Experimental Hematology 2009;37:1007–1015
Erythropoietin and erythropoiesis
Walter Fried
Oncology Specialists, Park Ridge, Ill., USA
(Received 20 April 2009; revised 20 May 2009; accepted 22 May 2009)
Although the concept of a humoral regulator of erythropoiesis was introduced more than 100
years ago, its existence was first firmly established a little more than 50 years ago. This review
briefly describes the historical development of information about erythropoietin. It then
describes our current understanding of where erythropoietin is produced; the factors that
regulate its rate of production; how erythropoietin acts at the cellular level to stimulate erythropoiesis; and its role in the regulation of the rate of erythropoiesis. Finally, it discusses the
clinical uses of erythropoietin in the diagnosis and therapy of hematopoietic diseases. Ó 2009
ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.
In 1882, Dr. Bert and his colleagues [1] took their microscope
high into the Mexican mountains and showed, for the first
time, that the amount of red blood cells is increased when
residing at a high altitude. They postulated that the rate of
erythropoiesis is regulated by the oxygen supply to the site
of red blood cell (RBC) production. This was the opening
salvo in the quest to understand regulation of erythropoiesis.
In 1906, when the concept of the humoral transmission
of information from one site to a distant one via the
bloodstream was new, two French scientists, Carnot and
DeFlandre [2], postulated that the effect of hypoxia on
erythropoiesis was mediated by a hormone, which they
named haemopoietin. To test this hypothesis, they bled
rabbits of 10 mL. Then they collected their plasma and
injected 10 mL intravenously into normal rabbits. They
claimed that this caused an increase in the recipient rabbits’
RBC count. These experiments were then, and are not
now, reproducible. However, the possibility of a hormonal
control of erythropoiesis was not rejected. In 1952,
Drs. Grant and Root [3] reviewed the studies on regulation
of erythropoiesis, including experiments that Dr. Grant
performed using an oxygen-sensitive electrode that was
implanted into the bone marrow of anemic animals. Despite
the anemia, the bone marrow oxygen tension remained
normal. They, therefore, concluded that the effect of
hypoxia on erythropoiesis could not be exerted directly
on the marrow. They considered a humoral mechanism to
be likely. In 1950, Dr. Kurt Reissman [4] parabiosed rats.
Offprint requests to: Walter Fried, M.D., Oncology Specialists, 1700
Luther Lane, Park Ridge, IL 60068; E-mail: fried_walt@hotmail.com
He then subjected one parabiont to hypoxia, but showed
that both rats became plethoric. He was able to document
that only one of the parabionts became hypoxic, and also
that there was capillary and small blood vessel communication between the parabionts that permitted transport of
humoral factors between them.
In 1953, Dr. Alan Erslev [5] provided the first direct and
reproducible evidence for a humoral factor in the plasma of
anemic rabbits that increased the reticulocyte count of
normal rabbits injected with the anemic rabbit plasma.
He bled rabbits of about 150 mL blood to reduce their
hematocrits to about 20%. Then he collected their plasma
and injected normal rabbits with 200 mL anemic rabbit
plasma. He documented a significant increase in the reticulocyte counts of the normal recipient rabbits. This was
essentially a repeat of the experiments of Carnot and
DeFlandre [2], which were performed 50 years previously;
however, with induction of much more severe anemia and
injection of 20-fold more anemic plasma into the normal
recipients. Also key to their success was the use of the
reticulocyte count, which was not available to the earlier
investigators, to assess erythropoiesis.
The year 1953 was also an auspicious one for me. As
a 4th-year premed student, I joined a classmate of mine,
Louis Plzak, in the laboratory of Dr. Leon O. Jacobson.
Dr. Jacobson was the Director of the Hematology Section
at the University of Chicago and the Director of the
Argonne Cancer Research Hospital (a hospital supported
by the Atomic Energy Commission to study the role of
radioactive isotopes in medicine). Dr. Jacobson was already
a renowned scientist whose studies on shielding spleens of
mice to protect their hematopoietic system from radiation
0301-472X/09 $–see front matter. Copyright Ó 2009 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.
doi: 10.1016/j.exphem.2009.05.010
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W. Fried/ Experimental Hematology 2009;37:1007–1015
damage led to the first evidence of the existence of multipotential hematopoietic stem cells that are capable of regenerating the total hematopoietic system after radiation damage.
