2026 Current Pharmaceutical Design, 2009, 15, 2026-2036

1381-6128/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.

Regenerative Medicine: Does Erythropoietin have a Role?

Michele Buemi1,*, Antonio Lacquaniti

1, Giulia Maricchiolo

2, Davide Bolignano

1, Susanna Campo

1,

Valeria Cernaro1, Alessio Sturiale

1, Giovanni Grasso

1, Antoine Buemi

1, Alessandro Allegra

1,

Valentina Donato1 and Lucrezia Genovese

2

1Chair of Nephrology, Department of Internal Medicine, University of Messina, Italy and

2Istituto per l'Ambiente

Marino Costiero (IAMC)-CNR, Sezione di Messina (Istituto Sperimentale Talassografico), Messina, Italy

Abstract: Regenerative Medicine, a recent new medical domain, aims to develop new therapies through the stimulation

of natural regenerative processes also in human beings. In this field, Erythropoietin (EPO) represents a significant subject

of research. Several studies allow the assertion that EPO, in different concentrations, has protective effects mainly on the

central nervous system, cardiovascular system and renal tissue. This action is carried out through one of few regenerative

activities of human beings: angiogenesis. This mechanism, which involves endothelial stem cells and VEGF (Vascular

Endothelial Growth Factor), has been experimentally demonstrated with Recombinant human erythropoietin (rHuEPO)

and Darbepoetin, a long-acting EPO derivate. Furthermore, the demonstration of a cardiac production of EPO in Fugu ru-

bripes and in Zebrafish has led cardiologists to “discover” Erythropoietin, postulating a hypothetical role in treatment of

cardiovascular disease for this hormone. This is some of the experimental evidence which demonstrates that EPO can be

in reason considered an important element of research of Regenerative Medicine and put in the network of drugs able to

regenerate tissues and organs.

Key Words: Regenerative medicine, erythropoietin, angiogenesis.

INTRODUCTION TO REGENERATIVE MEDICINE

Regeneration has always been deeply fascinating, inspir-ing many myths. Behind it lies the hope of immortality, of rejuvenation or at least of the reconstruction of injured or-gans. Among vertebrates, only teleost fishes and urodel am-phibians naturally keep their ability to regenerate injured or amputate parts for life. Recently, the understanding of the regeneration mechanisms of complex structures in the adult salamander has opened original lines of research. Regenera-tive Medicine, a new medical domain, tries to develop thera-peutic pathways through the stimulation of natural regenera-tive processes also in humans. The mechanisms for func-tional recovery are under the observational lens of research-ers, whose purpose is to know the natural network that regu-lates the regeneration process and the intricate connections that lead to the production of hormones and mediators fun-damental to the regeneration of different tissues [1].

During the last 20 years, the works of Brockes JP have validated some physiopathological assumptions of regenera-tive medicine. In a 1984 study [2], this author showed that the regeneration of a limb in amphibians after amputation depends on the division of progenitor cells called cells of the blastema. The cell division is controlled, at least in the early stages of the regeneration process, by the nerve fibres that innervate the blastema. Using a monoclonal antibody di-rected against the blastema cells of salamander, Brockes has also shown that Schwann cells and muscle fibres contribute

*Address correspondence to this author at the Via Salita Villa Contino, 30,

98100 Messina, Italy; Tel: +39090-2212265; Fax: +39090-2935162;

E-mail: buemim@unime.it

to form the blastema and that the Glial Growth Factor (GGF) is present in the blastema of salamander and is lost after den-ervation. In 2005, the same author studied the regeneration process of more complex structures using adult salamanders as a model, concluding that regeneration depends on mecha-nisms that include the plasticity of differentiated cells. The regeneration of limbs proceeds through the local formation of blastema, an area of growth of mesenchymal stem cells, at the level of the stump of amputated limb [3].

