Erythropoietin, Heart Attacks, and Beagles

Although the primary role of erythropoietin is to stimulate erythropoiesis, it has been shown to have a multitude of non-erythropoietic effects (Fisher, 2010). One such effect includes a cardioprotective mechanism that is activated by EPO during onset of cardiac ischemia (Hirata et. al, 2006). Ischemia refers to the restriction of blood flow to the tissues which in turn restricts the oxygen and glucose supply to these tissues, needed for cell metabolism (Silverthorn, 2009). A supplementary function of EPO during ischemia is the stimulation of endothelial progenitor cell (EPC) mobilization which is predicted to enhance neovascularization of ischemic regions (Hadley & Levine, 2007). “Erythropoietin Enhances Neovascularization of Ischemic Myocardium and Improves Left Ventricular Dysfunction after Myocardial Infarction in Dogs” is a journal article that pertains to a study conducted by Hirata et. al (2006), which aims to characterize the actions of erythropoietin on neovascularization and cardiac function following a myocardial infarction (heart attack). The investigators hypothesized that erythropoietin increases blood supply through ischemic regions by neovascularization and improving cardiac function after an ischemic event (Hirata et. al, 2006). The subsequent experiments were performed on beagle dogs due to similar cardiovascular homology to humans.   

*Article Link: http://www.sciencedirect.com/science/article/pii/S0735109706009156

 

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Figure 1 (A) & (B): (A) Adult grown Beagle dog. (B) Simplistic diagram depicting ischemia induced myocardial infarction (heart attack).

 

Images Retrieved from:

(A) – www.sfgate.com/blogs/images/sfgate/pets/2009/05/01/beagle-dog2500x375.jpg

(B) - myhealth.alberta.ca/health/_layouts/healthwise/media/medical/hw/h9991270_002.jpg

 

In this particular study, the investigators tested the acute effects that EPO administration has on the size of a myocardial infarction and also the effects of immediate or delayed EPO administration on neovascularization of the cardiac tissue and the resulting function.

 

 

In the immediate/delayed effects test, the investigators set up a control group (consisting of 8 dogs) and three experimental groups which received EPO at different time points following the LAD ligation. This enables the researchers to determine both short term and long term effects in one test. Similarly, myocardial blood flow and infarct size was measured at relevant time points. Cardiac function was determined using echocardiography, and tissue samples from both ischemic and non-ischemic regions were obtained and compared after all tests were conducted.

 

 

Figure 2A and 2B depicts the measurement protocols that would be followed for each of the two tests conducted, with the corresponding time interval. In the acute effects test, the investigators ligated the left anterior descending coronary artery (LAD) to ensure that all dogs relatively experience the same degree of ischemia. Both a control group, treated with saline, and an experimental group, treated with recombinant human EPO, was used for testing and both groups each consisted of 6 dogs. Blood flow to the heart and heart attack size was measured at 6 hours after dose administration.

For both experiments, hemodynamic parameters were measured at all relevant time intervals to monitor mean arterial blood pressure, heart rate, and left-ventricular end diastolic pressure.  
 

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Figure 2: (A) Measurement protocols to determine acute effects of EPO administration on infarct size. (B) Measurement protocols to determine effects of immediate and delayed administration of EPO on neovascularization and cardiac function.

 

http://www.sciencedirect.com/science/article/pii/S0735109706009156

 

 

Results Summary

Acute Effects Test;
 

- According to Figure 3, the researchers demonstrated that the myocardial infarct size was observed to be significantly smaller in dogs treated with EPO than the control group, treated with saline (Hirata et. al, 2006). However, there was little difference observed between the two groups regarding regional blood flow to the heart, and also the area affected by infarct-induced necrosis (tissue death). Referring to figure 3A and 3B, necrosis (blackened tissue) was present to the same degree in both control and EPO-treated groups (Hirata et. al, 2006).

 

 

 

Figure 3: (A) Left ventricular cross section of heart, treated with saline, at 6 hours following heart attack. (B) Left ventricular cross section of heart, treated with EPO, at 6 hours following heart attack. (C) Graph depicting infarct size at 6 hours, following heart attack, for control group and EPO-treated group (open circles indicate infarct size of individual animals).

http://www.sciencedirect.com/science/article/pii/S0735109706009156

 

 

EPO administration immediately following LAD ligation improved cardiac function within 90 minutes after the heart attack event, presumably because of reduced infarct size which prevented long-term dysfunction to develop afterwards (Hirata et. al, 2006).