Despite recognizing that the radioprotective effects of
spleen-shielding were due primarily to sparing of hematopoietic stem cells that could travel from the spleen to
repopulate sites in the marrow, Dr. Jacobson still had
a suspicion that there was a humoral factor released by
the shielded spleen that was instrumental in promoting
regeneration of hematopoiesis. Therefore, Dr. Jacobson
became very interested in the humoral factor that Dr. Erslev
had identified. At that time, Louis Plzak and I were
studying the uptake of radioactive iron into avian erythrocytes as a way of measuring erythropoiesis. Dr. Jacobson
sensed the importance of using radioactive iron uptake
into mammalian erythrocytes to develop a practical
bioassay for the humoral factor that Dr. Erslev had reported.
He asked Louis Plzak and I to work on developing such an
assay. He suggested that we use rats as the test animals.
In 1955, we [6] reported the development of a practical
but still crude assay for detecting erythropoietin (EPO).
By injecting 10 mL plasma from anemic rats into normal
rats, we were able to demonstrate a significant effect on
the rate of uptake of Fe59 into newly formed erythrocytes
of the recipients. In the subsequent year, we were able to
increase the sensitivity of the assay several fold by using
animals that had a plethora of oxygen compared to their
needs. The first such model was the hypophysectomized
rat [7] that had a markedly reduced metabolic rate and
therefore had more RBCs than needed for the first few
weeks postoperatively. Next we used transfusion-induced
plethoric rats. The concept was that in plethoric rats, EPO
production and consequently the serum EPO titer should
decrease to subnormal levels and then these animals
would become very sensitive to exogenous EPO. In 1962,
Dr. DeGowin et al. [8] described the posthypoxic mouse
model. Mice were made plethoric in a chamber evacuated
to one-half atmosphere. After 2 weeks, they were plethoric
and very sensitive to exogenous EPO. Compared to the
initial experiments that required 200 mL and then 10 mL
anemic plasma, these posthypoxic mice could detect
increased EPO titers in as little as 0.5 mL. Also, they could
detect EPO in rabbit, rat, mouse, and human plasma. This
bioassay became the standard for assaying EPO for 20 years
until development of a reliable immunoassay.
Using these newly available bioassays, we studied the
effect of a variety of factors on erythropoietin production,
including cobaltous chloride [9] and androgenic [10] and
anabolic steroids. In 1956, we began to investigate the
effect of removing various organs from rats on their ability
to produce EPO in response to various stimuli. We failed to
show any effect on EPO production of removing the various
endocrine glands. Then our classmate, Sandy Krantz,
suggested that we might consider the kidneys because
patients with renal failure were always anemic and some
with hypernephromas became plethoric. This called to
mind a study by Stohlman et al. [11]. He was called to
see a child who had a patent ductus that emptied into the
aorta below a coarctation. Consequently, the child was
pink above the diaphragm and blue below. Dr. Stohlman
aspirated bone marrow from both the sternum and the iliac
crest. Both sites had marked erythroid hyperplasia. The
implication was that erythropoiesis is regulated by
a humoral factor that originates from somewhere below
the diaphragm. Our next study was to perform bilateral
nephrectomies on one group of rats, ligate the ureters of
another group, and perform a laparotomy only on a third
group. Only the nephrectomized rats failed to produce
a significant amount of EPO in response to anemia, injection of cobaltous chloride, or hypoxia. Results of some of
these studies were first reported in 1957 [12] and provided
the first direct evidence for the role of the kidney in the
production of EPO.
In 1977, Drs. Miyake and Goldwasser [13] succeeded in
purifying EPO from the urine of patients with aplastic
anemia. This led to a logarithmic increase in the rate at which
new knowledge was acquired. The clumsy, technicianintensive bioassays were replaced by more-sensitive and
more-precise immunoassays. Scientists at Amgen [14],
working with antibodies against purified EPO, isolated
complementary DNA and were able to transfect this into
hamster ovary cells, grown en-masse to produce commercial
amounts of EPO for clinical use.
EPO is a 30.4-kD glycoprotein with four carbohydrate
residues [13]. The latter prevent EPO from being rapidly
cleared from the blood by the liver. EPO interacts as ligand
for a specific receptor [15] (EPO-R) found predominantly
on erythroid precursor cells. EPO-R is not found on multipotential hematopoietic stem cells and is not found on the
early burst-forming units-erythroid (earliest committed
erythroid precursors that form bursts of subcolonies in
culture). Therefore, EPO is not required for cell differentiation. It begins to appear on the later burst-forming unitserythroid and is found in highest concentration on
colony-forming units-erythroid (erythroid precursors that
are only one or two divisions upstream from the first
morphologically recognizable erythroid precursors, the
proerythroblasts) [16] and proerythroblasts. These cells
are the erythropoietin responsive cells (ERC).
