The ocular effects of erythropoietin.ABTRACT: Erythropoietin (EPO) is a hormone with hematopoietic functions, controlling red blood cell production in response to hypoxia. It stimulates erythropoiesis by preventing apoptosis of erythrocyte progenitors. In addition to hematopoiesis, EPO has also been shown to possess non-hematopoietic biological effects. These include neuroprotection, cardioprotection, renoprotection, and wound healing. Ocular effects include positive ones such as protection of photoreceptor, retinal ganglion, and retinal pigment epithelium cells, but also negative ones especially pathological neovascularization. There is growing interest in the use of EPO for treatments of various eye conditions including diabetic retinopathy, glaucoma, retinitis pigmentosa, age-related macular degeneration, and retinal detachment. In this paper we review the literature on EPO's effects on the eye, and its potential in the prevention and treatment of various ocular diseases.
KEY WORDS: erythropoietin, non-erythropoietic effects, ocular diseases
Erythropoietin (EPO) is a glycoprotein hormone that regulates erythropoiesis (red blood cell production). The primary source of production in adults is the kidney, with the interstitial cells of the renal cortex secreting the hormone (Zhong, L. et al., 2007); however, the liver, brain, and uterus also produce and secrete EPO (Maiese et al., 2008). Based on our literature search, there have been review articles on EPO's non-hematopoietic effects including on the renal (Fliser et al., 2006; Johnson et al. 2006; Bahlmann and Fliser, 2009), nervous (Genc et al., 2004; Hasselblatt et al., 2006; Rabie and Marti, 2008) and cardiovascular systems (Burger et al., 2009; Timmer et al., 2009; Vogiatzi et al., 2010), but there is no review on the ocular effects of EPO. In this paper, we review the literature on EPO's effects on the eye, as well as its potential in the prevention and treatment of various ocular disorders. (See Table 1 for abbreviations used in this review.)
The kidney produces EPO in response to hypoxia. EPO's hematopoietic role is to stimulate formation of red blood cells by inhibiting apoptosis of the erythroid progenitors in the bone marrow, as well as promoting their proliferation and differentiation (Krantz, 1991; Lacombe and Mayeux, 1998). Additionally, it has a non-hematopoietic protective role against tissue injury caused by ischemia. Its antiapoptotic, antioxidative, and anti-inflammatory capabilities provide direct protection against hypoxia, while its capacity for angiogenesis offers an indirect one by inducing oxygen supply to ischemic tissues (Paschos et al., 2008). EPO has also been shown to stabilize both actin and tubulin, components of the cytoskeleton. In the kidney, for example, Eto et al. (2007) proposed that EPO maintains the podocyte actin cytoskeleton in a rat model mimicking nephritic syndrome.
Moreover, in the brain, Sun et al. (2008) reported that EPO prevents hyperphosphorylation of tau (a microtubule-associated protein and major component of neurofibrillary tangles associated with Alzheimer's disease), thereby preventing microtubule destabilization, axonal transport impairment, and neuron death. Additionally, in their study of paclitaxel (an anticancer compound that binds to tubulin, inhibits microtubule disassembly and, in turn, cell division), Melli et al. (2006) demonstrated that recombinant human EPO (rhEPO) decreases the level of detyrosinated tubulin, and, thus, prevents distal axonal degeneration in sensory neurons in vitro.
Such findings suggest that EPO may even be involved in the stabilization of the cytoskeleton of erythrocytes during erythropoiesis. During non-mammalian erythrogenesis, nucleated erythrocytes transform from spheres to flattened discs to ellipsoids, with the cell shape determined by the organization of cytoskeletal elements, including actin and tubulin. Twersky et al. (1995) reported that pointed cells containing incomplete, pointed marginal bands of microtubules may represent intermediate stages in the formation of elliptical erythrocytes since the pointed cells were observed to be a consistent feature of Xenopus (clawed frog) cellular morphogenesis. As it did with the podocyte actin cytoskeletal arrangement, perhaps EPO also helps maintain the arrangement of cytoskeletal actin and tubulin so that mature elliptical erythrocytes are produced during erythropoiesis. Moreover, EPO may have this effect on mammalian erythrogenesis as well. EPO may perhaps play a role in the formation of the optic cup (forerunner of the retina) since the cytoskeleton is involved in this process. It has been reported that reorganization of the cytoskeleton is necessary for the early phases of eye morphogenesis (Cavodeassi et al., 2009).
EPO binds to its cell-surface EPO Receptor (EPOR), a cytokine receptor that is expressed in erythroid progenitor cells. Binding results in activation, then dimerization of the p66 chain on the receptor. One of the tyrosine kinases, Janus kinase-2 (JAK2), is then induced, leading to phosphorylation of EPOR and several proteins. Intracellular activation of the Ras/mitogen-activated kinase pathway (involved with cell proliferation), phosphatidylinositol-3 (PI-3) kinase, and signal transducer and activator of transcription (STAT) 1, 3, 5A, and 5B then occur. Additionally, EPO acts in synergy with growth factors: stem cell factor, granulocyte-macrophage colony-stimulating factor, interleukin 3, and insulin-like growth factor. Together, they cause maturation and proliferation of erythroid progenitor cells (Fisher, 2003).
EPOR has also been found to be expressed in many other cells and tissues, including in the brain, heart, and eye. Zhong, L. et al. (2007) believe such findings suggest that EPO possesses autocrine, paracrine, and endocrine functions.
Studies have shown that both EPO and EPOR are crucial in human development. Arcasoy (2008) reported that investigators have discovered that in mice the disruption of either leads to death in utero. Additionally, researchers found that EPO and EPOR are necessary for normal heart and brain development as well as blood vessel formation. Wu et al. (1999) observed that mouse embryos that lacked EPO-EPOR presented with cardiac ventricular hypoplasia. Studer et al. (2000) and Shingo et al. (2001) found that EPO signaling is crucial in embryonic neural development for it regulates the differentiation of neural progenitor cells. Additionally, Yu et al. (2001, 2002) reported that mouse embryos that did not have EPOR exhibited increased apoptosis in the myocardium and brain. Furthermore, Wu et al. (1999) and Kertesz et al. (2004) observed that mice lacking either EPO or EPOR displayed problems in physiological blood vessel formation that occurs actively during embryogenesis.
Due to its angiogenic potential, some believe EPO may promote tumor growth. There have been conflicting findings in terms of whether or not EPOR is expressed in tumor cells and if EPO even stimulates their growth. Jelkmann (2005) reported that early investigations, which used various tumor cell lines in vitro and a wide range of doses, found that rhEPO does not promote growth. Hassouna et al. (2008) support this theory as they believe that EPO is an unlikely factor in basal glioma growth. In their study, they found that EPO did not modulate basal glioma cell migration, stimulated proliferation in only one of four human glioblastoma cell lines, and, most importantly, did not enhance tumor growth in mouse brains. Meanwhile, Chan et al. (2005) discovered that both EPO and EPOR mRNA and protein were expressed in hemangioblastoma cells. They believe that such co-expression may not only mediate developmental stagnation, but may also induce proliferation of hemangioblastoma. Moreover, biopsies of human lung, breast, and cervical cancers have shown EPO-binding sites and EPOR proteins; and human hepatocarcinoma cell line Hep3B, renal carcinoma, various breast carcinoma cell lines, and malignant tumors of female reproductive organs exhibit EPOR mRNA and/or protein (Jelkmann, 2005). According to these studies, EPO can stimulate tumor cell proliferation in vitro and in nude mice in vivo. Furthermore, inhibition of EPO signaling via administration of anti-EPO antibody or soluble EPOR into tumor tissue can result in regression (Jelkmann, 2005).
Currently rhEPO is used as a therapeutic agent to treat patients with anemic conditions. These include anemias associated with chronic renal failure, AIDS treatment, chemotherapy, perioperative surgical patients, and autologous blood donation (Fisher, 2003). Epoetin alfa and epoetin beta are approved rhEPO analaogues, with plasma halflife of 6-8 hours.
However, darbepoetin alfa (hyperglycosylated version) and continuous EPOR activator (CERA), modified EPO analogues, are held in higher regard. Darbepoetin alfa has a plasma half-life of 24-26 hours, while CERA avoids degradation (fate of unmodified EPO after binding to receptor) by dissociating from receptor (Jelkmann, 2005). Thus, they have longer elimination half-life, which translates to less frequent dosing (Macdougall, 2008).
Another hopeful alternative is asialoerythropoietin (asialoEPO), which is produced by total enzymatic desialylation of rhEPO. This form has a very short plasma half-life and possesses the same high affinity for EPOR as rhEPO. Moreover, it is neuroprotective, fully protecting animal models of stroke, spinal cord injury, and peripheral neuropathy (Erbayrakatar et al., 2003; Wang et al., 2004; Grasso et al., 2006). Due to its extremely short half-life, unwanted erythropoiesis is generally avoided, allowing for multiple or chronic dosing. However, the risk of unwanted effects associated with the chronic overstimulation of EPOR still exists (Leist et al., 2004).
The most promising analogue seems to be carbamylated EPO (CEPO). This analogue does not bind to the classical EPOR, but instead engages an alternative receptor that then signals tissue protection (Leist et al., 2004). As a result, CEPO does not stimulate erythropoiesis. In addition, it was found to be as potent and effective a neuroprotectant as unmodified EPO against stroke, spinal cord compression, diabetic neuropathy, and experimental autoimmune encephalomyelitis (Leist et al., 2004). Additionally, according to Ramirez et al. (2009), CEPO does not stimulate angiogenesis. This is an important consideration for conditions like diabetic retinopathy (DR) and neovascular age-related macular degeneration (ARMD) in which EPO provides neuroprotection but where blood vessel formation is harmful. Thus, with CEPO, the benefit of tissue protection is provided (via reduction of apoptosis), while the unwanted erythropoietic and angiogenic effects are avoided (Leist et al., 2004; Ramirez et al., 2009).
It should be noted that though it offers benefits, the use of exogenous EPO, especially at high doses, is associated with hypertension and thromboembolism (Paschos et al., 2008). Athletes, for example, who use exogenous EPO to combat anemia, and, in turn, enhance their performance, are at risk for excessive erythrocytosis, which can lead to deep vein, coronary, and cerebral thromboses (Fisher, 2003). In a study using EPO to treat cancer-related anemia, Bennett et al. (2008) confirmed that administration of exogenous EPO, including darbepoetin, leads to increased risk for venous thromboembolism and increased mortality rates.
Due to its potential for neuroprotection, there are many current clinical trials studying the effects of EPO on various brain-related conditions. These include treatment for cerebral malaria, stroke, brain injury, Friedreich ataxia, periventricular leukomalacia, and schizophrenia (ClinicalTrials.gov; Rabie and Marti, 2008). EPO's cardioprotective potential has also led to various clinical studies concerning the heart. Trials are being conducted to see if EPO could he protective after myocardial ischemia and reperfusion (restoration of blood flow to an ischemic region), and lead to infarct size reduction and improvement in left ventricular function (ClinicalTrials.gov). In addition, there are clinical trials studying the efficacy of EPO on the kidney, including one on ischemia-reperfusion after kidney transplant and another oil acute kidney injury in those with chronic kidney disease (ClinicalTrials.gov).
Researchers have found in animal models that EPO can cause harmful blood vessel formation in the eye, and may be implicated in recurrent capsule opacity. However, along with these negative effects, investigators have also found that EPO offers protection for photoreceptor cells, retinal ganglion cells (RGCs), and retinal pigment epithelium (RPE) cells, and thus may be beneficial in the treatment of ocular diseases like DR, glaucoma, retinitis pigmentosa (RP), ARMD, and retinal detachment (RD). Moreover, Brines et al. (2000) and Grimm et al. (2002) discovered that therapeutic concentrations of EPO can cross the blood-brain barrier (BBB) and blood-retina barrier (BRB), respectively. Therefore, it can be administered systemically unlike other neuroprotectants. However, we have found only two clinical trials using EPO as a drug intervention for diseases of the eye. One trial is using EPO with early iron supplements for retinopathy of prematurity (ROP), and the other is studying the safety and efficacy of EPO as an add-on therapy to methylprednisolone for the treatment of acute optic neuritis (ClinicalTrials.gov).
With the potential for hypertension, thromboembolism, and pathological neovascularization in mind, we question if exogenous EPO can somehow still be used to combat conditions affecting the eye. Is there a way to reap the benefits of EPO while eliminating or at least minimizing the risks? Administration route (intravitreal, intraperitoheal, or retrobulbar), dosage, timing of administration, and the use of EPO analogues (e.g. CEPO or asialoEPO) should all be considered and further investigated.
NON-ERYTHROPOIETIC NON-OCULAR EFFECTS OF EPO
EPO and the Nervous System
EPO and EPOR are expressed in the cerebral cortex, cerebellum, hippocampus, pituitary gland, and spinal cord (Jelkmann, 2005). Researchers have found that EPO offers non-hematopoietic benefits in the nervous system, including protection and repair. Dreixler et al. (2009) reported that systemic injection of EPO protects neurons from ischemic damage, while Tsai (2008) reported that when EPO was injected 24 hours before ischemic injury (mouse model of stroke), 47% reduction of cerebral infarct size resulted. Tsai (2008) also reported that EPO was therapeutic in a mouse model of focal cerebral lesion leading to global degeneration. The hormone improved behavioral abnormality, cognitive dysfunction, and brain atrophy up to 8 months after the insult. Furthermore, Genc et al. (2004) reported that EPO may facilitate nerve regeneration after peripheral nerve injury. Also, intraperitoneal (systemic) injection of EPO was shown to be protective in a rat model of spinal cord injury (Tsai, 2008). Additionally, some patients who were given EPO while undergoing chemotherapy displayed improvement in cognitive function. However, it is unclear whether correction of anemia or neuroprotection, or both, led to the improvement (Tsai, 2008). Preliminary clinical trials have demonstrated EPO to be promising for the treatment of various neurological acute diseases such as stroke, and chronic/progressive ones, such as Parkinson's disease or schizophrenia (Rabie and Marti, 2008).
