Erythropoietin and Oxidative Stress

Abstract

Unmitigated oxidative stress can lead to diminished cellular longevity, accelerated aging, and accumulated toxic effects for an organism. Current investigations further suggest the significant disadvantages that can occur with cellular oxidative stress that can lead to clinical disability in a number of disorders, such as myocardial infarction, dementia, stroke, and diabetes. New therapeutic strategies are therefore sought that can be directed toward ameliorating the toxic effects of oxidative stress. Here we discuss the exciting potential of the growth factor and cytokine erythropoietin for the treatment of diseases such as cardiac ischemia, vascular injury, neurodegeneration, and diabetes through the modulation of cellular oxidative stress. Erythropoietin controls a variety of signal transduction pathways during oxidative stress that can involve Janus-tyrosine kinase 2, protein kinase B, signal transducer and activator of transcription pathways, Wnt proteins, mammalian forkhead transcription factors, caspases, and nuclear factor κB. Yet, the biological effects of erythropoietin may not always be beneficial and may be poor tolerated in a number of clinical scenarios, necessitating further basic and clinical investigations that emphasize the elucidation of the signal transduction pathways controlled by erythropoietin to direct both successful and safe clinical care.

OXIDATIVE STRESS

Initial work in pathways that can lead to oxidative stress by early investigators observed that increased metabolic rates could be detrimental to animals in an elevated oxygen environment. More current studies point to the potential aging mechanisms and accumulated toxic effects for an organism that are tied to oxidative stress (Maiese, et al., 2008a). Oxygen consumption in organisms, or at least the rate of oxygen consumption in organisms, has intrigued several investigators. Pearl proposed that increased exposure to oxygen through an increased metabolic rate could lead to a shortened life span (Pearl, 1928). Subsequent work by multiple investigators has furthered this hypothesis by demonstrating that increased metabolic rates could be detrimental to animals in an elevated oxygen environment (Muller, et al., 2007). When one moves to more current work, oxygen free radicals and mitochondrial DNA mutations have become associated with oxidative stress injury, aging mechanisms, and accumulated toxicity for an organism (Yui and Matsuura, 2006).

Oxygen free radicals can be generated in elevated quantities during the reduction of oxygen and subsequently lead to cell injury and apoptosis. Oxidative stress occurs as a result of the development of reactive oxygen species that consist of oxygen free radicals and other chemical entities. These agents can involve superoxide free radicals, hydrogen peroxide, singlet oxygen, nitric oxide (NO), and peroxynitrite (Chong, et al., 2005e). Most species are produced at low levels during normal physiological conditions and are scavenged by endogenous antioxidant systems that include superoxide dismutase, glutathione peroxidase, catalase, and small molecule substances such as vitamins C and E. Other closely linked pathways to oxidative stress may be tempered by different vitamins, such as vitamin D₃ (Regulska, et al., 2007) and the amide form of niacin or vitamin B₃, nicotinamide (Chlopicki, et al., 2007, Chong, et al., 2002d, Feng, et al., 2006, Hara, et al., 2007, Ieraci and Herrera, 2006, Lin, et al., 2000, Maiese and Chong, 2003).

Throughout the body, cell survival and lifespan is tied to the presence of oxidative stress and the subsequent induction of apoptotic cell injury (Chong, et al., 2006a, De Felice, et al., 2007, Lin and Maiese, 2001). It has recently been shown that genes involved in the apoptotic process are replicated early during processes that involve cell replication and transcription, suggesting a much broader role for these genes than originally anticipated (Cohen, et al., 2007). Apoptotic induced oxidative stress in conjunction with processes of mitochondrial dysfunction can contribute to a variety of disease states such as diabetes, ischemia, general cognitive loss, Alzheimer’s disease, and trauma (Chong, et al., 2005e, Chong, et al., 2005f, Harris, et al., 2007, Leuner, et al., 2007, Okouchi, et al., 2007). Oxidative stress can lead to apoptosis in a variety of cell types that involve neurons, endothelial cells (ECs), cardiomyocytes, and smooth muscle cells through multiple cellular pathways (Chong, et al., 2004a, Chong, et al., 2007b, Harris, et al., 2007, Kang, et al., 2003b, Karunakaran, et al., 2007, Verdaguer, et al., 2007).

Oxidative stress can impair mitochondrial permeability and function. Mitochondrial membrane transition pore permeability is increased (Chong, et al., 2003a, Di Lisa, et al., 2001, Kang, et al., 2003b, Lin, et al., 2000) and leads to a significant loss of mitochondrial NAD⁺ stores and subsequent apoptotic cell injury (Chong, et al., 2005g, Maiese and Chong, 2003). In addition, mitochondria are a significant source of superoxide radicals that are associated with oxidative stress (Chong, et al., 2005e, Maiese and Chong, 2004). Blockade of the electron transfer chain at the flavin mononucleotide group of complex I or at the ubiquinone site of complex III results in the active generation of free radicals which can impair mitochondrial electron transport and enhance free radical production (Chong and Maiese, 2007b, Li, et al., 2006a). Furthermore, mutations in the mitochondrial genome have been associated with the potential development of a host of disorders, such as hypertension, hypercholesterolemia, and hypomagnesemia (Li, et al., 2004b, Wilson, et al., 2004). Reactive oxygen species also may lead to the induction of acidosis-induced cellular toxicity and subsequent mitochondrial failure (Chong, et al., 2005f). Disorders, such as hypoxia (Roberts and Chih, 1997), diabetes (Cardella, 2005, Kratzsch, et al., 2006), and excessive free radical production (Ito, et al., 1997, Vincent, et al., 1999a, Vincent, et al., 1999b) can result in the disturbance of intracellular pH.

Apoptotic cell death is a dynamic process that entails both early and late events. Membrane phosphatidylserine (PS) externalization is an early event during cell apoptosis (Maiese, et al., 2000, Mari, et al., 2004) and can become a signal for the phagocytosis of cells (Chong, et al., 2005a, Li, et al., 2006b, Lin and Maiese, 2001). As an example, externalization of membrane PS residues occur in neurons during anoxia (Maiese, 2001, Maiese and Boccone, 1995, Vincent and Maiese, 1999a), nitric oxide exposure (Chong, et al., 2003f, Maiese, et al., 1997), and during the administration of agents that induce the production of reactive oxygen species, such as 6-hydroxydopamine (Salinas, et al., 2003). Membrane PS externalization on platelets also has been associated with clot formation in the vascular cell system (Leytin, et al., 2006). The translocation of membrane PS residues from the inner cellular membrane to the outer surface is a necessary component under most conditions for the removal of apoptotic cells (Maiese, et al., 2003, Maiese and Vincent, 2000a, Maiese and Vincent, 2000b). The loss of membrane phospholipid asymmetry leads to the externalization of membrane PS residues and assists microglia to target cells for phagocytosis (Chong, et al., 2003d, Kang, et al., 2003a, Kang, et al., 2003b, Maiese and Chong, 2003, Mallat, et al., 2005). This process occurs with the expression of the phosphatidylserine receptor (PSR) on microglia during oxidative stress (Li, et al., 2006a, Li, et al., 2006c), since blockade of PSR function in microglia prevents the activation of microglia (Chong, et al., 2003b, Kang, et al., 2003a).

