Conditions: Anemia CKD Atherosclerosis Cardiovascular
Keywords: erythropoietin
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 5
Healthy volunteers: no

Brief summary:
Although several large well designed clinical trials have shown that erythropoietin which is commonly used to treat anemia associated with kidney disease, increases the risk of stroke and heart disease, the mechanism for this increased risk is unknown. The investigators' preliminary studies show that the adverse effects of erythropoietin are from activation of the heterodimeric erythropoietin/ beta common receptor which only occurs with high doses of erythropoietin. The investigators propose a clinical trial of 120 patients assigned to low doses of erythropoietin given more frequently or the same cumulative dose of erythropoietin administered as a high dose once every two weeks and assess effects on the beta common receptor activation, inflammation and vascular disease as evidence by MRI of the carotid arteries.

Detailed description:
Erythropoietin (EPO) is the most widely prescribed cytokine, yet the benefits and potential side effects of different dosing regimens are poorly understood. It is now recognized that erythropoietin administered at high doses to patients with chronic kidney disease, results in an increased risk of morbidity and mortality from heart disease and stroke. However, the mechanisms that mediate this increased risk of cardiovascular disease is not known. There are two receptors for erythropoietin the homodimeric EPO receptor (EPOR) and the heterodimeric beta common receptor ( CR)/EPOR. The investigators have demonstrated that activation of the heterodimeric CR/EPOR only occurs with high doses of EPO. Our exciting, published, preliminary data also demonstrates that the CR is in a complex with vascular endothelial growth factor receptor-2 (VEGFR-2) and that high doses of EPO activate VEGFR-2 through the CR, resulting in the deleterious effects of VEGFR-2 activation on the cardiovascular system. This is particularly important in patients with kidney disease since they are already at a high risk of cardiovascular disease. Moreover in advanced kidney disease cyanate derived from the high urea levels can non-enzymatically form an amide bond with EPO. This carbamylated EPO (cEPO) has no effect on hemoglobin, but still activates the heterodimeric CR/EPOR. To date there have been no studies that have directly measured levels of cEPO or activation of the CR/EPOR in patients with kidney disease. Our hypothesis is that the administration of low-doses of EPO more frequently will result in lower levels of total and carbamylated erythropoietin decreased activation of VEGFR-2 via the heterodimeric CR/EPOR and consequently decreased inflammation and atherosclerosis. The investigators will directly test this hypothesis by randomly allocating 120 patients with chronic kidney disease to either low- dose EPO given thrice weekly or the same cumulative dose, a high-dose, administered once every 2 weeks. Our hypothesis predicts that low-dose EPO will be as effective at correcting anemia, but will demonstrate less progression of carotid artery plaque, as assessed by non-contrast magnetic resonance imaging, as compared to the high-dose, EPO given every 2 weeks. To delineate how EPO affects blood vessels, the investigators will isolate endothelial cells from blood vessels in 20 patients who are assigned to low-dose EPO and 20 allocated to high-dose EPO. Within these cells the investigators will investigate the signaling pathways that are triggered by activation of CR/EPOR. In a substudy of 20 subjects with kidney disease randomized to low-dose EPO or to high-dose EPO, as well as 20 healthy controls receiving a single dose of high- or low-dose EPO, the investigators will determine how kidney function and dosing affects levels of total and carbamylated erythropoietin. The investigators' study will not only provide us with a thorough understanding of the mechanism by which EPO mediates the increased risk of atherosclerosis, but a clinical strategy to avoid the side effects of EPO therapy and a tool to quantify the cardiovascular risk of EPO and newer erythropoiesis stimulating agents by assessing activation of the heterodimeric CR/EPOR.

Arms:
arm 1: Subjects randomized to this arm will receive low-dose of EPO administered thrice weekly arm 2: Subjects randomized to this arm will receive the same cumulative dose of EPO administered as a high-dose of EPO every 2 weeks

Interventions: intervention 1: Low dose erythropoietin intervention 2: High dose erythropoietin
Intervention descriptions: intervention 1: Subjects randomized to this arm will receive low-dose of EPO administered thrice weekly intervention 2: Subjects randomized to this arm will receive the same cumulative dose of EPO administered as a high-dose of EPO every 2 weeks

Protocol document:
Prot_SAP_000.pdf: 
ID: CARA-019-16F 
Frequent, Low-Dose Erythropoietin A Mechanistic Approach to Mitigate Adverse 
Cardiovascular Effects of Erythropoietin 
April 23, 2019


Protocol 
1. Project Title:  Frequent, Low-Dose Erythropoietin: A Mechanistic Approach to 
Mitigate Adverse Cardiovascular Effects of Erythropoietin Therapy in Patients with 
Chronic Kidney Disease 
2. Investigator(s): 
Mark S. Segal, MD/PhD 
Rajesh Mohandas, MD, MPH 
Demetra Christou, PhD 
John Forder, PhD 
Reginald Frye, PharmD, PhD 
Yan Gong, PhD 
 
3. Abstract:  Erythropoietin (EPO) is the most widely prescribed cytokine, yet the benefits and 
potential side effects of different dosing regimens are poorly understood. It is now recognized that 
erythropoietin administered at high doses to patients with chronic kidney disease, results in an 
increased risk of morbidity and mortality from heart disease and stroke. However, the 
mechanisms that mediate this increased risk of cardiovascular disease is not known. There are 
two receptors for erythropoietin the homodimeric EPO receptor (EPOR) and the heterodimeric 
beta common receptor (βCR)/EPOR. We have demonstrated that activation of the heterodimeric 
βCR/EPOR only occurs with high doses of EPO. Our exciting, published, preliminary data also 
demonstrates that the βCR is in a complex with vascular endothelial growth factor receptor-2 
(VEGFR-2) and that high doses of EPO activate VEGFR-2 through the βCR, resulting in the 
deleterious effects of VEGFR-2 activation on the cardiovascular system. This is particularly 
important in patients with kidney disease since they are already at a high risk of cardiovascular 
disease. Moreover in advanced kidney disease cyanate derived from the high urea levels can 
non-enzymatically form an amide bond with EPO. This carbamylated EPO (cEPO) has no effect 
on hemoglobin, but still activates the heterodimeric βCR/EPOR. To date there have been no 
studies that have directly measured levels of cEPO or activation of the βCR/EPOR in patients with 
kidney disease. Our hypothesis is that the administration of low-doses of EPO more frequently will 
result in lower levels of total and carbamylated erythropoietin decreased activation of VEGFR-2 
via the heterodimeric βCR/EPOR and consequently decreased inflammation and atherosclerosis. 
We will directly test this hypothesis by randomly allocating 120 patients with chronic kidney 


disease to either low- dose EPO given thrice weekly or the same cumulative dose, a high-dose, 
administered once every 2 weeks. Our hypothesis predicts that low-dose EPO will be as effective 
at correcting anemia, but will demonstrate less progression of carotid artery plaque, as assessed 
by non-contrast magnetic resonance imaging, as compared to the high-dose, EPO given every 2 
weeks. To delineate how EPO affects blood vessels, we will isolate endothelial cells from blood 
vessels in 20 patients who are assigned to low-dose EPO and 20 allocated to high-dose EPO. 
Within these cells we will investigate the signaling pathways that are triggered by activation of 
βCR/EPOR. In a substudy of 20 subjects with kidney disease randomized to low-dose EPO or to 
high-dose EPO, as well as 20 healthy controls receiving a single dose of high- or low-dose EPO, 
we will determine how kidney function and dosing affects levels of total and carbamylated 
erythropoietin. Our study will not only provide us with a thorough understanding of the mechanism 
by which EPO mediates the increased risk of atherosclerosis, but a clinical strategy to avoid the 
side effects of EPO therapy and a tool to quantify the cardiovascular risk of EPO and newer 
erythropoiesis stimulating agents by assessing activation of the heterodimeric βCR/EPOR. 
 
4.  
Background:  
A.1. Anemia and CKD: Progressive loss of renal function is associated with a reduced ability to 
synthesize EPO and progressive anemia. The decline in hemoglobin begins to occur when the 
creatinine clearance falls below 70 ml/min/1.73 m2 in men and 50 ml/min/1.73 m2 in women,25 
and worsens with further loss of renal function. In a cross-sectional, multicenter survey of 
patients with CKD, 9% had a hemoglobin of less than or equal to 10 g/dL, a level that qualifies 
for EPO treatment.25 Compared to those with mild CKD, the relative risk of having a hemoglobin 
of less than 10 increased from 2.6-fold in those with moderate CKD to 7.6-fold in those with 
severe CKD.25 Thus, anemia is common in CKD and with progression of renal disease, the 
majority of patients will require EPO therapy. 
 