 

Immediate/Delayed Effects Test;

 

 

The vascular endothelial growth factor (VEGF) levels in the blood was shown to be elevated in both the control and EPO (0 hr) groups, peaking around 6 hours following heart attack (Hirata et. al, 2006). Therefore, this finding indicates that EPO administration did not affect VEGF levels. It has been shown that both VEGF and EPO stimulate endothelial progenitor cell (EPC) mobilization and that these two factors may act synergistically (Hirata et. al, 2006). However, it is possible that the dose of EPO used for testing was not sufficiently high enough to promote VEGF proliferation and subsequent endothelial cell production, and it is further postulated that higher doses of EPO would produce different results (Hirata et. al, 2006).

 

According to Figure 4, there was no significant difference in the LCX (non-ischemic) region regarding capillary density and capillary to myocyte ratio when comparing the groups. However, in the LAD (ischemic) region both the capillary density and capillary to myocyte ratio was much higher in the EPO (0 hr) and EPO (6 hr) groups but not in the EPO (1 wk) group (Hirata et. al, 2006). This finding suggests that, during acute phase, EPO promotes neovascularization in the ischemic region. The mechanism by which this occurs is thought to be accomplished by mobilizing endothelial cells, and increases the amount of blood flow to the ischemic region by increasing the number of capillaries feeding into the region (Hirata et. al, 2006).

 

 

 

Figure 4: (A) Images depicting non-ischemic regions (LCX region, a-d) and ischemic regions (LAD region, e-h) in the control and experimental groups, using immunohistologic staining with an antibody. (B) Graph depicting capillary density in LCX and LAD regions. (C) Graph depicting capillary to myocyte ratio in LCX and LAD regions.

http://www.sciencedirect.com/science/article/pii/S0735109706009156
 

Graph A from Figure 5 depicts the relative number of mononuclear cells (CD-34 positive) detected within circulation. The number of mononuclear cells (MNC’s) increased in all groups after 1 week of heart attack (Hirata et. al, 2006). The EPO (0 hr) and EPO (6 hr) groups exhibited significantly higher levels of MNCs compared to the control group and EPO (1 wk) group. Similarly, at 2 weeks after heart attack event the MNC level for control group and EPO (1 wk) returned to baseline levels, whereas MNC levels for EPO (0 hr) and EPO (6 hr) groups remained high. All groups returned to baseline levels after 4 weeks of the heart attack event (Hirata et. al, 2006). MNC count was shown to correlate with endothelial cell number and that increased levels of MNC by EPO administration, indirectly relates to elevated endothelial cells.

 

 

 

Figure 5: (A) Changes of circulating mononuclear cells (CD-34 positive) after LAD ligation in control group and experimental groups.

http://www.sciencedirect.com/science/article/pii/S0735109706009156

 

 

According to Figure 6, there was no pertinent difference in myocardial blood flow to the ischemic region between any of the groups, at 90 minutes post heart attack event (Hirata et. al, 2006). However, at 4 weeks post heart attack event the blood flow increased markedly in the EPO (0 hr) and EPO (6 hr) groups but not in the control group or the EPO (1 wk) group (Hirata et. al, 2006). Because neovascularization was enhanced in both of these groups as well, it is thought that the increased blood flow maybe a secondary effect to the increased capillary density.

 

Figure 6: Trend of myocardial blood flow to the ischemic region (LAD region) at 90 mins to 4 weeks post myocardial infarction event, in control group and experimental groups. 

 

 http://www.sciencedirect.com/science/article/pii/S0735109706009156

 

There was no pertinent differences in baseline values for the left ventricular ejection fraction, end-diastolic dimension (in mm), and end-diastolic pressure (Hirata et. al, 2006). The ejection fraction decreased significantly for all groups except the EPO (0 hr) group, following the myocardial infarction event (Hirata et. al, 2006). It was also shown that pressure for the EPO (0 hr) group increased slightly more than baseline for the end-diastolic pressure, but was still lower than the control group and other experimental groups (Hirata et. al, 2006).

The same result was observed regarding the end-diastolic dimension parameter. Relative to the control, the EPO (0 hr) group is the only group that clearly demonstrates a lower infarct size percentage at 4 weeks post myocardial infarct event. The EPO (6 hr) and EPO (1 wk) groups’ showed similar infarct sizes to the control group (Hirata et. al, 2006). EPO treatment immediately following the heart attack event, induced by LAD ligation, was shown to reduce infarct size. Nevertheless, these size-limiting effects occurs rapidly and is possibly mediated by EPO’s non-erythropoietic actions such as anti-apoptosis and “scavenging” oxygen radicals which prevents tissue death (Fisher, 2010).