In contrast to the early externalization of membrane PS residues, the cleavage of genomic DNA into fragments (Maiese, et al., 1999, Maiese and Vincent, 2000a, Maiese and Vincent, 2000b) is considered to be a later event during apoptotic injury (Dombroski, et al., 2000, Jessel, et al., 2002, Kang, et al., 2003b, Maiese and Vincent, 2000b). Endonucleases lead to DNA degradation and have been differentiated based on their ionic sensitivities to zinc (Torriglia, et al., 1997), magnesium (Sun and Cohen, 1994), and calcium (Maiese, et al., 1999), an important regulator that can independently impair cell survival. In the nervous system, three separate endonuclease activities are present. These include a constitutive acidic cation-independent endonuclease, a constitutive calcium/magnesium-dependent endonuclease, and an inducible magnesium dependent endonuclease (Chong, et al., 2005f, Vincent and Maiese, 1999b, Vincent, et al., 1999a). In the consideration of oxidative stress-induced pathways (Arcasoy, 2008, Maiese, et al., 2008c), erythropoietin (EPO) offers a unique opportunity to prevent the exposure of membrane PS residues, inhibit the committed stages of genomic DNA destruction, and block cell injury.

EPO EXPRESSION, STRUCTURE, AND RECEPTOR ROLE IN CELLS AND TISSUES

EPO can be found in the breath of healthy individuals (Schumann, et al., 2006), suggesting its broad availability in the body (Maiese, et al., 2007a, Maiese, et al., 2007c). In addition, it has been suggested that EPO may provide developmental cognitive support in humans with the recent observations that elevated EPO concentrations during infant maturation have been correlated with increased Mental Development Index scores (Bierer, et al., 2006). The primary organs of EPO production and secretion are the kidney, liver, brain, and uterus. EPO production and secretion occurs foremost in the kidney (Fliser and Haller, 2007). The kidney peritubular interstitial cells are responsible for the production and secretion of EPO (Fisher, 2003). With the use of cDNA probes derived from the EPO gene, peritubular ECs, tubular epithelial cells, and nephron segments in the kidney also have been demonstrated to be vital cells for the production and secretion of EPO (Lacombe, et al., 1991, Mujais, et al., 1999). During periods of acute renal failure, EPO may provide assistance for the protection of nephrons (Sharples, et al., 2005, Sharples and Yaqoob, 2006). Secondary sites of EPO production and secretion occur in the liver and the uterus (Chong, et al., 2002a). Hepatocytes, hepatoma cells, and Kupffer cells of the liver can produce EPO (Fisher, 2003) and, in turn, EPO may provide a protective environment for these cells (Schmeding, et al., 2007). In relation to the uterine production of EPO, it is believed that the occurrence of neonatal anemia that can take place in the early weeks after birth may partly result from the loss of EPO production and secretion by placenta (Davis, et al., 2003).

The Food and Drug Administration has approved EPO for the treatment of anemia. However, recent work has shown that EPO is not only required for erythropoiesis, but also functions in other organs and tissues, such as the brain, heart, and vascular system (Chong, et al., 2003b, Chong, et al., 2002b, Chong and Maiese, 2007a, Mikati, et al., 2007, Moon, et al., 2006, Um, et al., 2007). It is the discovery of EPO and its receptor in the nervous and vascular systems that has resulted in great interest and enthusiasm for the potential clinical applications of EPO, such as in Alzheimer’s disease, cardiac insufficiency (Assaraf, et al., 2007, Palazzuoli, et al., 2006), and cardiac transplantation (Gleissner, et al., 2006, Mocini, et al., 2007). In the nervous system, the major sites of EPO production and secretion are in the hippocampus, internal capsule, cortex, midbrain, cerebral ECs, and astrocytes (Digicaylioglu, et al., 2004, Genc, et al., 2004, Maiese, et al., 2004, Maiese, et al., 2005c). Further work has revealed several other organs as secretory tissues for EPO that include peripheral ECs (Anagnostou, et al., 1994), myoblasts (Ogilvie, et al., 2000), insulin-producing cells (Fenjves, et al., 2003), and cardiac tissue (Fliser and Haller, 2007, Maiese, et al., 2005c).

The EPO glycoprotein is 30.4 kDa with approximately half of its molecular weight derived from carbohydrates that can vary among species (Maiese, et al., 2005c). EPO contains four glycosylated chains including three N-linked and one O-linked acidic oligosaccharide side chains. N-linked glycosylation sites occur at the positions 24, 38, and 83 of aspartyl residues, while the O-linked glycosylation site is at Serine¹²⁶. Three N-glycan chains of human EPO consist of the tetra-antennary structure with or without N-acetyllactosamine repeating units (Tsuda, et al., 1988). The O-linked sugar chain is composed of Gal-GalNAc and sialic acids (Sasaki, et al., 1987). The production and secretion of the mature EPO also relies upon the integrity of the N- and O-linked chains. The EPO gene is located on chromosome 7, exists as a single copy in a 5.4 kb region of the genomic DNA, and encodes a polypeptide chain containing 193 amino acids (Jacobs, et al., 1985). During the production and secretion of EPO, a 166 amino acid peptide is initially generated following the cleavage of a 27 amino acid hydrophobic secretory leader at the amino-terminal (Imai, et al., 1990). In addition, a carboxy-terminal arginine in position 166 is removed both in the mature human and recombinant human EPO (rhEPO) resulting in a circulatory mature protein of 165 amino acids (Chong, et al., 2002a). The glycosylated chains are important for the biological activity of EPO and can protect EPO from oxygen radical degradation. The carbohydrate chains stabilize EPO (Toyoda, et al., 2000) and the oligosaccharides in EPO may protect the protein from oxygen radical activity (Uchida, et al., 1997). The N-glycosylated chains are believed to contribute to the thermal stability of EPO (Tsuda, et al., 1988). In addition, the N- and O-linked chains may be necessary for the production and secretion of the mature EPO (Krantz, 1991). Replacement of asparagines 38 and 83 by glutamate or serine 126 by glycine can decrease the production and secretion of EPO (Dube, et al., 1988). The presence of the carbohydrates also are important in the control of the metabolism of EPO, since EPO molecules with high sialic acid content can be easily cleared by the body through specific binding in the liver (Tsuda, et al., 1990).

The biological activity of EPO also relies upon two disulfide bonds formed between cysteines at positions 7 and 160 and at positions 29 and 33 (Li, et al., 2004a). The requirement of these disulfide bridges has been demonstrated by the evidence that reduction of these bonds results in the loss of the biologic activity of EPO. Alkylation of the sulfhydryl groups results in irreversible loss of the biological activity of EPO. Re-oxidization of EPO after reduction by guanidine restores eighty-five percent of the biological activity of EPO (Wang, et al., 1985). Replacement of cysteine 33 with proline also reduces the biological function of EPO.

Following cloning of the EPO gene (Jacobs, et al., 1985, Lin, et al., 1985), the EPO receptor (EPOR) was found to be expressed in both normal and transformed erythroid cells (D’Andrea and Zon, 1990). The EPOR is part of the type 1 super-family of cytokine receptors and is activated via homodimerization (Bazan, 1990, Watowich, et al., 1994). This receptor family shares a common domain structure consisting of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain. The extracellular domain is necessary for the initial binding of EPO and the intracellular domain is responsible for the transduction of intracellular signaling (Mulcahy, 2001).

EPO controls bone marrow erythroid cell proliferation, differentiation, and survival through its binding to an erythroid progenitor cell surface EPOR. The EPOR also is expressed in numerous non-erythroid blood lines that include neurons, microglia, astrocytes, and in cerebral ECs (Anagnostou, et al., 1994, Fliser and Haller, 2007, Genc, et al., 2004, Maiese, et al., 2004, Maiese, et al., 2005c) as well as on myelin sheaths of radicular nerves in human peripheral nerves (Hassan, et al., 2004), suggesting both a developmental and potential protective role for EPO in the central and peripheral nervous systems. The EPOR also is expressed in primary cerebral ECs (Chong, et al., 2003a, Chong, et al., 2003c) as well as in human umbilical veins, bovine adrenal capillaries, and rat brain capillaries (Anagnostou, et al., 1994, Yamaji, et al., 1996).