In amphibians, regeneration is carried out through the dedifferentiation of cells (fibroblasts, muscle cells, os-teoblasts, Schwann cells, etc..) in stem cells. These cells mi-grate at the level of amputation and proliferate to form clus-ters of pluripotent cells, called blastema of regeneration, which will form the missing part. As has recently been dem-onstrated by Stocum [4], larval and adult urodels are able to readily regenerate their limbs through histolysis and dedif-ferentiation of mature cells adjacent to the surface of ampu-tation. These accumulate under the epithelium of the lesion to form a blastema of stem cells. These stem cells require growth and trophic factors arising from the epidermal sur-face and nerves that reinnervate the blastema ensuring their survival and proliferation. Stem cells derived from fibro-blasts and muscle cells can trans-differentiate into other types of cells during regeneration. The regeneration blastema is a self-organized system based on information of position inherited by limb precursor cells genetically addressed. The probable growth and proliferation factors involved include: members of the family of Fibroblasts Growth Factors (FGF) synthesized by the epidermal surface and nerves, Glial Growth Factors, substance P, nervous fibres transferrin. In addition, many molecules are involved in the network of

Regenerative Medicine Current Pharmaceutical Design, 2009, Vol. 15, No. 17 2027

cell-cell signals that allows regeneration of amputated struc-ture: FGF, Wnt / beta-catenin, TNF, BMP, IGF...

The study of the process of regeneration in amphibians through dedifferentiation is important for the development of Regenerative Medicine, since the understanding of the un-derlying mechanisms can offer pharmacological indications on the regeneration of tissues in mammals [5].

ERYTHROPOIETIN AND REGENERATIVE MEDI-CINE

In the field of Regenerative Medicine Erythropoietin rep-resents a particularly significant subject of research. In fact, besides the ability of stimulating red blood cell production, this hormone has many pleiotropic effects; that is to say that it can act on several types of cells through different mecha-nisms, carrying out functions additional to the role which is classically attributed to it. For example, it contributes to modulation of inflammatory and immune process enhancing phagocytic function of the polymorphonuclear cells and re-ducing the activation of macrophages [6]. Moreover, EPO has an important direct hemodynamic and vasoactive action, which does not depend exclusively on any increase in hema-tocrit and viscosity [7].

Several studies allow the assertion that EPO, in different concentrations, has protective effects mainly on the central nervous system and cardiovascular system through various mechanisms, among which a key role seems to be held by the ability to stimulate angiogenesis and to modify the apop-totic kinetics.

The ontogenetic development in nature is due to the in-crease, in millennium, of oxygen concentration in the atmos-phere. This is the main element which regulates the secretion of EPO. After all, in physiological condition there is a per-fect balance between contribution and consumption of oxy-gen; this equilibrium is maintained through adaptation mechanisms which intervene in conditions of critical oxy-genation. In this sense EPO secretion, glycoproteic hormone mainly produced by kidney and in minor quantities by the liver, is able to expand erythropoiesis, preventing CFU-E and proerythroblast apoptosis and stimulating erythroid cells proliferation and maturation.

At the level of endothelial cells of peritubular interstitial spaces and of proximal tubule cells, there is a "sensor" for oxygen, represented by a protein containing a heme group. In conditions of normoxia, oxygen interacts with the sensor and this triggers a cascade of reactions catalyzed by prolyl hydroxylase that culminate in the degradation of alpha subunit of HIF (Hypoxia-inducible factor), with consequent reduction of transcription, at nuclear level, of the gene cod-ing for erythropoietin [8]. On the contrary, in a state of hy-poxia, through a protein ROS the cascade of prolyl hydroxy-lase is inactivated and alpha subunit can bind to beta subunit to form a transcription factor (HIF alpha) that enters the nu-cleus and induces the transcription of the gene for erythro-poietin, resulting in the stimulation of erythropoiesis and increasing the capacity of the blood to meet the demands of oxygen [9].

However, it has been shown that the condition of hy-poxia, leading to the synthesis of erythropoietin, stimulates

not only erythropoiesis but also other processes such as an-giogenesis, anaerobic glycolysis, transport of glucose, the regulation of cell cycle and of apoptosis (Fig. 1).

