Extrahematopoietic Actions of Erythropoietin

1.1.1

The Integrated Response to Systemic Hypoxia Involves Multiple Cell Types

The maintenance of adequate tissue O ₂ concentration to support cellular metabolism is a crucial physiological function for oxygen-utilizing animals. In vertebrates, delivery of O ₂ to tissues occurs via a tightly orchestrated negative feedback control process in which oxygenated hemoglobin within erythrocytes is delivered to tissue beds by the systemic circulation. The key hormone responsible for maintaining adequate circulating hemoglobin concentrations is EPO (see Chapter 11, Chapter 12 ). In the normal adult mammal, for example, hypoxia is detected within the cortex and outer medulla of the kidney by peritubular fibroblast-like type-1 interstitial cells with the resultant synthesis of hypoxia inducible factor (HIF). In turn, HIF activates a wide variety of responses adaptive for hypoxic conditions, including the transcription of the EPO gene. EPO is subsequently released into the circulation to activate the survival of erythrocyte precursors (colony forming units erythroid; CFU-E) residing within the bone marrow. It is important to note that the erythropoietic response depends only in part on adequate circulating EPO concentrations, as the cognate receptor is restricted to a subset of erythroid precursors at a specific stage of differentiation. Therefore, receptor expression is a critical driver of the biological response. This is also a feature of EPO’s nonerythropoietic activities.

EPO-responsive immature red cells express a receptor for EPO that consists of two identical protein monomers (EPOR ₂ ) present at relatively low concentrations (∼1000/cell). When inserted into the cell membrane, these subunits migrate to lipid rafts, and if at a sufficient receptor density, spontaneously associate through a leucine zipper mechanism within the cell membrane spanning region. When an EPO molecule bridges across the assembled receptor monomers using distinct recognition binding sites, a conformational change occurs in EPOR ₂ that results in autophosphorylation of janus kinase-2 (JAK2). This molecular signal in turn activates multiple secondary cascades that include the (1) signal transducer and activator of transcription (STAT), (2) phosphatidyl inositol-3 kinase (PI-3K), (3) mitogen-activated protein kinase (MAPK), (4) protein kinase C (PK-C) and (5) phospholipase C (PLC) pathways ( Fig. 23.1 ). As a group, the activity of these molecular cascades antagonizes the ongoing programmed cell death of immature erythrocytes, allowing maturation to proceed (see Chapter 11 ). As discussed below, except for PLC, each of these pathways has been documented to transduce, sometimes in a tissue-specific manner, the nonerythropoietic actions of EPO.

Systemic hypoxia also activates other EPO-dependent biological responses that function to improve or maintain the delivery of O ₂ to tissues. Thus, EPO jointly activates adhesion molecules, e.g., endothelial cell selectin expression and vascular cell adhesion molecule, while in synergy with thrombopoietin stimulates the production rate and reactivity of platelets. Together, these actions are strongly prothrombotic, resulting in a state which potentially prevents blood flow into damaged tissues and also reduces the probability of hemorrhagic blood loss. Additionally, EPO improves local tissue oxygen delivery by modulating regional blood flow via stimulating endothelial nitric oxide production. EPO also directly interacts with the neural control of ventilation so as to increase the intake of O ₂ during hypoxia. On a longer time scale, EPO improves tissue oxygenation by activating neoangiogenesis via stimulating the mitosis, migration, and differentiation of endothelial cells as well as synergizing with angiogenic factors, e.g., vascular endothelial growth factor (VEGF). Thus, in its hematopoietic role EPO mediates cell growth, survival, differentiation, migration, and function of a variety of cell types in addition to the CFU-E.

Besides the kidney, other tissues and organs synthesize HIF when subjected to hypoxic stress under a wide variety of pathological and physiological conditions. However, mere expression of HIF does result in EPO synthesis, as there is also control provided by tissue-specific repressive factors, e.g., via the GATA box. However, results of study have shown that similar to renal production of EPO, increases in HIF in some nonrenal tissues is accompanied by EPO synthesis. For example, glial cells maintained in vitro produce EPO following exposure to hypoxia, which is abolished by HIF neutralization. Similar to EPO expression, EPO receptor is also upregulated by HIF. In sum, a number of studies have shown that oxygen-deprived cells can produce both EPO and its receptor, thus constituting an autocrine/paracrine system distinct from the hormonal hematopoietic system.

