents based on the EPO molecule and actions.

Anemia is a very frequent clinical finding in elderly people. There are several factors contributing to this condition, namely nutritional deficiencies, inflammation and chronic renal insufficiency. The etiology of chronic kidney disease related anemia is multifactorial, but a relative decrease of erythropoietin production is a major cause. Accordingly, the use of erythropoietic stimulating agents (ESA) is the cornerstone of the therapy in pre-dialysis elderly patients. Resistance or hyporesponsiveness to the treatment happens when the ESA dose needed to produce an otherwise expected increase in hemoglobin is particularly high. Iron deficiency is the commonest cause of resistance to ESA. Inflammation, malnutrition, hyperparathyroidism, drugs and vitamin B12 and folate deficiencies are other important causes of ESA resistance. The clinical investigation of ESA hyporesponsiveness is mandatory, since the correction of chronic kidney disease related anemia is cost saving and decreases the morbidity and mortality in the long term.

The extracellular matrix (ECM) is composed of fiber proteins (collagen, elastin, and fibrillin), specialized proteins (fibronectin and laminin), and ground substances such as glycosaminoglycan (GAG) and proteoglycan (PG). PGs are complexes of core proteins and GAG. GAGs such as heparin-like substances, except hyaluronic acid, are abundantly sulfated and interact with arginine-rich motifs including the heparin-binding domains (HBD) of cytokines, chemokines, and growth factors. The existence of an optimal dose is expected for the cardiovascular effects of erythropoietin (EPO)-derivatives. Asialoerythropoietin (AEPO), intermediate products of EPO in the Golgi, as well as the metabolic products of EPO by desialylation are expected to play roles in tissues as paracrine cytokines that protect organs from damage. Like VEGF, AEPO expresses tissue affinity, and tissues can maintain a constant concentration of AEPO. However, AEPO is not suitable for the treatment of anemia or endothelial cells injury. We have synthesized a chimeric derivative of EPO, heparin-binding erythropoietin (HEPO), introduced with the HBD of human PLGF-2. HEPO expressed a long-acting nature in erythropoiesis in vivo as expected. Surprisingly, the natural recovery of blood flow in a mouse limb ischemia model was inhibited by the administration of HEPO. PLGF shares the same receptors with VEGF-A, and expresses stronger angiogenic activity than VEGF-A. HBD of PLGF-2 is richer in arginine and lysine than VEGF-A. HEPO may inhibit the angiogenic effects of intrinsic VEGF by occupying tissue GAG with the HBD of PLGF-2.

Pyroglutamate helix B surface peptide (pHBSP) is an 11 amino acid peptide based on the tertiary structure of erythropoietin (EPO). EPO is released from the kidney and its primary function is to control the production of erythrocytes in response to hypoxia. More recently, EPO has been found to possess many pleiotropic actions, in particular its ability to protect tissues against injurious stimuli. However, its use in patients is associated with an increased risk of thrombotic events due to the stimulation of erythropoiesis, this has led to the development of multiple EPO analogues including pHBSP. pHBSP has demonstrated its tissue-protective capability in many pre-clinical studies, with effects comparable to those of EPO. pHBSP is proposed to act via a receptor structurally distinct to the classical EPO receptor homodimer; the β-common receptor (βcR), therefore it does not induce erythropoiesis. pHBSP is a novel and attractive therapeutic intervention for the treatment of many disease states including acute kidney injury, stroke and myocardial infarction, however there are currently no clinical studies using this peptide.

Familial amyloidosis ATTR V30M is an hereditary disorder, the most frequent type of transthyretin related amyloidosis. The main manifestation of the disease is a sensory-motor and autonomic polyneuropathy. Other manifestations occur such as cardiovascular, gastrointestinal, ocular, renal and hematological disorders. Anemia is a common feature, and occurs late in the disease course. It is associated with low erythropoietin production. Decreased production can start early in the course of the disease and precede clinical symptoms. The possible underlying pathogenic mechanisms are discussed.

Lately erythropoietin (EPO), a hematopoietic substance, has been used increasingly to support, regenerate or repair tissue and organ functions. As the prevalence of chronic wounds is considerably increasing in an aging population, there is the need for supportive therapies in wound care management. The use of this pleiotropic glycoprotein hormone could therefore present widespread non-hematopoietic tissueprotective effects, which have also been evaluated in physiologic and pathologic skin wound healing. EPO interacts with different pathways relevant in wound healing, i.e., decrease of inflammation and apoptosis, enhancement of cell migration, as well as proliferation and maturation of microvessels. Despite ample experimental data referring to the tissue-protective effects of EPO, severe side effects occurred in clinical studies. Accordingly, the use of EPO beyond the treatment of chronic anemia and significant blood loss has again been questioned, also in skin wound healing. The challenge for the future with regard to skin regeneration and repair will therefore be (I) to improve the efficiency of alternative routes of EPO application, (II) to improve and engineer several well-tolerated EPO-like molecules, which induce all EPO-effects but erythropoiesis (III) to process and evaluate solid acting antibodies which are able to reliably provide evidence of EPO receptor on the respective tissues or cells and (IV) finally to promote randomized double blind controlled clinical studies which are able to describe potential safety concerns of EPO and so determine about its use in daily clinical routine. This book chapter therefore aims at describing the current knowledge on the use of EPO in different problems of skin wound healing in detail, critically discusses the potential underlying mechanisms and highlights the problems in its clinical use.