The induced-production of erythropoietin happens not only in the kidney and partly in the liver, but also in other tissues. This has enabled the demonstration of the pleiotropic ability of the hormone. The first evidence came in 1989 when it was noted that the administration in bolus of rHuEPO is associated with dose-dependent increase of blood pressure [10].

Then, the existence of an Erythropoietin receptor at the level of endothelial cells of brain capillaries was demon-strated [11]. The presence of erythropoietin was highlighted in the cerebrospinal fluid in humans, demonstrating an in-trinsic production of this hormone in the brain [12].

These surprising data made it possible to better define the physiological activity of EPO. Red blood cells and endothe-lial cells derive from a single precursor, the hemangioblast which, if stimulated by Erythropoietin gives rise to red blood cells, but if it is subjected to the action of VEGF (Vascular Endothelial Growth Factor) differs into endothelial cell: fun-damental angiogenic element, which continues to own the receptor sensitive to Erythropoietin [13].

In this context, Ribatti et al. were among the first to ex-plore the angiogenic potential of Erythropoietin, showing among other things that the administration of rHuEpo (re-combinant human Erythropoietin) in vivo induces a powerful angiogenic response in chorioallantoic membrane of chick [14].

As demonstrated by Buemi et al., there are no-erythroid receptors that, depending on the type of cell, mediate the different biological activities of Erythropoietin [15]. For ex-ample, the receptors present on the stromal cell membrane of liver, if activated by binding with erythropoietin, have a mi-togenic action; at the level of testicular Leydig's cells, Erythropoietin stimulates testosterone production; receptors present on enterocytes, if activated, stimulate cell migration and play an anti-apoptotic action [16]. In addition, the erythropoietin production and the number of receptors in-crease progressively during embryonic development of the nervous system [17]. While in an experimental model of myocardial infarction, Parsa et al. have demonstrated a better functional recovery of the cardiac area periinfarctual after rHuEPO administration [18].

Erythropoietin appears to play a protective role at renal level too. Indeed, Aydin et al. have recently found that rHuEpo protects the kidney from ischemic and cytotoxic damage slowing the progression of renal failure [19].

The protective tissue action of Erythropoietin is dose- and receptor-dependent. This has led to pharmacological synthesis of various types of erythropoietin as the Asialo-erythropoietin, which has a half-life of less than 2 minutes (erythropoietin has a half-life of 5-10 hours), and also the CEPO (Carbamyl-erythropoietin), which owns fewer free NH2 groups. Just CEPO has shown to play a more effective protective action in damage associated with spinal cord compression, diabetic neuropathy [20] or experimental auto-immune encephalitis without an erythropoietic effect. This will allow in the future to exploit exclusively pleiotropic

2028 Current Pharmaceutical Design, 2009, Vol. 15, No. 17 Buemi et al.

tissue protective action even in patients with normal values of red blood cells, thereby avoiding the polyglobulia and increasing plasma viscosity.

This admirable not erythropoietic effect involves various natural mechanisms of action that on the whole, through a particular receptor, may carry the protective action of injured tissues: Proliferation, Anti-apoptosis, mobilization of stem cells and increase of VEGF expression, Angiogenesis [21] (Fig. 2).

ERYTHROPOIETIN AND ANGIOGENESIS

Angiogenesis is a set of functional processes appointed to the formation of new blood vessels starting from the pre-existent ones [22]. This is one of the few natural regenerative activities of humans. The angiogenic process is carried out through different stages: production and release of the angi-ogenic factor, link to endothelial cell receptor and conse-quent activation and proliferation of the latter, directional migration, remodelling of the extracellular matrix, training and stabilization of vessels.

Angiogenesis is realized in many physiological proc-esses: embryonic development, wound healing, menstrua-tion, pregnancy, etc., and is designed to provide oxygen and nutrients and then remove products of tissue metabolism [23-24].

Experimental data of Buemi et al. have suggested and demonstrated that rHuEPO, in a rat model of angiogenesis, is

able to stimulate the proliferation of endothelial cells thus promoting healing of wounds [25].