EPO-R contains two polypeptide chains on its extracellular component, which become homodimerized after
interaction with their ligand [17]. This, in turn, activates
the receptor and results in phosphorylation of Jak-2, which
is either in close proximity or attached to the intracellular
portion of EPO-R. This results in transmission of the signal
via the Jak/Stat pathway and possibly also via Ras [18,19].
The signal that is transmitted to the nucleus prevents
apoptosis and permits the cell to complete its terminal
maturation into a reticulocyte and subsequently into an
erythrocyte. EPO also increases the size of the ERC
W. Fried/ Experimental Hematology 2009;37:1007–1015
population by stimulating proliferation of these cells.
Accordingly, in an intact erythropoietic system, one in
which the hematopoietic microenvironment, the multipotential stem cells and the early erythroid precursors
(burst-forming units-erythroid) are intact and the nutrients
required for terminal maturation are not limited, the rate
of erythropoiesis is a function of the number of intact
ERC and the amount of circulating EPO. Therefore, one
would expect that as the ERC population decreases
in numbers and/or function, the dose of EPO required to
maintain the rate of erythropoiesis will increase. This has
been demonstrated in mice exposed to increasing doses of
irradiation [20] and is compatible with what is observed
in patients with refractory anemias that are treated with
EPO [21]. A corollary of this is that the amount of EPO
required to maintain a given rate of erythropoiesis should
be less if the ERC are increased. This is likely to be the
explanation for the compensated hemolytic state. People
with a chronic hemolytic condition, such as hereditary
spherocytosis, often maintain an increased rate of erythropoiesis to compensate for their shortened RBC lifespan,
with minor or no anemia and no increase in the EPO titer.
The primary site of EPO production in adult mammals is
the kidney [12]. In experimental animals, the site of EPO
production in the kidney is a specialized peritubular cell
found mainly in the proximal tubular region [22]. In fetal
and neonatal life [23,24], the hepatocyte is the prime source
of EPO production and this may continue, to a minor
extent, into adult life [23,25]. In rodents, procedures that
result in regeneration of hepatocytes increase hepatic
EPO production [26]. In humans with end-stage renal
failure, the hemoglobin and hematocrit sometimes increase
during episodes of hepatitis [27]. EPO gene expression
has been demonstrated in various extrarenal sites, including
even the hematopoietic tissue of the bone marrow [28,29].
The physiologic significance of these extrarenal sites
requires clarification. EPO is also produced by some
neoplasms. The most common is the hypernephroma [30]
and the next most common is the hepatoma [31]. However,
a variety of other neoplasms have also been reported to
produce paraneoplastic EPO [32,33]. The paraneoplastic
production of EPO frequently results in erythrocytosis
without hypoxia and with increased serum EPO levels
that persist despite the erythrocytosis.
EPO production is primarily regulated by the oxygen
supply to a sensor in the kidney relative to its oxygen
requirements [34]. Conditions that result in a decrease in
oxygen delivery to the renal sensor will stimulate EPO
production. This includes residence at high altitude,
pulmonary disease that affects the diffusing capacity of
the lungs and results in decreased uptake of oxygen, admixture of arterial and venous blood through a right to left
shunt, decreased renal blood flow, etc. Conditions that
increase oxygen delivery, such as erythrocytosis or
breathing a gas mixture that contains an increased oxygen
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pressure, will decrease production of EPO. In conditions
that decrease the oxygen needs, such as hypothyroidism
and hypopituitarism, a normal rate of oxygen delivery
represents plethora and causes EPO production and the
serum EPO titer to decline. The oxygen sensor probably
resides in proximity to the site of EPO production and
may consist of a heme-containing protein [35].