EPO and the Cardiovascular System
EPORs have been shown to be present on vascular smooth muscle cells, endothelial cells, and cardiomyocytes (Mehta, 2008), and EPO has been found to offer cardioprotection. Mehta (2008) reported that after ischemia, administration of EPO reduced the myocardial infarct size. In addition, it stimulated new vessel formation in the ischemic heart, and Mehta (2008) proposed that it did so by stimulating EPORs on cardiomyocytes. Furthermore, in Aracasoy's (2008) review of Silverberg et al. (2000, 2001, and 2003), he reported that rhEPO improved cardiac and renal functions when used to treat anemia in congestive heart failure patients. EPO treatment resulted in an improvement of New York Heart Association function class, an increase in left ventricular ejection fraction, and a reduction of the need for diuretics.
EPO and the Renal System
EPORs are expressed on tubular epithelial, mesangial, and endothelial cells of the kidney (Johnson et al., 2006), and multiple studies have found that EPO offers renal protection. In renal tissue, EPO exerts not only an antiapoptotic effect but also a regenerative one (Fliser et al., 2006). Johnson et al. (2006) discovered that EPO protects in experimental ischemic and acute renal failure, independent of its hematopoietic effects. It not only suppresses apoptosis of tubular epithelium, but also enhances its proliferation. Additionally, it accelerates the functional recovery of the tubular epithelium. They also reported that others have found similar findings of improved renal histological and functional recovery in animal models. Fliser et al. (2006) reported that rhEPO analogue darbepoetin alpha is renoprotective in chronic renal failure as well. It offers vascular and tissue protection and preserves function in a rat model that features progressive injury to the renal microvascular endothelium (leads to glomerulosclerosis and tubulointerstitial damage) (Fliser et al., 2006). EPO treatment resulted in reduction of renal dysfunction as well as significantly improved survival of uremic rats. Furthermore, it was discovered that darbepoetin protected podocytes, epithelial cells of Bowman's capsule (important in filtration), that, when injured, contributes to proteinuria and glomerulosclerosis (Eto et al., 2007). Rats treated with puromycin (nephritic syndrome model) caused increased proteinuria, podocyte foot process retraction, actin cytoskeletal rearrangement, and deranged nephrin distribution. However, Eto et al. (2007) reported that treatment with darbepoetin reversed all of these effects, and they postulate that it did so by maintaining the podocyte actin cytoskeleton and nephrin expression. All of these findings suggest that EPO may be therapeutic against acute renal failure and progressive chronic kidney disease (Fliser et al., 2006).
EPO and Wound Healing
EPO has also been shown to be effective in the wound healing process. Haroon et al. (2003) associated EPO's effectiveness to promote healing with its ability to promote blood vessel formation during granulation tissue formation. They proposed that it mobilizes endothelial cells to participate in restorative neoangiogenesis. Also, Aracasoy (2008) reviewed the work of Agnello et al. (2002), Villa et al. (2003), Zhang et al. (2005), and Mitsuma et al. (2006) and reported that EPO reduces the levels of proinflammatory cytokines, and, thus, their harmful effects. Furthermore, abundant expression of EPOR protein has also been found in macrophages, cells that play a pivotal role during wound healing (Haroon et al., 2003).
EPO AND THE EYE
During eye development, specifically human retinal vascular development, EPO seems to play a crucial role. Patel et al. (2008) found increasing concentrations of both EPO mRNA and protein with increasing gestation in the human fetal eye. They speculated that EPO concentration increases to protect against retinal damage, mainly by inhibiting gene expression of apoptotic enzymes.
In another study, Wu et al. (2008) examined the protein expression of EPO and EPOR, STAT5, and pro-apoptotic protein Bcl-2-associated X (BAX) across various developmental stages of retinal neuron and lens cell differentiation during eye development. They detected EPO throughout the entire process, and their observed results of EPOR, STAT5 and BAX expressions during retinal and lens differentiation suggest that EPOR may play an important part in the normal development of the eye via apoptosis (Wu et al., 2008).
Retinopathy of Prematurity
After birth, the eyes of premature babies are vulnerable to the potentially blinding condition, ROP, where blood vessels grow abnormally in the infant's eye.
Due to prematurity, infants must be treated for anemia. Exposure to high levels of oxygen was thought to be the therapeutic approach. However, as Dorfman et al. (2008) reported, postnatal hyperoxia causes an arrest in growth of retinal blood vessels, as well as severe damage to retinal structure and function. It causes significant thinning of the outer plexiform layer, lowering of the horizontal cell count, and significant reduction in amplitude of photopic and scotopic electroretinographies. Alternatively, to deliver oxygen, rhEPO is used, and the need for blood transfusions is reduced (Romagnoli et al., 2000). However, EPO possesses properties similar to vascular endothelial growth factor (VEGF), an angiogenic factor most implicated in the pathogenesis of ROP (Suk et al., 2008). Sato et al. (2009) investigated the vitreous levels of endogenous EPO as well as VEGF in eyes with ROP. They found a significant positive correlation between EPO and VEGF levels in moderately and mildly vascular-active ROP eye, suggesting that both VEGF and EPO may contribute to the pathogenesis of the condition. Furthermore, Morita et al. (2003) believe that HLF (HIF-1[alpha]-like factor)/ (HIF-2[alpha] hypoxia-inducible factor-2[alpha], involved in transcription during embryonic vascularization, is involved in ROP.
Given that EPO stimulates angiogenesis, many have explored whether a correlation exists between treatment with rhEPO and development of ROP. After observing 85 very low birthweight infants (<1500 g), Shah et al. (2010) found that no association existed between rhEPO treatment (200-250 units/kg/dose three times/week for 10 doses) and the development of the condition. Similarly, Schneider et al. (2008) found that rhEPO treatment for anemia did not increase the frequency or severity of ROP in a study comparing 138 infants who received rhEPO and 138 who did not.
However, other studies showed contradictory findings. Romagnoli et al. (2000), for example, discovered that treatment with rhEPO and iron supplementation increased the risk of ROP, as incidence of the condition was significantly higher in the treated group compared to control. Moreover, Brown et al. (2006) found that exposure to rhEPO (total 6-week dose) was linked to an increased risk of ROP progression. They, therefore, suggested that there exists an association between cumulative rhEPO exposure and an increased risk for ROP. Furthermore, Suk et al. (2008) studied 264 infants with birth weights less than 1500 g who were administered 400 IU/kg of rhEPO intravenously or subcutaneously three times a week, along with 5 to 6 mg/kg/day of enteral iron. They found that infants who received more than 20 doses of rhEPO had an increased risk of developing ROP in comparison to those who received 20 or fewer doses. Moreover, the age at which treatment was started was also a significant risk factor. Infants who started rhEPO treatment after 20 days of age were almost four times more likely to develop ROP compared with those starting on or before 20 days of age. Thus, higher doses of EPO are associated with a higher risk of developing ROP in an age-dependent manner (Chen and Smith, 2008).
Chen and Smith (2008) explained that after premature birth, the infant is exposed to a relatively hyperoxic environment compared with that in utero. The hyperoxic environment suppresses the production of oxygen-regulated growth factors like VEGF. Consequently, retinal vascular growth is inhibited (normally would occur in the third trimester), and an avascular peripheral zone results. However, the nonvascularized retina becomes more metabolically active as the infant matures. This leads to ischemia, resulting in the release of hypoxia-induced growth factors which include VEGF and EPO. Abnormal angiogensis then occurs at the area between the vascularized retina and the avascular region. Such blood vessel formation may lead to retinal detachment and blindness.
Thus, the determining factor whether treatment with rhEPO plays a favorable or detrimental role in ROP is the timing (Chen and Smith, 2008). Though EPO may have a positive effect against anemia, administration at the wrong time (phase of retinopathy) may actually be destructive. EPO given at the neovascular stage (late) may worsen retinal blood vessel formation since EPO concentration is already elevated. Instead its suppression may be the proper approach. Early treatment with rhEPO is recommended since it will both combat anemia and prevent ROP (Chen and Smith, 2008).
Individuals with long-term diabetes often encounter the complication of DR, one of the major causes of visual impairment in Western countries. In this condition, pericytes and endothelial cells undergo apoptosis and vessel basement membranes thicken (Garcia et al., 2008). Retinal vasopermeability also increases, resulting in retinal hemorrhages, swelling, and formation of exudates that can impair vision. Thus, intraretinal or subretinal edema occurs with the disease (Jonas and Neumaier, 2007). In the advanced stage, proliferative diabetic retinopathy (PDR), neovascularization occurs and the newly formed blood vessels can invade and bleed into the vitreous. This can then produce a tissue that can result in retinal detachment and blindness (Garcia et al., 2008).
There have been many studies conducted to ascertain the role of EPO in DR. Inomata et al. (2004) report that EPO concentration is elevated in the vitreous of those with ischemic retinal diseases like DR. Many have found that the EPO levels in the eyes of diabetic subjects are considerably higher than in those without diabetes. For example, Watanabe et al. (2005) and Takagi et al. (2007) found that the vitreous EPO level in 73 patients with PDR was significantly higher than in 71 nondiabetic patients (464.0 vs. 36.5 mIU/ml). Asensio-Sanchez et al. (2008) also observed that the vitreous EPO levels of patients with PDR were much higher than those who were nondiabetic, observing concentrations of 512 mU/mL in PDR patients and 25.1 mU/mL in those without diabetes. Katsura et al. (2005) obtained similar results. Moreover, Asensio-Sanchez et al. (2008) observed that EPO values in patients with active PDR were significantly higher than in those with less active PDR. Furthermore, Jonas and Neumaier (2007) found that EPO levels in the aqueous humor were present in considerably higher concentrations in the eyes with diabetic macular edema than in eyes with exudative age-related macular degeneration, a condition exhibiting similar characteristics (macular edema and neovascularization). The 28 patients with diabetic macular edema had a concentration of 60.1 [+ or -] 46.7 mUnits/mL while the 59 patients with ARMD had a concentration of 22.9 [+ or -] 23.2 mUnits/mL. Finally, Garcia-Ramirez et al. (2008) also detected higher expression of EPO in the retinas of diabetics than nondiabetics. However, they also observed that EPOR expression was similar in both groups. Since overexpression of EPO appears to be an early occurrence in the retina of diabetic patients, and this is not associated with any change in EPOR, they proposed that at this early stage, other factors apart from hypoxia may be the cause of the EPO upregulation.
Researchers believe that the retina locally produces EPO. For example, since Watanabe et al. (2005) were not able to observe any significant correlation between the vitreous and plasma levels of EPO, they postulated that the increased local production of EPO in the retina is the source of the increased EPO levels in the vitreous fluid. This supports the findings of Hernandez et al. (2006) who, in a study with 12 PDR patients, found EPO concentrations in the vitreous 30 times higher than in serum. Such a discovery suggests that EPO is produced locally in the retina, with Fu et al. (2008) pointing to Muller cells (primary glial cells in the retina), specifically, as the main source.
Zhang et al. (2008), after inducing diabetes in Sprague-Dawley rats via intraperitoneal injection of streptozotocin, observed at the onset of diabetes a breakdown in the BRB, a significant reduction in retinal thickness and number of cells in the outer nuclear layer (ONE), and vascular and photoreceptor cell death. However, they found that a single intravitreal (local) injection of EPO (0.05ng-200ng) at the onset of diabetes largely prevented all these changes. For the BRB, EPO dosages of 5, 20, and 50 ng/eye offered similar protection in the first two weeks. After three weeks, 5 ng/eye demonstrated loss of its protective effect. At four weeks, 50 ng/eye still showed significant protection. The highest concentration of 200 ng/eye was less effective but still significantly protective. Thus, the optimal dosage of EPO for BRB protection was observed at 50 to 200 ng/eye. This discovery that EPO protects the BRB supports the theory of Hernandez et al. (2006) that the increased EPO concentration they found in those with diabetic macular edema serves to protect by acting as an antipermeability factor in the retina. For the upkeep of retinal thickness and number of cells in the ONE, 50 ng/eye was best; and for protection of retinal neurons against apoptosis 50 ng/eye was also the optimal dosage. They speculated that EPO interacts with EPOR in the photoreceptor inner segment, protecting retinal photoreceptors from light-induced apoptotic pathways. Such findings suggest that EPO may serve as a therapy or a prophylactic measure in the treatment of DR in its earliest stages, as it protects both vascular and neuronal cells.
Zhu et al. (2008) discovered similar results, finding that EPO protects retinal neurons and glial cells in early-stage diabetes in SpragueDawley rats. After inducing diabetes in the rats via intraperitoneal injection of streptozotocin, they treated some with rhEPO while leaving others untreated. After four weeks, the untreated group displayed a decrease in the amplitudes of b-wave and oscillatory potentials (OPs) (commonly seen in the early stage of diabetes), vacuolation and swelling in the mitochondria in RGCs (a dysfunction in early diabetes), and retinal glutamate increase (excess glutamate is toxic to neuronal cells and glial cells usually remove them). However, rats that were intraperitoneally injected with rhEPO (5000 IU/kg of body weight) 3 times per week for 2 weeks after diabetes induction showed no decrease in amplitudes of b-wave and OPs, no mitochondrial metamorphosis in ganglion cells, and no statistically significant increase in glutamate content in the retinas.
Also, in a study conducted by Friedman et al. (2003) in which five azotemic anemic pre-end stage renal disease diabetic subjects were treated with EPO for anemia for one year, three patients reported substantial improvement in vision. These three patients with macular edema displayed retention of vision and resolution of exudates. EPO was administered intravenously in doses of 50-150 U/kg 3 times weekly until the mean hematocrit increased to 40%. Maintenance doses of EPO were given to sustain the hematocrit above 32%. Friedman et al. (2003) speculated that the improvements result from increased hematocrit due to the administration of EPO. One of the patients, a 63-year-old woman, was treated with EPO at a dose of 4000IU injected subcutaneously twice weekly. After 8 months of treatment with EPO, hematocrit increased from 22% to 33%. The patient's visual acuity went from 20/200 in the right eye to 20/60. Additionally, the dense lipid exudates at the foveal center in her right eye went from an Early Treatment Diabetic Retinopathy Study (ETDRS) grade of 5 to 3. ETDRS was a multicenter randomized clinical trial that tested visual acuity, assessing the effects of argon laser photocoagulation and aspirin treatment on the course of DR in patients with mild to severe nonproliferative or early PDR (Fong et al., 1999). The second patient, a 60-year-old woman, was treated with EPO at a dose of 4000IU injected subcutaneously twice weekly. Her hematocrit went from 21% to 31%. Her DR caused diminished visual acuity of 20/400, but EPO treatment improved it to 20/200. Moreover, her ETDRS grade went from 5 to 4. Finally, the third patient, a 55-year-old woman, was treated with EPO at a dose of 4000IU injected subcutaneously twice weekly. After 11 months of treatment with EPO, her hematocrit increased from 24% to 32%. Her visual acuity improved, substantial resorption of her macular edema and hard exudates occurred, and her ETDRS grade went from 5 to 3.