After this initial report, other authors have confirmed in several experimental models of ischemic, traumatic, toxic and inflammatory damage that tissue protective action of Epo is mediated by proliferative angiogenic action after the activation of an endothelial receptor, different from the erythropoietic one [26].

Then Epo has been experimentally used for the treatment of heart, neurological and kidney disease including cerebral and traumatic ischemia, myocardial infarction, congestive heart disease and drug cardiotoxicity and nephrotoxicity [27- 32] (Table 1).

Efthimiadou et al. have recently assessed the angiogenic potential of rHuEPO on cyclosporine-induced nephrotoxicity in rats, comparing it with a well known angiogenic factor, the b-FGF (basic-fibroblast growth factor). This study showed that Epo has a significant angiogenic effect, similar to the effect of the other angiogenic factor, b-FGF [33].

Epo also seems to have an angiogenic potential of entity overlapping to that of VEGF in inducing the formation of new vessels in human myocardial tissue [34].

But what are the Interactions between Erythropoietin

and VEGF?

Some authors have shown that erythropoietin can act as an angiogenic factor independently of VEGF. Inducing in

Fig. (1). Oxygen governs the activity of protein HIF-1 alpha. The protein HIF-1 alpha is very unstable in the presence of oxygen, it is rapidly

degraded in 5 minutes from proteasome. In a condition where prolyl hydroxylase is inactive, the protein HIF-1 alpha is quickly stabilised and

migrates in the nucleus where it binds to protein HIF-1 beta. The activation of HIF-beta, stimulated by hypoxia, causes the synthesis of pro-

tein factors involved in erythropoiesis, angiogenesis and anaerobic glycolysis.

Regenerative Medicine Current Pharmaceutical Design, 2009, Vol. 15, No. 17 2029

mice a myocardial infarction, Darbepoietina-alpha and VEGF-165 stimulate formation of new vessels starting from the Aortic arch independently of one another, even if Epo angiogenic action would be dose-dependent. In fact, the ad-ministration of moderate doses is not angiogenic but anti-apoptotic [35].

Moreover, in experimental models of both mice and pri-mates, the strong inhibition of VEGF led to an increase of hematocrit (60-75%) through induction of hepatic synthesis

of erythropoietin mediated by HIF-1-alpha, in parallel to suppression of kidney Epo mRNA. The induction of hepatic erythropoietin depends on VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. Consequently, VEGF can be considered a previously unsuspected negative regulator of hepatic Epo synthesis [36] (Fig. 3).

Another example of interaction between erythropoietin and VEGF is represented by models of renal damage from

Fig. (2). Molecular biology of the erythropoietic and tissue-protective effects of erythropoietin. The erythropoietic response to EPO (left) is

fostered through activation of EPO-R and downstream signal transduction pathways. Erythropoietin regulates the red blood cell mass in a

negative-feedback control system that involves the kidneys and the bone marrow. Upon binding to performed EPO-R homodimers, EPO

causes activation of the JAK2 kinases through transphosphorylation. The major pathways that facilitate the hematopoietic response to EPO

are the JAK2-STAT5, RAS-MAPK and P13K-AKT pathways. Unlike erythropoiesis, the EPO tissue-protective molecular pathway (right)

operates via an autocrine/paracrine mode. Tissue injury, local inflammation, hypoxia and metabolic stress induce the synthesis of HIF which

increases the production of EPO locally. The tissue-protective effects of EPO are fostered through a distinct receptor complex which is an

EPO-R/bCR heterotrimer. Activation of this heteroreceptor requires high concentrations of EPO, as its binding affinity is much lower than

the circulating concentration of EPO. The interaction between the intracellular domains of the EPO-R and bCR subunits leads to activation of

multiple downstream signalling pathways. Besides the JAK2-STAT5, RAS-MAPK and P13K-AKT cascades, the NF-kB and HSP70 path-ways are involved in tissue protection. Unlike erythropoiesis, the signalling pathways implicated in tissue-protection are not redundant.