 

 

Figure 7: (A) Change in left ventricular ejection fraction (LVEDF), (B) end diastolic dimension, (C) end diastolic pressure, and (D) infarct size during the course of test period in control group and experimental groups.

http://www.sciencedirect.com/science/article/pii/S0735109706009156

 

In all tested groups, there were no significant differences observed in hemodynamic parameters such as arterial blood pressure and heart rate. This finding suggests that EPO actions selectively affected certain cardiac parameters and did not completely alter the manner in which the heart functions.

 

Through this study, it was shown that erythropoietin can have a cardioprotective effect in dogs and reduce the damage that results from a heart attack, only if the hormone is administered within a short time window. It is further suggested that recombinant human EPO may be used as a supplement for enhancing recovery in patients who have suffered a myocardial infarction (Hirata et. al, 2006).

Although this study yielded intriguing results and the investigators clearly tested multiple parameters to support their conclusions, I believe there are some concerns regarding their experimental protocols.

 

Firstly, the entire study was conducted using 47 dogs. For the two experimental protocols; each group, whether a control or EPO group, only consisted of 6-8 dogs. I believe this sample size is too restricted for testing an event (heart attack) that can have such very diverse effects. Furthermore, the investigators state that four dogs were excluded from the data analysis due to excessive regional myocardial blood flow following the coronary artery ligation (Hirata et. al, 2006). This further indicates that certain blood parameters can be very different between dogs, even dogs of the same breed and relative size.

 

Secondly, all dogs tested were from the beagle breed. Evidently, this was done in order to account for any differences between dog breeds and also because beagles are notably the dog breed most often used in animal testing, due to their passivity and size (Carlson, 2012). Although commonly used as surrogates for human testing, I believe the researchers should have included dogs from different breeds as differences between dog breeds may also reflect the differences between humans. Also, supplementary testing on miniature pigs may be appropriate due to the higher organ homology with humans (Carlson, 2012).

 

Based on these results, the investigators suggest that exogenous EPO may be used in long term treatment for its cardioprotective effects but they do not account for any of the erythropoietic effects. Using recombinant human erythropoietin to treat ischemia induced damage may disrupt the production of natural EPO. Lastly, the testing period was 4 weeks long which may be too short to discount long term effects of EPO administration.

 

Despite this, I believe the investigators carried out their experiments thoroughly and the included results is sufficient to support the authors’ claims that immediate administration of erythropoietin, following an ischemia-induced heart attack, may reduce infarct size and promote neovascularization in the ischemic region.

 

Future Experiments

 

In this study, the investigators wanted to determine whether erythropoietin has cardioprotective effects in ischemic regions. The results of the study supported their claims, albeit with some limitations.

It was previously mentioned that EPO may have neuroprotective effects; I would like to conduct a study to determine whether the same cardioprotective effects of EPO can also act as protectants for neurons in the brain, during ischemia. Ischemic stroke is the condition which results when the brain does not receive sufficient blood to support its metabolic requirements (Silverthorn, 2009).

 

 

 

Figure 8: CT Scan of brain section showing cerebral infarct in the right hemisphere.

http://en.wikipedia.org/wiki/File:INFARCT.jpg

 

For the experiments I would use a rat model, because of our high understanding of the underlying connections, and I would perform similar experiments to the ones conducted by Hirata et. al, 2006. By severing a major cerebral artery in the brain, the resulting ischemia would induce an infarction (heart attack-like event). Administration of recombinant human EPO at different time points and concentrations following ligation, will allow us to monitor the effects at different parameters. EPO can be directly administered to the ventricles using a cannula implantation, via stereotaxic surgery, because as a peptide hormone it will be unable to cross the blood brain barrier.

 

 

It has already been implicated that EPO has neovascularization effects. I expect (and hope) that the increase in capillary formation will be able to perfuse the ischemic regions and reduce any subsequent necrosis. Using a rat model will also allow us to use a larger sample size for the study due to the availability and ethical approval for use in experimentation (Carlson, 2012). A large sample size in a study like this is critical as different rats (and humans) respond to ischemic events in different ways. These studies will allow us to better understand the non-erythropoietic effects of erythropoietin and may also serve as a possible conduit for future treatments.  