Hypoxia is a contributing factor to organ, tissue, and/or cellular injury progression in diabetic patients (Friedman et al., 2003). Brownlee (2001) believes that a single process of overproduction of superoxide by the electron transport chain of the mitochondria (induced by hyperglycemia) is the underlying mechanism of microvasculopathy like retinopathy. If true, Friedman et al. (2003) propose that raising tissue oxygen tension (e.g. by increasing blood hemoglobin level via rhEPO administration) might be the proper approach. Sinclair et al. (2003), however, state that there are many postulates concerning the mechanisms by which treatment of anemia with rhEPO improves lesions caused by DR and slows tile progression of the disease. They add that there are no large studies which specifically address DR and correction of anemia.
Although many have found promise in the potential of EPO as a therapeutic agent, other researchers have also reported EPO's potential for harm, specifically pathological neovascularization in the retina. VEGF has been known to be a primary angiogenic factor. In retinal neovascularization, however, its inhibition reduces blood vessel formation, but does not completely inhibit it. Watanabe et al. (2005), through multivariate logistic regression analyses, discovered that EPO is an angiogenic factor that acts during retinal angiogenesis during PDR, and does so independently of VEGF. They found that exposure to EPO stimulated the growth of bovine retinal endothelial cells in a dose-dependent manner. Maximal cell growth occurred at 20 IU of erythropoieitin per milliliter. However, they found that intraretinal injections of soluble EPOR reduced neovascularization in 19-day-old mice in a dose dependent manner (25 ng resulted in 65% reduction, 62.5 ng resulted in 59% reduction, and 250 ng resulted in 55% reduction). By blocking EPO, they observed that blood vessel formation in the retina is inhibited in vivo and endothelial cell proliferation in the vitreous of patients with DR is also inhibited in vitro (Watanabe et al., 2005; Takagi et al., 2007). Thus, they proposed that inhibiting EPO could be effective against retinal angiogenesis. Moreover, Chen et al. (2009) found that when EPO mRNA is expressed with small interfering RNA (siRNA), blood vessel formation in the retina is suppressed. By intravitreally injecting EPO siRNA, approximately 60% of retinal EPO mRNA expression was inhibited. In turn, retinal neovascularization was suppressed by approximately 40%. Therefore, EPO siRNA may be useful for treating PDR and similar diseases.
Watanabe et al. (2005) and Takagi et al. (2007) also found that blockage of EPO was as efficient in inhibiting the stimulation of cell growth in vitro as blockage of VEGF. This suggests that EPO may be just as angiogenically powerful as VEGF in patients with PDR. However, they noted that a stimulant other than ischemia, such as high glucose levels, oxidative stress, intraocular inflammation, or the presence of other cytokines may also affect EPO expression.
In contrast to Watanabe et al. (2005) and Takagi et al. (2007), Song et al. (2008) believe that EPO does not cause harmful blood vessel formation. Specifically, they found that short-term elevation of EPO did not result in angiogenic changes in the rabbit eye. However, they did not determine whether or not chronic exposure to EPO would cause neovascularization. They also reported that other studies propose that EPO is not involved in angiogenic activity. For example, an experiment using rats with tumors that expressed EPOR found no evidence of angiogenesis or tumor growth after multiple injections of rhEPO. In addition, Ysai (2008) found that single intravitreal dosing does not cause blood vessel formation in the retina, and that administration does not cause adverse effects on retinal function. They observed no qualitative changes in the structure, morphology, and thickness of the individual retinal nerve layers among the rats exposed to dosages of 50 ng, 100 ng, and 250 ng.
Prevention of DR necessitates strict control of blood glucose and blood pressure (Porta and Allione, 2004). Currently, laser photocoagulation (burns choroidal neovasculature) and vitrectomy (surgical removal of vitreous) are the standard treatment for the condition (El-Asrar et al., 2009). However, studies for new treatments for DR are being conducted, including those that oppose excessive retinal vasopermeability and angiogenic responses (Garcia et al., 2008). As shown in Table 2, exogenous EPO has positive effects on the diabetic eye. It should therefore be considered as a potential prophylactic and/ or therapeutic agent. However, EPO's angiogenic ability should also be a concern. Perhaps even its inhibition could be the proper treatment in some cases.
Glaucoma is often a major cause of preventable blindness. It is associated with high intraocular pressure (IOP), due to reduced drainage of aqueous humor. Moreover, the condition causes optic nerve damage, visual field defects, and RGC death. The increased IOP and/or retinal ischemia in glaucoma leads to RGC loss, and in turn, visual impairment (Zhong, L. et al., 2007).
Many have reported the potential beneficial role of EPO in the treatment of glaucoma. Studies have shown EPO to be protective of RGCs against acute ischemia injury. For example, Junk et al. (2002) demonstrated that rhEPO administration (via intraperitoneal injection before or immediately after acute ischemia-reperfusion injury) resulted in both reduction of histopathological damage and promotion of functional recovery. After elevating the IOP (120 mm Hg for 45 or 60 minutes) in the eyes of rats, they observed 35-40% reduction in retinal thickness as compared with control. However, when rhEPO was administered immediately after 45 minutes of ischemia, significant preservation in thickness and histoarchitecture of the inner retina as well as functional improvement were observed. It should be noted that rhEPO treatment did not lead to such results in rats exposed to 60 minutes of ischemia. Additionally, rhEPO decreased the number of terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL)-positive neurons. Twenty-four hours after retinal ischemia, TUNEL-positive cells (indicator of apoptosis) were found in the ganglion cell layer and inner nuclear layer (INL). However, pretreatment with rhEPO resulted in significantly fewer TUNEL-positive cells in these layers. Such a discovery further suggests that rhEPO protects by inhibiting apoptosis.
EPO has been shown to reduce apoptosis of RGCs after ocular hypertension and transection of the optic nerve. For example, two weeks after inducing ocular hypertension (an injury that mimics glaucoma) via laser coagulation, Fu et al. (2008) observed a significant increase of EPO and EPOR proteins in the rat retina. When they tried to neutralize the endogenous EPO by intravitreally injecting 20 ng of soluble EPOR at 0, 4, 7, and 11 days after the injury, they found that the ocular hypertensive injury was exacerbated as RGCs died. There were 13.9 [+ or -] 1.8% total RGCs lost 2 weeks following ocular hypertension. However, 20.7 [+ or -] 3.7% RGC loss were observed after treatment with soluble EPOR. They hypothesize that soluble EPOR may neutralize the endogenous EPO to worsen the injury. This supports the findings of Junk et al. (2002) who observed that intravitreal administration of soluble EPOR (2 or 20 ng) exacerbated experimental ischemic injury in rats.
In addition, Fu et al. (2008) found that administration of rhEPO rescued RGCs after chronic ocular hypertension. After intravitreal injection of 2U rhEPO at 0, 4, Z and 11 days after the first laser coagulation, survival of RGCs was enhanced by -1.5 [+ or -] 3.08% in comparison to control eyes on day 14 injury. Intraperitoneal injection of rhEPO at 5,000 U [(42 [micro]g, 1 ml)/kg 24 h before or 30 min before first laser coagulation injury] also enhanced the survival of RGCs. RGC loss in the control group was 12.8 [+ or -] 4.2% weeks after ocular hypertension, while no loss of RGCs occurred in the rhEPO-treated group.
They also found that after optic nerve transection (mimics acute injury), intraperitoneal injection of rhEPO at 5,000 U (42 [micro]g, 1 ml)/ kg enhanced the survival of RGCs. After 1 week, the control group lost 44.0 [+ or -] 2.4% RGCs, while rhEPO treated group lost only 22.6 [+ or -] 4.3%. Thus, there were significantly more surviving RGCs.
The findings of Fu et al. (2008), therefore, suggest that there exists an endogenous EPO/EPOR system which participates in recovery after retinal injury. Moreover, exogenous EPO has a significant neuroprotective effect on the survival of RGCs in an ocular hypertension model of glaucoma (chronic injury) and optic nerve transection (acute injury).
Moreover, Tsai et al. (2005) found that one 200 ng dose of EPO administered intravitreally protects RGC viability in an in vivo rat model of glaucoma (induced by episcleral vessel cautery). This dosage was based on the study of Sakanaka et al. (1998) in which in vivo evidence was presented that EPO protects neurons from ischemic damage. Tsai et al. (2005) separated the animals into three experimental groups: episcleral vessel cautery only (EVC), episcleral vessel cautery with intravitreal normal saline injection (EVC-NS), and episcleral vessel cautery with intravitreal EPO treatment (EVC-EPO). In comparison to the retinas of control rats (RGC count of 12,619 [+ or -] 310), after 21 days the RGC counts were 9116 [+ or -] 273 for the EVC and 9489 [+ or -] 293 for the EVC-NS. However, the count for the EVC-EPO was 11,212 [+ or -] 414, a non-significant decrease to the control.
Additionally, Zhong, L. et al. (2007) found that DBA/2J mice models of glaucoma that were intraperitoneally injected with EPO displayed significant prevention of RGC loss in comparison to untreated mice, without affecting IOP. The mice were injected control substances or various doses of EPO, starting at the age of 6 months and continuing for an additional 2, 4, or 6 months. Treatment with EPO at doses of 3000, 6000, and 12,000 U/kg body weight per week all prevented significant RGC loss, compared with untreated DBA/2J control animals, without affecting IOP. Relative to untreated mice, at 3000 U/kg (similar results at 6000, and 12,000 U/kg) number of viable RGCs increased by 23.90% after four months and number of viable RGCs increased by 41.15% after six months. These further support the idea that systemic administration of EPO prevents the loss of RGCs, and does so without affecting IOP. However, tile researchers note that EPO increases the number of circulating erythrocytes. It can therefore improve the oxygenation of the retina in the treated mice, indirectly improving the survival of the RGCs. Nevertheless, treatment with systemic EPO significantly prevents RGC loss, whether directly or indirectly. Therefore EPO should be considered in the treatment of glaucoma in addition to lOP-lowering medications (Zhong, L. et al., 2007).
Weishaupt et al. (2004) injected EPO into the vitreous body to investigate neuroprotection of axotomized rat RGCs. They found that after axotomy (via optic nerve transection), approximately 85% of all RGCs die within 14 days by apoptosis. However, they observed that intraocular injections of 2 U EPO/eye on days 0, 4, 7, and 10 after axotomy enhanced RGC survival by 92%, from 455 [+ or -] 95.7 surviving RGCs/[mm.sup.2] to 872 [+ or -] 243.5. These data again provide support of the idea that EPO could be beneficial against RGC death in the context of glaucoma. They note that the animal model examined in this study lacked important aspects of the disease including elevated lOP.
Kilic et al. (2004) examined the effect of EPO on the retrograde degeneration of RGCs following optic nerve transection in vivo. They used their transgenic mouse line tg21 that constitutively expresses rhEPO in neuronal cells without inducing polycythemia. Compared to the wild-type, the RGCs of EPO transgenic tg21 mice were protected against degeneration after axotomy. EPO considerably increased the density of surviving RGCs. Fourteen days after lesioning in wild-type mice, only 21.3 [+ or -] 7.6% of RGCs were viable. On the other hand, 14 days after lesioning in transgenic mice, 61.4 [+ or -] 20.6% of RGCs were viable. Also, they injected selective inhibitors of extracellular-signal-regulated kinases -1/-2 or Akt pathways into the vitreous space and found that transgenic EPO protected the RGCs by a pathway involving extracellular-signal-regulated kinases -1/-2 but not Akt.
King et al. (2007) also observed that EPO was neuroprotective against optic nerve transection in adult rats. Immediately after intraorbital optic nerve transection, rats were administered a single intravitreal injection of EPO (5, 10, 25, and 50 units). Four weeks later, the RGC concentration was 253 RGCs/[mm.sup.2] in control rats. Only the treatments with 5 units and 10 units significantly increased RGC concentrations (to 333 RGCs/[mm.sup.2] and 344 RGCs/[mm.sup.2], respectively). Treatments with 25 and 50 units of EPO led to concentrations of only 293 and 233 RGCs/[mm.sup.2], respectively.
For studies on neuroprotection, administration of EPO was via intravitreal or intraperitoneal injections. However, the safer and more common approach in most clinical settings is the retrobulbar injection. Zhong et al. (2008) found that retrobulbar administration of EPO could protect RGCs from acute elevated lOP as well. After raising the IOP of the right eye to 70 mm Hg for 60 min, 1000 U of rhEPO was immediately administered via retrobulbar injection. They observed that the number of surviving RGCs per square millimeter in the eyes of the acute elevated IOP + rhEPO retrobulbar injection group (2142 [+ or -] 98) was significantly higher than in the eyes of the acute elevated IOP (1733 [+ or -] 62) and acute elevated IOP + vehicle solution (1724 [+ or -] 102) retrobulbar injection groups. Concerning the RGC ultrastructure, organelle number decreased, but some intact mitochondria still existed. Also, there was an increase in the densities of EPO and EPOR expression in the RGC layers in the eyes of acute elevated lOP subjects. This injection method, therefore, should be considered for the treatment of retinal neuropathy.
Studies have also implicated excess glutamate and nitric oxide (NO) in ischemic retinal degenerations like DR and glaucoma because they promote oxidative damage which, in turn, leads to cell death (Yamasaki et al., 2005; Zhong, Y. et al., 2007). Yamasaki et al. (2005) investigated whether EPO might be beneficial in protecting RGCs from glutamate and NO toxicity using 3-day-old female Wistar rats. Both glutamate and a NO-generating reagent caused RGC death. However, EPO significantly increased RGC survival. RGCs were cultured for 48 h with glutamate at low concentrations ([less than or equal to] 100 [micro]M) or for 24 h at high concentrations ([greater than or equal to] 1mM). Pretreatment with EPO (12 hours at .5 U/ml) significantly prevented the toxic effects of 1 mM glutamate. Also, a high concentration of EPO (1.5 U/ml) prevented glutamate-induced toxicity as well.