The EPO gene is activated by a hypoxia-inducible factor
(HIF) [36,37], which attaches to the hypoxia-sensitive
region of the EPO gene to activate it. Because commercial
EPO assays are not sensitive enough to detect small
changes in EPO titer, it is not possible to reliably determine
how great a hypoxic stimulus is required to increase EPO
production. There is evidence that after phlebotomizing
someone without a known erythropoietic defect of
500 mL whole blood, the erythropoietin titer does not
increase detectably [38]. This is not surprising because
there are so many ways in which our bodies can compensate for a small change in the oxygen-carrying capacity
of the blood. Even if the rate of erythropoiesis remains
constant and does not increase, the red cell mass will slowly
recover (after losing 500 mL blood [240 mL RBCs] about
2 mL less RBCs will become senescent every day for
120 days. Accordingly, 2 mL of the lost 240 mL RBCs
will be recovered daily).
Although the ratio of oxygen delivery to oxygen
requirements is the primary physiologic regulator of EPO
production, there are others. Androgenic steroids
[10,39,40], anabolic steroids [41], and cobaltous chloride
[9] stimulate EPO production by an unknown mechanism.
Protein deprivation [34] and inflammatory cytokines,
such as interleukin-6 and tumor necrosis factor [42,43],
decrease EPO production. This contributes to the anemia
of chronic inflammatory disease and the anemia of cancer,
as will be discussed later.
There is also evidence in experimental animals that an
increase in the RBC mass can decrease production of
EPO independent of its effect on oxygen delivery [44,45].
Perhaps this effect is related to the resultant increase in
whole blood viscosity. The plasma EPO titer at any given
severity of anemia is inversely proportional to the effective
rate of erythropoiesis. This has been observed under several
conditions [46,47]. In rats exposed to hypoxia, plasma EPO
titer peaks within 6 to 8 hours, but after 72 hours of
exposure, when the RBC mass is not yet detectably
increased, it is almost back to the prehypoxic level. In
contrast, the rate of erythropoiesis, as measured by Fe59
uptake into newly formed erythrocytes, is increased after
24 hours, peaks after 48 hours of hypoxic exposure, and
persists at a maximum level for 7 days (this is compatible
with the explanation that less EPO is required to maintain
the rate of erythropoiesis if the ERC population is
enlarged). Three possible explanations have been proposed.
The EPO titer may exert a negative feedback effect on EPO
production. This was tested in an animal model and denied
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[48]. The increase in ERC results in consumption of
EPO [47]. This also was tested and found not to be the
case [46,49,50]. Therefore, the most likely possibility is
that the effective ERC population somehow exerts
a negative feedback on EPO production. The phenomenon
of the inverse effect of the ERC population on the plasma
EPO titer has been observed in various situations in
humans. Following intensive chemotherapy, the serum
EPO titer rises before the hemoglobin and the hematocrit
falls [51,52]. Treatment of patients with vitamin B-12
deficiency and with iron deficiency results in a fall in serum
EPO titer before the hemoglobin and the hematocrit
rise [53]. Serum EPO levels of patients with aplastic
anemia and with myelodysplastic syndromes are higher
than in those with comparable anemias associated with
active erythropoiesis [54,55]. However, more direct data
on EPO clearance rates in humans are needed before the
hypothesis that ERC exert a negative feedback effect on
EPO production is proven. It might be of interest to screen
the EPO titers of patients who are considered to be hematologically normal for those that might have an EPO titer
that is O2 standard deviations beyond the normal for the
laboratory. A follow-up of such patients might indicate an
occult marrow dyscrasia.
Once EPO has interacted with its receptor on the
erythroid progenitor cell, that cell can go on to express its
terminal maturation program if the conditions required for
the synchronous synthesis of hemoglobin and DNA are
adequate. As stated earlier, under those conditions, the
rate of erythropoiesis is a function of the serum EPO level
and the size and integrity of the ERC population.
Let us begin with a discussion of the factors that
determine the ability of the erythroid precursors to synthesize hemoglobin. Hemoglobin molecules consist of four
globin chains, each of which has a heme moiety attached.
Defective globin synthesis generally is the result of
mutations of the promoter portion that adversely affect
one or more of the a- or the b-globin genes to produce
a variety of thalassemia. The synthesis of heme occurs in
the mitochondria of the erythropoietic precursor cells and
is dependent on insertion of iron into preformed protoporphyrin molecules. Defective heme synthesis occurs because
of iron deficiency, because iron is not released in sufficient
quantity from its storage sites in the reticuloendothelial
system as occurs in the anemia of chronic disease, or
because there is a defect in either the structure of the
mitochondria or in the ferrochetalase enzyme that catalyzes
the insertion of iron into protoporphyrin. The resultant
accumulation of iron in the mitochondria gives rise to the
ringed sideroblasts, which are the diagnostic feature of
sideroblastic anemias.