 

References

- Carlson, N. R. (2012) Physiology of Behavior (11^th ed.), Pearson Education, Inc. Upper Saddle River, NJ

- Fisher, J. W. (2010) Landmark advances in the development of erythropoietin. Experimental Biology and Medicine. 235 (12): 1398-1411

 

- Hirata, A., Minamino, T., Asanuma, H., Fujita, M., Wakeno, M., Myoishi, M., Tsukamoto, O., Okada, K., Koyama, H., Komamura, K., Takashima, S., Shinozaki, Y., Mori, H., Shiraga, M., Kitakaze, M., and Hori, M. (2006) Erythropoietin Enhances Neovascularization of Ischemic Myocardium and Improves Left Ventricular Dysfunction after Myocardial Infarction in Dogs. Journal of the American College of Cardiology; 48 (1): 176-184

- Silverthorn, D. U. (2009). Human Physiology, An integrated approach (5^th ed.). Benjamin Cummings, Pearson Education : San Francisco, CA

Erythropoietin - Function

Principally, the primary function of erythropoietin is to induce erythropoiesis, red blood cell production, in hematopoietic tissues but also to maintain hemoglobin concentrations within a normal range during steady-state conditions (Hadley & Levine, 2007). The release of erythropoietin is stimulated by a reduction of oxygen (O₂) content in the blood (Hadley & Levine, 2007). EPO is essential for the proliferation and survival of erythrocyte progenitor (erythroid) cells in the bone marrow or fetal liver (Ng et. al, 2003). Furthermore, the glycoprotein has also been implicated in having immunomodulatory effects in the body (Ng et. al, 2003).

Figure 1: EPO action on erythroid cells of hematopoietic bone marrow.

http://sickle.bwh.harvard.edu/epoironmodel.gif

Studies have shown that circulating EPO increases exponentially following a sharp decrease in hemoglobin levels, which may occur in the case of ischemia (Hadley & Levine, 2007). The feedback regulator of EPO production however, is not mediated by hemoglobin concentration or red blood cell concentration in the body but by oxygen pressure in the tissues (pO₂) (Hadley & Levine, 2007). Tissue oxygen pressure therefore depends on hemoglobin concentration which corresponds to erythrocyte level in the body (Silverthorn, 2009). It is because of this feedback mechanism that habitation at higher altitudes, where atmospheric oxygen pressure is low, EPO production is stimulated to increase the production of red blood cells and hence the carrying capacity of oxygen to the body (Silverthorn, 2009).

Figure 3: Pictographic description of the primary function of erythropoietin in the body.

http://pharamcytimes.com/category/human-anatomy-physiology/the-blood/

 

It is postulated that oxygen sensing in the body is carried out by a heme protein, however recent evidence implicate that reactive oxygen species (ROS) triggers the hypoxia-induced transcription of the EPO gene (Hadley & Levine, 2007). The hypoxia-induced factor (HIF) is a family of proteins which serve as transcriptional factors to mediate oxygen homeostasis (Hadley & Levine, 2007). HIF-2 is the thought to be the major transcriptional factor that activates EPO gene expression (Hadley & Levine, 2007).

 

When erythrocyte level is low in the body, peritubular fibroblasts in the kidneys are stimulated and release erythropoietin into the peritubular capillaries which transports the hormone into the general systemic circulation. EPO then enters the vascular system which supports the hematopoietic bone marrow (red marrow) and binds to specific erythroid precursor cells that expresses the EPO receptor (Ng et. al, 2003). The erythropoietin receptor is a member of the superfamily of cytokine receptors (e.g. GH receptor) and characterized as a 72-78 kDa glycosylated/ phosphorylated transmembrane polypeptide (Ng et. al, 2003). There are two distinct erythroid progenitor cells contained within the erythroid cell compartment of the bone marrow; the burst forming unit-erythroid (BFU-E), and the colony forming unit-erythroid (CFU-E) (Fisher, 2010). Initially, the BFU-E does not appear to be sensitive to EPO but gains sensitivity as it matures, whereas the CFU-E is inherently a more mature cell and serves as the primary target of EPO (Fisher, 2010). CFU-E differentiation is completely dependent on erythropoietin.

 

 

Binding of EPO to its receptor in the membrane of erythroid cells induces a dimerization of the receptor with another erythropoietin receptor (homodimer), causing a conformational change that allows Janus family tyrosine protein kinase 2 (JAK2) molecules to associate with the receptor and become activated by trans-phosphorylation (Fisher, 2010). JAK2 molecules in turn phosphorylate tyrosine residues of the EPO receptor in the cytoplasmic domain (Fisher, 2010). These residues serve as a docking site for various homologous intracellular proteins which leads to the activation of STAT5A and STAT5B, as well as the Ras/MAP kinase pathway and other kinases, all of which are involved in gene activation (Fisher, 2010). The STAT5A and STAT5B molecules associate following activation and translocate to the nucleus where it binds specific hormone response elements that leads to transcription and translation of proteins which triggers cellular proliferation (Fisher, 2010). The acute of effects of EPO lasts between 30 and 60 minutes (Hadley & Levine, 2007).