A.2. Cardiovascular disease (CVD) and CKD: Approximately 13% of the general population of 
the US and 20% of Veterans have CKD.26 CKD is associated with exponential increases in 
morbidity and mortality and patients with advanced CKD, especially those with end stage kidney 
disease (ESKD), carry annual mortality rates 5-25 times higher than the age and gender 
matched general population.27 CVD is the major contributor to mortality in patients with CKD 
and ESKD, with nearly half of all deaths attributed to CVD.28 CKD and ESKD are also major risk 
factors for accelerated atherosclerosis29 and increased risk of premature30 and recurrent 
atherosclerotic CVD.31 Patients with CKD are also at a substantial risk for non-atherosclerotic 
CVD, which is in large part believed to be related to vascular stiffness/endothelial dysfunction, 
and inflammation.28, 32  
 
A.3. Erythropoiesis stimulating agents and increased risk of CVD and death from cancer 
recurrence: The landmark clinical trials, Correction of Hemoglobin and Outcomes in Renal 
Insufficiency (CHOIR),33 The Cardiovascular Risk Reduction by Early Anemia Treatment with 
Epoetin Beta (CREATE),34 and the Trial to Reduce Cardiovascular Events with Aranesp 
Therapy (TREAT)35 all demonstrated that targeting a higher hemoglobin, with increased doses 
of an erythropoiesis stimulating agent, led to an increase in the primary composite endpoint of 
death, myocardial infarction, and stroke in CHOIR, more angina and peripheral vascular disease 
in CREATE, and increased incidence of stroke and cardiac revascularization in TREAT. We 
demonstrated that mobilization of bone marrow derived angiogenic cells (BMDAC) by EPO is 
dependent on the heterodimeric βCR/EPOR signaling though the VEGFR-2.1 Since the Kd of 


the heterodimeric βCR/EPOR is ~9-times higher than that of the 
homodimeric EPOR, we believe the clinical trials targeting a higher hemoglobin level, using 
higher doses of erythropoiesis stimulating agents, led to activation of the heterodimeric 
βCR/EPOR and in turn VEGFR-2. Activation of VEGFR-2 leads to an increase in inflammation36 
and downregulation of VEGFR-2 diminishes the risk of atherosclerosis.37 Thus activation of the 
heterodimeric βCR/EPOR may explain the cardiovascular side effects seen with high-doses of 
EPO. This is supported by a recent meta-analysis of 31-randomized control trials demonstrating 
that the dose of EPO correlated strongly with cardiovascular adverse events, even after 
controlling for the target hemoglobin.38 Our proposal directly tests this hypothesis and if true 
would provide a mechanism to increase hemoglobin levels without increasing the risk of 
adverse cardiovascular events. 
 
A.4. The benefit of higher hemoglobin levels on quality of life: In response to the CHOIR,33 
CREATE,39 and TREAT35 studies, in 2007 the FDA required a black box warning on 
erythropoiesis stimulating agents to avoid targeting a hemoglobin level greater than 11 g/dL. 
However, in a retrospective analysis, a time-averaged hemoglobin less than 12 g/dL in patients 
with CKD was associated with higher mortality and faster progression to ESKD.40 This 
observation was confirmed in a subsequent retrospective study, specifically in Veterans, which 
demonstrated a slower decline in renal function after initiation of erythropoiesis stimulating 
agents.41 Studies that have examined the quality of life and hemoglobin level have consistently 
demonstrated an improvement in the quality of life42 as well as work capacity43 with increasing 
hemoglobin. So limiting the use of EPO threatens to hasten the progression of kidney disease 
and need for dialysis, increase mortality, and decrease quality of life. If our hypothesis is correct, 
we would be able to obtain the benefits of a higher hemoglobin level on quality of life and 
slowing progression of CKD, while avoiding the increased risk of cardiovascular events. 
 
A.5. Hypothesis to be tested and its significance: As previously discussed there are two 
EPO receptors: 1) the homodimeric EPOR that has a Kd of ~6 mIU/ml; and 2) the heterodimeric 
βCR/EPOR that has a Kd of ~50 mIU/ml. Activation of the heterodimeric βCR/EPOR only 
occurs with high-doses of EPO. The βCR is in a complex with VEGFR-2 and thus 
supraphysiologic doses of EPO activate VEGFR-2 through the βCR, resulting in all the 
deleterious effects of excessive VEGFR-2 activation. The erythropoietic effect of EPO is 
inactivated by carbamylation, a process in which the high urea levels in CKD patients leads to 
production of cyanate which modifies EPO through an amide bond can modify EPO (Figure 2). 
However, cEPO can still activate the heterodimeric βCR/EPOR.19 Our hypothesis is that 
frequent administration of low-doses of EPO, as compared to the administration of 
infrequent, high-dose EPO, will result in lower levels of total and carbamylated EPO, 
decreased activation of VEGFR-2 via the heterodimeric βCR/EPOR, and consequently 
decreased inflammation and atherosclerosis (Figure 3). By understanding the complex 
interaction between EPO dosing, carbamylation, and half-lives of EPO and cEPO we will be 
able to develop optimal dosing strategies for EPO and erythropoietin stimulating agents, 
minimizing adverse events.  


 
 
 
 
A.6. Relevance of hypothesis to a new generation of erythropoiesis stimulating agents: 
For the last thirty years, the basis of erythropoiesis stimulating agents has been on directly 
stimulating the homodimeric EPOR, with either EPO or modified EPO (darbepoetin alfa an 
engineered EPO with extra glycosylation to increase its half-life). EPO is regulated 
predominantly by oxygen sensing by hypoxia-inducible factor (HIF) prolyl hydroxylases. 
Mimicking hypoxia by inhibiting HIF prolyl hydroxylases, leads to HIF stabilization and leads to 
an increase in endogenous EPO production. Recent studies3, 4 have demonstrated that at doses 
that lead to an increase in hemoglobin levels, prolyl hydroxylase inhibitors will result in EPO 
levels well above 50 mIU/ml, the Kd of the heterodimeric βCR/EPO. Our hypothesis predicts 
that endogenous EPO levels above the Kd of the heterodimeric βCR/EPOR, would lead to the 
same side effects as exogenous EPO. The cardiovascular and cancer risk of EPO was not 
recognized until 17 years after EPO came to market and only because of large clinical trials 
powered to look at cardiovascular endpoints. The new erythropoiesis stimulating agents which 
inhibit prolyl hydroxylase may have the same cardiovascular side effects, which if not dosed 
properly may go unrecognized. This study will not only provide us with a thorough 
understanding of the mechanism by which EPO mediates the increased risk of 
atherosclerosis, but will also offer a clinical strategy to avoid the side effects of EPO 
therapy and a tool to quantify the cardiovascular risk of EPO and newer erythropoiesis 
stimulating agents by assessing activation of the heterodimeric βCR/EPOR.  
 
A.7. Relevance to the Veteran population: The prevalence of CKD in the Veteran population 
is estimated to be 34% higher than in the general population due to demographic differences 
and the prevalence of co-morbidities, such as diabetes mellitus and hypertension. The Veterans 
Administration (VA) currently cares for over 600,000 Veterans with kidney disease and 15,000 
Veterans receive dialysis treatments at the 70 VA dialysis units or in the community under VA 
contracted care. Of Veterans with CKD initiating dialysis, 36% are estimated to be receiving 
EPO and another 31% would benefit from EPO therapy.44 However EPO use could increase the 
risk of CVD, which is the largest cause of morbidity and mortality among the aging US Veteran 
population and the top expenditure among chronic conditions for the VA healthcare system. A 
therapeutic strategy that will improve the quality of life of Veterans with kidney disease, slow 
down progression of kidney disease, and decrease the risk of heart disease will be of immense 
benefit.  
 
B. PRELIMINARY STUDIES 
The foundation for our hypothesis was our work on the mechanism by which EPO mobilized 
BMDAC. We had previously shown nitric oxide (NO) generation was critical in BMDAC 
mobilization and NO deficiency impaired BMDAC mobilization.2 We confirmed the observation 
by others45 that EPO mobilizes BMDAC and wanted to determine if EPO mobilization was NO 
dependent and which EPO receptor mediated mobilization.  


 
B.1. EPO increases CD34+ BMDAC migration via an NO dependent mechanism: It has 
previously been demonstrated that EPO lea to an increase in NO within endothelial cells.46 We 
have demonstrated that CD34+ BMDAC migration was dependent on NO.2 We have also 
shown that EPO stimulates chemokine mediated CD34+ BMDAC migration (Figure 4). 
Importantly, the ability of EPO to stimulate CD34+ BMDAC migration is NO dependent. If 
CD34+ BMDAC are preincubated with 10 µM of a NO synthase inhibitor, NG-Nitro-L-Arginine 
Methyl Ester (L-NAME), a concentration that inhibits the ability of CD34+ BMDAC to increase 
the level of NO, but does not alter basal levels of NO (data not shown), EPO’s ability to 
stimulate CD34+ BMDAC migration is markedly reduced (Figure 4).  
 