Production of EPO and the expression of its receptor are altered during development. Elevated expression of the EPOR occurs in early embryonic neuronal tissues at levels similar to that observed in the adult spleen and bone marrow (Liu, et al., 1994). Yet, the level of endogenous EPOR expression is significantly reduced following the maturation of the brain (Liu, et al., 1997). During gestation, EPO production is increased, but later becomes suppressed following birth to be regulated by the tissue oxygen supply (Chong, et al., 2002c). A deficiency in tissue oxygen results in the production of EPO and an increase in the expression of the EPOR not only in peripheral organs (Fliser and Haller, 2007, Li, et al., 2004a, Maiese, et al., 2004, Maiese, et al., 2005c), but also in the brain (Li, et al., 2007a) that may be responsible for hypoxic tolerance in some species (Ravid, et al., 2007). EPO secretion in the brain appears to be more sustained than in peripheral organs such as the kidney (Chikuma, et al., 2000), suggesting that EPO production may originate in the brain and possibly cross the blood-brain barrier to reach the blood and peripheral organs (Li, et al., 2004a). Work performed in vivo with subjects exposed to hypoxia also demonstrates an increase in expression of EPO and EPOR mRNA following reduced oxygenation (Marti, et al., 1996). Furthermore, both primary neurons (Chikuma, et al., 2000, Liu, et al., 2006) and neuronal cell lines (Stolze, et al., 2002) have been found to retain the capacity to express EPO in an oxygen-dependent manner. Although EPO is recognized as a critical modulator of erythropoiesis, a low concentration of red blood cells alone does not directly stimulate EPO production, but requires the presence of a diminished oxygen tension. Once a hypoxic stimulus is received, EPO is subsequently released into the peripheral blood circulation and upon arrival in the bone marrow, EPO binds to its receptor that is highly expressed on the surface of erythroid progenitor cells and leads to erythropoiesis (Broudy, et al., 1991). This results in an elevation in the number of mature erythrocytes and the improvement of oxygen supply. EPO also functions to stimulate colony-forming erythroid cells to induce these cells to proliferate, mature into erythrocytes, and possibly assist with reticulocyte release to the blood (Sathyanarayana, et al., 2007).

Hypoxia-inducible factor 1 (HIF-1) controls expression of EPO and EPOR during periods of reduced oxygen content. HIF-1 is essential for the production and secretion of EPO in response to hypoxia (Ikeda, 2005). At the transcriptional level, the hypoxia- dependent gene transcription of EPO and EPOR directly results from the activation of the HIF-1 pathway under hypoxic conditions. Gene transcription of EPO is mediated by the transcription enhancer located in the 3′-flanking region of the EPO gene that specifically binds to HIF-1 (Wang and Semenza, 1995). HIF-1 is a basic helix-loop-helix heterodimeric transcription factor containing two subunits, HIF-1α and HIF-1β. HIF-1β is a constitutively expressed 91–94 kDa subunit that was characterized previously as aryl hydrocarbon receptor nuclear translocator (ARNT) (Hoffman, et al., 1991). HIF-1α is a 120 kDa oxygen-labile subunit that is degraded through the ubiquitin-proteasome pathway under normoxic conditions (Huang, et al., 1998). During hypoxia or conditions such as iron chelation that can mimic hypoxia, blocking HIF-1α association with von Hippel-Lindau protein impairs degradation of HIF-1α (Maxwell, et al., 1999). HIF-1α subsequently translocates to the nucleus and heterodimerizes with HIF-1β to form a stable HIF-1 complex. The HIF complex then binds to the conserved sequence (5′RCGTG3′) near the 5′ end of the hypoxia-responsive enhancer of the EPO gene to up-regulate EPO gene transcription (Bunn, et al., 1998). Increased DNA binding activity of HIF-1 occurs in rat cortical neurons during oxidative stress, suggesting that HIF-1 may function as oxygen sensor regulating adaptive gene transcription and resulting in the production and secretion of the EPO protein during hypoxia in the nervous system (Maiese, et al., 2004, Maiese, et al., 2005c). It is important to note that each of the HIF family members HIF-1α, HIF-1β, and HIF-3α play important roles in regulating the expression of EPO and the EPOR to foster protection against hypoxic cell injury (Heidbreder, et al., 2003).

Reduced oxygen content is not the only factor responsible for the expression of EPO and the EPOR. The production and secretion of EPO in female reproductive organs is estrogen-dependent. During the cyclic development of the uterine endometrium, 17β-estradiol can lead to a rapid and transient increase in EPO mRNA in the uterus (Yasuda, et al., 1998), oviducts, and ovaries (Masuda, et al., 2000). Hypoxic induced EPO mRNA expression in uterine tissue occurs only in the presence of 17β-estradiol. EPO mRNA expression by hypoxia in the uterus is less pronounced than the EPO expression that occurs in the kidney and the brain (Chikuma, et al., 2000). Interestingly, a variety of cellular disturbances may lead to either increased or decreased EPO expression through the control of HIF, such as hypoglycemia, cadmium exposure, raised intracellular calcium, or intense neuronal depolarizations generated by mitochondrial reactive oxygen species (Chong, et al., 2002c, Genc, et al., 2004, Obara, et al., 2003). Anemic stress, insulin release, and several cytokines, including insulin-like growth factor, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) (Nagai, et al., 2001) also can lead to increased expression of EPO and the EPOR (Maiese, et al., 2004, Maiese, et al., 2005c).

EPO AND CELLULAR SIGNAL TRANSDUCTION

EPO cellular signal transduction requires the activation of the EPOR. Once EPO is bound to the EPOR, the EPOR activates Janus-tyrosine kinase 2 (Jak2) through phosphorylation. Jak2 is a member of a family of Janus-type proteintyrosine kinases including Jak1, Jak2, Jak3, and Tyk2 that are characterized by a kinase domain in the carboxyl portion, a kinase-like domain, and a large amino-terminal domain (Wilks, et al., 1991). The amino-terminal domain of Jak2 is responsible for the binding of Jak2 with the β-subunit of the EPOR at a region proximal to the membrane that contains the Box 1 sequence (Zhao, et al., 1995). EPO can prevent apoptotic injury through its reliance on Jak2 phosphorylation (Kawakami, et al., 2001, Sharples, et al., 2004), since loss of Jak2 activity reduces protection by EPO (Digicaylioglu, et al., 2004, Lipton, 2007).

The signal transducer and activator of transcription (STAT) proteins are direct substrates of Janus kinases. Seven mammalian STAT genes encoding proteins exist and are considered to be latent DNA binding factors that can be activated by tyrosine phosphorylation (Reich, 2007). Activation of Janus kinases results in tyrosine phosphorylation and dimerization of STATs. Once activated, STATs translocate to the nucleus and bind to specific DNA sequences in the promoter regions of responsive genes to lead to gene transcription. Closely linked to these transcription pathways are the mitogen-activated protein kinases that include the extracellular signal-related kinases (ERKs), the c-Jun-amino terminal kinases, and p38 MAP kinase that can oversee erythroid proliferation and differentiation (Nagata, et al., 1998). However, in regards to cytoprotection, EPO has been shown to not only activate STAT 3 (Asaumi, et al., 2007, Chong and Maiese, 2007a, Parsa, et al., 2003), STAT 5 (Chong and Maiese, 2007a, Menon, et al., 2006b, Moon, et al., 2006, Um and Lodish, 2006, Wei, et al., 2006), and ERK 1/2 (Bullard, et al., 2005, Menon, et al., 2006a), but also to employ these pathways for cell development and cell protection. EPO activates STAT3, STAT5, and ERK 1/2 in primary cerebral vascular cells, suggesting that EPO may require these cellular pathways to confer EC cytoprotection during oxidative stress (Chong and Maiese, 2007a). In addition, activation of STAT5 also can modulate EPO proliferation as well as protection against cellular apoptosis (Damen, et al., 1995). In erythroleukemic cell lines, EPO-dependent cell survival is accompanied by sustained STAT5 DNA-binding activity. Stable expression of the truncated STAT5a has been shown to enhance STAT5-DNA binding activity and reduce the induction of apoptosis (Bittorf, et al., 2000). In contrast, induction of apoptosis can be observed in cells that lack STAT5 (STAT5a^−/−/5b^−/) function (Socolovsky, et al., 2001). For example, STAT5a^−/−5b^−/− fetal liver erythroid progenitors show higher levels of apoptosis and are less responsive to the presence of EPO (Socolovsky, et al., 1999).