To maintain a normal rate of erythropoiesis, the erythron
requires an amount of iron that is equal to the amount
required to replace the RBCs that become senescent daily.
In an average adult 70-kg male, this would amount to about
20 mL RBCs or 20 mg iron. Females require 18 mg.
Assuming 1 mg insensible loss daily, about 17 to 19 mg
iron should become available from senescent RBCs, which
were scavenged by the macrophages that day. If there is no
known source of blood loss, only 1 mg iron should be
required from food sources daily. Females, during their childbearing years, however, lose approximately 30 mg iron
monthly during normal menses; and lose about 750 mg
iron during a 250-day pregnancy. Therefore, premenopausal
females require about 2 mg iron from food sources and
during pregnancy about 4 mg (this assumes that they still
have a 1-mg daily insensible loss).
Most diets contain about 7 mg iron per 1,000 calories.
Worldwide, the average caloric intake is about 2000
calories. Food iron comes in three varieties: ferrous, ferric,
and heme. Iron is absorbed in the proximal duodenum by
specialized cells called enterocytes [56]. Heme iron is
readily absorbed into these cells, whereas ferric and ferrous
iron are subject to chelation by a variety of substances in
various foods. Absorption of ferric iron is also influenced
by the pH of the intestinal lumen, because it is insoluble
in an alkali medium. The divalent metal transporter located
on the luminal surface of enterocytes, in proximity to an
enzyme that reduces ferric iron to ferrous, facilitates
transport of ferrous iron into the enterocyte [57]. The diet
of more prosperous peoples contains a larger amount of
heme iron, which is derived mainly from muscle tissue;
whereas poorer diets include a larger percentage of
inorganic iron.
Once in the enterocyte, iron is either incorporated into
ferritin and sloughed with the aging enterocyte or is
transported through the basolateral surface of the enterocyte
and enters the bloodstream, where it is attached to transferrin
(TF1) for transport to the developing erythroid cells. There
the iron-saturated TF1 interacts with the transferrin receptor
(TFR1) for transport into the cell where the divalent metal
transporter facilitates the release of iron from the TF1 and
its transport to the mitochondria for synthesis of heme.
Regulation of the amount of iron that is transported
from the enterocyte into the bloodstream is critical, as
only 1 or 2 mg is required by the erythroid precursors
and, once their TFR1 is saturated, the remainder is likely
to be taken up into the hepatocytes, the cardiac myocytes,
etc. Accumulation of iron in these cells can cause serious
tissue damage. Iron is transported from the basolateral
surface of the enterocyte through a tubular transmembrane
protein, ferroportin. (FPN1) [58,59]. This gateway protein
is regulated by a hepatocyte generated protein, hepcidin
[60,61] that binds to FPN1 and causes the phosphorylation
of several amino acids on the intracellular portion of FPN1.
This then causes the internalization and ubiquitization of
the FPN1-hepcidin complex. Therefore, an increase in
hepcidin production will decrease iron absorption from
the gastrointestinal tract, whereas a decrease will have the
opposite effect.
W. Fried/ Experimental Hematology 2009;37:1007–1015
Synthesis of hepcidin is regulated by both a storage
regulator and an erythropoietic regulator. The storage
regulator controls production of hepcidin in accord with
the body’s iron needs as determined by the body’s iron
stores. This involves the complex interaction of the atypical
class I major histocompatibility complex protein, HFE (the
hemochromatosis gene), transferrin receptor 2 (TFR2). and
hemojuvalin (HJV). HJV is a glycophosphoinositide linked
protein on the hepatocyte membrane, which functions as
a coreceptor with the receptor for the bone morphogenic
proteins (BMP) [62]. The BMP-HJV receptor, when activated by interaction with its ligand, transmits a signal, by
phosphorylation of SMAD, to the nucleus to activate the
hepcidin gene and thereby increase the synthesis of hepcidin. The activation of the BMP-HJV receptor is postulated
to be linked to the status of the body’s iron stores as
follows: the HFE protein competes with diferric (saturated)
transferrin for a binding site on TFR1. If, as occurs in iron
overload states, there is an excess of diferric transferrin,
then HFE binds to TRF2. The TRF2-HFE complex interacts
with the BMP-HJV receptor to sensitize it and increase the
synthesis of hepcidin. In the case of hypoferremia, on the
other hand, HFE binds preferentially with TRF1. The
TRF1-HFE complex cannot interact with the BMP-HJV
receptor and hepcidin production is not increased.