 
 
 
 
 
 
 
 
 
B.2. EPO induction of NO within CD34+ BMDAC is likely to occur via the heterodimeric 
βCR/EPOR: cEPO is only able to stimulate the heterodimeric βCR/EPOR. However, cEPO is 
able to stimulate CD34+ BMDAC migration to an equal extent as EPO (Figure 5A) and cEPO is 
able to stimulate NO within CD34+ BMDAC as effectively as EPO (Figure 5B). The implication 
of these results is that the heterodimeric βCR/EPOR may be mediating EPO induced stimulation 
of BMDAC. To determine the mechanism of this stimulation in NO, we examined 
phosphorylation of eNOS, a major mechanism of control of eNOS activity, and found that EPO 
induces phosphorylation of eNOS on serine 1177.1 
 
 
 
 
 
B.3. The heterodimeric βCR/EPOR is expressed in CD34+ BMDAC and mediates the 
increase in NO within BMDAC: By real time-PCR analysis, CD34+ BMDAC as well as human 
BMDAC colonies appear to express the βCR and the EPOR (Figure 6A). The CR is required for 
EPO/cEPO induction of NO, since mouse BMDAC colonies from a βCR knockout (-/-) mice did 
not increase their intracellular NO in response to EPO or cEPO (Figure 6B). Importantly, 


bradykinin could stimulate NO in these mouse colonies as well as in BMDAC colonies isolated 
from wild-type mice, indicating the NO synthase system was intact (Figure 6C).  
 
 
 
 
 
 
 
 
 
 
 
B.4. Vascular endothelial growth factor (VEGF)-induced NO production is dependent on 
the heterodimeric βCR/EPOR: If EPO is able to mobilize BMDAC and increasing BMDAC is 
beneficial,47 why did the CHOIR,33 CREATE,41 and TREAT33 studies demonstrate adverse 
events when a higher hemoglobin level was targeted? To investigate this question, we studied 
the downstream signaling of EPO. We found that EPO requires the presence of VEGFR-2 
(Figure 7A) and that paracrine production of VEGF was not involved, since anti-VEGF 
antibodies added to the media did not prevent EPO mediated increase in NO (Figure 7B). In 
addition, VEGF stimulated an increase in NO that was dependent on the presence of the βCR, 
since bone marrow cells from βCR knockout animals had no increase in NO in response to 
VEGF treatment (Figure 7C). This suggested an interaction between the βCR with VEGFR-2. 
 
 
 
 
 
 
 
 
 
 
 
B.5. The βCR and VEGFR-2 colocalize and co-immunoprecipitate: Since the heterodimeric 
βCR/EPOR required VEGFR-2 and the VEGFR-2 required the βCR for stimulation of NO, this 


suggested an intimate interaction between the two receptors. When examined by 
immunofluorescence, the two receptors colocalized and stimulation with EPO or VEGF did not 
affect the colocalization (Figure 8A). When we immunoprecipitate the βCR from endothelial cell 
extracts and immunoblot with an antibody against VEGFR-2, VEGFR-2 was detected. Again 
stimulating with EPO or VEGF did not influence the interaction (Figure 8B) 
 
 
 
 
B.6. Activation of VEGFR-2 or the heterodimeric βCR/EPOR 
results in inflammation: Since a previous study demonstrated that activation of VEGFR-2 
results in an increase in inflammation, as evidenced by an increase in IL-636, we hypothesized 
that treatment with EPO at a dose to stimulate the heterodimeric βCR/EPOR would also lead to 
an increase in IL-6. RNA was isolated from endothelial cells treated overnight with either 50 
mIU/ml EPO, a dose that stimulates the hetero-dimeric βCR/EPOR, or 100 ng/ml of VEGF, and 
real-time PCR performed. EPO treatment resulted in an increase in IL-6 (Figure 9).  
 
B.7. Ability to capture and analyze an individual’s endothelial cells: In order to examine the 
effect of the different EPO dosing regimens on endothelial activation in-vivo, in Specific Aim 2 
we will use a translational, physiological approach to capture and study endothelial cells, as we 
have previously published.48-50 Briefly, 2 sterile J-shaped guidewires will be sequentially 
advanced ~10 cm through an 18-gauge antecubital intravenous catheter and retracted. Cells 
will be recovered by washing the wires with a dissociation buffer and centrifugation. After fixing 
the cells with 4% paraformaldehyde, the cells will be washed, plated on poly-L-lysine coated 
slides, and stored at -80°C until immunofluorescence staining is performed. Based on studies 
on human subjects over a wide range of age and chronic medical conditions an adequate 
number of endothelial cells are obtained via venous biopsies to allow for quantification of protein 
levels by immunofluorescence.48, 51, 52 For immunofluorescence staining, the fixed, vascular, 
endothelial cells will be rehydrated and the slides incubated with the appropriate primary 
antibodies followed with a corresponding Alexa Fluor 488 conjugated secondary antibody. 
Slides will also be incubated with a primary antibody for VE Cadherin (Abcam, Cambridge, MA) 
and corresponding secondary antibody conjugated with Alexa Fluor 555 (Life technologies, 
Grand Island NY) to allow identification of endothelial cells. Finally, slides will be mounted with 
Vectashield with nuclear DAPI stain (Vector Laboratories, Burlingame, CA). To minimize the 


potential confounding of inter-batch variability in staining, 2 slides of control cells (i.e., human 
umbilical vein endothelial cells; HUVEC) will be stained in each batch and endothelial cell 
protein levels will be reported relative to average HUVEC intensity in that batch. For analysis, 
cells will be examined with a fluorescence microscope at 100 X magnification using a fixed 
exposure time (Figure 10). We have previously demonstrated, in a pilot study, that exercise 
training resulted in a decreased expression of nicotinamide adenine dinucleotide phosphate-
oxidase (NADPH oxidase), nitrotyrosine, and tumor necrosis factor alpha (TNF-α) and 
upregulation of eNOS phosphorylation at serine 1177 (Figure 11). 
  
 
 
 
 
 
 
 
 
B.8. Ability to detect cEPO: Although the concept of inactivation of the erythropoietic actions 
of EPO was first described by Mun and Golper in 2000,53 to date, there has been no publication 
demonstrating the ability to directly detect cEPO. A key feature of our hypothesis is that cEPO 
can lead to activation of the heterodimeric βCR/EPOR, while not aiding erythropoiesis. To 
determine whether the levels of cEPO are affected by different dosing strategies (frequent, low-
dose versus infrequent, high-dose), we need to determine the level of cEPO in blood and for the 
first-time determine its half-life. Using in-vitro cEPO, we were able to capture cEPO on a 
standard EPO ELISA plate (R&D Systems, Inc., Minneapolis, MN) and then using a primary 
biotinylated antibody that recognizes carbamoyl groups (MyBioSource, Inc., San Diego, CA) 
and streptavidin poly-horseradish peroxidase (Pierce), to amplify the signal, we were able to 
detect 1 mIU/ml of cEPO (Figure 12).  
 
 
 
Implications of the preliminary data: We have demonstrated that EPO mobilization of 
BMDAC is dependent on the heterodimeric βCR/EPOR, whose activation requires the presence 
of VEGFR-2. EPO, like VEGF, induces IL-6 in endothelial cells. This preliminary data provides a 
mechanism by which higher doses of EPO can mediate atherosclerotic events via activation of 


the heterodimeric βCR/EPOR, likewise lower, more frequent doses of EPO may be able to 
increase the hemoglobin levels without untoward side effects. In addition, we have 
demonstrated that we have the ability to monitor the direct, in-vivo effects of EPO on biopsied 
endothelial cells. Lastly, we can accurately detect and quantify cEPO and thus we will be able to 
determine whether the carbamylation and the half-life of cEPO are dependent on the dosing 
regimen of EPO.  
 