Downstream from Janus kinases, STATS, and the ERKs are the apoptotic pathways of the caspase family. Caspases are composed of three domains including an N-terminal prodomain, a large subunit, and a small subunit (Earnshaw, et al., 1999). As a result of their activation sequence, caspases are classified as either initiator caspases (also known as apical caspases) or effector caspases (Li, et al., 2006a, Maiese, et al., 2005a, Okouchi, et al., 2007). An initiator caspase cleaves and subsequently activates an effector caspase. The apoptotic-associated caspases include initiator caspases, such as caspase 2, 8, 9, and 10, that activate downstream effector caspases, resulting in an amplification of cascade activity. The initiator caspases consist of long N-terminal prodomains that contain caspase recruitment domains (CARDs) in caspase 2 and caspase 9 or death effector domains (DEDs) in caspase 8 and caspase 10 (Hofmann, et al., 1997). The effector caspases consist of caspase 3, 6, and 7 that function to directly cleave crucial cellular protein substrates to result in cell destruction. The caspases 1 and 3 have each been linked to the independent apoptotic pathways of genomic DNA cleavage and cellular membrane PS exposure (Chong, et al., 2003a, Chong, et al., 2003e, Takahashi, et al., 1999). These caspases, in addition to caspase 8 and 9, are also tied to the direct activation and proliferation of microglia (Chong, et al., 2003b, Kang, et al., 2003a, Kang, et al., 2003b). Caspase 1 is believed to be principally responsible for the externalization of membrane PS residues in several cell systems that can subsequently activate microglial phagocytosis (Maiese and Vincent, 2000b, Vanags, et al., 1996). Furthermore, caspase 9 is activated through a process that involves the cytochrome c -apoptotic protease-activating factor-1 (Apaf-1) complex (Chong, et al., 2004b, Li, et al., 1997). In addition, caspase 8 serves as an upstream initiator of executioner caspases, such as caspase 3, and also leads to the mitochondrial release of cytochrome c (Engels, et al., 2000, Stegh, et al., 2002). Following caspase 8 and caspase 9 activation, caspase 3 directly leads to genomic DNA degradation.

The ability of EPO to prevent specific caspase 1 and caspase 3-like activities appears to play a significant role in its cellular protection (Chong, et al., 2003b, Chong, et al., 2002b, Chong, et al., 2003e, Digicaylioglu, et al., 2004, Li, et al., 2007a, Okutan, et al., 2007, Wu, et al., 2007a). In regards to caspase 1, EPO prevents PS externalization primarily through the inhibition of caspase 1 -like activity and, to a lesser degree, through other caspases such as 3, 8, and 9 (Chong, et al., 2003a, Chong, et al., 2003b, Chong, et al., 2002b, Chong, et al., 2003e). EPO also can block genomic DNA degradation through the inhibition of cytochrome c and the subsequent inhibition of caspase 3 - like activity (Chong, et al., 2003b). EPO prevents cellular apoptosis through parallel pathways that prevent the induction of Apaf-1, caspase 8, and caspase 9 as well as by preserving mitochondrial membrane potential in conjunction with enhanced Bcl-x_L expression (Chong, et al., 2003a, Chong, et al., 2003b, Chong, et al., 2003e, Sharples, et al., 2004)

The ability of EPO to enhance cell survival during injury also directly relies upon the phosphatidylinositol 3-kinase (PI 3-K) pathway through the serine-threonine kinase protein kinase B (Akt). Phosphorylation of Akt in conjunction with EPO administration leads to its activation and protects against genomic DNA degradation and membrane PS exposure (Chong, et al., 2003a, Chong, et al., 2003b, Chong, et al., 2003e). Up-regulation of Akt activity during multiple injury paradigms, such as vascular and cardiomyocyte ischemia (Miki, et al., 2006, Parsa, et al., 2003), free radical exposure (Chong, et al., 2003b, Matsuzaki, et al., 1999), matrix detachment (Rytomaa, et al., 2000), neuronal axotomy (Namikawa, et al., 2000), N-methyl-D-aspartate toxicity (Dzietko, et al., 2004), hypoxia (Chong, et al., 2002b, Zhang, et al., 2007), β-amyloid toxicity (Chong, et al., 2005d, Martin, et al., 2001), DNA damage (Chong, et al., 2004a, Chong, et al., 2002b, Henry, et al., 2001, Kang, et al., 2003a), metabotropic ligand (Anjaneyulu, et al., 2008, Maiese, et al., 2005a) and receptor signaling (Chong, et al., 2005a, Chong, et al., 2006b, Maiese, et al., 2005a), cell metabolic pathways (Chong, et al., 2005g, Maiese and Chong, 2003), and oxidative stress (Chong, et al., 2004a, Kang, et al., 2003a, Kang, et al., 2003b) increases cell survival. Akt also can directly control microglial activation through the prevention of Bcl-x_L degradation (Chong, et al., 2004a) and the inhibition of caspase 1-, 3-, and 9-like activities (Chong, et al., 2005a, Kang, et al., 2003a, Kang, et al., 2003b).

Akt also appears to be a vital component for EPO cytoprotection especially during inflammatory cell activation, since inhibition of Akt activity blocks cellular protection and anti-inflammatory mechanisms by EPO (Chong, et al., 2003a, Chong, et al., 2003b, Chong, et al., 2003e). Activation of Akt is usually cytoprotective, such as during free radical exposure (Chong, et al., 2003b, Matsuzaki, et al., 1999), hyperglycemia (Anitha, et al., 2006), endothelial cell hypoxia (Chong, et al., 2002b), β-amyloid toxicity (Chong, et al., 2007a, Chong, et al., 2005d), cardiomyopathy (Kim, et al., 2008), and oxidative stress (Chong, et al., 2004a, Kang, et al., 2003a, Kang, et al., 2003b). EPO uses the PI 3-K/Akt pathway in a variety of experimental models of injury (Bahlmann, et al., 2004, Chong, et al., 2003b, Chong, et al., 2002b, Chong, et al., 2003e, Chong and Maiese, 2007a, Li, et al., 2006b, Miki, et al., 2006, Parsa, et al., 2003, Sharples, et al., 2004, Um, et al., 2007, Um and Lodish, 2006, Wu, et al., 2007b). These can involve transcription factor regulation (Chong and Maiese, 2007a), maintenance of ΔΨ_m, prevention of cytochrome c release (Chong, et al., 2003a, Chong, et al., 2003b, Chong, et al., 2003e), and blockade of caspase activity (Chong, et al., 2003a, Chong, et al., 2003b, Chong, et al., 2002b).