Additionally, Yamasaki et al. (2005) examined whether EPO could inhibit apoptosis brought on by NO, a compound that has been shown to be a downstream mediator of glutamate-induced cytotoxicity. RGCs were pretreated with EPO for 12 h. They were then treated with different concentrations of a NO-generating agent called sodium nitoprusside (SNP) for 6 h. SNP resulted in RGC death in a dose-dependent manner. However, prevention of toxicity (induced by 100 and 500 1aM of SNP) was achieved by EPO at 1.5 U/ml.
In short, EPO seems to be protective of RGCs from glutamate-induced cytotoxicity, partly by preventing NO-induced toxicity.
To explore the mechanism of neuroprotection from NO-induced toxicity, Yamasaki et al. (2005) examined whether EPO could reverse the expression of proapoptotic B-cell lymphoma (Bcl-2) superfamily members. In the absence of EPO, mRNA levels of Bcl-2 were markedly downregulated in RGCs cultured with SNP (500 [micro]M) for 6 h. Meanwhile, the expression levels of Bcl-2 were significantly reversed in the presence of EPO in a dose-dependent manner. These suggest a possible involvement of Bcl-2 in the mechanism through which EPO protects RGCs from NO-induced toxicity.
In a related study, Zhong, Y. et al. (2007) investigated whether EPO had a positive effect on neurite outgrowth and if it could protect cultured neurocytes suffering from glutamate-induced cytotoxicity. They cultured the retinal cells of Sprague-Dawley rat pups for 48 hours. Afterwards, the cells were cultured in serum-free media containing 5 mM or 10 mM glutamate. The cells were then incubated in the presence or absence of EPO (1.0 U/ml, 3.0 U/ml, 6.0 U/ml respectively) for another 48 hours. They found that neurite outgrowth length of the retinal neurocytes improved/increased as a result of EPO exposure in a dose-dependent manner. 1.0 U/ml of EPO led to 135% increase; 3.0 U/ml of EPO led to 155.7% increase; and 6.0 U/ml of EPO led to 162.8%.
Furthermore, increased apoptosis in the cultured retinal neurocytes occurred after exposure to glutamate toxicity. 5 mM/1 or 10 mM/1 glutamate significantly increased apoptosis rates. However, EPO promoted increased survival and decreased early and total apoptosis. For early apoptosis, 1 U/ml, 3 U/ml, and 6 U/ml of EPO improved the survival of the cultured neurocytes incubated with 5 mM/1 glutamate. However, only 6 U/ml EPO significantly improved the survival of the cultured neurocytes incubated with 10 mM/L glutamate. In terms of late apoptosis, EPO did not significantly decrease the apoptosis rates of the cultured neurocytes. Regarding total apoptosis, 3 U/ml and 6 U/ml EPO significantly decreased the total apoptosis rates of the cultured neurocytes incubated with 5 mM/1 glutamate. However, only 6 U/ml EPO significantly decreased the total apoptosis rates of the cultured neurocytes incubated with 10 mM/l glutamate.
In summary, EPO, in a dose-dependent manner, resulted in an improvement of neurite outgrowth of dissociated retinal neurocytes in vitro. Additionally, against glutamate-induced cytotoxicity, it promoted survival and decreased the cell death rates.
Current treatments for glaucoma, which include medications and surgeries, are aimed towards reduction of lOP by improving aqueous outflow and/or reducing the production of aqueous humor (Soltau and Zimmerman, 2002). By lowering lOP, RGC loss is inhibited. However, no sort of treatment for direct protection of RGCs has yet been established (Song et al., 2008). There are ongoing studies to more clearly identify lOP-independent mechanisms of damage and to find treatments for neuroprotection to prevent RGC death (Desai and Caprioli, 2008). Perhaps, EPO can be used for therapy for glaucoma since numerous studies have demonstrated its ability to protect RGCs from apoptosis (Table 3).
Retinitis Pigmentosa and Age-Related Macular Degeneration
RP is an inherited progressive disease in which bony spicule pigmentation appears in the mid/far peripheral retina. It primarily affects the rod photoreceptors and the RPE cells. Impaired visual loss or even blindness results usually after midlife (Pagon, 1988).
ARMD is another retinal disorder of which there are two major forms: atrophic/nonexudative (dry) and neovascular/exudative (wet), with atrophic ARMD usually developing first. In the dry form, photoreceptors in the central part of the retina, the RPE, and neuroretina slowly undergo apoptosis leading to irreversible vision loss (Wang et al., 2009b). Oxidative stress, inflammatory injury, formation of drusen (small yellow deposits), and accumulation of the pigment lipofuscin are all believed to insult RPE, which then triggers dry ARMD (Kourlas and Abrams, 2007; Wang et al., 2009b). In wet ARMD, the more severe form of the disease, abnormal vessels in the choroid capillary matrix proliferate. This leads to subretinal hemorrhage and leakage, exudative RPE detachment, disciform scarring, and fibrosis (Kourlas and Abrams, 2007).
In both RP and ARMD, apoptosis occurs in photoreceptors as well as in the RPE. Reactive oxygen species (ROS) are believed to cause oxidative damage, and, in turn, degeneration of retinal pigment epithelium. RPE cells form the outer BRB. One of their functions is to remove the byproducts of photoreceptor turnover, resulting in a lifetime of constant exposure to ROS like hydrogen peroxide [H.sub.2][O.sub.2] (Wang et al., 2009a). Eventually, RPE cell junctions are disrupted and BRB integrity is compromised. This breakdown may lead to exposure of the retina to various proteins and passage of immune cells into the retina, which will consequently result in the release of inflammatory cytokines (Wang et al., 2009a). Wang et al. (2009a) demonstrated that [H.sub.2][O.sub.2] increased the expression of tumor necrosis factor-[alpha] and interleukin-1[beta] (inflammatory cytokines) in RPE cells, but administration of EPO resulted in reduction of such expression. They also reported that administration of rhEPO could attenuate the breakdown of the BBB, which is similar to BRB. Furthermore, as previously discussed, Zhang et al. (2008) found that intravitreal administration of rhEPO may protect the BRB in a rat model of early diabetes. Wang et al. (2009a) also discovered that RPE cells cultured with [H.sub.2][O.sub.2] exhibited significantly reduced changes in permeability when these cells are pretreated with EPO (1 IU/ml two hours before [H.sub.2][O.sub.2] treatment). Pretreatment resulted in significant reduction of ROS production stimulated by [H.sub.2][O.sub.2] . As reported in earlier studies, these investigators observed an antioxidant function of EPO as it seeks ROS from the surroundings. According to Wang et al. (2009b) its antioxidant capability may directly regulate heine oxygenase-1 and glutathione peroxidase, and induce iron depletion, lessening oxidative injury caused by iron. Indirectly, the increased hematocrit caused by EPO may reduce cellular oxidative stress since red blood cells carry plenty of antioxidative enzymes.
Since the mitochondria of RPE cells consume molecular oxygen, they are one of the most important sources of ROS. The membrane permeability of the mitochondria is compromised as a result of oxidative injury as it leads to depolarization. This type of injury results in leakage of cytochrome c, which ultimately leads to apoptosis. However, Wang et al. (2009a) found that pretreatment with EPO effectively protected the mitochondria as it maintained membrane potential and prevented cytochrome c release. Their finding supports previous studies which have demonstrated that EPO preserves mitochondrial function.
Moreover, the depolarization of the mitochondria and subsequent release of cytochrome c stimulates caspase-3-like activity, which leads to DNA fragmentation and membrane phosphatidylserine exposure (end point of apoptosis that triggers phagocytosis). However, Wang et al. (2009a) found that, during oxidant injury, EPO directly inhibited such activity, providing protection for the RPE via the PI-3 kinase pathway.
In summary, Wang et al. (2009a) found that EPO can impede or prevent cell death in the RPE. They observed positive responses in RPEs that were administered 1 IU/ml of EPO following oxidant-treatment. These include, among others, a significant increase in viability, a reduction in the release of tumor necrosis factor-[alpha] and interleukin-1][beta] (inflammatory cytokines), recovery of the RPE cells' barrier integrity, prevention of cell DNA fragmentation, a decrease in the levels of intracellular ROS, and restored cellular antioxidant potential. In addition, Wang et al. (2009b) reported that EPO boosts expression of cytokines that function in protection and has neuroprotective effects in the retina.
Furthermore, light seems to accelerate disease progression in both RP and ARMD (Grimm et al., 2004). High levels of light damage the retina, whose purpose is to transform light into an electrophysiological signal (Ranchon Cole et al., 2007). Ultimately, the light causes cell death. However, Grimm et al. (2002) found that protection of photoreceptor cells against apoptosis (caused by exposure to damaging light levels) is achieved through pretreatment with hypoxia. They preconditioned mice by exposing them to varying low levels of oxygen for six hours followed by four hours of reoxygenation. They found that 6% and 10% oxygen protected both retinal morphology and retinal function most substantially. They believe that hypoxic preconditioning stabilizes in the retina the [alpha]-subunit of the hypoxia-inducible transcription factor-1 (Grimm et al., 2002), which controls the expression of the EPO gene in response to reduced oxygen tension (Halvorsen and Bechensteen, 2002). This supports the findings of Becerra and Amaral (2002) who discovered that EPO is an endogenous retinal survival factor to the photoreceptors against photochemical injury.
Grimm et al. (2002) also found that before or after light insult, systemically applied rhEPO protects against retinal degeneration. They injected rhEPO intraperitoneally (5,000 IU/kg) and found that there was a robust protection of photoreceptors in the retina when EPO was applied within a timeframe of one hour before and one hour after onset of light exposure. Thus, such findings indicate EPO's potential for retinal diseases in which photoreceptors undergo apoptosis.
In a separate study, Grimm et al. (2004) observed that in the retina of transgenic (tg6) mice, which constitutively overexpressed rhEPO, the photoreceptors were protected against degeneration caused by light. After exposure to 13 klux of white light for 2 hours, they found that wild type mice exhibited photoreceptor apoptosis in a large central area, with almost all photoreceptor nuclei pyknotic (condensed chromatin) after 24 hours, which indicates ongoing cell death. Meanwhile, in the tg6 mice, not only was the affected area not as great, but not all photoreceptor nuclei were pyknotic as well (even in the most affected region). Thus, apoptosis was significantly reduced in tg6 mice. Also, 12 days after light exposure, analysis showed that within the retina, wild type mice had 1-2 rows of surviving photoreceptor nuclei, while tg6 had 4-5 rows in the most affected region. The inner and outer segments of the photoreceptors were better preserved in tg6 mice as well.
They also found that in a light-independent and a light-accelerated mouse model of RP, high levels of rhEPO affected neither the course nor extent of retinal degeneration. Repetitive intraperitoneal injections of rhEPO did not protect the retina either. Such findings indicate that apoptotic mechanisms during acute, light-induced photoreceptor cell death differ from those in genetically based retinal degeneration. Consequently, inherited retinal degeneration may require different therapies (Grimm et al., 2004). This discovery is contradictory to the studies of Rex et al. (2004) who proposed that EPO can protect RGCs from both light and genetic-induced degeneration.
Thus, EPO has been shown to have neuroprotective promise on retinal models that have undergone short period of insult (1 hour ischemia or 2 hour light damage). However, short-term and longterm light exposure differs in the molecular pathways that lead to photoreceptor cell death. Ranchon Cole et al. (2007) tested EPO's protective effect on a rat model of retinal degeneration caused by a long period of exposure to a moderate light intensity. They administered an intraperitoneal injection of 30,000 U/kg of rhEPO in rats, 1 hour or 4 hours before exposure to damaging light (24 hours; 2200 lux). They discovered that rhEPO injected 1 hour before light insult preserved retinal function significantly in comparison to the control but much less significantly than when administered 4 h before. Moreover, it did not protect retinal structure. Meanwhile, EPO injected 4 h before light exposure, protected both retinal function and structure, with the ONL being significantly thicker. They propose that in order to achieve significant protection of retinal function and structure, rhEPO has to be injected 4 hours before exposure to damaging light. Moreover, they found that EPO induced an increase in caspase-9 activity and expression 4 hours after EPO injection, corresponding to the start of light exposure. This suggests that caspase-9 plays a role in neuroprotection (Ranchon Cole et al., 2007). Thus, in addition to the findings of Grimm et al., 2002 that EPO has a protective effect on short-term injury, Ranchon Cole et al. (2007) demonstrated that it can also protect against long-term light damage.
Currently, there are no means of prevention or cure for RE and therapy is aimed towards slowing down the degenerative process, including sunlight protection, vitaminotherapy, and treating complications like cataract and macular edema. However, researchers are studying new therapeutic strategies including gene therapy, neuroprotection, and retinal prosthesis (Hamel, 2006).
As for ARMD, some of the suggested therapeutic approaches for the atrophic form include using antioxidants, controlling local inflammation, decreasing the lipofuscin formation, decreasing drusen formation, obtaining RPE transplants, protecting photoreceptors against apoptosis, and using immunomodulators (Wang et al., 2009b). The proliferation of new blood vessels in wet ARMD is thought to be stimulated by various angiogenic molecules, but VEGF has been found to be a major factor. And so, in addition to laser photocoagulation (burns choroidal neovasculature) and photodynamic therapy (occludes vessels), treatments to inhibit VEGF are being utilized. These include pegaptanib, ranibizumab, and bevacizumab (avastin), which are all administered by intravitreal injection (Avery et al., 2006; Kourlas and Abrams, 2007).
EPO's ability to protect photoreceptor cells and RPE (refer to Table 4) warrants its consideration as a possible therapy for both RP and atrophic ARMD. However, its potential as an angiogenic agent should be taken into account for the possible treatment of conditions like neovascular ARMD. In such cases, inhibition of EPO may be the more appropriate treatment route.
In RDs, photoreceptor apoptosis occurs causing vision loss. Greater vision loss results especially when the central macula is involved. When the retina detaches from the RPE, the outer layers of the retina suffer from hypoxia and later experience more severe ischemic insult to photoreceptors. The photoreceptors degenerate because the detachment results in the cutoff of nutrient supplies to the retina. After inducing RD in albino rats by injecting them with 1.4% sodium hyaluronate, Xie et al. (2007) observed that the mRNA and protein EPO/EPOR levels increase with the duration of detachment, reaching their peaks at 48 hours after RD. They observed strong expression of EPO and EPOR in the normal retina 48 hours after RD. These include the ganglion cell layer, inner plexiform layer, INL, outer plexiform layer, ONL, and photoreceptor inner segments (and outer segment for EPO expression). It is believed that the EPO/EPOR system serves in protection of retinal neurons during RD. Xie et al. (2007) proposed that exogenous EPO might promote the survival of retinal neurons during RD. Moreover, since EPO and EPOR are expressed all over the neurosensory retina, they suggested that the EPO/EPOR system may be protective in ischemic retinopathies like acute glaucoma, DR, hypertensive vascular disease, and ROP.