 
5. Specific Aims: 
Erythropoietin (EPO) is the most widely prescribed cytokine, yet the benefits and potential side 
effects of different dosing regimens are poorly understood. Unfortunately, in an effort to save 
nursing visits and administration costs, there has been a clinical shift in this country to give 
fewer doses of EPO, but at supraphysiologic doses. This was done without regard to the 
presence of two EPO receptors: 1) the homodimeric EPO receptor (EPOR) that has high affinity 
for EPO [Kd of ~6 milli-International Units per milliliter (mIU/ml)] and is responsible for 
erythropoiesis; and 2) the low affinity [Kd of ~50 mIU/ml] heterodimeric beta common receptor 
(βCR)/EPOR. We have demonstrated that activation of the heterodimeric βCR/EPOR only 
occurs with high doses of EPO. Our exciting, published, preliminary data demonstrates that the 
βCR is in a complex with vascular endothelial growth factor receptor-2 (VEGFR-2) and that 
supraphysiologic doses of EPO activate VEGFR-2 through the βCR, resulting in all the 
deleterious effects of excessive VEGFR-2 activation, such as increased inflammation and 
atherosclerosis. Clinically, it is now recognized that EPO administered at high doses to cancer 
patients results in an increased probability of cancer recurrence and patients with chronic kidney 
disease (CKD) randomized to a higher hemoglobin target and receiving elevated doses of EPO 
have an increased risk of morbidity and mortality from heart disease and stroke. We believe that 
our novel observation of VEGFR-2 activation by high doses of EPO explains these clinical 
findings. Importantly in patients with kidney disease, cyanate, derived from the high urea levels, 
can non-enzymatically form an amide bond with EPO. This carbamylated EPO(cEPO) lacks 
erythropoietic activity, but is still able to activate the heterodimeric βCR/EPOR. Consistent with 
this, levels of carbamylated albumin are increased in CKD and correlates with EPO resistance. 
However, no study has directly measured levels of cEPO or activation of the βCR/EPOR in 
CKD. The activation of the heterodimeric βCR/EPOR is dependent on a complex interaction 
between EPO dosing, carbamylation, and the half-lives of EPO and cEPO. Our hypothesis is 
that frequent administration of low-doses of EPO, as compared to the administration of 
infrequent, high-dose EPO, will result in lower levels of total and carbamylated EPO, 
decreased activation of VEGFR-2 via the heterodimeric βCR/EPOR, and consequently 
decreased inflammation and atherosclerosis. We will directly test this hypothesis by the 
following specific aims: 
 
Specific Aim 1: Determine if administration of more frequent, low-doses of EPO as compared 
to the same, cumulative dose given every 2 weeks, will be as effective at stimulating 
erythropoiesis, but have less progression of carotid artery plaque in subjects with CKD and 
anemia requiring EPO therapy.  
A total of 100 subjects with stage 3 or 4 CKD, estimated glomerular filtration rate between 15-59 
ml/min/1.73 m2, and requiring EPO therapy will be randomized to receive either low-dose EPO 
(25 IU/kg/dose) three-times a week, or the same, cumulative dose administered every two 


weeks (i.e., 150 IU/kg/2 weeks; high-dose). The progression of carotid artery plaque will be 
determined by magnetic resonance imaging (MRI) at baseline and after 12 months of receiving 
EPO.  
 
Specific Aim 2: Determine whether receiving high-dose EPO results in VEGFR-2 activation and 
increased endothelial activation, as measured by phosphorylated VEGFR-2, vascular cell 
adhesion molecule (VCAM) and interleukin-6 (IL-6) expression, as compared to low-dose 
EPO. 
In 20 subjects randomized to low-dose EPO three-times a week and 20 subjects randomized to 
high-dose EPO every 2 weeks, we will capture endothelial cells using a vascular endothelial cell 
biopsy technique, at baseline and after 12 months. Endothelial cell activation will be quantified 
by immunofluorescence.  
  
Specific Aim 3: Determine the interaction of renal function and dosing of EPO on a) the rate of 
carbamylation; and b) the length of time that total and cEPO exceed the Kd of the 
βCR/EPOR. 
In a subset of 5 subjects randomized to low-dose EPO, 5 subjects randomized to high-dose 
EPO, as well as 5 healthy subjects who will receive a single, low-dose of EPO, and 5 healthy 
subjects who will receive a single, high-dose of EPO, we will determine the pharmacokinetics of 
EPO and cEPO. We predict that high-dose EPO given every 2 weeks will result in increased 
time with total serum levels of EPO exceeding 50 mIU/ml, the Kd of the βCR/EPOR.  
 
The cardiovascular and cancer risk of EPO was not recognized until 17 years after EPO came 
to market and only because of large clinical trials powered to look at cardiovascular endpoints. 
Although new erythropoiesis stimulating agents which inhibit prolyl hydroxylase are being 
developed, they stimulate endogenous EPO production and may have the same cardiovascular 
side effects, which if not dosed properly may go unrecognized. This study will not only provide 
us with a thorough understanding of the mechanism by which EPO mediates the increased risk 
of atherosclerosis, but a clinical strategy to avoid the side effects of EPO therapy and a tool to 
quantify the cardiovascular risk of erythropoiesis stimulating agents.  
6. Research Plan: 
Please note that only some of the study subjects enrolled into Aim 1, will be asked to enroll into 
the sub-studies for aims 2 and 3.  In addition, only aim 3 will have a healthy control group.  
 
C.1. Specific Aim 1: Determine if administration of thrice weekly, low-doses of EPO as 
compared to the same, cumulative dose given every 2 weeks, will be as effective 
at stimulating erythropoiesis, but slow progression of carotid artery plaque in 
subjects with CKD and anemia requiring EPO therapy. 
Rationale: Several well-designed, large, randomized, control trials have demonstrated that 
EPO therapy targeting a higher hemoglobin level, is associated with adverse cardiovascular 
outcomes in patients with CKD.33-35 A recent meta-analysis of 31 prospective studies found that 
adverse effects are related to the dose of EPO, rather than targeted or attained hemoglobin 
levels.38 Consistent with this observation, increased levels of endogenous EPO have been 
associated with adverse cardiovascular events in diverse patient populations.54-56 While EPO 
has been associated with endothelial dysfunction in patients with CKD57 and in-vitro EPO 
stimulates proliferation of vascular smooth muscle cells,58 the mechanistic basis of accelerated 
atherosclerosis with EPO therapy remains unknown. Our preliminary data demonstrates that 
high-doses of EPO stimulates the heterodimeric βCR/EPOR and in turn VEGFR-2. Persistent 
activation of VEGFR-2 has been associated with inflammation36 and atherosclerosis.37 The 


stimulation of the heterodimeric βCR/EPOR does not occur with low-doses of EPO. Thus our 
novel observation not only suggests an underlying mechanism for atherosclerosis associated 
with EPO therapy, but also provides an inexpensive way to mitigate cardiovascular side-effects 
of EPO therapy by administering more frequent, lower-doses of EPO. 
 
Study design: This is an open label, randomized, clinical trial comparing progression of 
carotid atherosclerosis in subjects with stage 3 -5  CKD (estimated glomerular filtration rate of 
<30 ml/min/1.73 m2) and anemia requiring EPO therapy assigned to thrice weekly, low-dose 
EPO or to same, cumulative high-dose of EPO administered every 2 weeks. 
 
Primary outcome measure: Change in carotid total plaque volume from baseline to 
approximately 1 year, as assessed by non-contrast MRI. 
 
Secondary outcome measures: Severity of maximal stenosis, percentage of total plaque area, 
and the characteristics of plaques (soft or fibrous and stable or unstable) at baseline and upon 
follow-up.  
 
Investigators and sites: Mark S. Segal, MD/PhD is an expert in molecular biology 
particularly of endothelial cells and EPO signaling and has been the principal investigator of 
several National Institute of Health funded clinical trials. Rajesh Mohandas, MD, MPH has 
training in epidemiology and is an expert in clinical trials and data analysis. Drs. Segal and 
Mohandas are physicians at the Malcom Randall VA Medical Center (VAMC), a large, 
academic VAMC within the North Florida/South Georgia Veterans Health System 
(NF/SGVHS) where they study will be conducted. The NF/SGVHS consists of two VAMCs, 
three large multi-specialty outpatient clinics, and eight small community-based primary care 
outpatient clinics. In 2013, NF/SGVHS was the busiest health care system in the VA system, 
treating 131,000 unique patients, with 1.6 million outpatient visits. It currently has more than 300 
active research projects and is home to four VA Research Centers.  
 
Study population: We will enroll Veterans who fulfill the following criteria: 1) age greater 
than 18; 2) stage 3, 4, or 5 CKD (estimated glomerular filtration rate of less than 60 ml/min/1.73 
m2) on at least two separate occasions greater than 3 months apart; and 3) candidates for EPO 
therapy as per the National Kidney Foundation’s Kidney Disease Outcomes Quality Initiative 
guidelines (hemoglobin < 10 gm/dL and anemia of CKD).  For specific aim 3, we will also enroll 
age- and sex-matched control Veterans.  These subjects can have chronic medical conditions, 
like hypertension, that are treated, but will be required to have an estimated glomerular filtration 
rate of greater than 60ml/min/1.73 m2. 
 