Several novel pathways that may mediate the ability of EPO to prevent cellular apoptosis are intimately tied to Akt. Akt is a primary mediator of phosphorylation of the mammalian forkhead transcription factors of the O class (FoxOs), FoxO1, FoxO3a, and FoxO4 (Chong, et al., 2005b, Maiese, et al., 2007b). More than 100 forkhead genes and 19 human subgroups that range from FOXA to FOXS are now known to exist since the initial discovery of the fly Drosophila melanogaster gene forkhead (Maiese, et al., 2007b). The fork-head box (FOX) family of genes is characterized by a conserved forkhead domain commonly noted as a “forkhead box” or a “winged helix” as a result of the butterfly-like appearance on X-ray crystallography (Clark, et al., 1993) and nuclear magnetic resonance (Jin, et al., 1998). All Fox proteins contain the 100-amino acid winged helix domain, but it should be noted that not all winged helix domains are Fox proteins (Larson, et al., 2007). FoxO proteins are expressed throughout the body and are found in the ovary, prostate, skeletal muscle, blood vessels, brain, heart, lung, liver, pancreas, spleen, thymus, and testis (Maiese, et al., 2008b, Maiese, et al., 2007b). Of the FoxOs, FoxO3a is one member that has emerged as a versatile target for a number of disorders. Akt can prevent cellular apoptosis through the phosphorylation of FoxO proteins. Post-translational phosphorylation of FoxO proteins will maintain FoxO transcription factors in the cytoplasm by association with 14-3-3 proteins and prevent the transcription of proapoptotic target genes (Chong and Maiese, 2007a, Maiese, et al., 2005c). In the absence of inhibitory Akt1 phosphorylation, FoxO3a is activate, can translocate to the nucleus, and controls a variety of functions that involve cell cycle progression, cell longevity, and apoptosis (Lehtinen, et al., 2006, Li, et al., 2006a, Maiese, et al., 2007a). As a result, control of FoxO3a is considered to be a viable therapeutic target for agents such as metabotropic glutamate receptors (Chong, et al., 2006b, Maiese, et al., 2005a), neurotrophins (Zheng, et al., 2002), and NAD⁺ precursors (Chong, et al., 2004c, Li, et al., 2006a, Li, et al., 2006b) to increase cell survival. In addition, FOXO3a interfaces with several pathways that regulate cellular lifespan and function to control neoplastic growth (Li, et al., 2007b). EPO controls the phosphorylation and degradation of FOXO3a to retain it in the cytoplasm through binding to 14-3-3 protein and foster vascular cell protection during oxidative stress (Chong and Maiese, 2007a). Regulation of caspase 3 - like activity by EPO also has recently been linked to a unique regulatory mechanism that blocks the proteolytic degradation of phosphorylated forkhead transcription factors by caspase 3. Given that specific proapoptotic transcription factors, such as FoxO3a, have been shown to be a substrate for caspase 3-like proteases at the consensus sequence DELD³⁰⁴A (Charvet, et al., 2003), studies have shown that blockade of caspase 3 - like activity prevents the destruction of the inactive phosphorylated FoxO3a during oxidative stress to increase cell survival (Chong and Maiese, 2007a).

Akt also is associated with proteins are derived from the Drosophila Wingless (Wg) and the mouse Int-1 genes (Chong, et al., 2007a, Chong, et al., 2007c, Li, et al., 2006c, Speese and Budnik, 2007). The Wnt proteins are secreted cysteine-rich glycosylated proteins that can control cell proliferation, differentiation, survival, and tumorigenesis (Chong and Maiese, 2004, Li, et al., 2006c). More than eighty target genes of Wnt signaling pathways have been demonstrated in human, mouse, Drosophila, Xenopus, and zebrafish. These genes are present in several cellular populations, such as neurons, cardiomyocytes, endothelial cells, cancer cells, and pre-adipocytes (Maiese, 2008b). At least nineteen of twenty-four Wnt genes that express Wnt proteins have been identified in the human (Li, et al., 2005, Li, et al., 2006c, Maiese, et al., 2008d).

Wnt proteins are generally divided into functional classes based on their ability to induce a secondary body axis in Xenopus embryos and to activate certain signaling cascades that consist of the Wnt1 class and the Wnt5a class (Maiese, 2008b, Maiese, et al., 2008d). These involve intracellular signaling pathways are critical for Wnt signal transduction (Maiese, 2008a, Maiese, 2008b). One Wnt pathway involves intracellular calcium release and is termed the non-canonical or Wnt/calcium pathway consisting primarily of Wnt4, Wnt5a, and Wnt11. The non-canonical system functions through non-β-catenin-dependent pathways and also includes the planar cell polarity (PCP) pathway or the Wnt-calcium-dependent pathways (Li, et al., 2005, Li, et al., 2006c, Maiese, et al., 2008d). A second pathway controls target gene transcription through β-catenin, generally referred to as the canonical pathway that involves Wnt1, Wnt3a, and Wnt8. Wnt signaling can prevent cell injury through a variety of mechanisms. Wnt prevents apoptosis through β-catenin/Tcf transcription mediated pathways (Chen, et al., 2001) and also can protect cells against c-myc induced apoptosis through cyclooxygenase-2 and Wnt induced secreted protein (You, et al., 2002). Wnt signaling also can inhibit apoptosis during oxidative stress (Chong and Maiese, 2004) and β-amyloid toxicity that may require modulation of glycogen synthase kinase-3β (GSK-3β) and β-catenin (Chong, et al., 2007a).

Abnormalities in the Wnt signaling pathways, such as with transcription factor 7-like 2 gene, may impart increased risk for type 2 diabetes in some populations (Grant, et al., 2006, Lehman, et al., 2007, Scott, et al., 2006) as well as have increased association with obesity (Guo, et al., 2006). Yet, intact Wnt family members may offer glucose tolerance and increased insulin sensitivity (Wright, et al., 2007) as well as protect glomerular mesangial cells from elevated glucose induced apoptosis (Lin, et al., 2006). These observations suggest a potential protective cellular mechanism for EPO through Wnt signaling to improve clinical cardiac function in diabetic patients (Silverberg, et al., 2006) and decrease complications in woman with diabetic pregnancies (Teramo, et al., 2004). Cell culture studies demonstrate that the Wnt1 protein is necessary and sufficient to impart cellular protection during elevated glucose exposure (Chong, et al., 2007c). Administration of exogenous Wnt1 protein can significantly prevent apoptotic EC injury during elevated glucose exposure. EPO maintains the expression of Wnt1 during elevated glucose exposure and prevents loss of Wnt1 expression that would occur in the absence of EPO during elevated glucose. Blockade of Wnt1 with a Wnt1Ab also can neutralize the protective capacity of EPO, illustrating that Wnt1 is a critical component in the cytoprotection of EPO during elevated glucose exposure (Chong, et al., 2007c).

In the Wnt pathway, modulation of glycogen synthase kinase-β (GSK-3β) and nuclear factor-κB (NF-κB) activity can affect cell survival during oxidative stress (Li, et al., 2005, Maiese, 2008b, Maiese, et al., 2008d). GSK-3β is considered to be a therapeutic target for some neurodegenerative disorders (Balaraman, et al., 2006, Chong, et al., 2005e, Nurmi, et al., 2006, Qin, et al., 2006) and also may to influence inflammatory cell survival (Chong, et al., 2007b) and activation (Tanuma, et al., 2006). During models of diabetes, inactivation of GSK-3β by small molecule inhibitors or RNA interference prevents toxicity from high concentrations of glucose and increases rat beta cell replication, suggesting a possible target of GSK-3β for pancreatic beta cell regeneration (Mussmann, et al., 2007). Clinical applications for GSK-3β are attractive, especially in concert with EPO. For example, both the potential benefits of EPO to improve cardiovascular function in diabetic patients (Silverberg, et al., 2006, Silverberg, et al., 2001) and the positive effects of exercise to improve glycemic control during DM (Maiorana, et al., 2002) appear to rely upon the inhibition of GSK-3β activity. EPO blocks GSK-3β activity (Chong, et al., 2005e, Chong, et al., 2007b, Rowe, et al., 2007, Wu, et al., 2007a) and combined with exercise may offer synergistic benefits, since physical exercise also has been shown to phosphorylate and inhibit GSK-3β activity (Howlett, et al., 2006). Expression and cytoprotection of EPO is dependent, in part, upon Akt and NF-κB. The phosphorylation of IκB proteins by the IκB kinase (IKK) and their subsequent degradation lead to the release of NF-κB for its translocation to the nucleus to initiate gene transcription (Hayden and Ghosh, 2004). Dependent upon Akt controlled pathways, the transactivation domain of the p65 subunit of NF-κB is activated by IKK and the IKKα catalytic subunit to lead to the induction of protective anti-apoptotic pathways (Chong, et al., 2005b). Increased expression of NF-κB during injury models can occur in inflammatory microglial cells (Chong, et al., 2005d, Chong, et al., 2007b, Guo and Bhat, 2006) and in neurons (Sanz, et al., 2002). NF-κB represents a critical pathway that is responsible for the activation of inhibitors of apoptotic proteins (IAPs), the maintenance of Bcl-x_L expression, (Chen, et al., 2000, Chong, et al., 2005f), and protection against cell injury during oxidative stress (Chong, et al., 2005d). NF-κB also is strongly associated with the cytopro-tection of trophic factors that includes EPO (Chong, et al., 2005d, Nakata, et al., 2004, Sae-Ung, et al., 2005). NF-κB also plays a key role in the expression of EPO during HIF-1 induction. Akt can significantly increase NF-κB and HIF-1 activation resulting in the enhancement of EPO expression. Although NF-κB has not consistently been found to be beneficial in all cell systems (Esposito, et al., 2006, Jacobsen, et al., 2006) and may sometimes not be cytoprotective (Nurmi, et al., 2006, Xu, et al., 2005), EPO subsequently uses NF-κB to prevent apoptosis through the enhanced expression and translocation of NF-κB to the nucleus to elicit anti-apoptotic gene activation (Bittorf, et al., 2001, Chong, et al., 2005d, Li, et al., 2006b, Spandou, et al., 2006).