Recurrent Capsule Opacity
Recurrences in capsule opacity usually occur in children or those with uveitis or vitroretinal pathology. However, Kelly (2003) observed this condition in his 82-year-old patient who had cataracts, an eye condition in which opacity of the lens leads to visual impairment. The patient had bilateral uneventful phacoemulsification, implantation of intraocular lenses, and, after three years, bilateral capsulotomies. Ten years later, the patient returned with complaints of visual acuity loss in the right eye, and so received another capsulotomy. EPO may be associated in this unusual recurrence. It was believed that the patient may have had a predisposing factor since the individual had been treated with weekly doses of EPO for myelodysplasia (clonal disorder of the bone marrow) for 6 years. EPO may have increased the cortical proliferation since lens epithelial cells (LECs) can react to cytokines similar to EPO. Kelly (2003), therefore, proposed that the recurrent capsular opacity may have been due to long term EPO treatment and stimulation of LEC proliferation. However, he also noted that it is difficult to point to EPO as the definitive cause of the proliferation since the patient had a history of alcoholism and had been on other treatments including ones for systemic hypertension. Additionally, the occurrence may have been related to the underlying myelodysplasia.
Researchers have discovered that EPO is capable of much more than regulating erythropoiesis. The hormone possesses antiapoptotic, antioxidative, anti-inflammatory, and proangiogenic capabilities (Paschos et al., 2008), and is involved in cytoskeleton stabilization. Also, EPO has been found to be beneficial for the nervous, cardiovascular, and renal systems, and for wound healing. After ischemia, EPO protects neurons from damage, stimulates repair in nerve injury, reduces myocardial infarct size, suppresses renal tubular epithelium apoptosis, augments renal tubular epithelium functional recovery, and stimulates restorative angiogenesis in the heart. Additionally, after injury, its ability to reduce inflammation and promote favorable blood vessel formation makes it very effective in the healing process.
As shown in Table 5, endogenous EPO is elevated in the eyes of those with ocular diseases. Moreover, a multitude of studies using animal models have demonstrated that exogenous EPO is protective against the progression of various ocular disorders. For instance, a single intravitreal (local) injection of EPO (0.05ng-200ng) at the onset of diabetes appears to prevent the complications associated with DR which include BRB breakdown, retinal thickness and ONE cell number reduction, and apoptosis of vascular and photoreceptor cells (Zhang et al., 2008). In addition, it seems to facilitate improvement of vision and resorption of macular edema and hard exudates (Friedman et al., 2003).
EPO may also play a critical role in the treatment of glaucoma for it seems to reduce RGC death in animal models of the disease. Tsai et al. (2005) discovered that a single intravitreal dose (200 ng) of EPO is protective of RGC, while Zhong, L. et al. (2007) found that an intraperitoneal (systemic) injection (3200 U) did the same, without affecting IOP. Also, Fu et al. (2008) showed that rhEPO rescued RGCs after chronic ocular hypertension via intravitreal (2U) and intraperitoneal (5,000 units) injections. Meanwhile, Zhong et al. (2008) demonstrated that a retrobulbar administration of EPO also protects RGCs from acute elevated IOP, and suggested it is a much safer technique. Upon optic nerve transection, both Kilic et al. (2004) and Weishaupt et al. (2004) found that EPO enhanced RGC survival, with the latter utilizing an intravitreal injection of 2 U. Furthermore, Yamasaki et al. (2005) demonstrated that pretreatment with EPO reduced RGC death caused by excess glutamate and nitric oxide, which are both implicated in glaucoma. Zhong, Y. et al. (2007) also found that EPO promoted survival and decreased cell death in cultured neurocytes that were suffering from cytotoxicity caused by glutamate.
EPO's antiapoptotic effect in photoreceptors as well as RPE cells makes it a potential therapeutic agent for atrophic ARMD and RP also. Wang et al. (2009a), for instance, found that EPO can delay or prevent degeneration of RPE caused by ROS' oxidative damage. They believe that it acts as an antioxidant, decreases the production of the pro-inflammatory cytokines, increases the expression of protective cytokines, and serves as a survival factor for retinal neuronal and vascular cells. Additionally, several studies have found that rhEPO protects against retinal degeneration caused by light, a factor, when at high levels, seems to accelerate both diseases. Grimm et al. (2002) discovered that intraperitoneal injection of EPO (5,000 U/kg) is protective of retinal photoreceptors when applied within a time frame of one hour before and one hour after onset of light exposure. Also, the photoreceptors in the retina of transgenic (tg6) mice that constitutively overexpressed rhEPO were protected against light-induced degeneration (Grimm et al., 2004). Furthermore, Ranchon Cole et al. (2007) found that rhEPO provides significant neuroprotection if injected 4 hours before exposure to damaging light.
Finally, since photoreceptors undergo apoptosis in RD, due to nutrient supply cutoff to the retina, EPO may be beneficial for this condition as well. In a RD rat model, Xie et al. (2007) found that mRNA and protein EPO/EPOR levels increase with the duration of detachment. They propose that the EPO/EPOR system is protective during RD, and that rhEPO may promote the survival of retinal neurons.
As we have reported, many studies have demonstrated that EPO has the potential to be beneficial in combating various eye disorders. However, EPO has also been implicated in pathological neovascularization in the retina. Its use may thus be harmful in conditions like ROP, PDR, and neovascular ARMD. Watanabe et al. (2005) found that EPO is an angiogenic factor in PDR that acts independent of VEGF. When they injected soluble EPOR in the retina of mice, neovascularization was reduced in a dose-dependent manner. Thus, inhibition of EPO could be the proper approach in conditions where retinal angiogenesis occurs. Yet, not all are coming to the same conclusion, as other researchers believe that EPO does not cause harmful blood vessel formation. Song et al. (2008), for example, found that short-term elevation of EPO does not result in angiogenic changes in the rabbit eye. Similarly, Tsai (2008) found that single dosing of EPO (via intravitreal injection) in rats does not result in blood vessel formation in the retina. Both recognize their shortcomings as Song et al. (2008) did not determine whether or not chronic exposure to EPO would cause neovascularization, while Tsai (2008) did not address the potential consequences of multiple intravitreal injections.
As Kelly (2003) reported EPO may also be associated with recurrent capsule opacity. As he observed in his patient, long-term EPO treatment may increase cortical proliferation of LECs and cause the abnormal recurrence.
Researchers are not completely sure about the exact mechanism by which EPO/EPOR bestows its neuroprotective effect. Some hypothesize that they reduce the release of ROS and glutamate. Others believe that they reverse vasospasm, attenuate apoptosis, modulate inflammation, and recruit stem cells. Nevertheless, when EPO binds to EPOR, apoptosis of neurons is prevented or retarded. Seemingly, there is cross-talk between the JAK2 and nuclear factor kappaB signaling cascades, which leads to transcription of various neuroprotective factors. Additionally, when the JAK2 pathway is activated, release of glutamate from surrounding ceils may also be inhibited. Furthermore, activation of phosphatidylinositol-3 (PI-3) kinase may also be critical. In a rat focal ischemic model, neuroprotection was stopped by PI-3 kinase inhibitor Wortmannin. Also, activation of PI-3 kinase may inhibit cell death by deactivating the proapoptotic molecules Bcl-xL/ Bcl-2-associated death promoter and/or caspase-3. (Tsai, 2008)
The promising results obtained by all the previously discussed investigators warrant the consideration of EPO as a possible treatment or prophylactic measure against various eye disorders. Further studies need to be conducted in order to ascertain the conditions in which EPO can be most effective. For instance, which method for delivery of EPO leads to the best results? As we reported, intravitreal and intraperitoneal injections seem to be effective. Lagreze et al. (2009) examined three patients receiving EPO for acute vascular occlusion, and assessed the practicality of administering EPO intravitreally in humans. They found that a single injection of 2000 U (a dose used in many in vivo studies) does not result in injection-related toxicity nor does it cause any obvious detrimental effect in visual acuity, visual fields, IOP, electroretinogram, and hematocrit and serum EPO levels. Although EPO is able to cross the BBB, systemic use has to be closely monitored since it can cause known side effects polycythemia vera and vascular thrombosis (Tsai, 2008). Perhaps retrobulbar injection is the better approach since it is safer and more commonly utilized in most clinical settings (Zhong et al., 2008). In their study, Zhong et al. (2008) found that administration of EPO using this method protects RGCs from acute elevated IOP and keeps some mitochondria intact. On the other hand, Tsai (2008) would contend that a single intravitreal injection does not have a negative effect on retinal function. They later add that intravitreal sustained-release devices may be an effective delivery method as well since preliminary studies using this approach to deliver fluocinolone have been promising for the treatment of posterior uveitis (Tsai, 2008).
Moreover, what is the appropriate dosage? Researchers have used varying amounts, but several studies seem to find that 200 ng or 2 U is the effective dose for intravitreal injections, and 3,000-5,000 U for systemic administration. However, Tsai et al. (2008) found in their study that one of the rats exposed to the high dose of 625 ng exhibited reduction in retinal thickness and cell density. As such, the possibility of adverse effects due to dosage should be of concern.
Furthermore, at what point in the course of a particular eye disease should EPO be administered? Some have found that early administration can be preventive. For example, Zhang et al. (2008) observed that EPO can be preventive when administered early, specifically at the onset of diabetes; Yamasaki et al. (2005) demonstrated that pretreatment with EPO reduced RGC apoptosis caused by glutamate and NO toxicity; Zhu et al. (2008) found that rats injected with rhEPO 3 times per week for 2 weeks after diabetes induction showed no decrease in amplitudes of b-wave and OPs, no changes in the mitochondria of ganglion cells, and no significant increase in retinal glutamate content; and Grimm et al. (2002) discovered that EPO is protective of retinal photoreceptors when applied one hour before and one hour after onset of light exposure. Others also observed that EPO can be therapeutic after various kinds of insults. For instance, Fu et al. (2008) found that EPO rescued RGCs after chronic ocular hypertension, while Kilic et al. (2004) and Weishaupt et al. (2004) discovered that EPO enhanced RGC survival upon axotomy.
Many studies have found that EPO may serve as a neuroprotectant, protecting photoreceptor cells, RGCs, and RPE from apoptosis. Therefore, it may be beneficial in the treatment of ocular diseases like DR, glaucoma, RP, ARMD, and RD. Yet we have found no clinical trials that test or have tested EPO's value in the treatment of such diseases. We question why it has not yet been tested on patients who suffer from such conditions. We postulate that it may have been overlooked or it has been considered, but its use carries side effects that may outweigh its benefits. Its potential for hypertension and/or thromboembolism may be a cause for concern, especially using the systemic injection approach. Perhaps asialoEPO, an EPO derivative that is protective but does not stimulate erythropoiesis, should be used when using the systemic administration route. It should be noted that the Food and Drug Administration has recently warned against the use of erythropoiesis-stimulating agents (ESAs) like EPO, citing increased mortality, tumor promotion, and thromboembolic events in some non-myelodysplastic syndromes patients who were receiving ESAs (FDA.gov). On the other hand, no adverse effects of ESAs on survival were observed in other studies of patients with solid tumors receiving chemotherapy.
Moreover, EPO's potential to promote pathological angiogenesis in the retina may yet serve as a deterrent. As such, the use of CEPO may be the safer approach. This EPO analogue provides the desired protective, antiapoptotic property without causing unwanted hematopoietic side effects or stimulating pathological blood vessel formation. Montero et al. (2007) found that, in terms of neuroprotection, CEPO is at least as efficient as EPO. An alternative may be the use of EPO in conjunction with EPO siRNA or a similar compound or drug that inhibits neovascularization.
More research is necessary, and Tsai et al. (2008) propose that rats be used instead of other experimental animal models since the retinal vascular system and optic nerve head structure of rats more closely resemble humans. Different methods of delivery should be considered. Proper dosage or administration at a specific window of time during the development of the disease may also be the key. The various forms of EPO should also be examined. These areas should be the central focus of further studies. By doing so, ideal conditions for which EPO can be preventive, protective, and/or therapeutic could finally be determined.
AGNELLO, D., P. BIGINI, P. VILLA, T. MENNINI, A. CERAMI, M.L. BRINES, AND P. GHEZZI. 2002. Erythropoietin exerts an anti-inflammatory effect on the CNS in a model of experimental autoimmune encephalomyelitis. Brain Res. 952: 128-134.
ARCASOV, M.O. 2008. The non-haematopoietic biological effects of erythropoietin. Br. J. Haematol. 141(1): 14-31.
ASENSIO-SANCHEZ, V.M., V. GOMEZ- RAMIREZ, AND I. MORALES- GOMEZ. 2008. Erythropoietin concentrations in the vitreous body from patients with proliferative diabetic retinopathy. Arch. Soc. Esp. Oftalmol. 83(3): 169-172.
AVERY, R.L., D.J. PIERAMICI, M.D. RABENA, A.A. CASTELLARIN, M.A. NASIR, AND M.J. GIUST. 2006. Intravitreal bevacizumab (avastin) for neovascular age-related macular degeneration. Ophthalmol. 113(3): 363-372.
BAHLMANN, F.H. AND D. FLISER. 2009. Erythropoietin and renoprotection. Curr. Opin. Nephrol. Hypertens. 18(1): 15-20.
BECERRA, S.P., AND J. AMARAL. 2002. Erythropoietin--an endogenous retinal survival factor. N. Engl. J. Med. 347(24): 1968-1970.
BENNETT, C.L., S.M. SILVER, B. DJULBEGOVIC, A.T. SAMARAS, C.A. BLAU, K.J. GLEASON, S.E. BARNATO, K.M. ELVERMAN, D.M. COURTNEY, J.M. MCKOY, B.J. EDWARDS, C.C. TIGUE, D.W. RAISCH, P.R. YARNOLD, D.A. DORR, T.M. KUZEL, M.S. TALLMAN, S.M. TRIFILIO, D.P. WEST, S.Y. LAI, AND M. HENKE. 2008. Venous thromboembolism and mortality associated with recombinant erythropoietin and darbepoetin administration for the treatment of cancer-associated anemia. JAMA. 299: 914-924.