Erythropoietin (EPO) stimulates erythropoiesis through binding to and activation of homodimeric receptors, comprised of two ~59 kDa transmembrane proteins (EPO-R). Preclinical studies purported pleiotropic cytoprotective roles for the EPO/EPO-R system in tissues and organs, and the potential benefits of erythropoiesisstimulating agents (ESAs) for cardiovascular diseases are a focus of current research. This article summarizes putative actions of ESAs in the cardiovascular system, with emphasis on the human responses. The potential for EPO-mediated mobilization of stem cells into the blood stream has attracted wide interest because of the possibility that ESAs may promote the neovascularization of ischemic tissues. Supporting the possibility, EPO-R mRNA is detectable in vascular endothelium, and EPO has been reported to stimulate angiogenesis in some preclinical models. However, in most such studies very high concentrations of EPO were applied. Moreover, recent well-designed studies have failed to show direct effects of ESAs on endothelial cells. By use of a specific anti-EPO-R antibody very little EPO-R protein was detected on immunoblots of extracts from normal cardiovascular tissues. While in preclinical studies high-dosed ESAs reduced the myocardial infarct volume and improved contractile properties following ischemia, human placebo-controlled clinical trials failed to demonstrate clear beneficial effects of ESAs in patients with coronary syndrome or myocardial infarct. ESA therapy is currently used to prevent red blood cell transfusions in anemic patients with chronic kidney disease or chemotherapy for cancer and is being explored as an anti-anemia treatment in patients with heart disease. However, because blood viscosity increases and blood pressure may rise with hematocrit, hemoglobin concentrations should not be raised above indicated levels.

It is commonly believed that mammalian cardiomyocytes withdraw from the cell cycle shortly after birth, and postnatal growth of the heart is by hypertrophy rather than by hyperplasia. Although proliferating myocytes have been detected in the heart in ischemia and acute myocardial infarction, recent experimental evidence strongly suggests that these cells derive from circulating and locally released precursor cells. These discoveries in conjunction with increased incidence and prevalence of heart disease and few new classes of pharmacologic agents for cardiovascular disease have paved the way for numerous clinical trials of stem and progenitor cell therapy for acute myocardial infarction and congestive heart failure. Nearly all trials have demonstrated safety and feasibility of stem cell therapies for cardiovascular disease. Many have suggested that the survival of the transplanted cells is improved when applied with erythropoietin (EPO), resulting in improved clinical outcomes. Besides its hematopoietic effects erythropoietin has anti-apoptotic effects especially under ischemic conditions and attenuates oxidative stress. EPO receptors are expressed on erythroid precursors, megakaryocytes, vascular smooth muscle cells, endothelial cells, skeletal myoblasts, neurons, nephrons, and cardiac myocytes. Augmentation of this self-repair process through application of EPO and transplantation of exogenous endothelial progenitor cells is coming closer and closer to clinical reality. Clinicians will need to understand the fundamental concepts underlying the preclinical and clinical applications of EPO and EPCs. This chapter provides a primer on those basic principles.

Cerebral malaria a severe complication of an infection caused by the parasite Plasmodium falciparum. It is an acute encephalopathy and currently the adjunctive therapy supporting the anti-malarial treatment consists of glucose and oxygen supplementation. Wishing to improve the adjunctive therapy, we have studied the use of erythropoietin (EPO) and its possible neuroprotective effects in a murine model of cerebral malaria. EPO reduces the mortality and neuropathology in this model and is well tolerated. Our research is discussed in relation to the current literature and the potential as using EPO as adjunct therapy in humans is discussed.

Erythropoiesis-stimulating agents (ESAs) are approved worldwide for treating anemia in cancer patients receiving chemotherapy. ESA-safety issues include thromboembolic events and there have been concerns about a possible induction of disease progression or increased mortality in cancer patients treated with these agents. This chapter discusses available evidence regarding this question. Clinical data and preclinical studies provide no convincing evidence that ESAs increase any risk related to the patient’s cancer when used within label. Assessment and definition of disease progression vary across studies, and preclinical work has often been conducted with inadequate methodology.