EPO AND CLINICAL ENTITIES

Cardiac and Vascular Integrity

The control of angiogenesis by EPO offers an important level of cytoprotection (Chong, et al., 2002a, Walshe and D’Amore, 2008). In neonatal mesenteric microvascular ECs, EPO leads to vasculogenesis (Ashley, et al., 2002). In clinical studies, EPO serum levels are significantly associated with the number and function of circulating endothelial progenitor cells and EPO can stimulate postnatal neovascularization by increasing endothelial progenitor cell mobilization from the bone marrow (Heeschen, et al., 2003). Angiogenesis also has been observed in endothelial samples derived from human adult myocardial tissue following treatment with EPO (Jaquet, et al., 2002). In addition, the uterine endometrium and the ovaries are dependent upon EPO for the induction of angiogenesis to compensate for lost vessels during the estrus cycle. EPO has been shown to be necessary to foster blood vessel formation in the endometrium in ovariectomized mice and to be required for the formation of a capillary network for the development of follicles and the corpora lutea (Yasuda, et al., 1998). In models of cerebral ischemia, EPO promotes factors for angiogenesis such as Tie-2 and Angiopoietin-2 that may assist with the restoration of cerebral blood flow to pre-ischemic levels (Li, et al., 2007a). EPO controlled angiogenesis also may play a significant role during renal inflammation and prevention of allograft rejection (Reinders, et al., 2006). Yet, it is important to consider the inhibition of angiogenesis. Although EPO induced angiogenesis may impart beneficial effects to ischemic cells of the nervous and cardiovascular systems for nutrient and oxygen supply, other scenarios that involve ocular neovascularization may seek to block or limit angiogenesis by EPO to prevent disease progression (Zhang and Ma, 2007).

In the vascular system, EPO also offers direct preservation of EC integrity (Chong, et al., 2002a, Chong, et al., 2003a, Chong, et al., 2002b). EPO has both a mitogenic and chemotactic effect that can lead to matrix metalloproteinase-2 production, cell proliferation, and vessel formation in EC lines (Maiese, et al., 2004, Maiese, et al., 2005c). In cultured human and bovine ECs, EPO stimulates EC proliferation and fosters the migration of ECs (Anagnostou, et al., 1990). It is important to note that as a large molecule, EPO may maintain the establishment of EC communication and function that could become crucial in a number of scenarios, such as repair of the blood-brain barrier during injury (Martinez-Estrada, et al., 2003). In addition, by assuring EC integrity, EPO prevents ischemic cardiac demise by reducing myocardial injury and cardiomyocyte apoptosis (Burger, et al., 2006), lessening myocardial ischemia (Bullard, et al., 2005), modulating cardiac remodeling (Miki, et al., 2006, Toma, et al., 2007), reducing ventricular dysfunction (Parsa, et al., 2004, Parsa, et al., 2003), and improving cardiac function (Gao, et al., 2007, Westenbrink, et al., 2007). Therefore, EPO plays a critical role in the vascular and renal systems with the maintenance of erythrocyte (Foller, et al., 2007) and podocyte (Eto, et al., 2007) integrity, regulates the survival of ECs (Chong, et al., 2003a, Chong, et al., 2002b), and may act as a powerful endogenous protectant during cardiac injury (Asaumi, et al., 2007). EPO can protect against myocardial cell apoptosis and decrease infarct size, resulting in improved left ventricular contractility. In isolated rat heart preparations following ischemia/reperfusion experiments, beneficial effects of treatment with EPO have been shown to significantly improve post-ischemic recovery of left ventricular pressure (Moon, et al., 2003, van der Meer, et al., 2004a). EPO treatment also can prevent myocardial cell apoptosis and decrease infarct size, resulting in enhanced cardiac function and recovery (Parsa, et al., 2004). At the onset of coronary artery occlusion, EPO administered also can significantly inhibit apoptosis in the central region of myocardial ischemia (Tramontano, et al., 2003). Even in acute scenarios following coronary artery ligation, EPO leads to a decrease in apoptotic cells by fifty percent in the myocardium and significantly improves cardiac function (Moon, et al., 2003, Parsa, et al., 2003).

Although some cardiac injury experimental models do not consistently demonstrate a benefit with EPO (Olea, et al., 2006), initial studies in patients with anemia or on chronic hemodialysis have suggested a direct cardiac benefit from EPO administration (Goldberg, et al., 1992, Silverberg, et al., 2001). Randomized control studies with EPO administration in patients with congestive heart failure or diabetes combined with congestive heart failure also demonstrate an improved cardiac output and a decrease in medical resource utilization (Maiese, et al., 2005c, Silverberg, et al., 2006). Subsequent work has demonstrated that EPO administration can improve exercise tolerance either during cardiac or renal insufficiency in patients with anemia and congestive heart failure (Mancini, et al., 2003, Palazzuoli, et al., 2006) and that this may be tied to improved pulmonary function (Wu, et al., 2006). Other studies have shown that patients with acute myocardial infarction have increased plasma EPO levels within seven days of the cardiac insult, suggesting a possible protective response from the body (Ferrario, et al., 2007). Serum levels of EPO also may function as a biomarker for cardiovascular injury (Fu and Van Eyk, 2006) as well as improve survival following out of hospital cardiac arrest (Cariou, et al., 2008). A randomized, concealed, multicenter trail of 1460 patients who received 40,000 U of epoetin alfa up to a 3 week maximum following intensive care unit admission also demonstrated a reduced mortality in patients with trauma (Corwin, et al., 2007).

Unfortunately, agents such as EPO may not be well tolerated by all individuals, especially those with comorbid conditions such as congestive heart failure and hypertension. Some studies suggest that elevated plasma levels of EPO independent of hemoglobin concentration can be associated with increased severity of disease in individuals with congestive heart failure (van der Meer, et al., 2004b) and that EPO may contribute to vascular stenosis with intima hyperplasia (Reddy, et al., 2007). Adverse effects during treatment with EPO are not uncommon, such as an increased incidence of thrombotic vascular effects (Corwin, et al., 2007) or the use of EPO in cancer patients receiving chemotherapy that has been associated with nonfatal myocardial infarction, pyrexia, vomiting, shortness of breath, paresthesias, and upper respiratory tract infection (Henry, et al., 2004). In addition, the use of EPO in patients with hypertension must proceed with caution, since both acute and long-term administration of EPO can significantly elevate mean arterial pressure (Kanbay, et al., 2007).