BRINES, M.L., P. GHEZZI, S. KEENAN, D. AGNELLO, N.C. DE LANEROLLE, C. CERAMI, L.M. ITRI, AND A. CERAMI. 2000. Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proc. Natl. Acad. Sci. 97(19): 10526-10531.
BROWN, M.S., A.E. BARON, E.K. FRANCE, AND R.F. HAMMAN. 2006. Association between higher cumulative doses of recombinant erythropoietin and risk for retinopathy of prematurity. J. AAPOS. 10(2): 143-149.
BROWNLEE, M. 2001. Biochemistry and molecular cell biology of diabetic complications. Nature. 13: 813-820.
BURGER, D., A. XENOCOSTAS, AND Q.P. FENG. 2009. Molecular basis of cardioprotection by erythropoietin. Curr. Mol. Pharmacol. 2(1): 56-69.
CAVODEASSI, F., K. IVANOVITCH, AND S. WILSON. 2009. Cytoskeletal reorganisation at the edge of the eye field is required for early stages of eye morphogenesis. Mech. Develop. 126: 89.
CHAN, C., E.Y. CHEW, D. SHEN, J. HACKETT, AND Z. ZHUANG. 2005. Expression of stem cells markers in ocular hemangioblastoma associated with von Hippel-Lindau (VHL) Disease. Mol Vis. 11: 697-704.
CHEN, J., K.M. CONNOR, C.M. ADERMAN, K.L. WILLETT, O.P. ASPEGREN. AND L.E.H. SMITH. 2009. Suppression of retinal neovascularization by erythropoietin siRNA in a mouse model of proliferative retinopathy. Invest. Ophthalmol. Vis. Sci. 50(3): 1329-1335.
CHEN, J. AND L.E. SMITH. 2008. A double-edged sword: Erythropoietin eyed in retinopathy of prematurity. J. AAPOS. 12(3): 221-222.
DESAI, P. AND J. CAPRIOLI. 2008. The treatment of normal-tension glaucoma. Prog. Brain Res. 173: 195-210.
DORFMAN, A., O. DEMBINSKA, S. CHEMTOB, AND P. LACHAPELLE. 2008. Early manifestations of postnatal hyperoxia on the retinal structure and function of the neonatal rat. Invest. Ophthalmol. Vis. Sci. 49(1): 458-466.
DREIXLER, J.C., S. HAGEVIK, J.W. HEMMERT, A.R. SHAIKH, D.M. ROSENBAUM, AND S. ROTH. 2009. Involvement of erythropoietin in retinal ischemic preconditioning. Anesthes. 110: 774-780.
EL-ASRAR, A., M. AHMED, H.S. AL-MEZAINE, AND M.S. OLA. 2009. Changing paradigms in the treatment of diabetic retinopathy. Curr. Opin. Ophth. 20(6): 532-538.
ERBAYRAKTAR, S., G. GRASSO, A. SFACTERIA, Q.W. XIE, T. COLEMAN, M. KREILGAARD, L. TORUP, Z. SAGER, Z. ERBAYRAKTAR, N. GOKMEN, O. YILMAZ, P. GHEZZI, P. VILLA, M. FRATELLI, S. CASAGRANDE, M. LEIST, L. HELBOE, J. GERWEIN, S. CHRISTENSEN, M.A. GEIST, L.O. PEDERSEN, C. CERAMI-HAND, J.P. WUERTH, A. CERAMI, AND M. BRINES. 2003. Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo Pro Natl Acad Sci USA 100:6741-6746
ETO, N., T. WADA, R. INAGI, H. TAKANO, A. SHIMIZU, H. KATO, H. KURINHARA, H. KAWACHI, S.J. SHANKLAND, T. FUJITA, AND M. NANGAKU. 2007. Podocyte protection by darbepoetin: preservation of the cytoskeleton and nephrin expression. Kidney Int. 72(4): 455-463.
FISHER, J.W. 2003. Erythropoietin: physiology and pharmacology update. Exp. Biol. Med. (Maywood) 228(1): 1-14.
FLISER, D., F.H BAHLMANN, AND H. HALLER. 2006. EPO: renoprotectinn beyond anemia correction. Ped. Nephrol. 21(12): 1785-1789.
FONG, D.S., F.B BARTON, AND G.H. BRESNICK. 1999. Impaired color vision associated with diabetic retinopathy: early treatment diabetic retinopathy study report no. 15. Am. J. of Opthalmol.. 128(5): 612-617.
FRIEDMAN, E., F.A. L'ESPERANCE, C.D. BROWN, AND D.H. BERMAN. 2003. Treating azotemia-induced anemia with erythropoietin improves diabetic eye disease. Kidney Int. 64(87): 57-63.
FU, Q.L., W. WU, H. WANG, X. LI, V.W.H. LEE, AND K.F. So. 2008. Up-regulated endogenous erythropoietin/erythropoietin receptor system and exogenous erythropoietin rescue retinal ganglion cells after chronic ocular hypertension. Cell. Molec. Neurohiol. 28(2): 317-329.
GARCIA, C., J. ARANDA, E. ARNOLD, S. THEBAULT, Y. MACOTELA, F. LOPEZ-CASILLAS, V. MENDOZA, H. QUIROZ-MERCADO, H.L. HERNANDEZ-MONTIEL, S.H. LIN, G. MARTINEZ DE LA ESCALERA, AND C. CLAPP. 2008. Vasoinhibins prevent retinal vasopermeability associated with diabetic retinopatby in rats via protein phosphatase 2A-dependent eNOS inactivation. J Clin. Invest. 118(6): 2291-2300.
GARCIA-RAMIREZ, M., C. HERNANDEZ, AND R. SIMO. 2008. Expression of erythropoietin and its receptor in the human retina. Diabetes Care. 31(6): 1189-1194.
GENC, S., T.F. KOROGLU, AND K. GENC. 2004. Erythropoietin and the nervous system. Brain Res. 1000: 19-31.
GRASSO, G., A. SFACTERIA, S. ERBAYRAKTAR, M. PASSALACQUA, F. MELI, N. GOKMAN, O. YILMAZ, T.D. LA, M. BUEMI, D.G. IACOPINO, T. COLEMAN, A. CERAMI, M. BRINES, AND F. TOMASELLO. 2006. Amelioration of spinal cord compressive injury by pharmacological preconditioning with erythropoietin and an nonerythropoietic erythropoietin derivative. J Neurosurg Spine 4: 310-318.
GRIMM, C., A. WENZEL, M. GROSZER, H. MAYSER, M. SEELIGER, M. SAMARDZIJA, C. BAUER, M. GASSMANN, AND C.E. REME 2002. HIF-l-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration. Nat. Med. 4(7): 718-724.
GRIMM, C., A. WENZEL, D. STANESCU, M. SAMARDZIJA, S. HOTOP, M. GROSZER, M. NAASH, M. GASSMANN, AND C. REME. 2004. Constitutive overexpression of human erythropoietin protects the mouse retina against induced but not inherited retinal degeneration. J. Neurosci. 24(25): 5651-5658.
HALVORSEN, S. AND A.G. BECHENSTEEN. 2002. Physiology of erythropoietin during mammalian development. Aura. Paediatr. 438:17-26.
HAMEL, C. 2006. Retinitis pigmentosa. ORPHANET. J. RARE. DIS. 1: 40.
HAROON, Z.A., K. AMIN, X. JIANG, AND M.D. ARCASOY. 2003. A novel role for erythropoietin during fibrin-induced wound-healing response. Amer. J Path. 163: 993-1000.
HASSELBLATT, M., H. EHRENREICH, AND A.L. SIREN. 2006. The brain erythropoietin system and its potential for therapeutic exploitation in brain disease. J. Neurosurg. Anesthesiol. 18(2): 132-138.
HASSOUNA, I., S. SPERLING, E. KIM, W. SCHULZ-SCHAEFFER, M. RAVE-FRANK, M. HASSELBLATT, W. JELKMANN, A. GIESE, AND H. EHRENREICH. 2008. Erythropoietin augments survival of glioma cells after radiation and temozolomide. Int J Radiat Oncol Biol Phys. 72(3): 927-934.
HERNANDEZ, C., A. FONOLLOSA, M. GARCIA-RAMIREZ, M. HIGUERA, R. CATALAN, A. MIRALLES, J. GARCIA-ARUMI, AND R. SIMO. 2006. Erythropoietin is expressed in the human retina and it is highly elevated in the vitreous fluid of patients with diabetic macular edema. Diab. Care. 29(9): 2028-2033.
INOMATA, Y., A. HIRATA, E. TAKAHASHI, T. KAWAJI, M. FUKUSHIMA, AND H. TANIHARA. 2004. Elevated erythropoietin in vitreous with ischemic retinal diseases. Neuroreport. 15: 877-879.
JELKMANN, W. 2005. Effects of erythropoietin on brain function. Curr. Pharm. Biotechnol. 6: 65-79.
JOHNSON, D.W., C. FORMAN, AND D.A. VESEY. 2006. Novel renoprotective actions of erythropoietin: New uses for an old hormone. Nephrology. 11(4): 306-312.
JONAS, J., AND M. NEUMAIER 2007, Erythropoietin levels in aqueous humour in eyes with exudative age-related macular degeneration and diabetic retinopathy. Clin. Exp. Opth. 35: 186-193.
JUNK, A.K., A. MAMMIS, S.I. SAVITZ, M. SINGH, S. ROTH, S. MALHOTRA, P.S. ROSENBAUM A. CERAMI, M. BRINES, AND D.M. ROSENBAUM. 2002. Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury. Proc. Natl. Acad. Sci. USA. 99(16): 10659-10664.
KATSURA. Y., T. OKANO, K. MATSUNO, M. OSAKO, M. KURE T. WATANABE, Y. IWAKI, M. NORITAKE, H. KOSANO, H. NISHIGORI, AND T. MATSUOKA. 2005. Erythropoietin is highly elevated in vitreous fluid of patients with proliferative diabetic retinopathy. Diab. Care. 28: 2252-2254.
KELLY, J.S. 2003. Recurrent capsule opacity and erythropoietin. Cataract Refract Surg. 29(2): 415--417.
KERTESZ, N., J. WU, T.H. CHEN, H.M. Sucov, AND H. WU. 2004 The role of erythropoietin in regulating angiogenesis. Develop. Biol. 276, 101-110
KILIC, U., E. KILIC, J. SOLIZ, C.I. BASSETTI, M. GASSMANN, AND D.M. HERMANN. 2004. Erythropoietin protects from axotomy-induced degeneration of retinal ganglion cells by activating ERK-1/-2. FASEB J. 19(2): 249-251.
KING, C.E., J. RODGER, C. BARTLETT, T. ESMAILI, S.A. DUNLOP. AND L.D. BEAZLEY. 2007. Erythropoietin is both neuroprotective and neuroregenerative following optic nerve transection. Exp Neurol. 205(1): 48-55.
KOURLAS, H. AND P. ABRAMS. 2007. Ranibizumab for the treatment of neovascular age-related macular degeneration: a review. Clin. Therapeutics. 29(9): 1850-1861
KRANTZ, S.B. 1991. Erythropoietin. Blood. 77: 419-434.
LACOMBE, C. AND P. MAYEUX. 1998. Biology Of Erythropoietin. Haematologica. 83: 724-732.
LAGREZE, W.A., N. FELTGEN, M. BACH, AND T. JEHLE. 2009. Feasibility of intravitreal erythropoietin injections in humans. Br J Ophthalmol. 93(12): 1667-1671.
LEIST, M., P. GHEZZI, G. GRASSO, R. BIANCHI, P. VILLA, M. FRATELLI, C. SAVINO, M. BIANCHI, J. NIELSEN, J. GERWIEN, P. KALLUNKI, A.K. LARSEN, L. HELBOE, S. CHRISTENSEN. L.O. PEDERSEN, M. NIELSEN, L. TORUP, T. SAGER, A. SFACTERIA, S. ERBAYRAKTAR, Z. ERBAYRAKTAR, N. GOKMEN, O. YILMAZ, C. CERAMI-HAND, Q.W. XIE, T. COLEMAN, A. CERAMI, AND M. BRINES. 2004. Derivatives of erythropoietin that are tissue protective but not erythropoietic. Science. 305(5681): 239-242.
MACDOUGALL, I.C. 2008. Novel erythropoiesis-stimulating agents: a new era in anemia management. Clin. J. Am. Sac. Nephrol. 3: 200-207.
MAIESE, K., Z. CHONG, AND Y. SHANG. 2008. Raves and risks for erythropoietin. Cytokine Growth Factor Rev. 19(2): 145-155.
MEHTA, J. 2008. Erythropoietin in cardioprotection: does it have a future or is it all in the past? Cardiovasc. Res. 79: 549-550.
MELLI, G., C. JACK, G.L. LAMBRINOS, M. RINGKAMP. AND A. HOKE. 2006. Erythropoietin protects sensory axons against paclitaxel-induced distal degeneration. Neurobiol Dis. 24(3): 525-530.
MITSUMA, W., M. ITO, M. KODAMA, K. FUSE, K. OKAMURA, S. MINAGAWA, K. KATO, H. HANAWA, K. TOBA, M. NAKAZAWA, AND Y. AIZAWA. 2006. Cardioprotective effects of recombinant human erythropoietin in rats with experimental autoimmune myocarditis. Biochem. and Biophys. Res. Comm. 344: 987-994.
MONTERO, M., F.R. POULSEN, J. NORABERG, A. KIRKEBY, J. VAN BEEK,M. LEIST, AND J. ZIMMER. 2007. Comparison of neuroprotective effects of erythropoietin (EPO) and carbamylerythropoietin (CEPO) against ischemia-like oxygen-glucose deprivation (OGD) and NMDA excitotoxicity in mouse hippocampal slice cultures. Exp. Neurol. 204(1): 106-117.