Table 1.

Indication Results

Ischemic stroke Improvement in 1 month follow-up and outcome scales

Reduction in infarct size compared to control

Acute myocardial infarction

Darbepoetin was safe and well tolerated

Possible positive effect of darbepoetin treatment on longer-term infarct healing and cardiac

remodelling

CHF and kidney disease

Ongoing study

Goal to determine the impact of darbepoetin therapy on mortality and nonfatal cardiovascular

events in patients with chronic kidney disease and CHF

Non-diabetic kidney disease (predialysis) Early-EPO treatment group had a 60% reduction of the risk of initiating renal replacement or

death compared to the deferred-EPO treatment group

2030 Current Pharmaceutical Design, 2009, Vol. 15, No. 17 Buemi et al.

Ischemia/Reperfusion. Under these conditions a complete structural and functional recovery through several mecha-nisms usually occurs: the regeneration of tubular epithelial cells, the recruitment of immature cells mainly located in the medulary portion and a small number of stem cells that are derived from bone marrow [37].

On the whole there are now convincing data that erythro-poietin exerts a protective action in relation to its prolifera-tive and anti-apoptotic effects but also for the ability to mo-bilize stem cells and increase plasma levels and renal expres-sion of VEGF (Fig. 4).

The administration in the mouse brain of stem cells to-gether with rHuEPO significantly increases the expression and secretion of VEGF through the activation of signal trans-lation systems PI3K/Akt and ERK1 / 2; these cells increase the expression of VEGFR2 by brain endothelial cells thereby promoting the formation of new vessels [38].

Similarly, combining the infusion of Epo with the plant-ing of mesenchymal stem cells from bone marrow in rat in-farcted heart, better results are obtained in terms of angio-myogenesis and cardiac function recovery compared with an monotherapy approach with Epo or stem cells [39].

The myocardial Epo-EpoR system mediates activation of STAT3 and p38, VEGF production and development of cap-illaries, thus preserving the contractile function. The endo-thelial Epo-EpoR system can also induce coronary angio-genesis in hypertrophic myocardium too [40].

These several findings have led many cardiologists to "discover" erythropoietin, postulating for this hormone a possible role in the treatment of cardiovascular disease. This surprising link between erythropoietin and the heart is fur-ther defined in fish Fugu rubripes and Zebrafish where physiological production of this hormone is also of cardiac origin.

THE REGENERATIVE AND ANGIOGENETIC EX-PERIENCE IN FISH

The zebrafish or Danio rerio, is taking an increasingly dominant role in the study of the possible mechanisms that underlie the processes of tissue regeneration. This is a small freshwater fish that reaches 4-6 cm in length in adulthood. In recent years, it has been increasingly used as a model for studying the development of vertebrates and for many hu-man diseases: pathogenesis of some bacterial infections, neu-rodegenerations, hereditary and congenital diseases. Small size, big proliferative capacity, short interval between one generation and the next, embryo transparency make this fish an experimental model increasingly used in Regenerative Medicine.

The final site of hematopoiesis, as for all fish, is the kid-ney since seventh day from fertilization. Cheng-Ying and his team have recently cloned the gene coding for erythropoietin in zebrafish, highlighting in the codified protein a sequence homology equal to 90%, 55% and 32% respectively with the carp, humans and fugu erythropoietin.

Fig. (3). Pharmacologic (soluble receptor, VEGFR2-specific antibody) or genetic (hepatocyte VEGF-A) deletion inhibits VEGFR2 on liver

sinusoidal endothelial cells, resulting in active production of factor(s) by liver sinusoidal endothelial cells, which stimulate production of Epo by hepatocytes, with subsequent erythrocytosis.

Regenerative Medicine Current Pharmaceutical Design, 2009, Vol. 15, No. 17 2031

It was also shown that the main sites of expression of the gene for erythropoietin are represented by heart and liver [41].