Immune Function and the Nervous System

Given the impact that inflammatory cells may have upon the progression or resolution of degenerative insults throughout the body, it becomes essential to consider agents that can control inflammatory pathways (Chong, et al., 2005a, Chong, et al., 2004a, Kang, et al., 2003b). Therefore, cytoprotective agents that are known to modulate inflammatory cell function may offer attractive therapeutic considerations (Chong, et al., 2007b, Li, et al., 2006b). EPO appears to fill such a need in regards to its role during periods of cellular inflammation. EPO can reduce cytokine gene expression in endothelial cells exposed to tumor necrosis factor (Avasarala and Konduru, 2005), prevent ulcer progression in cases of scleroderma (Ferri, et al., 2007), and block primary microglial activation and proliferation during oxidative stress (Chong, et al., 2003b, Chong, et al., 2005d). Furthermore, EPO can block microglial cell activation and proliferation to prevent phagocytosis of injured cells through pathways that involve cellular membrane PS exposure, protein kinase B (Chong, et al., 2004a), and the regulation of caspases (Chong, et al., 2003a, Chong, et al., 2003b, Wu, et al., 2007a). EPO can directly inhibit several pro-inflammatory cytokines, such as IL-6, TNF-α, and monocyte chemoattractant protein 1 (Li, et al., 2004a, Maiese, et al., 2005c), as well as reduce leukocyte inflammation (Contaldo, et al., 2007). In addition, EPO may foster the preservation of microglial cells for neuronal and vascular restructuring by preventing apoptotic injury in microglia (Li, et al., 2006b, Vairano, et al., 2002). In regards to the capacity of EPO to maintain microglial cellular integrity, EPO retains its capacity to prevent early apoptotic injury with membrane PS externalization as well as later stages of apoptotic injury involving DNA fragmentation in microglia (Li, et al., 2006b) similar to other cell systems of neurovascular origin (Chong, et al., 2003b, Chong, et al., 2002b, Chong, et al., 2005d, Parsa, et al., 2003, Sharples, et al., 2004).

As a robust cytoprotectant, EPO can enhance the survival of cells during several types of injury models in the nervous system (Lykissas, et al., 2007, Maiese, et al., 2004, Maiese, et al., 2005c). In cells that involve the brain or the retina, EPO can prevent injury from hypoxic ischemia (Chong, et al., 2003b, Chong, et al., 2002b, Liu, et al., 2006, Meloni, et al., 2006, Wei, et al., 2006, Yu, et al., 2005), excitotoxicity (Montero, et al., 2007, Yamasaki, et al., 2005), infection (Kaiser, et al., 2006), free radical exposure (Chong, et al., 2003a, Chong, et al., 2003e, Yamasaki, et al., 2005), staurosporine (Pregi, et al., 2006), and dopaminergic cell injury (Demers, et al., 2005, McLeod, et al., 2006). In addition, administration of EPO also represents a viable option for the prevention of retinal cell injury during glaucoma (Tsai, et al., 2007). Systemic application of EPO also can improve functional outcome and reduce cell loss during spinal cord injury (King, et al., 2007, Okutan, et al., 2007), traumatic cerebral edema (Verdonck, et al., 2007), cortical trauma (Cherian, et al., 2007), and epileptic activity (Mikati, et al., 2007, Nadam, et al., 2007).

EPO also may improve cognition, such as memory loss or psychiatric illness. In particular, Alzheimer’s disease has become a prime consideration for the applications of EPO. Alzheimer’s disease leads to a progressive deterioration of cognitive function with memory loss and injury to hippocampal neurons. The generation of extracellular plaques of amyloid-β (Aβ) peptide aggregates composed of a 39-42 amino acid peptide are considered to be one of the pathological mechanisms that may promote the development of Alzheimer’s disease (Chong, et al., 2005f). Accumulation of Aβ accumulation can lead to apoptotic injury with chromatin condensation, DNA fragmentation, and cellular membrane PS exposure (Chong, et al., 2005c, Chong, et al., 2005f). Aβ also can release reactive oxygen species and lead to toxicity in neurons. In addition, Aβ can not only precipitate a significant inflammatory response with microglial activation and the secretion of TNF-α (Bornemann, et al., 2001), but also Aβ can elicit the neuronal expression of inducible nitric oxide synthase, peroxinitrite production, and neuronal apoptosis during an acute inflammatory response (Chong, et al., 2005e, Combs, et al., 2001). Furthermore, Aβ may lead to the induction of caspase mediated pathways (Nakagawa, et al., 2000, Troy, et al., 2001) that work in concert with oxidative stress (Tamagno, et al., 2003). As a result, therapeutic strategies that address the toxicity of Aβ as a result of oxidative stress may foster novel developments for the treatment of Alzheimer’s disease. EPO appears to be both necessary and sufficient to protect neurons from Aβ toxicity. Application of a blocking antibody of EPO, which can bind to EPO and block its biological activities in cells (Koshimura, et al., 1999), can otherwise negate the protective effects of EPO to increase neuronal hippocampal cell survival and prevent apoptotic injury during Aβ exposure (Chong, et al., 2005d).

Enhanced survival by EPO also extends to afford protection of the neurovascular unit during cerebral vascular disease (Demers, et al., 2005, Dzietko, et al., 2004, Maiese, et al., 2004, Wei, et al., 2006). In addition, EPO can protect sensitive hippocampal neurons from both focal and global ischemic brain injury (Keogh, et al., 2007, Wei, et al., 2006, Yu, et al., 2005, Zhang, et al., 2006). Systemic administration of EPO also represents a viable option for several other disorders. EPO administration for retinal cell injury can protect retinal ganglion cells from apoptosis (Grimm, et al., 2002), EPO can improve functional outcome and reduce lipid peroxidation during spinal cord injury (Kaptanoglu, et al., 2004), and EPO can maintain autoregulation of cerebral blood flow, reverse basilar artery vasoconstriction, and enhance neuronal survival and functional recovery following subarachnoid hemorrhage (Olsen, 2003).

However, it should be noted that the biological effects with EPO administration may not always be entirely beneficial during cytoprotective therapy. Irrespective of the problems associated with EPO abuse and gene doping (Baoutina, et al., 2007, Diamanti-Kandarakis, et al., 2005, Segura, et al., 2007), EPO has been correlated with the alteration of red cell membrane properties leading to a cognitive decrement in rodent animal models (Li, et al., 2004a, Maiese, et al., 2004, Maiese, et al., 2005c). In addition, development of potentially detrimental side-effects during EPO therapy, such as for cerebral ischemia with increased metabolic rate and blood viscosity (Frietsch, et al., 2007), could severely limit or halt the use of EPO for neurovascular diseases. As a result, alternate strategies have been suggested. New investigations are studying the role of targeted bioavailability for EPO such as in bone marrow stromal cells genetically engineered to secrete EPO (Eliopoulos, et al., 2006) and controlled release of EPO from encapsulated cells (Orive, et al., 2005, Ponce, et al., 2006). The passage of EPO entry into the central nervous system continues to attract significant interest (Doolittle, et al., 2007) as well as does the use of novel intranasal routes for EPO administration (Yu, et al., 2005). Other avenues of study are directed to the development of derivations of EPO to reduce erythropoietic activity and the potential associated vascular complications (Montero, et al., 2007). Yet, these lines of investigation are not without limitations, since chemical derivatives of EPO can become absent of clinical efficacy (Maiese, et al., 2004, Maiese, et al., 2005c) as well as possibly loose the ability to promote sustainable cytoprotective effects, such as neurogenesis (Gonzalez, et al., 2007) and angiogenesis (Li, et al., 2007a, Reinders, et al., 2006, Slevin, et al., 2006, Zhang and Ma, 2007).