MORITA, M., O. OHNEDA, T. YAMASHITA, S. TAKAHASHI, N. SUZUKI, O. NAKAJIMA, S. KAWAUCHI, M. EMA, S. SHIBAHARA, T. UDONO, K. TOMITA, M. TAMAI, K. SOGAWA, M. YAMAMOTO, AND Y. FUJII-KURIYAMA. 2003. HLF/HIF-2[alpha] is a key factor in retinopathy of prematurity in association with erythropoietin. The EMBO J. 22(5): 1134-1146.
PAGON, R.A. 1988. Retinitis pigmentosa. Survey of Ophthalmology. 33(3): 137-177.
PASCHOS, N., M.G. LYKISSAS, AND A.E. BERIS. 2008. The role of erythropoietin as an inhibitor of tissue ischemia. Int. J. of Biol. Sci. 4: 161-168.
PATEL, S., M.J. ROWE, S.A. WINTERS, AND R.K. OHLS. 2008. Elevated erythropoietin mRNA and protein concentrations in the developing human eye. Pediatr. Res. 63(4): 394-39Z
PORTA, M. AND A. ALLIONE. 2004. Current approaches and perspectives in the medical treatment of diabetic retinopathy. Pharm. Therap. 103(2): 167-177.
RABIE, T., AND H.H. MARTI. 2008. Brain protection by erythropoietin: a manifold task. Physiol. (Bethesda). 23: 263-274.
RAMIREZ, R., J. CARRACEDO, S. NOGUERAS, P. BUENDIA, A. MERINO, S. CANADILLAS, M. RODRIGUEZ, C. TETTA, A. MARTIN-MALO, AND P. ALJAMA. 2009. Carbamylated darbepoetin derivative prevents endothelial progenitor cell damage with no effect on angiogenesis. J Mol Cell Cardiol. 47(6): 781-788.
RANCHON COLE, I., B. BONHOMMIE, AND M. DOLY. 2007. Pre-treatment of adult rats with high doses of erythropoietin induces caspase--9 but prevents light-induced retinal injury. Exp. Eye Res. 85(6): 782-789.
REX, T.S., M. ALLOCCA, L. DOMENICI, E.M. SURACE, A.M. MAGUIRE, A. LYUBARSKY, A. CELLERINO, J. BANNETT, AND A. AURICCHIO. 2004. Systemic but not intraocular Epo gene transfer protects the retina from light-and genetic-induced degeneration. Mol. Ther. 10(5): 855-861.
ROMAGNOLI, C., E. ZECCA, F. GALLINI, P. GIRLANDO, AND A.A. ZUPPA. 2000. Do recombinant human erythropoietin and iron supplementation increase the risk of retinopathy of prematurity? Eur J Pediatr. 159:627-634
SAKANAKA, M., T.C. WEN, S. MATSUDA, S. MATSUDA, E. MORISHITA, M. NAGAO. AND R. SASAKI. 1998. In vivo evidence that erythropoietin protects neurons from ischemic damage. Pro. Natl. Acad. Sci. USA. 95: 4635-4640.
SATO, T., S. KUSAKA, H. SHIMOJO, AND T. FUJIKADO. 2009. Vitreous levels of erythmpoietin and vascular endothelial growth factor in eyes with retinopathy of prematurity. Oph. 116(9): 1599-1603.
SCHNEIDER, J.K., D.K. GARDNER, AND L. CORDERO. 2008. Use of recombinant human erythropoietin and risk of severe retinopathy in extremely low-birth-weight infants. Pharmacother. 28(11): 1335-1340.
SHAH, N., P. JADAV, D. JEAN-BAPTISTE, J. WEEDON, L.M. COHEN, AND M.R. KIN. 2010. The effect of recombinant human erythropoietin on the development of retinopathy of prematurity. Am. J. Perinatol. 27(1): 67-71.
SHINGO, T., S.T. SOROKAN, T. SHIMAZAKI, AND S. WEISS. 2001. Erythropoietin regulates the in vitro and in vivo production of neuronal progenitors by mammalian forebrain neural stem cells. J Neurosci. 21(24): 9733-9743.
SILVERBERG, D.S., D. WEXLER, M. BLUM, G. KEREN, D. SHEPS, E. LEIBOVITCH, D. BROSH, S. LANIADO, D. SCHWARTZ, T. YACHNIN, I. SHAPIRA, D. GAVISH, R. BARUCH, B. KOIFMAN, C. KAPLAN, S. STEINBRUCH, AND A. IAINA. 2000. The use of subcutaneous erythropoietin and intravenous iron for the treatment of the anemia of severe, resistant congestive heart failure improves cardiac and renal function and functional cardiac class, and markedly reduces hospitalizations. J Am Coll Cardiol. 35: 1737-1744.
SILVERBERG, D.S., D. WEXLER, M. BLUM, J.Z. TCHEBINER, D. SHEPS, G. KEREN, D. SCHWARTZ, R. BARUCH, T. YACHNIN, M. SHAKED, I. SCHWARTZ, S. STEINBRUCH, AND A. IAINA. 2003. The effect of correction of anaemia in diabetics and non-diabetics with severe resistant congestive heart failure and chronic renal failure by subcutaneous erythropoietin and intravenous iron. Neph., Dial., Transplant. 18: 141-146.
SILVERBERG, D.S., D. WEXLER, D. SHEPS, M. BLUM, G. KEREN, R. BARUCH, D. SCHWARTZ, T. YACHNIN, S. STEINBRUCH, I. SHAPIRA, S. LANIADO, AND A. IAINA. 2001. The effect of correction of mild anemia in severe, resistant congestive heart failure using subcutaneous erythropoietin and intravenous iron: a randomized controlled study. J Am Coll Cardiol. 37: 1775-1780.
SINCLAIR, S.H., C. DELVECCHIO, AND A. LEVIN. 2003. Treatment of anemia in the diabetic patient with retinopathy and kidney disease. Am J Opthalmol. 135:740-743.
SOLTAU, J. AND T. ZIMMERMAN. 2002. Changing paradigms in the medical treatment of glaucoma. Survey Opht. 47(1): 2-5.
SONG, B.J., H. CAI, I.J.C. TSAI, S. CHANG, M. FORBES AND L. V. DEL PRIORE. 2008. Intravitreal recombinant human eryhtropoietin: a safety study in rabbits. Curr. Eye Res. 33: 750-760.
STUDER, L., M. CSETE, S.H. LEE, N. KABBANI, J. WALIKONIS, B.WOLD, AND R. MCKAY. 2000. Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in oxygen. J Neurosci. 20(19): 7377-7383.
SUK, K.K., J.A. DUNBAR, A. LIU, N.S. DAHER, C.K. LENG, J.K. LENG, P. LIM, S. TELLER, AND E. FAYARD. 2008. Human recombinant erythropoietin and the incidence of retinopathy of prematurity: a multiple regression model. J AAPOS. 12(3): 233-238.
SUN, Z.K., H.Q. YANG, J. PAN, H. ZHEN. Z.Q.WANG, S.D. CHEN, AND J.Q. DING. 2008. Protective effects of erythropoietin on tau phosphorylation induced by beta-amyloid. J Neurosci Res. 86(13): 3018-3027.
TAKAGI, H., D. WATANABE, K. SUZUMA, M. KURIMOTO, I. SUZUMA, H. OHASHI, T. OJIMA, AND T. MURAKAMI. 2007. Novel role of erythropoietin in proliferative diabetic retinopathy. Diabetes Res. Clin. Pract. 77 Suppl 1: S62-64.
TIMMER, S.A., K. DE BOER, P. KNAAPEN, M.J. GOTTE, AND A.C. VAN ROSSUM. 2009. The potential role of erythropoietin in chronic heart failure: from the correction of anemia to improved perfusion and reduced apoptosis.J. Card. Fail. 15(4): 353-361.
TSAI, J.C. 2008. Safety of intravitreally administered recombinant erythropoietin (an AOS thesis). Trans. Am. Ophthalmol. Soc: 106: 459-472.
TSAI, J.C., W. LI, B. WORGUL, M. FORBES, AND C. JINGTAI. 2005. Intravitreal administration of erythropoietin and preservation of retinal ganglion cells in an experimental rat model of glaucoma. Curr Eye Res. 30(11): 1025-1031.
TSAI, J.C., B.J. SONG, L. WU, AND M. FORBES. 2007. Erythropoietin: a candidate neuroprotective agent in the treatment of glaucoma. J Glaucoma.16(6): 567-571.
TWERSKY, L.H., A.D. BARTLEY, N. RAVOS, AND W.D. COHEN. 1995. Immature erythroid cells with novel morphology and cytoskeletal organization in adult Xenopus. Protoplasma. 185: 37-49.
VILLA, P., P. BIGINI, T. MENNINI, D. AGNELLO, T. LARAGIONE, A. CAGNOTTO, B. VIVIANI, M. MARINOVICH, A. CERAMI, T.R. COLEMAN, M. BRINES, AND P. GHEZZI. 2003. Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis.J. Expt. Med. 198: 971-975.
VOGIATZI, G., A. BRIASOULIS, D. TOUSOLIS, N.PAPAGEORGIOU, AND C. STEFANADIS. 2010. Is there a role for erythropoietin in cardiovascular disease? Expert Opin. Biol. Ther. 10(2): 251-264.
WANG, X., C. ZHU, X. WANG, J.G. GERWIEN, A. SCHRATTENHOLZ, M. SANDBERG, M. LEIST, AND K. BLOMGREN. 2004. The nonerythropoietic asialoerythropoietin protects against neonatal hypoxia-ischemia as potently as erythropoietin. J Neurochem. 91: 900-910.
WANG, Z.Y., L.J. SHEN, L. TU, D.N. HU, G.Y. LIU, Z.L. ZHOU, Y. LIN, L.H. CHEN, AND J. QU. 2009A. Erythropoietin protects retinal pigment epithelial cells from oxidative damage. Free Radic. Biol. Med. 46(8): 1032-1041.
WANG, Z.Y., K.K. ZHAO, Z.M. SONG, L.J. SHEN, AND J. QU. 2009B. Erythropoietin as a novel therapeutic agent for atrophic age-related macula degeneration. Med. Hypotheses. 72(4): 448--450.
WATANABE, D., K. SUZUMA, S. MATSUI, M. KURIMOTO, J. KIRYU, M. KITA, I. SUZUMA, H. OHASHI, T. OJIMA, T. MURAKAMI, T. KOBAYASHI, S. MASUDA, M. NAGAO, N. YOSHIMURA, AND H. TAKAGI. 2005. Erythropoietin as a retinal angiogenic factor in proliferative diabetic retinopathy. N Engl J Med. 353(8): 782-792.
WEISHAUPT, J.H., G. ROHDE, E. POLKING, A. SIREN, H. EHRENREICH, AND M. BAHR. 2004. Effect of erythropoietin axotomy-induced apoptosis in rat retinal ganglion cells. Inv. Oph. Vis. Sci. 45:1514-1522.
WU, H., S.H. LEE, J. GAO, X. LIU, AND M.L. IRUELA-ARISPE. 1999. Inactivation of erythropoietin leads to defects in cardiac morphogenesis. Development. 126(16): 3597-3605.
WU, L., S. CHANG, Y. CHEN, Q. XIA, M. FORBES, AND J.C. TSAI. 2008. Erythropoietin receptor plays a role in the cell differentiation of retina and lens during eye development. Invest. Oph. Vis. Sci. 49: 3078-D736.
XIE, Z., X. WU, Q. QIU, Y. GONG, Y. SONG, Q. GU, AND C. LI. 2007. Expression pattern of erythropoietin and erythropoietin receptor in experimental model of retinal detachment. Curr. Eye Res. 32(9): 757-764.
YAMASAKI, M., H.K. MISHIMA, H. YAMASHITA, K. KASHIWAGI, K. MURATA, A. MINAMOTO, AND T. INABA. 2.005. Neuroprotective effects of erythropoietin on glutamate and nitric oxide toxicity in primary cultured retinal ganglion cells. Brain Res. 1050(1-2): 15-26.
YU, X., C.S. LIN, F. COSTANTINI, AND C.T. NOGUCHI. 2001. The human erythropoietin receptor gene rescues erythmpoiesis and developmental defects in the erythropoietin receptor null mouse. Blood. 98(2): 475-477.
YU, X., J.J. SHACKA, J.B. EELLS, C. SUAREZ-QUIAN, R.M. PRZYGODZKI, B. BELESLIN-COKIC, C.S. LIN, V.M. NIKODEM, B. HEMPSTEAD, K.C. FLANDERS, F. COSTANTINI, AND C.T. NOGUCHI. 2002. Erythropoietin receptor signalling is required for normal brain development. Development. 129(2): 505-516.
ZHANG, J., Y. LI, Y. CUI, J. CHEN, M. LU, S.B. ELIAS, AND M. CHOPP. 2005. Erythropoietin treatment improves neurological functional recovery in EAE mice. Brain Res. 1034: 34-39.
ZHANG, J., Y. Wu, Y. JIN, F. JR, S.H. SINCLAIR, Y. LUO, G. XU, L. LU, W. DAI, M. YANOFF, W. LI, AND G.T. XU. 2008. Intravitreal injection of erythropoietin protects both retinal vascular and neuronal cells in early diabetes. Inv. Oph. Vis. Sci. 49(2): 732-742.
ZHONG, L,J. BRADLEY, W. SCHUBERT, E. AHMED, A.P. ADAMIS, D.T. SHIMA, G.S. ROBINSON, AND Y.S. NG. 2007. Erythropoietin promotes survival of retinal ganglion cells in DBA/2J glaucoma mice. Inv. Opt. Vis. Sci. 48(3): 1212-1218.
ZHONG, Y.S., X.H. LIU, Y. CHENG, AND V.J. MIN. 2008. Erythropoietin with retrobulbar administration protects retinal ganglion cells from acute elevated intraocular pressure in rats. J. Ocul Pharmacol Thor. 24(5): 453-459.
ZHONG, Y., H. YAO, L. DENG, Y. CHENG, AND X. ZHOU. 2007. Promotion of neurite outgrowth and effect of erythropoietin on the retinal neurons of rats. Graefes Arch Clin Exp Ophthalmol. 245(12): 1859-1867.
ZHU, B., W. WANG, Q. GU, AND X. XU. 2008. Erythropoietin protects retinal neurons and glial cells in early-stage streptozotocin-induced diabetic rats. Exp. Eye Res. 86(2): 375-382.