1.1.2

The Innate Immune Response: A Balance Between Protection and Injury

The IIR is an ancient, stereotyped, localized reaction to molecular signals arising from tissue injury and/or pathogen invasion. The key effector molecules are proinflammatory cytokines, e.g., TNFα, which although potentially toxic for foreign invaders, also can damage normal tissues. One important control molecule of inflammation is NFkB which is ordinarily under chronic inhibition by the inhibitor of IkB kinase (IKK). In response to cellular stress, the inhibitor of IKK is itself antagonized and NFkB becomes functional through disinhibition of IKK. Following triggering, proinflammatory cytokines are generated, which if significant concentrations are achieved, kill surrounding normal and abnormal tissues. Once activated, the IIR tends to operate via a positive feedback control system. Too vigorous an IIR is not beneficial, e.g., sustained TNFα production is a common feature of many diseases.

1.1.3

The Hypoxic Stress and Innate Immune Responses are Interrelated

HIF functions as a heterodimer with a hypoxia-sensitive HIF alpha subunit and a constitutively expressed beta subunit (see Chapter 11 ). Under normoxic conditions, HIF is continuously degraded by the O ₂ -dependent prolyl hydroxylases (PHD). Tissue hypoxia causes a decrease in PHD activity, which in turn stabilizes HIF heterodimer formation ( Fig. 23.2 ). As a result, a myriad of genes involved in metabolism, angiogenesis, and cytoprotection are activated, including EPO . However, PHD is also a molecular control of the IIR, as IKK is also inhibited by PHD. Specifically, under hypoxic conditions, the resultant PHD inhibition results in activation of IKK, and thus also NFkB, which in turn acts as a transcriptional activator of HIF. In this manner, innate immunity and hypoxic responses are interlinked, a relationship that is evolutionarily ancient, as homologs for HIF and NFkB are expressed by invertebrates and interact in similar ways.

In the complex setting of tissue injury in which both hypoxia and inflammation play important pathophysiologic roles, EPO may be expressed in a distinct temporal pattern by a variety of cells that do not respond to hypoxia alone. As an important example, noninjury-related HIF expression by capillary endothelial cells does not result in EPO expression. In contrast, capillary endothelial cells within the immediate vicinity of an ischemic brain infarct strongly turn on EPO production with 24 h, followed with a delay by microglia (3 days) and reactive astrocytes (7 days). Therefore, capillary endothelial cells within close proximity to a region of injury are a potential source of EPO for any tissue.

1.1.4

EPO Modulates Immunity

As the above discussion has summarized, cellular stress activates a positive feedback system that turns on inflammation. To prevent catastrophic collateral damage there must be control elements capable of containing these processes. Work performed over the last 20 years has shown that EPO is the key negative modulator of these immune responses ( Fig. 23.2 ). EPO synthesis is controlled by the hypoxia response element (HRE) within its structural gene to which HIF binds. However, NFkB also has a binding site adjacent to the HRE that inhibits EPO expression. In addition, products of NFkB activation, e.g., TNFα, can directly inhibit EPO production. At the same time, EPO itself inhibits both HIF and NFkB production. In contrast to the effector molecule EPO, its receptor is strongly upregulated by both HIF and proinflammatory cytokines. Thus, there is a complex interplay between the expression of proinflammatory and protective molecules that are produced in a staggered temporal manner at spatially distinct sites such that the receptor concentration may be high when the ligand concentration is low ( Fig. 23.2 ).

1.1.5

EPO Is Essential for Normal Tissue Development and Repair

Activity of the paracrine EPO system is critical not only for protection from a wide variety of stressors but also for angiogenesis during development, which is driven in part by hypoxia-induced EPO expression. This is evidenced by the observation that either EPO or EPOR gene knockout results in a dramatic reduction in the number and complexity of vessels in mouse embryos beginning at day 10.5 in development, followed 2 days later by cardiac ventricular hypoplasia, and ultimately death 1 day later. Additionally, local EPO production an important signal in the cyclical angiogenic changes in the adult of the uterus and endometrium as shown by disruption of angiogenesis following administration of a soluble EPOR that complexes and inactivates endogenous EPO. EPO is also required for stem cell differentiation in some tissues. For example, EPO mRNA is detectible and increases as a function of time in the developing human brain and subsides to a much lower level in the adult. Proof of the indispensability of EPO in this setting is demonstrated by gene knockout of EPO which severely disrupts brain development and results in massive losses of neuronal progenitor cells through apoptosis.

In addition to providing a multitude of direct and indirect cytoprotective roles, EPO also participates in all phases of tissue repair and regeneration. Normal healing requires both an inflammatory stage, characterized by programmed cell death, followed by remodeling driven by the growth of new blood vessels. Similar to its function in development, a major function of EPO in repair is to mediate (in conjunction with VEGF) neoangiogenesis resulting in the generation of the microvascular network that underlies wound healing. In this function, EPO activates the homing of endothelial progenitor cells (EPCs) which differentiate to form new vessels and stimulates nitric oxide production which enhances blood flow. EPO also turns on metalloproteinases which reorganize the intercellular matrix, allowing tissue-specific stem cells to migrate within the region of injury and tissue remodeling to occur.