Increased hepcidin results in decreased iron absorption
and decreased release of iron from the macrophages and
visa versa [63].
The erythropoietic regulator is sensitive to changes in
the rate of erythropoiesis and particularly in the rate of ineffective erythropoiesis [64,65]. An increase in the amount of
ineffective erythropoiesis results in decreased production of
hepcidin. This effect is more potent than that of the storage
regulators and if both iron overload and increased
ineffective erythropoiesis coexist, the erythropoietic effect
will override that of the storage regulator and hepcidin
production will decline and iron absorption will increase
despite iron overload. The mechanism by which increased
ineffective erythropoiesis causes a decrease in hepcidin
synthesis is the subject of active research. Both EPO and
the Hippel von Landau HIF have been shown to decrease
hepcidin production by hepatocytes in vitro [66,67].
GDF15, a member of the transforming growth family
(TGF-b) has been found, by transcription profiling, to be
upregulated during accelerated erythropoiesis and is
capable of suppressing hepcidin production. Therefore,
it is also a potential mediator of the erythropoietic
regulator [68].
Hepcidin is critically involved in regulating the amount
of iron absorbed from the gastrointestinal tract. It is,
however, also involved in regulating the availability of
iron from the reticuloendothelial system. FPN1 is present
on the membrane of the macrophages that phagocytose
senescent RBCs and is the site through which iron is
transported from the macrophages into the bloodstream.
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In addition to the storage regulators and the erythropoietic
regulators of hepcidin production, there is also an
inflammatory regulator, which is mediated through the
inflammatory cytokine interleukin-6 [69]. This acts on
the hepcidin gene in the hepatocyte and possibly also on
a hepcidin gene in the monocyte to stimulate hepcidin
production and thereby to inhibit iron absorption from the
gastrointestinal tract and also to inhibit the release of iron
from the macrophages. Interleukin-6 induced hepcidin
production causes the characteristic changes of the anemia
of chronic inflammatory and/or malignant disease.
The increased hepcidin decreases the number of FPN1
molecules available to recycle iron from senescent erythrocytes and, therefore, patients with malignancy and/or
inflammatory diseases have increased iron stores, decreased
serum iron, and cannot compensate for the loss of RBCs
that become senescent. They, therefore, become anemic.
The anemia of chronic disease has been regarded by
many as a virtual iron-deficiency anemia because there
is a deficiency of iron being delivered to the erythroid
precursors to satisfy their EPO receptors. As mentioned
here, inflammatory cytokines also cause a decrease in
EPO production, which contributes to the anemia.
Let us return now to our average 70-kg male who
requires 20 mg iron daily to compensate for the daily loss
of senescent RBCs. This person requires 1 mg of iron
from food sources, and he will have to recycle 19 mg
iron from his senescent RBCs. Iron derived from newly
phagocytosed RBCs is readily recycled to the erythroid
precursors if the regulatory mechanism for hepcidin
production is functioning normally and an adequate, but
not excessive, amount of FPN1 molecules are present on
the basolateral surface of the enterocytes and on the
membranes of the macrophages. However, if there is an
excess of hepcidin and excessive FPN1 have been internalized and ubiquitized, then there will be a decrease in the
rate of iron absorption from the gastrointestinal tract and
the newly deposited iron from senescent RBCs will not
be completely recycled. The iron then is deposited in
ferritin and with time it is converted to hemosiderin.
The longer the iron is deposited, the less available it
becomes [70]. In experimental animals, newly deposited
iron is most readily mobilized from the macrophages. In
humans, this concept has also been supported by the
following clinical observations: Eschbach et al. [71,72]
noted in their classical study on the treatment of the anemia
of chronic renal failure with EPO that patients who
responded with a brisk increase of their RBC mass often
stopped responding and developed hypoferremia before
their iron stores were exhausted. Similarly, patients with
hemochromatosis who regenerate their RBCs rapidly after
repeated phlebotomies often stop regenerating before their
iron stores are totally exhausted. Conversely, if hepcidin
production is inhibited, as in patients with hemochromatosis, iron uptake from the gastrointestinal tract increases
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and the iron deposited in the macrophages is more
readily released. This results in an increase in the iron-saturated TF1 and consequently in the uptake of iron into
hepatocytes. This also explains why patients with hemochromatosis have less than the expected amount of iron
stored in their macrophages. Persons with increased
ineffective erythropoiesis due to thalassemia intermedia
or myelodysplastic syndromes are prone to develop organ
damage due to iron deposition into their hepatocytes and
cardiac myocytes [73,74]. They absorb excess iron from
the gastrointestinal tract. In addition, the iron that is
deposited into their macrophages after blood transfusion
is rapidly released because the erythropoietic regulator
overpowers the iron storage regulator and the resulting
decrease in hepcidin allows the iron to be transported out
of the macrophages and become deposited in the
hepatocytes.