Exclusion criteria: We will exclude any Veteran who meets any of the following criteria: a) 
pregnant, planning to become pregnant in the next year, or breast feeding; b) uncontrolled 
hypertension (blood pressure > 180/100 mm Hg despite optimal antihypertensive medications); 
c) active gastrointestinal bleeding (visible blood or positive tests for stool occult blood 
accompanied by a decrease in hemoglobin); d) likely to have EPO resistance (serum ferritin < 
200 ng/ml, iron saturation < 20% despite iron replacement, intact PTH ≥ 600 pg/ml, alkaline 
phosphatase ≥ 160 IU/L or serum albumin < 3.0 g/dL); e) an adverse cardiovascular event in the 
prior three months; f) active or recent (within the last 3 months) severe, systemic infection; g) 
active inflammatory disease such as lupus, rheumatoid arthritis, or vasculitis requiring 
immunosuppressive or immunomodulatory medications; h) history of solid organ 
transplantation; i) expected off-dialysis survival of less than one year (as determined by the 
estimated glomerular filtration slope and the treating physician; j) active cancer (undergoing 
chemotherapy or radiation within the last 3 months) or primary bone marrow disease such as 


myelofibrosis; or k) a contraindication for an MRI or individuals who cannot comply with the 
study protocol.  We will exclude healthy subjects you meet a, b, f, g, h, or j. 
 
Rationale for the study population: Five percent of patients with stage 3 CKD and almost half 
(44%) of patients with stage 4 CKD have anemia, which often requires EPO therapy.25 Cross-
sectional studies have shown that patients with CKD stage 3, 4 or 5 have significant carotid 
atherosclerosis.59 Since EPO therapy increases the risk of adverse cardiovascular events by 
30%,33 it is likely these atherosclerotic lesions will progress during the proposed 1-year follow-
up period. Thus, this patient population is uniquely suited to study the effect on EPO on 
atherosclerosis. We excluded those on dialysis and those expected to require dialysis during the 
follow up period, to avoid confounding by factors that are inherent to renal replacement therapy, 
such as access, infection, adequacy of dialysis etc.  
 
Recruitment strategy: Any new patient referred to the EPO clinic at NF/SGVHS will be 
eligible for screening for recruitment. Veterans that are referred to the clinic will be mailed the 
recruitment letter and will be followed up as below.   
 
We will also utilize a ‘source cohort strategy’ to identify and enroll subjects that have a 
hemoglobin of less than 10 gm/dL and CKD stage 3, 4, or 5 who may not have been referred 
to the EPO clinic. This technique has been successfully employed by a number of clinical 
researchers at NF/SGVHS and the University of Florida (UF) with excellent enrollment rates. 
If found eligible, the Veteran will receive a letter from our research team, signed by the 
Veteran’s primary care provider, informing them that a study coordinator will contact them by 
phone in approximately 2 weeks to discuss the possibility of participation. A toll-free number will 
be provided for the Veteran to call in to “opt-out” of participation. Those who do not opt-out will 
be contacted by telephone. We will ensure that subjects are not coerced and know they have 
the option of not participating if they so desire. Once patients agree to participate, we will carry 
out a phone screen to make sure our EMR screen did not miss any exclusion criteria.  All 
Veterans in the source cohort will be evaluated consecutively for eligibility by chart review. This 
process will continue until the target enrollment of 100 is achieved. 
 
 
 
Control subjects for specific aim 3 will be via a flyer as well as we will ask those participants in 
the study if any of their friends of the same sex and same age, who are Veterans, who have 
normal kidney function would want to participate.   
 
Recruitment feasibility: CKD is prevalent and it is estimated that 4-5% of the general 
population has CKD. The prevalence is higher (greater than 2-fold) among Veterans.60-62 The 
NF/SGVHS has one of the largest populations of CKD patients within the VA and has a 
proven track record of supporting clinical and basic research. In preparation for this study, we 
have obtained the ‘numbers’ of our source cohort available through a de-identified query of 
‘VINCI’ and found that we have a total of 1,568 patients with stage 3 or 4 CKD, within our 
VAMC, who have a hemoglobin of less than 10 gm/dL. All have at least one outpatient visit 


over the last 18 months and appear to fulfill our inclusion and exclusion criteria. Even with a 
conservative enrollment rate of 7%, we should have an adequate number of subjects to 
achieve our recruitment goals. A number of ongoing or recently concluded clinical studies 
performed at the NF/SG-VAMC (Ongoing Institutional Review Board -protocols # UF142-2009, 
148-2012) have consistently demonstrated recruitment rates in the 10-15% range. Figure 13 is 
a graph of our recruitment goals and expected rate of subject retention over the life of the 
study. We will assess the enrollment and targets, in our monthly milestone meetings. In the 
unlikely scenario that recruitment is lagging, we will consider extending recruitment to the Bay 
Pines VAMC.  
 
 
Randomization: Studies have demonstrated that cardiovascular outcomes are worse in 
patients with proteinuria regardless of the cause of underlying kidney disease.63 Thus, we will 
employ block randomization stratified by albuminuria >300 mg/gm of creatinine. A complete list 
will be maintained by the investigators and will be periodically reviewed by the Data Safety and 
Monitoring Board (DSMB), per protocol. The pharmacy will dispense EPO and maintain records. 
 
Intervention: Subjects will be randomized to a low-dose of EPO administered thrice weekly or 
the same cumulative dose of EPO administered as a high-dose of EPO every 2 weeks. The 
starting dose of EPO will be approximately 25 IU/kg given subcutaneously thrice weekly or 
approximately 150 IU/kg given every 2 weeks. These dosing regimens have been validated in 
large clinical trials and have been shown to be equally efficacious in treating anemia.11 While 
titrating the EPO dose, hemoglobin measurements will be performed approximately every 2 
weeks.  However patient or clinical circumstances may dictate obtaining hemoglobin more or 
less frequently and approximately every 4 weeks, however patient or clinical circumstances may 
dictate obtaining hemoglobin more or less frequently, once therapeutic levels of hemoglobin are 
attained. The pharmacists at the EPO Clinic will follow the hemoglobin level and titrate the dose 
per a clinical protocol.  
  
 
 
Rationale for EPO dosing regimens: To test our 
hypothesis we have chosen two clinically utilized, well-validated, EPO dosing regimens with the 
same cumulative dose of EPO. We used pharmacokinetic modeling to determine if the serum 
concentrations differed. The results of our modeling, shown in Figure 14, demonstrated that 25 
IU/kg/dose of EPO administered subcutaneously every 48 hours will result in a peak serum EPO 
level of only 17.8 mIU/ml.64 This level is well below the Kd of the heterodimeric βCR/EPOR. In 
addition, we determined that any EPO dose below 67 IU/kg administered subcutaneously every 
48 hours would not attain serum EPO levels above 50 mIU/ml. Alternatively, subcutaneous dosing 
of EPO at 150 IU/kg every other week, results in peak levels of EPO of 81.7 mIU/ml, a 
concentration above the Kd of heterodimeric βCR/EPOR (50 mIU/ml).65 In addition, the area 
under the curve of EPO dosed at 25 IU/kg three times a week is only 736, nearly six-times less 


than the area under the curve for EPO dosed at 150 IU/kg every other week of 4,413. In fact, a 
subject dosed at 150 IU/kg every other week will have an EPO level above 50 mIU/ml for 60 hours 
a month, compared to 0 hours spent above 50 mIU/ml when EPO is dosed at 25 IU/kg three times 
a week. Thus we are confident that these dosing regimens will afford us the ability to study the 
effects of activation of heterodimeric βCR/EPOR on carotid atherosclerosis.  
 
Duration of intervention: Each subject will be treated with low- or high-dose EPO for 
approximately 12 months or until they reach ESKD or death. In some circumstances the subject 
may be on EPO for over 14 months until the MRI could be scheduled at a time convenient for 
the subject.  In previous studies, of comparable sample sizes,66, 67 this duration has proven to be 
sufficient to detect a clinically significant change in progression of atherosclerosis. Subjects who 
develop ESKD as well as subjects in the low-dose EPO group who require EPO doses above 
65 IU/kg subcutaneously thrice weekly will be censored for analysis. If they have completed at 
least 6 months of therapy, we will perform a carotid MRI and endothelial cell sampling prior to 
censoring. 
 