Figure 4: Simplistic diagram of the main signal transduction pathways activated by EPO receptor.

http://pmj.bmj.com/content/79/933/367.full

 

Termination of EPO’s effects is accomplished by a hematopoietic cell phosphatase (HCP) which catalyzes the de-phosphorylation of JAK2 (Hadley & Levine, 2007). Mutations of the HCP enzyme or the erythropoietin receptor leads to erythrocytosis, an abnormally high red blood cell level (Hadley & Levine, 2007).

Erythropoietin exerts several non-erythropoietic actions including stimulation of mitosis along with cardioprotective and neuroprotective effects (Fisher, 2010). EPO increases the number of endothelial progenitor cells, as well as migration of the mature forms (Hadley & Levine, 2007). Hepcidin suppression by EPO also increases iron absorption in the body, which is a required building block for erythrocytes (Silverthorn, 2009). A complete knock out of the EPO receptor gene results in severe cardiac malformations and fetal death, possibly due to tissue hypoxia and anemia (Hadley & Levine, 2007).


It has been demonstrated that EPO can act as a neuroprotective whereby it inhibits the death of neurons due to lack of blood supply and oxygen (Fisher 2010). This is accomplished by preventing the formation of oxygen radicals due to nitrogen oxide accumulation, or by antagonizing the effects of these radicals (Fisher, 2010). Preliminary studies have also shown that recombinant human EPO produces a protective effect in animal models for multiple sclerosis (Fisher 2010). In addition, EPO may act to inhibit apoptosis in cardiomyocytes and reduce the deterioration of these cells following an ischemic event, such as a myocardial infarction (Fisher, 2010). It has been postulated that non-hematopoietic effects of EPO may be regulated by heteromers of the erythropoietin receptor (Hadley & Levine, 2007).

Figure 5: Non-hematopoietic effects of EPO on cells of the nervous system.

http://www.drjen4kids.com/soap%20box/426744_330923730280269_134844053221572_895462_222560101_n.jpg

Figure 6: (a) Coronal section of mouse brain following blunt force trauma and treated with saline solution(above) compared to coronal section of mouse brain following similar trauma but treated with recombinant human erythropoietin (below). (b) Bar graph depicting the relative degree of necrosis, following blunt force damage, in the brain region in a sham control and a EPO treated subject.

http://ebm.rsmjournals.com/content/235/12/1398.full

Chronic renal failure can lead to anemia, lower than normal red blood cell levels, which is caused by an insufficient amount of EPO required for new erythrocyte production (Fisher, 2010). This insufficiency is perpetuated by a decrease in renal function. Another proposed cause is resistance to endogenous EPO (Fisher, 2010). Studies on mice have shown that a knockout of the EPO gene during embryological development is lethal, caused by anemia and heart hypoplasia, therefore the same knockout effect in adults can only be studied using a gene silencer such as Cre recombinase (Zeigler et. al, 2010). Following induction of Cre, mutant mice exhibited a much lower serum EPO level and consequently developed chronic, normocytic, anemia (Zeigler, 2010). It was also demonstrated that target genes, which is acted upon by EPO, is significantly reduced in the bone marrow. These observations are similar to the clinical signs of chronic kidney disease (Zeigler, 2010).


Systemic overexpression of EPO results in erythrocytosis and the subsequent high erythrocyte level increases the hematocrit. However, drugs are currently under development that will act enhance the protective effects of EPO without increasing the hematocrit (Jelkmann, 2007).

Figure 7: Hematocrit levels for different levels of erythrocytes; affected by EPO conc.

http://encyclopedia.lubopitko-bg.com/Blood_Studies.html

 

References

-  Hadley, M. E. & Levine, J. E. (2007). Endocrinology (6^th ed.). Prentice Hall, Pearson Education: Upper Saddle River, NJ

- Fisher, J. W. (2010) Landmark advances in the development of erythropoietin. Experimental Biology and Medicine. 235 (12): 1398-1411

 

- Ng, T., Marx, G., Littlewood, T., & Macdougall, I. (2003) Recombinant erythropoietin in clinical practice, Postgraduate Medical Journal, 79: 367-376

 

- Silverthorn, D. U. (2009). Human Physiology, An integrated approach (5^th ed.). Benjamin Cummings, Pearson Education : San Francisco, CA

- Jelkmann, W. (2007). Erythropoietin after a century of research: younger than ever. European Journal of Haematology. Institute of Physiology, University of Luebeck: Luebeck, Germany

- Zeigler, B.M. Vajdos, J. Qin, W. Loverro, L. Niss, K. (2010) A mouse model for an erythropoietin-deficiency anemia, Disease models and mechanisms, 3(11-12); 763-772