To complete the discussion of the nutrients required for
the synchronous production of hemoglobin and for
sufficient DNA to complete three terminal divisions,
I will just mention the need for vitamin B-12 and folic
acid to generate endogenous thymidine.
The study of the role of EPO in human disease began in
1957, when Dr. Gurney and his colleagues [75] bioassayed
plasma EPO levels of a large variety of anemic patients and
observed that only those with renal failure regularly
contained reduced EPO levels. This was subsequently
also observed by Gallagher et al. [76]. In 1986, Cotes
et al. [77] developed a reliable immunoreactive assay for
EPO, which used recombinant DNA to produce pure EPO
to obtain a specific anti-EPO antibody. This is the prototype
for the commonly available commercial assays for serum
EPO that are now available. For the most part, serum
EPO in various anemias increases proportionally to the
severity of the anemia [78–80]. However, as mentioned
previously, serum EPO titer of patients with inflammatory
states or malignancies is lower than that of comparably
anemic patients with other anemias [78]. Also serum EPO
titer of patients with anemias due to marrow failure are
higher than those of comparably anemic patients with other
anemias [79,80]. Also, the assay is best able to detect
elevated EPO titers. Because the range of normal EPO titer
is large, identification of reduced EPO titers is often
tenuous [78–80].
Where then is the assay of the EPO titer clinically
useful? It is essential to assay the endogenous EPO
titer before beginning therapy with EPO in patients with
myelodysplasia. If the serum EPO titer is excessive
(O500 U), then it is unlikely that the person will respond
because the very high endogenous EPO level probably is
indicative of a very small effective ERC population. In
patients with erythrocytosis, diagnosis of polycythemia
rubra vera was usually made clinically using the criteria
developed by the Polycythemia Study Group, because
decreased serum EPO titers are not reliably detected [81].
Now, with the discovery of the role of the JAK-2 mutation
in the pathogenesis of polycythemia rubra vera [82], the
determination of EPO titer is even less useful. However,
in patients with erythrocytosis without the common
mutation in the JAK-2 gene, serum EPO titers are still
useful [83]. I have found, however, that it is most useful
to phlebotomize patients to a normal hematocrit prior to
assaying the EPO titer because the plethoric condition
might reduce serum EPO titer of patients with any of the
forms of hypoxia-induced erythrocytosis. If serum EPO
titer is still normal after the hematocrit is normalized, one
might consider one of the less common forms of congenital
erythrocytosis, or a less common mutation of the JAK-2
gene, detection of which requires a different probe [83].
The first clinical use of EPO was reported by Winearls
et al. [84] in the United Kingdom and by Eschbach et al.
[71] in the United States. These were combined phase
I and II trials of patients with chronic renal failure on
hemodialysis. Since then, extensive phase III trials have
documented the efficacy of EPO to correct the anemia of
renal failure in patients both on and off dialysis. Complications are few. However, one must be observant for development of functional iron deficiency, as the available iron
stores are utilized by the rapid increase in erythropoiesis.
Also, hypertension is observed to increase in some patients
and must be treated. More recently, the optimum targeted
hematocrit has been reduced to 11 g because of a suggestion
that there is an increased incidence of thromboembolic
complications if the hematocrit is completely normalized.
Use of EPO has had a marked beneficial effect on the
well-being of most patients with renal failure.
Following the initial studies documenting the
effectiveness of EPO in correcting the anemia of chronic
renal failure, EPO has been used in the management of
a variety of refractory anemias. These include, the anemia
of chronic disease [85]; anemia of patients with various
neoplasms that are undergoing chemotherapy [86], anemias
of patients with myeloma [87]; anemia of some patients
with myelodysplasia and aplastic anemia [88,89], and
anemia associated with the use of antivirals to treat HIV
[90]. The dosage of EPO required to treat these anemias
must be significantly increased relative to that is effective
in correcting the anemia of patients with renal failure.