Subject follow-up and retention: We will ensure compliance by having the study staff contact 
the subjects approximately every 2-4 weeks. This frequent contact will reinforce medication 
compliance, make sure that any subject’s concerns are answered, and ensure all side effects 
are reported. Study visits will occur at enrollment and at the conclusion of the study at 1 year 
(Table 1). Subjects will be reimbursed for their travel costs and their time and effort. We will use 
pharmacy refill data (medication possession ratio)68 to categorize subject adherence. As per 
consensus in the literature, subjects with a ratio greater than 80% will be considered adherent.69 
 
Table 1. Schedule of Visits and Procedures 
VISIT 
Visit 1 
(Baseline) 
Visit 2 (12 months) 
Screening Medical Records 
X 
 
Informed Consent  
X 
 
Physical Examination 
X 
X 
Routine Laboratory Data 
X 
X 
MRI of Carotid vessels 
X 
X 
Endothelial Cell Harvest (n=20) 
X 
X 
Pharmacokinetic study (n=10) 
X 
 
Labs: Reticulocyte count, vitamin B12, folate, iron profile (serum iron, iron binding capacity and 
ferritin) thyroid stimulating hormone levels, intact parathyroid hormone levels will be performed 
at baseline. Iron profile will be repeated approximately every 3 months, as per standard-of-care. 
 
Subject safety and monitoring for adverse events: EPO will be prescribed as per current 
guidelines for clinical use. Adverse events will be recorded locally and serious adverse events 
will be communicated with the DSMB within 24 hours of occurrence. The DSMB will meet 
quarterly, as per the Institutional Review Board requirements, and will have full access to all 
adverse events and randomization codes. Outcomes will be ascertained by examination of VA 
medical records approximately every 6 months during the duration of study. In addition, if during 
the routine phone calls an adverse event is noted, an experienced nurse or physician will 
contact the subject to capture care that might have been received outside of the VA system. We 
will ascertain the following outcomes: death, major cardiovascular event (hospitalization for 
myocardial infarction or angina, congestive heart failure or stroke), and composite of death or 
major cardiovascular events. The frequency of death is expected to be low in this population for 
the proposed follow-up period. However, when a subject is identified as deceased, all 
information on the circumstances surrounding death, including best estimate of cause of death, 
will be documented and classified as cardiovascular or non-cardiovascular related. For subjects 


admitted to the VA system for cardiovascular reasons, we will collect documentation from the 
emergency room and inpatient chart to classify hospitalizations as myocardial infarction, angina, 
congestive heart failure or stroke. Events will be adjudicated by a committee that is blinded to 
the randomization. If a Veteran has a non-fatal cardiac event, they will continue to be followed 
according to the protocol. While we are not powered to see differences in adverse events, for 
safety, we will perform an interim analysis by an independent statistician blinded to the 
treatment allocation when 50% subjects have been randomized and completed 1 year of follow 
up. We will compare the adjusted event-free times to death, non-fatal myocardial infarction or 
major cardiovascular event using the using a two-sided significance test with the O’Brien–
Fleming spending function and a type I error rate of 5 percent. If there is a statistical difference 
between the two arms, the study will be terminated by the DSMB. 
 
Rationale for carotid MRI: Non-contrast MRI of carotid for total plaque area has numerous 
advantages compared to available alternatives, particularly in the CKD population (Table 2). 
MRI is non-invasive, provides superior images and minimizes inter-observer variations.70, 71 The 
use of non-contrast MRI to identify and follow the progression of atherosclerotic plaques is well 
established both in the general population and in CKD patients.72, 73 Several studies have 
validated that carotid MRI provides excellent distinction of normal from pathological carotid 
arterial walls,74, 75 allows quantiﬁcation of plaque size and type,73, 76 and assessment of integrity 
of the ﬁbrous cap.77 Recently, morphological features such as ﬁbrous cap thinning or rupture on 
MRI has been linked to clinical outcomes such as transient ischemic attacks and stroke.77 Thus 
MRI not only quantitates the severity of stenotic lesions and their spatial distribution, but the 
varying composition and stability of the plaques as well. 
Table 2: Comparison of Different Imaging Modalities for Atherosclerosis 
 
Location 
Intimal Lesions 
Plaque 
Contrast 
Invasive 
Reproducibility 
Ultrasound-Carotid 
Carotid 
Intermediate 
+ 
None 
No 
Poor 
Intravascular Ultrasound 
Coronary 
Detailed 
+++ 
Yes 
Yes 
Very good 
MRI 
Carotid 
Detailed 
+++ 
None 
No 
Very good 
 
Statistical considerations:  
Power Analysis: The primary objective of Specific Aim 1 is to test the hypothesis that change 
in total plaque volume in patients randomized to high-dose EPO is significantly different from 
that of those in the low-dose EPO group. Previous studies, which have investigated changes in 
plaque volume, have shown an average standard deviation of 3%.66, 72-77 We will recruit a 
pragmatic sample of 100 patients. Even with a generous 30% subject attrition, we expect to 
have a total of 70 subjects or 35 in each group complete the study. Using a conservative 
assumption that the repeated measures correlation is at least 0.50 (explains 25% of variance or 
less), the study of 35 evaluable per group has 95% power to detect a difference of 0.87 
standard deviations or 2.6% difference in plaque volume. With 30 evaluable per group, we will 
have 95% power to detect 0.95 standard deviations (2.85% difference) in plaque volume and 
with 25 evaluable per group, we will have 95% power to detect 1.04 standard deviations (3% 
difference in plaque volume). These changes, although small, are associated with a clinically, 
significant increase in adverse cardiovascular outcomes.19, 22, 67, 73  
 
Initial Data Analysis: An initial data analysis will be performed to detect any differences in 
distribution of characteristics measured at baseline and at the end of the study between groups. 
Measures of central tendency and variability will be reported for continuous variables and 
frequency distributions for categorical variables. This preliminary analysis will provide us with 
insight into data, distribution of variables and refine further analysis.  
 


Statistical Analysis Plan: We will use analysis of covariance with baseline plaque volume as 
the covariate, treatment groups as the independent variable, and change in plaque at 12 
months as the dependent variable. Even though in this randomized controlled trial study, the 
simple analysis of variance (ANOVA) model would offer unbiased treatment effect estimate, the 
analysis of covariance models are in general more efficient than ANOVA models. We will 
approximate intent-to-treat as closely as possible in the following ways: subjects with data at 
only one time-point (baseline or 12 months) will contribute their data to the model. The MRIs of 
subjects that adhered to the study protocol as well as those who did not will be included in the 
group they were randomized to for analysis.  
 
Sensitivity analysis & incomplete data: While the analysis presumes a completely at random 
model for missing variables, we shall conduct a sensitivity analysis that will impute a final result 
from the pooled baseline verse the 12-month result for those with complete data, and use the 
predicted final value as the dependent variable (missing values behave as null hypothesized). 
 
Model assumptions and alternate analysis: Model assumptions will be checked using validated 
informal (inspection of residuals) and formal methods (score tests for extra parameter or 
likelihood displacement). The influence of outliers will be evaluated using influence diagnostic 
methods, comparing estimates from models with and without outlying values. If the data is not 
normally distributed, non-parametric methods or log transformation of variables will be 
considered. To determine heterogeneity of the effect in response to EPO, a subgroup analysis 
will be performed on those who were on EPO prior to randomization and those with and without 
pre-existent atherosclerosis. We will also analyze the effect of EPO on composition of plaques 
(fibrous cap, lipid core, hemorrhage and calcification). These additional analyses will be 
considered exploratory.  
 
Data management: The McKnight Brain Institute (MBI) at UF routinely performs MRIs for 
Veterans enrolled in VA studies and so there are established Institutional Review Board 
protocols for obtaining and storing MRI data securely at this facility. In brief, all MRI data will be 
collected anonymously and subjects will be identified only by the study ID. The images will be 
stored in a DICOM format to enable analysis to be performed blinded to randomization and to 
protect all protected health information. A secure ftp site has been constructed and resides 
behind firewalls of the Computer Information Technology (IT) section at the MBI and passwords 
will be provided only to the MRI personnel. Two computers will be dedicated for the sole use of 
this project at the MRI Core Laboratory. One will be dedicated to the plaque analysis processing 
of MRI data sets, while the other will serve as a secure ftp and PACS server. VA IT security 
director will have oversight of the protocols to ensure data security and regulatory compliance. 
All data will be available to the DSMB for quality control. 
 
Expected results: Successful completion of this aim will determine if we can use more frequent 
low-doses of EPO to mitigate cardiovascular adverse effects associated with EPO therapy. Our 
hypothesis predicts that subjects randomized to low-dose, thrice weekly EPO will demonstrate 
less progression of carotid plaque volume by MRI, have less carotid stenosis, plaque area and 
more stable plaques as compared to those randomized to high-dose, biweekly EPO. 
 
 
Methods: 
MRI protocol: Subjects will be scanned using a 3T Philips Achieva MRI system located in the 
MBI at UF. Following successful screening, a double inversion fast spin echo imaging sequence 
will be used to acquire thirty contiguous image planes centered about the carotid bifurcation on 
each side of the neck. Slice thickness will be 3 mm, field of view 25 x 25 cm, and matrix of 1024 


x 1024. T1-weighted images will be acquired with TR 1 sec and TE 11 ms; while T2-weighted 
images will utilize TR 500 msec and TE 40 msec. Proton density images will be acquired with a 
TR 5 sec and TE 11 msec. In our experience total exam time is ~20 minutes.  
 