Other authors, on the other hand, believe that in some fish the kidney is not only the main erythropoietic organ but also the largest source of erythropoietin. Wickramasinghe has in fact shown that supernatants prepared by kidney ho-mogenates of rainbow trout (Salmo gairdneri) contain more immunoreactive erythropoietin compared with autologous plasma, serum and supernatants obtained by homogenates of liver or spleen [42].

The mechanisms that underlie the production of erythro-poietin are similar in humans and in fish and are recognized as the main factor hypoxia. In fact, in fish, there is a relation-ship between the increase of renal erythropoietin levels, re-duced levels of splenic erythropoietin and spleen somatic index. Under conditions of hypoxia, spleen contraction and the subsequent release of erythrocytes justify the initial in-

crease of oxygen in the blood, while the erythropoietin ac-tion is probably responsible for increasing levels of haemo-globin which occur at a later stage [43].

Even the work of Rojas et al. highlighted the existence of an important sequence homology between Hypoxia-inducible growth factors (HIF) 1 and 2 between zebrafish and vertebrates on confirmation of a pathophysiological identity [44].

However, one of the differentiative foundations between zebrafish, vertebrates and humans is the natural regenerative capacity involving fins, skin, heart and brain in the larval stage of these primordial living beings. Regeneration seems an archaic tract of the animal world: it was kept in some metazoaria organisms and secondly lost in others for un-known reasons. However, the study of regenerative capacity of zebrafish is an experimental basis to study and understand why human beings have lost this extraordinary ability and what routes should be given to "recall the past." Bayliss et al.

Fig. (4). Possible mechanisms for the protective roles of the endogenous Epo-EpoR system in hypertrophied hearts. The myocardial Epo-

EpoR system mediates STAT3 and p38 activation, VEGF production, and capillary growth, resulting in the preservation of contractile func-tion. The endothelial Epo-EpoR system also may play a role in coronary angiogenesis in hypertrophied hearts.

2032 Current Pharmaceutical Design, 2009, Vol. 15, No. 17 Buemi et al.

have examined the role of angiogenesis and receptor signals that modulate this process through chemical inhibition of VEGF in the caudal fin of adult zebrafish, showing that tis-sues can regenerate without direct interaction with endothe-lial cells and to a certain distance from blood supply; moreo-ver adult specimen of zebrafish, heterozygous for a mutation in effector receptor phospholipase C 1, show greater sensi-tivity to chemical inhibition [45].

Recently, our group has used the Dicentrarchus Labrax (or Sea Bass), a saltwater teleost, as experimental angioge-netic model (unpublished data). The effects of rHuEPO in the hematopoietic process were assessed, especially in re-generation of caudal fin (Fig. 5).

These data show that even a saltwater fish, Dicentrarchus labrax, can be regarded as an angiogenetic tissue regenera-tion model. Indeed, in a group of Control (5), cutting of the caudal fin, stimulated the partial structural regrowth of the organ in a period of 30 days. Administration of recombinant human erythropoietin (3000 IU every week) has allowed the demonstration of the regenerative angiogenetic capacity of the hormone even in this experimental model (Table 2). The speed of growth and the size of caudal fin after 15 and 30 days of initiation of therapy was statistically higher in fish treated (5) compared to the control (5). Therefore, this ex-periment shows that the spontaneous regenerative angioge-netic capacity of caudal fin in fish can be pharmacologically

Fig. (5). The caudal fin of the bass (Dicentrarchus labrax) was cut along its middle.

A represents the bass control while B the bass treated with recombinant human erythropoietin alfa (3000UI/week subcutaneously). The ob-

servation period lasted 30 days. Fifteen days after cutting, an increase greater than the tail in treated fish compared to the control was noticed,

forming buds of capillaries on the entire surface of the cutting. In 30 days, a more obvious growth of the tail in terms of length and from the point of view of structural complexity is appreciated.

Regenerative Medicine Current Pharmaceutical Design, 2009, Vol. 15, No. 17 2033

increased with recombinant erythropoietin, as demonstrated in zebrafish with VEGF [46].