However, EPO has been successful in reducing the transfusion requirements and improving the quality of life of
many patients with these disorders. It has also been used in
patients without anemia that must undergo frequent phlebotomies to accumulate blood for autologous blood transfusions
at the time of orthopedic surgery [91].
In 2003, Henke and coworkers [92] presented data that
looked at the effect of EPO on the long-term survival
of patients with head and neck cancers undergoing radiotherapy in a randomized double-blind placebo-controlled
trial. Prior to that time, the endpoint of studies on the use of
EPO in patients with the anemia of cancer were its effects
W. Fried/ Experimental Hematology 2009;37:1007–1015
on the blood hemoglobin concentration, transfusion requirements, and effect on quality of life. In Dr. Henke’s study, EPO
raised the hemoglobin, as expected and also the quality of life
of the patients. However, the unexpected and very disturbing
finding was that the patients who received EPO had a significantly shorter overall survival than did those in the placebo
group. Leyland-Jones and colleagues [93] reported that
administration of EPO to maintain normal hemoglobin levels
in patients with metastatic breast cancer also shortened the
overall survival of the EPO recipients. There are shortcomings to both of these studies, which I will not discuss here.
However, there have also been other trials since with similar
results [86,94]. The explanation is uncertain. One possibility
is that in these studies, EPO was given to reach a higher-target
hemoglobin than is now considered to be desirable; thereby
increasing the incidence of thromboembolic events [94].
Another explanation is that EPO interacts with EPO receptors, which have been identified on the tumor cells. There
is, however, uncertainty whether the investigators, studying
these receptors had sufficiently specific antibodies to
separate those that react with EPO receptors from those
that react with heat shock proteins [95]. There is also
evidence suggesting that EPO can react with receptors on
endothelial cells to stimulate angiogenesis [96]. At this
time, the reason(s) for the observed decreased survival of
patients with cancer that receive EPO is unclear. However,
the results of these studies have led to a change in the guidelines for use of EPO to treat the anemia of cancer. These new
guidelines limit the use of EPO to the anemia of patients with
cancer that are undergoing chemotherapy; and reduce the
target blood hemoglobin concentration to 12 g/100 mL.
Conclusions
Since 1953, when Erslev [5] first demonstrated, in
a convincing and reproducible way, the existence of EPO,
much has been learned about the major sites of EPO
production and the conditions that control its rate of
production and the rate of erythropoiesis. Successful
extraction of pure EPO from the urine of aplastic
patients by Miyake and Goldwasser [13] in 1977 facilitated
production of specific antibodies to EPO. Subsequently, it
became possible to more accurately identify the cellular
site of EPO production and its action at the cellular level.
Also, this made possible the development of commercially
available accurate and specific iimmuunoassays [77] for
diagnostic purposes and the production of commercial
amounts of EPO for therapeutic use [14].
There are, however, also new questions that have arisen
and that are now the target of investigation. The success in
treating patients with chronic renal failure and a variety of
refractory anemias has been gratifying in that it has
decreased the transfusion requirements and increased the
quality-of-life of many patients. However, in the past
6 years, we have also been made aware of important
1013
complications of EPO therapy. EPO increases the incidence
of thromboembolic disease in patients with malignancy and
with renal failure who already have a hypercoagulable
state [94]. This may be particularly pertinent if the
blood hemoglobin concentration is raised to normal or
near-normal levels. Also, there is evidence that patients
with a variety of cancers who receive EPO may have
a shortened overall survival [92,93]. This has led to development of new guidelines regarding who should receive
EPO and what the optimal target blood hemoglobin concentration should be [86]. Trials in patients with breast cancer
that reported that EPO shortened the overall survival suggested that the cancer cells contain EPO receptors that
are activated by EPO. There is also data suggesting that
endothelial cells contain EPO receptors and that EPO has
angiogenic activity [96]. EPO receptors have been
described on a number of nonerythroid cells. Their physiologic significance requires clarification. Also, extrarenal
EPO production has been reported in a number of sites [28,29]
(nonparaneoplastic). The significance of this extrarenal EPO
also requires clarification. A great deal has been learned
about EPO and erythropoiesis in the past 50 years. As is
the case in all important research areas, important new
knowledge generates important new questions.
Conflict of Interest
No financial interest/relationships with financial interest relating
to the topic of this article have been declared.
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