Sensitivity to changes in plaque volume has been reported at 7% using a 1.5T MRI platform.73, 
76 By moving to a higher field (i.e., 3T) we can increase the spatial resolution by a factor of 4 (in-
plane resolution at 1.5T was 475 µm2, while at 3T we routinely achieve 250 µm2), with only a 
modest increase in acquisition time. Thus, we will be able to detect changes in plaque volume of 
1.75%. Field of view and matrix size will be adjusted to provide an in-plane resolution of 250 
µm2. Three complete data sets will be acquired, comprised of T1-weighted, T2-weighted, and 
proton density weighted images. In addition to estimating atherosclerotic lesion size, these data 
sets will also permit the characterization of the plaque that comprises the lesion.73, 76 Although 
these are the parameters we expect to need, the precise spatial dimensions and imaging parameters 
may need to be modified to provide the best image quality. 
 
Carotid total plaque volume estimation: Studies will be performed only after passing a pre-
study quality control. All studies will be read by a single, experienced observer (Dr. Forder) at 
UF. The plaque characteristics will be analyzed by MRI-PlaqueViewTM (VP diagnostics Inc., 
WA). Planimetry will be performed on the image data sets, using histogram equalization to 
improve edge detection for plaque, arterial wall and lumen. Differences in image contrast 
between T1-weighted, T2-weighted, Time of Flight, and proton density will be used to 
characterize the plaque as fibrous, stable, or unstable.  
 
Complicated plaque will be defined by areas of hyperintensity within the plaque (defined as 
intensity exceeding that of the surrounding muscle), and will be quantitated using thresholding 
and planimetry.78 Volume areas for lipid-, necrotic-, and hemorrhagic-cores, luminal diameter, 
and fibrotic cap thickness will be determined for each of the slices. Vulnerable plaque will be 
assessed by progression from baseline exam to follow-up exam. Stability will be determined by 
thickness of fibrotic cap over the lipid or necrotic core.  Although these are the parameters we 
expect to need, the precise spatial dimensions and imaging parameters may need to be modified to 
provide the best image quality. 
 
C.2. Specific Aim 2: Determine whether receiving high-dose EPO results in VEGFR-2 
activation and increased endothelial activation, as measured by VCAM and IL-6 
expression, as compared to low-dose EPO. 
Rationale: While there is a preponderance of clinical trial data linking EPO to adverse 
cardiovascular events,33-35 particularly in high doses,38 the basic mechanism underlying this 
increased risk remains unknown. This has hampered development of therapeutic options to 
mitigate the cardiovascular risk associated with EPO therapy. Our novel, preliminary, in-vitro 
data demonstrates that EPO activates endothelial cells via the βCR/EPOR and this is 
dependent on VEGFR-2 tyrosine kinase activity.1 Published studies have demonstrated that 
administrating EPO to healthy volunteers at doses that would result in serum levels markedly 
above 50 mIU/ml (levels that would activate the βCR/EPOR) leads to an increase in circulating 
levels of E-Selectin79 and P-selectin,80 suggesting endothelial cell activation. However, signaling 
within endothelial cells, particularly with long-term administration of EPO has not been studied. 
We will, for the first time, use our ability to capture endothelial cells to elucidate the exact, 
molecular, downstream effects of elevated EPO levels.  
 
Study design: We will attempt to recruit 30 subjects in the low-dose, EPO group and thirty 
subjects in the high-dose, EPO group, who have not previously received EPO.  We will attempt 
to isolate endothelial cells from each subject before initiation of EPO therapy and at some point 


near the conclusion of the study. For the collection after the initiation of EPO therapy the 
subjects will be asked to come to the VAMC approximately 18-24 hours after administration of 
their EPO dose, coinciding with peak serum levels of EPO after subcutaneous administration.81 
We will use an intravenous catheter and guidewire to collect venous endothelial cells. 
Approximately 30 cells will be fixed and stained with an antibody to VCAM, phosphorylated 
VEGFR-2, or IL-6 and counterstained with appropriate fluorescent secondary antibodies. 
Fluorescence intensity will be quantified after correcting for background fluorescence and 
normalized to fluorescent intensity of HUVEC to normalize for inter-batch variability in staining.  
 
Sample size and feasibility: We will recruit a pragmatic sample of 30 patients in each arm for 
this aim. Since the procedure is brief, low risk, and we are selecting from motivated subjects 
who have already enrolled in the study we do not anticipate difficulties in meeting recruiting 
goals for this aim. The power calculations are based on the mean and standard deviation from 
our own preliminary studies. The percentage detectable change for a power of 80 and 90%, as 
well as beta error of 5 or 1%, is summarized in Table 3. Assuming a dropout rate of 30% or 
even 50%, we will have 21 or 15 subjects per group, respectively with complete data for 
analysis. The detectable differences with 14 or 10 subjects are shown in parenthesis.  Based on 
our previous studies as well as that of others differences of the magnitudes presented below are 
attainable and clinically significant.  
Table 3: Power Calculations for Specific Aim-2 (Detectable Change in %) 
Outcome 
Mean 
SD 
80% Power 
90% Power 
 
 
 
 
 
 
 
VEGFR2 
802 
221 
25 (30,36) 
30 (39,47) 
30 (39,47) 
16 (44,52) 
VCAM 
1012 
115 
10 (13,15) 
13 (16,19) 
13 (16,19) 
15 (18,22) 
IL-6 
0.274 
0.021 
7 (8,10) 
8 (11,13) 
8 (11,13) 
10 (12,15) 
 
Data analysis and interpretation: The primary analysis is to determine the difference in 
endothelial cell activation (levels of VEGFR-2 phosphorylation, VCAM and IL-6) between 
subjects randomized to low- or high-dose of EPO using analysis of covariance adjusted for 
baseline levels of phosphorylated VEGFR2, VCAM and IL-6. Fluorescence intensity will be 
compared using procedures with appropriate transformation or nonparametric alternatives when 
normality assumptions are not met. Missing values will be addressed as outlined in Specific 
Aim 1.  
 
Additional analysis: While Aim 1 has an analysis that has large enough sample sizes for the 
central limit theorem to apply, making the analysis virtually assumption-free, the small sample 
size for Aim 2 requires a sensitivity analysis. All of the data will be pooled to obtain a least 
squares line of month 12 data on baseline (ignoring for the moment the treatment group). The 
resulting residuals from the line (observed minus fitted) will be analyzed by a 2-sample 
permutation test to compare the treatment group. We will also perform additional analysis 
adjusting for covariates such as age and renal function. 
 
Expected results: This aim will elucidate the basic mechanism underlying the increased 
cardiovascular risk associated with EPO therapy. Our hypothesis predicts that baseline levels of 
IL-6, VCAM, and phosphorylated VEGFR-2 will be the same in both groups and subjects 
randomized to high-dose EPO will have significantly higher levels of IL-6, VCAM, and 
phosphorylated VEGFR-2 at the end of the study. 
 
 
Methods: 
Harvesting endothelial cells: As previously described by our group and others, endothelial 
cells will be collected from an antecubital vein by sequentially advancing and retracting two 


sterile, ~10cm, J-shaped guidewires (Daig, Inc., Minnetonka, MN) through an 18 gauge 
intravenous catheter, as previously described by our group and others.49, 82, 86 Cells will be 
recovered from the wires by washing with a dissociation buffer and centrifugation. Cells will be 
fixed with 4% paraformaldehyde (USB corporation, Cleveland, OH), washed thoroughly with 
PBS, plated on poly-l-lysine coated slides (Sigma Chemical, St. Louis, MO), and stored at -80°C 
until the immunofluorescence staining will be performed. 
 
Immunofluorescence: Fixed vascular endothelial cells will be rehydrated with PBS containing 
50 mmol/L glycine. After blocking non-specific sites with 5% donkey serum (Jackson 
Immunoresearch, West Grove, PA), slides will be incubated with a primary antibody (rabbit) for 
phosphorylated VEGFR-2 (Santa Cruz, Dallas, TX) or IL-6 (Abcam, Cambridge, MA) or VCAM 
(Abcam) followed with a secondary antibody with Alexa Fluor 488 goat anti-rabbit IgG 
(Invitrogen, Carlsbad, CA). To allow identification of endothelial cells, slides will also be stained 
with a primary antibody for VE cadherin (Abcam) and a secondary antibody with Alexa Fluor 
555 (Life technologies, Grand Island, NY). Finally, to determine nuclear integrity, slides will be 
mounted with Vectashield containing the nuclear stain DAPI (Vector Laboratories, Inc., 
Burlingame, CA). To minimize the potential confounder of inter-batch variability in staining, 2 
slides of HUVEC will be stained in each batch and endothelial cell protein levels will be reported 
relative to average HUVEC intensity in that batch. 
 