PATHOLOGICAL ANGIOGENESIS

Besides physiological angiogenesis, there is a pathologi-cal one. The excessive formation of new vessels can induce blindness, rheumatoid arthritis, cancer, AIDS complications and psoriasis. On the contrary a deficient angiogenesis can induce stroke, ischemic heart disease, ulcers, scleroderma and infertility.

Several studies show that EPO can play a role in the pro-liferative phase of diabetic retinopathy. We can distinguish two forms of diabetic retinopathy: not proliferative diabetic retinopathy, characterized by the presence of microaneu-rysms, haemorrhages and/or hard exudates, cottony nodules, anomalies of venous calibre and intravascular retinal anoma-lies; proliferative diabetic retinopathy, to which the not pro-liferative form evolves as a direct consequence of occlusion of retinal vessels, with formation of retinal ischemic areas that can lead to formation of new retinal vessels with the consequent risk of pre-retinal and intravitreous haemor-rhages and secondary retinal detachment.

It seems that EPO has, in proliferative diabetic retinopa-thy, a greater angiogenic effect than VEGF [47]. The vitre-ous EPO levels in patients with proliferative diabetic reti-nopathy are higher than that in nondiabetic patients; Epo and VEGF are independently associated with proliferative reti-nopathy; Epo is more strongly associated than VEGF; block-ade of Epo inhibits retinal neovascularization in vivo and endothelial cell proliferation in vitro [48]. On the other hand, taking into consideration models of diabetic mice, Epo proves to be able to promote repair of wounds stimulating several processes, like epidermis regeneration, angiogenesis and granulation tissue production [49].

ANGIOGENESIS AND CANCER

Proliferation and growth (expansion) of small colonies of metastatic cells can happen only if adequate quantities of oxygen and nutritious substances are provided; then, new vessel formation is a crucial event for the development of metastasis. Chemotactic factors, growth factor and prote-olytic enzymes contribute to the recruitment of endothelial cells, which from their sites migrate toward metastatic cells crossing vascular wall and degrading extracellular matrix.

The first draft of endothelial cells migrates and degrades the extracellular matrix giving rise to a lumen. Subsequently, cell proliferation supported by hormones and growth factors determines the formation of a capillary network that finally get organized in a real vascular structure. Several angiogenic molecules produced by both cancer cells and host have been identified, including TGF- , the basic Fibroblast Growth Factor (bFGF), the VEGF and angiogenin. Is this perhaps the ancestral regenerative "but alas pathological" memory which remains in humans [50] ?

And Erythropoietin?

We have already seen how experimental evidence for an angiogenic action of Erythropoietin is greater. The conse-quent problem that we have to face is that anaemia therapy with recombinant human Epo in patients with cancer may accelerate the progression of neoplastic disease by promot-ing tumour angiogenesis and thus, metastasization [51-57]. (Table 3)

Ribatti et al. [58] have shown that the Epo / EpoR system is involved in the angiogenesis that promotes hepatocellular carcinoma (HCC) in humans. Looking immunohistochemi-cally with anti-CD31, anti-Epo and anti-EpoR antibodies HCC primitive samples obtained from 50 patients undergo-ing curative hepatectomy, these authors have found that the poorly differentiated HCC had a high degree of vascularisa-tion compared to other stadiums and that the expression of Epo / EpoR increased both in cancer cells and in endothelial ones in parallel with the degree of malignancy and was highly correlated with the angiogenesis extent. In conclu-sion, this study has shown that levels of Epo / EpoR correlate with angiogenesis and the progression of neoplastic disease in patients with HCC and this suggests the existence of a "loop" in the Epo / EpoR system: Epo is secreted by cancer cells and acts with paracrine mechanism on vascular endo-thelium through its receptors promoting angiogenesis. This means that Epo plays an important role in angiogenesis of liver cancer. The same authors have shown that the Epo / EpoR system is involved in human neuroblastoma [59].