LAURA H. TWERSKY, AUGUST J. GENEROSO, WENDY FLORES, AND E. REGINA GIULIANI
DEPARTMENT OF BIOLOGY, SAINT PETER'S COLLEGE, JERSEY CITY, NJ 07306, LTWERSKY@SPC.EDU
Table 1. Abbreviations ARMD Age-Related Macular Degeneration asialoEPO Asialoerythropoietin BAX Bcl-2-associated X BBB Blood-Brain Barrier Bcl-2 B-Cell Lymphoma-2 BRB Blood-Retina Barrier CEPO Carbamylated Erythropoietin DR Diabetic Retinopathy EPO Erythropoietin EPOR Erythropoietin Receptor ESA Erythropoiesis-Stimulating Agent ETDRS Early Treatment Diabetic Retinopathy Study INL Inner Nuclear Layer IOP Intraocular Pressure JAK2 Janus Kinase-2 LEC Lens Epithelial Cell NO Nitric Oxide ONL Outer Nuclear Layer OP Oscillatory Potential PDR Proliferative Diabetic Retinopathy PI-3 Phosphatidylinositol-3 RD Retinal Detachment RGC Retinal Ganglion Cell rhEPO Recombinant Human Erythropoietin ROP Retinopathy of Prematurity ROS Reactive Oxygen Species RP Retinitis Pigmentosa RPE Retinal Pigment Epithelium siRNA Small Interfering RNA SNP Sodium Nitroprusside STAY Signal Transducer and Activator of Transcription TUNEL Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling VEGF Vascular Endothelial Growth Factor Table 2. Effects of Exogenous EPO on the Diabetic Eye CONDITION MODEL TREATMENT RESULTS Patient 1: Right eye Diabetes Human 5 azotemic anemic acuity went from pre-end stage renal 20/200 to 20/60; disease diabetic exudates went from subjects were ETDRS grade of 5 treated with EPO for to 3 1 year for anemia. Maintenance doses of Patient 2: Right eye EPO were given to acuity went from 20/ sustain the 400 to 20/200; hematocrit above exudates went from 32%. ETDRS grade of 5 to 4. Patient 3: Visual acuity improved, substantial resorption of macular edema and hard exudates, and ETDRS grade went from 5 to 3. substantial resorption of macular edema and hard exudates, and ETDRS grade went from 5 to 3. Early Rat EPO via intravitreal A single Diabetes injection (0.05ng- intravitreal 200ng) at onset of injection of EPO induced diabetes (0.05ng-200ng) at the onset of diabetes prevented breakdown in the BRB, a significant reduction in retinal thickness and number of cells in the ONL, and vascular and photoreceptor cell death. Early Rat EPO via No decrease in Diabetes intraperitoneal amplitudes of b- injection (5,000 IU/ wave and OPs, no kg of b.w.) 3 times/ mitochondrial week for 2 weeks metamorphosis in after diabetes ganglion cells, and induction statistically significant increase in retinal glutamate content. CONDITION MODEL CONCLUSION REFERENCE The three patients Friedman et al., Diabetes Human with macular edema 2003 displayed retention of vision and resolution of exudates possibly as a result of increased hematocrit due to the administration of EPO. Early Rat EPO can serve as Diabetes prophylaxis against Zhang et al., 2008 diabetic complications in the eye. Early Rat EPO protects retinal Zhu et al., 2008 Diabetes and glial cells in early stage diabetes. Table 3. Effects of Exogenous EPO on Glaucoma CONDITION MODEL TREATMENT Glaucoma Mouse Intraperitoneal (via raised injection of various IOP) doses of EPO starting at the age of 6 months and continuing for an additional 2, 4, or 6 months Glaucoma (via Rat Retrobulbar raised IOP) injection of 1000 U of rhEPO immediate immediately a after onset of acute elevated IOP Glaucoma (via Mouse Transgenic mouse optic nerve line tg21 that transection) constitutively expresses human EPO Glaucoma (via Rat Intravitreal optic nerve injections of 2 U transection) EPO/eye on days 0, 4, 7, and 10 after axotomy Glaucoma (via Rat Intravitreal optic nerve injection of EPO (5, transective 10, 25, and 50 U) immediately after transection Glaucoma Rat Single intravitreal (induced by 200ng dose of EPO episcleral vessel cautery) Glaucoma Rat Intravitreal (via ocular injection of 2U hyperten- rhEPO at 0, 4, 7, sion by laser and 1 days after coagulation first laser coagulation; Intraperitoneal injection of rhEPO at 5,000 units 24 h before or 30 min before first laser coagulation injury Glaucoma (via Rat Cultured retinal glutamate and cells were nitric oxide pretreated with EPO toxicity) for 12 hrs. Glaucoma (via Rat Cultured retinal glutamate- cells were incubated induced with various doses cytotoxicity) of EPO Glaucoma (via Rat Cultured retinal glutamate- cells were incubated induced with various doses cytotoxicity) of EPO. CONDITION MODEL RESULTS Glaucoma Mouse Relative to (via raised untreated mice, at IOP) 3000 U/kg (similar results at 6000, and 12,000 U/kg) number of viable RGCs increased by 23.90% after 4 months and number of viable RGCs increased by 41.15% after 6 months Glaucoma (via Rat Number of surviving raised IOP) RGCs per square millimeter: Acute elevated IOP + rhEPO retrobulbar injection group = 2142 [+ or -] 98; Acute elevated IOP = 1733 [+ or -] 62; Acute elevated IOP + vehicle solution = 1724 [+ or -] 102 Glaucoma (via Mouse 14 days after optic nerve lesioning: transection) Transgenic wild- type mice displayed 61.4 [+ or -] 20.6% viable RGCs. Nontransgenic wild- type mice displayed only 21.3 [+ or -] 7.6% viable RGCs Glaucoma (via Rat Enhanced RGC optic nerve survival by 92%, transection) from 455 [+ or -] 95.7 surviving RGCs- [mm.sup.2] to 872 [+ or -] 43.5 Glaucoma (via Rat After four weeks: optic nerve Control = 253 RGCs/ transective [mm.sup.2]; 5 U = 333 RGCs/ [mm.sup.2], 10 U = 344 RGCs/[mm.sup.2] Glaucoma Rat RGC count in retinas (induced by after 21 days: episcleral Episcleral Vessel vessel cautery) Cautery only = 9116 [+ or -] 273; Episcleral Vessel Cautery Normal Saline = 9489 [+ or -] 293; Episcleral Cautery EPO treated = 11,212 [+ or -] 414, a non- significant decrease to control (12,619 [+ or -] 310) Glaucoma Rat Intravitreal (via ocular injection resulted hyperten- in enhanced survival sion by laser of RGCs by -1.5 [+ coagulation or -] 3.08% in comparison to control eyes on day 14 injury. Intraperitoneal injection resulted in no loss of RGCs, while control group lost 12.8 [+ or -] 4.2%. Glaucoma (via Rat Pretreatment with glutamate and EPO for 12h at a nitric oxide concentration of .5 toxicity) U/ml (or high dose of 1.5 U/ml) significantly prevented the toxic effects of 1 mM glutamate. Glaucoma (via Rat Neurite outgrowth glutamate- length of the induced retinal neurocytes: cytotoxicity) 1.0 U/ml of EPO = 135% increase; 3.0 U/ml of EPO = 155.7% increase; 6.0 U/ml of EPO =1 62.8% Glaucoma (via Rat 1 U/ml, 3 U/ml, and glutamate- 6 U/ml of EPO induced improved early/ cytotoxicity) total apoptosis rates of the cultured neurocytes incubated with 5 mM/ l glutamate. (Only 6 U/ml EPO significantly improved the early/ total apoptosis of the cultured neurocytes incubated with 10 mM/L glutamate.) CONDITION MODEL CONCLUSION REFERENCE Glaucoma Mouse (via raised Systemic Zhong, L. et IOP) administration of al., 2007 EPO prevents the loss of RGCs,and does so without affecting IOP Glaucoma (via Rat Retrobulbar Zhong et al., 2008 raised IOP) administration of EPO should be considered because it is not only effective in protecting RGCs, but the procedure also benefits RGC ultrastructure. Glaucoma (via Mouse rhEPO protects RGCs Kilic et optic nerve after axotomy. al., 2004 transection) Glaucoma (via Rat Intravitreal Weishaupt et al., optic nerve injection of EPO has 2004 transection) a protective effect on RGC. Glaucoma (via Rat Intravitreal King et al., 2007 optic nerve injection of EPO has transective a protective effect on RGC. Glaucoma Rat Intravitreal Tsai et al., 2005 (induced by injection of EPO has episcleral a protective effect vessel cautery) on RGC. Glaucoma Rat Intravitreal and Fu et al., 2008 (via ocular intraperitoneal hyperten- injections of EPO sion by laser rescues RGCs after coagulation chronic ocular hypertension. Glaucoma (via Rat EPO significantly Yamasaki et glutamate and reduced RGC death al., 2005 nitric oxide induced by glutamate toxicity) and NO. Glaucoma (via Rat EPO, in a dose- Zhong, Y. et glutamate- dependent man-ner, al., 2007 induced results in an cytotoxicity) improvement of neurite outgrowth of dissociated retinal neurocytes in viva. Glaucoma (via Rat EPO promotes Zhong, Y. et glutamate- increased survival al., 2007 induced and decreased early cytotoxicity) and total apoptosis caused by glutamate toxicity. Table 4. Effects of Exogenous EPO on Retinitis Pigmentosa (RP) and Age- Related Macular Degeneration (ARMD) CONDITION MODEL TREATMENT RESULTS RP and ARMD Mouse Intraperitoneal Robust protection of injection of rhEPO photoreceptors in (5,000 IU/kg) within the retina a timeframe of 1 hour before and 1 hour after onset of light exposure Transgenic (tg6) After exposure to 13 RP and ARMD Mouse mice, which klux of white light constitutively for 2 h, wild type overexpressed rhEPO exhibited photoreceptor cell death in a large central area, while the affected area in tg6 mice was smaller and not all photoreceptor nuclei were pyknotic. Also, 12 d after fight exposure, wild type had 1-2 rows of surviving photoreceptor nuclei, while tg6 had 4-5 rows. RP and ARMD Rat Intraperitoneal rhEPO injected 1 h injection of 30,000 before light insult U/kg of rhEPO, 1 hr preserved retinal or 4 firs before function but not exposure to damaging structure. rhEPO light (24 hours; injected 4 h before 2200 lux) light exposure, protected both retinal function and structure. Administration of 1 Among others, RP and ARMD Human IU-ml of EPO in significant increase cultured oxidant- in viability of RPE, treated retinal a reduction in the pigment epithelia. release of inflammatory cytokines, and restored cellular antioxidant potential CONDITION CONCLUSION REFERENCE RP and ARMD Systemic administration Grimm et al., 2002 of EPO protects photoreceptor cells. RP and ARMD rhEP0 protects Grimm et al., 2004 photoreceptor cells from light-induced degeneration. RP and ARMD EPO also can Ranchon Cole et protectagainst long al., 2007 term light damage. RP and ARMD EPO can delay or Wang et al., 2009a prevent apoptosis of the retinal pigment epithelium caused by oxidative damage caused by reactive oxygen species. Table 5. Endogenous EPO in Variuos Ocular Diseases CONDITION MODEL OBSERVATION CONCLUSION ROP Human Significant positive Both VEGF and EPO correlation exists may contribute to between the the pathogenesis of endogenous EPO and ROP. VEGF levels in moderately and mildly vascular- active ROP eyes. PDR Human EPO level in Vitreous EPO levels vitreous: 73 are higher in patients with PDR = patients with PDR 464.0 mIU/mL vs. 71 than in non- patients w/o diabetic patients. diabetes = 36.5 mIU/ mL PDR Mouse Intraretinal Inhibition of injections of endogenous EPO in soluble EPOR the retina may stop resulted in harmful blood vessel reduction of formation in the neovascularization. eye. 25 ng = 65% reduction; 62.5 = 59%ng reduction; 250 ng = 55% reduction. PDR Human EPO level in Vitreous EPO levels vitreous: patients are higher in with PDR= 512.0 mU/ patients with PDR mL vs. patients w/o than in non- diabetes = 25.1 mU/ diabetic patients. mL (the more active the PDR the higher the EPO concentration) Diabetic Human EPO level in EPO concentrations Macular vitreous: 12 are higher in Edema patients w/ DME patients with DME. 12.4mU/mL; 12 patients w/ PDR =10 mU/ml; 10 patients w/o diabetes = 8.7mU/ml Diabetic Human EPO level in aqueous EPO level in the Macular humor:28 patients aqueous humor are Edema vs. with DME = 60.1 f higher in patients Exudative ARMD 46.7 mU/mL vs. 59 with diabetic and Cataracts patients w/ARMD = macular edema than 23.2 [+ or /] 23.2 in those with mU/mL; 49 patients exudative ARMD and with cataracts = cataracts. 22.0 [+ or /] 21.0 mU/mL Glaucoma Rat 2 weeks after ocular Soluble EPOR (via ocular hypertension, 13.9 exacerbated ocular hypertension [+ or -] 1.8% total hypertensive injury, by laser RGCs were lost. indicating it coagulation) After treatment with neutralized soluble EPOR (via endogenous EPO intravitreal (shows EPO serves to injection), 20.7 protect RGCs after [+ or -] 3.7% RGCs injury). were lost. RP and ARMD Mouse Pretreatment with 6% Pretreatment with and 10% oxygen hypoxia protects protected both photoreceptors from retinal morphology apoptotic cell death and retinal function after exposure to substantially. damaging light levels in viva Possibly by stabilizing HIF-1, which controls EPO (endogenous) expression. RD Rat mRNA and protein EPO/EPOR system may EPO/EPOR levels serve in protection increase with the of retinal neurons duration of during RD, and detachment. They exogenous EPO may reach their peaks at promote survival. 48 hours after RD. CONDITION MODEL REFERENCE(S) ROP Human Sato et al., 2009 PDR Human Watanabe et al., 2005; Takagi et al., 2007 PDR Mouse Watanabe et al., 2005 PDR Human Asencio-Sanchez et al., 2008 Diabetic Human Hernandez et Macular al., 2006 Edema Diabetic Human Jonas and Macular Neumaier, 2007 Edema vs. Exudative ARMD and Cataracts Glaucoma Rat Fu et al., 2008 (via ocular hypertension by laser coagulation) RP and ARMD Mouse Grimm et al., 2004 RD Rat Xie et al., 2007