For analysis, cells will be examined with a fluorescence microscope (Eclipse 80i, Nikon 
Instruments, Inc., Melville, NY) at 100 X magnification using the same exposure time. Images of 
endothelial cells (identified by the presence of von VE cadherin staining) with intact nuclei 
(confirmed by DAPI staining) will be digitally captured by a coolSNAP ES2 camera 
(Photometrics, Tun, AR). Endothelial cell VEGFR-2, Il-6 or VCAM levels will be measured by 
quantifying Alexa Fluor 488 intensity while correcting for background fluorescence (NIS 
Elements software (version 3.2), Nikon Instruments, Inc., Melville, NY) and reported as intensity 
normalized to the intensity of the HUVEC fluorescence. 
 
C.3. Specific Aim 3: Determine the interaction of renal function and dosing of EPO on a) the 
rate of carbamylation; and b) the length of time that total and cEPO exceed the 
Kd of the βCR/EPOR. 
Rationale: Our hypothesis predicts that administration of low-doses of EPO three times weekly, 
as compared to the same cumulative dose administered as a high-dose every 2 weeks, will 
result in lower peak EPO levels and less activation of the heterodimeric βCR/EPOR. Key to our 
hypothesis is that the Kd of heterodimeric βCR/EPOR is ~50 mIU/ml, 10-fold higher than that of 
the homodimeric EPOR. This hypothesis is supported by a recent meta-analysis of all 
prospective EPO trials that found that adverse cardiovascular effects from EPO were strongly 
associated with EPO dose even when controlled for target hemoglobin levels.38 Studies have 
also found a strong association between endogenous EPO levels and cardiovascular events.54-
56 Complicating the situation is that EPO, in CKD patients, is carbamylated, a process where 
cyanate derived from elevated urea levels can non-enzymatically bind to proteins. cEPO lacks 
erythropoietic activity, but can still bind and activate the heterodimer βCR/EPOR. Levels of 
carbamylated albumin, a surrogate for cEPO, are increased in patients with kidney disease and 
levels correlate with EPO resistance.87 However protein levels and amino acid composition 
affects levels of carbamylation88 and the carbamylation of albumin could be vastly different from 
that of EPO. In addition, cEPO may have a very different half-life than EPO itself. We have 
devised a novel ELISA that will enable us to directly measure total and cEPO levels 
simultaneously. Our ability to directly measure cEPO gives us the ability to determine how EPO 
levels and CKD affect the kinetics of carbamylation. This has not been previously described!  


 
Study design: We will perform a pharmacokinetic study to assess the total levels of EPO and 
cEPO in approximately 10 subjects with CKD randomized to low-dose EPO administered three 
times a week and 10 subjects randomized to approximately the same cumulative dose of EPO 
administered as a high-dose once every 2 weeks. In subjects given low dose EPO, we will 
perform the pharmacokinetic studies after at least 2-4 weeks of continuous EPO therapy, 
ensuring ensure steady state EPO levels and stable hemoglobin levels. Previous studies have 
shown that those assigned to high-dose EPO every 2 weeks will never be in steady state, since 
EPO levels return to baseline by 7-10 days.89 Since we are unsure about the half-life of cEPO, 
we will study these patients after at least 6-8 doses of EPO. We will exclude smokers, since 
smoking has been has been shown to increase cyanate and carbamylation through 
myeloperoxidase-catalyzed oxidation of thiocyanate.90 To isolate the effects of renal function on 
carbamylation and EPO levels, we will also include approximately 20 age- and sex-matched, 
nonsmoking, healthy subjects, 10 of whom will be given a single low-dose of EPO and 10 given 
a single high-dose of EPO. Short-term administration of EPO to healthy controls is safe and has 
been used in multiple studies.64, 65, 81, 89, 91 
 
Subjects will be brought into the VAMC for the pharmacokinetic study. Venous blood (3 ml) will 
be attempted to be collected in heparinized tubes before and at approximately 1, 3, 6, 9, 12, 15, 
24, 36, and 48 hours after subcutaneous administration of EPO.  Those subjects receiving EPO 
chronically will receive their regular EPO dose, those healthy subjects will receive approximately 
25 IU/kg or 300 IU/kg. Since the time that the blood will be collected will be recorded, the time of 
the blood draws are not critical and are just approximate and clinical circumstances may 
necessitate missing some of the blood draw times or delaying others.  Subjects will be housed 
near the research unit to allow for the timed blood draws. In some circumstances, the subjects 
may need to be taken to the Clinical Research Center at UF to complete the lab draws.  Serum 
will be separated and stored at -80°C till analysis. Carbamylated and total EPO will be 
measured simultaneously using a sandwich ELISA as described in detail in the methods 
section.64, 89 
 
Sample size and feasibility: The aim is to determine a) the length of time that total and cEPO 
exceeds the Kd of the βCR/EPOR and b) the interaction of renal function (normal and CKD) and 
dosing of EPO (low vs. high dose) on the rate of carbamylation. With 20 subjects we will have 
80% power to detect a 36 or 47% difference in AUC > 50 between groups and at 90% power a 
difference of 54 or 69% at =0.05 and 0.01 respectively. Our modeling studies predict an AUC > 
50 of 4,413 and 736 in the high and low EPO groups. While we will have enough power to 
detect the effect of dosing regimens on EPO levels that exceed 50 mIU/ml, the power to detect 
an interaction will be insufficient and analysis will be exploratory. This will be informative in 
terms of variance estimation for future studies. Because these observations are outlier prone, 
we shall use natural logs. 
 
Data analysis and interpretation: A one compartment, open model, assuming first order 
absorption and a constant endogenous production of EPO, will be used to determine the plasma 
concentration-time profile of EPO and cEPO as per the published literature.92 The 
pharmacokinetic parameters and the proportion of AUC above 50 mIU/ml will be calculated for 
each subject using WinNonlin software. The key interaction estimate is the AUC above 50 
mIU/ml in patients with kidney disease (low-dose vs.high-dose) and the response for controls 
(low-dose vs. high-dose). Because these observations are outlier prone, we shall use natural 
logs. 
 


Expected results: This aim will characterize the pharmacokinetics of EPO and cEPO. Our 
hypothesis predicts low-dose EPO will significantly decrease total and cEPO levels and the 
length of time that the total serum levels of EPO are above the Kd of the heterodimeric 
EPOR/βCR. 
 
Methods: 
EPO ELISA: EPO levels will be determined by a solid phase sandwich ELISA (R&D). This 
assay has a sensitivity of 0.6 mIU/ml and an assay range of 2.5-200 mIU/ml.  
 
cEPO ELISA: We will perform a sandwich ELISA using a primary, capture, anti-EPO antibody 
(R&D biosystems), a secondary biotinylated anti-carbamylated lysine antibody (MyBioSource, 
Inc.) and Streptavidin conjugated poly horse radish peroxidase (Pierce) to increase the 
sensitivity of the assay. cEPO levels will be determined using a standard curve using known 
amounts of cEPO made from known amounts of EPO. We will confirm complete carbamylation 
by the lack of any erythropoietic activity, as previously described.1 
7. Possible Discomforts and Risks: 
The potential risks to subjects include: 
a) Risks related to EPO: EPO has been associated with high blood pressure, swelling, fever, 
dizziness, nausea and pain at the site of the injection. However, we will use EPO as per 
standard clinical standard guidelines issued by Kidney Dialysis Outcomes Quality Initiative 
(KDOQI). As such the risks relate to that of EPO and no excess risk should result from 
participating in this study.  
b) Risks of MRI: we will not use contrast. Non contrast MRI is not associated with serious 
untoward effects. Some subjects may feel claustrophobic. 
 
c) Risks of endothelial cell capture: Endothelial capture can result in endothelial dysfunction, 
vessels spasm and bleeding. However, in all the collections done to date, in multiple clinical 
conditions there is no report of any serious adverse effects.  
 
d) Risks related to venipuncture: There may be minimal risk of bruising, bleeding or infection at 
the site of puncture or psychological stress associated with the lab draw itself. However we will 
utilize an experienced phlebotomist for blood draws and we do not anticipate any problems. 
 
e) Risks related to potential breach of confidentiality: There is minimal risk of breach of 
confidentiality. 
8. Possible Benefits: There is potential for study subjects to benefit from this study. Subjects 
enrolled in this study may benefit from diagnosis of subclinical carotid atherosclerosis that may 
otherwise have been missed. These results will be communicated to the primary physician caring 
for the subjects. 
9. Conflict of Interest: None. 
 
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