Metabolic Disease

Clinical work indicates that EPO has a significant role during diabetes mellitus (DM). DM is a significant health concern for both young and older populations (Maiese, et al., 2007a, Maiese, et al., 2007c). Approximately 16 million individuals in the United States and more than 165 million individuals worldwide suffer from DM. By the year 2030, it is predicted that more than 360 million individuals will be afflicted with DM and its debilitating conditions (Wild, et al., 2004). Type 2 DM represents at least 80 percent of all diabetics and is dramatically increasing in incidence as a result of changes in human behavior and increased body mass index (Laakso, 2001). Type 1 insulin-dependent DM is present in 5–10 percent of all diabetics (Maiese, et al., 2007c), but is increasing in adolescent minority groups (Dabelea, et al., 2007). Furthermore, the incidence of undiagnosed diabetes, impaired glucose tolerance, and fluctuations in serum glucose in the young raises additional concerns (Jacobson, et al., 2007). Both type 1 and type 2 DM represent important health concerns whether they begin early or later in life (Maiese, et al., 2007a), since each can result in long-term complications throughout the body (Daneman, 2006). In regards to the vascular and nervous systems, patients with DM can develop severe neurological and vascular disease (Donahoe, et al., 2007, Singh, et al., 2008) that can lead to an increased risk for cognitive decline especially from vascular disease (Chong, et al., 2005e, Li, et al., 2006a Schnaider Beeri, et al., 2004). Disease of the nervous system can become the most debilitating complications for DM and affect sensitive cognitive regions of the brain, such as the hippocampus that modulates memory function, resulting in significant functional impairment and dementia (Awad, et al., 2004, Gerozissis, 2003). DM also has been found to increase the risk for vascular dementia in elderly subjects (Schnaider Beeri, et al., 2004, Xu, et al., 2004). DM also may affect the course of Alzheimer’s disease. Although some studies have found that diabetic patients may have less neuritic plaques and neurofibrillary tangles than non-diabetic patients (Beeri, et al., 2005), contrasting work suggests a modest adjusted relative risk of Alzheimer’s disease in patients with diabetes as compared with those without diabetes to be 1.3 (Luchsinger, et al., 2001).

Both insulin resistance and the complications of DM are closely linked to the occurrence of cellular oxidative stress (Maiese, 2008a, Maiese, et al., 2007a, Maiese, et al., 2007c). In patients with DM, elevated levels of ceruloplasmin are suggestive of increased reactive oxygen species (Memisogullari and Bakan, 2004) and acute glucose fluctuations may promote oxidative stress (Monnier, et al., 2006). Hyperglycemia can lead to increased production of reactive oxygen species in endothelial cells, liver and pancreatic β-cells (Ceriello, et al., 1996, Ihara, et al., 1999, Ling, et al., 2003, Yano, et al., 2004). Prolonged duration of hyperglycemia is not necessary to lead to oxidative stress injury, since even short periods of hyperglycemia, generate reactive oxygen species, such as in vascular cells (Yano, et al., 2004). Clinical correlates support these experimental studies to show that acute glucose swings in addition to chronic hyperglycemia can trigger oxidative stress mechanisms during type 2 DM, illustrating the importance for therapeutic interventions during acute and sustained hyperglycemic episodes (Monnier, et al., 2006).

Since administration of antioxidants during elevated glucose concentrations can block free radical production and prevent the production of advanced glycation endproducts known to produce reactive oxygen species during DM (Giardino, et al., 1996), EPO may offer an attractive alternative therapy to maintain proper cellular metabolism and mitochondrial membrane potential during DM. In clinical studies with DM, plasma EPO is often low in diabetic patients with anemia (Mojiminiyi, et al., 2006) or without anemia (Symeonidis, et al., 2006). Furthermore, the failure of these individuals to produce EPO in response to a declining hemoglobin level suggests an impaired EPO response in diabetic patients (Thomas, et al., 2005). Yet, increased EPO secretion during diabetic pregnancies may represent the body’s attempt at endogenous protection against the complications of DM (Teramo, et al., 2004). Similar to the potential protective role of insulin (Duarte, et al., 2006), EPO administration has been shown both in diabetics as well as non-diabetics with severe, resistant congestive heart failure to decrease fatigue, increase left ventricular ejection fraction, and significantly decrease the number of hospitalization days (Silverberg, et al., 2006).

Cell culture studies with vascular cells exposed to elevated glucose also have elucidated a strong cytoprotective effect of EPO (Maiese, et al., 2004). Administration of EPO can significantly improve EC survival in a 1.0 ng/ml range (Chong, et al., 2007c). EPO administration in patients also can significantly increase plasma levels of EPO well above this range of 1.0 ng/ml that has been associated with potential EPO cellular protection in patients with cardiac or renal disease (Mason-Garcia, et al., 1990, Namiuchi, et al., 2005), suggesting that the effects of EPO observed during in vitro studies may parallel the cellular processes altered by EPO in patients with DM (Bierer, et al., 2006). Furthermore, EPO can block apoptotic DNA degradation in ECs during elevated glucose similar to other models of oxidative stress in cardiac and vascular cell models (Avasarala and Konduru, 2005, Chong, et al., 2003a, Chong, et al., 2002b, Chong and Maiese, 2007a, Moon, et al., 2006). The preservation of cellular energy reserves is dependent upon the maintenance of mitochondrial integrity during DM (Newsholme, et al., 2007). Cytoprotection by EPO also is closely related to the maintenance of mitochondrial membrane potential (ΔΨ_m). Loss of ΔΨ_m through the opening of the mitochondrial permeability transition pore represents a significant determinant for cell injury and the subsequent induction of apoptosis (Leuner, et al., 2007, Maiese and Chong, 2004). EPO has the capacity to prevent the depolarization of the mitochondrial membrane that also affects the release of cytochrome c (Chong, et al., 2002b, Chong, et al., 2003e, Miki, et al., 2006).

Cancer

The possible induction or progression of cancer represents a significant concern with EPO administration (Kokhaei, et al., 2007, Maiese, et al., 2005b). Not only has both EPO and its receptor been demonstrated in tumor specimens, but under some conditions EPO expression has been suggested to block tumor cell apoptosis through Akt (Maiese, et al., 2008a, Maiese, et al., 2008c), enhance tumor progression, increase metastatic disease, (Lai and Grandis, 2006), and negate the effects of radiotherapy by assisting with tumor angiogenesis (Ceelen, et al., 2007). In studies of patients with head and neck cancer, EPO decreased disease progression-free survival and overall survival (Henke, et al., 2003). Similar results were reported in trials with metastatic breast cancer (Leyland-Jones, et al., 2005) and the expression of the EPOR in tumors appeared to suggest a worse prognosis (Henke, et al., 2006). Yet, the potential risk of EPO administration to either initiate tumor growth or lead to tumor progression is not entirely understood. In regards to the possible tumor promoting ability of EPO (Rades, et al., 2007), a number of competing factors must be considered that include the possible benefits of EPO administration in patients with cancer that involve the synergistic effects of EPO with chemotherapeutic modalities (Ning, et al., 2005, Sigounas, et al., 2004) and the treatment of cancer-related anemia. New large scale prospective trials are necessary that can more clearly examine the attributes and contraindications for EPO.

CONCLUSIONS

EPO is a unique agent in many ways offering potential clinical treatment for a diverse range of disorders that can range from anemia to the restoration of cardiovascular and cognitive function. Yet, treatment applications for EPO are not without controversy especially in regards to the potential of this growth factor to promote tumorigenesis. Future cell and animal investigations that parallel new clinical trials are surely warranted with a strong emphasis upon the elucidation of the signal transduction pathways controlled by EPO to direct both successful and safe clinical care.