Hardee et al. [60], using a model of rodent breast cancer rodent, have determined that Epo is an important angiogenic factor regulating the induction of neovascularisation led by tumour cells and growth during the early stages of carcino-genesis. The suppression of angiogenesis and neoplastic pro-gression through blockade of Epo suggests that this hormone

Table 2.

rHuEPO

(UI)

FIN

(cm)

EPO

(mU/ml)

rHuEPO

(UI)

FIN

(cm)

EPO

(mU/ml)

rHuEPO

(UI)

FIN

(cm)

EPO

(mU/ml)

*A

(CONTROLS)

0

28,3

16,7

0

29,4

17,4

0

30,1

17,3

*B

(TREATED FISH )

3000

29,5

17,3

3000

33,6

2240

3000

34,9

2340

DATE BASAL 15 DAYS 30 DAYS

*average of values of 5 fishes.

2034 Current Pharmaceutical Design, 2009, Vol. 15, No. 17 Buemi et al.

may represent a potential target for therapeutic modulation of tumour angiogenesis.

Even for multiple myeloma there is experimental evi-dence demonstrating the pathological regenerative activity of recombinant Erythropoietin which could affect the progres-sion of neoplastic disease [61].

The study of angiogenic process as a factor promoting development and metastasization led to the synthesis of drugs that, blocking VEGF, exert an anti-angiogenic action, contrasting cancer spread.

It was, however, noted that benefits in terms of survival are relatively modest, with the possible exception of patients with renal cell carcinoma.

Is Erythropoietin Perhaps the Further Angiogenic Hor-mone to Block in Tumour Pathology?

The increased mortality observed in some trials in pa-tients with cancer treated with erythropoetin has been also correlated with the high incidence of arterial and venous thromboembolic events (VTEs). The Agency for Healthcare Research and Quality recently published data suggesting that the Hb level at which treatment with ESAs is stopped in clinical trials may relate to relative risk for a thromboem-bolic event [62]. A 2006 Cochrane meta-analysis of data from 38 trials involving epoetin or darbepoetin supports the role of the Hb level in determining the risk for a VTE [63].

The high incidence of thromboembolic events has been observed particularly in trials that targeted Hb levels higher than those recommended by current ESA labeling and trials that enrolled patients who were nonanemic at baseline. The exact mechanism underlying the VTE effect of

ESAs is not clear yet: it may involve the Hb itself, or an interaction of ESAs with endothelial cells or platelets.

CONCLUSIVE CONSIDERATIONS

Epo plays a role in Regenerative Medicine since it inter-venes in a persistent natural regenerative activity of humans: angiogenesis.

This capacity can be considered negative in the develop-ment of a cancer, or positive when involved in protection of heart tissue, brain and kidney.

Tissue regeneration, ancestral memory of a lost Eden, in the animal world would be preserved in some metazoaria organisms and subsequently lost in others for unknown rea-sons [64].

Epo seems to be a privileged protagonist in the regenera-tive dynamics, given the role that haematopoietic stem cells are taking in this area. The new knowledge on hematopoiesis organisation, in the past considered a hierarchical system, suggest that in reality there is no clear distinction between multipotent cells and commissioned ones, but that, in a "stem cell continuum", the various cellular types can represent dif-ferent functional stages (been) of an element capable of evolving in different ways depending on the different micro-environmental stimuli [65].

Similar concepts can be applied to tissues other than he-mopoietic one, thus enabling us to modify significantly the concept of cellular plasticity, that is the capacity of different cells to give rise to different tissue elements through an ap-propriate modulation of their genetic program. There is probably in the various organs - perhaps in the kidney in particular - a reservoir of dormant stem cells, contained in special "niches" that can acquire different transcriptional ability when properly stimulated by cell-specific transcrip-tion factors with proteins expressed in a ubiquitous way [66].

There is also a clear need to assess carefully in the near future, the effects of Erythropoietin on at least two funda-mental cell types in regenerative medicine: embryonic stem cells and mesenchymal stem cells. The former are now a consolidated technology platform on which to design the action of various substances able to promote or block cell differentiation. In fact, these cells in culture sum up most of the development programmes concerning embryo during development, intervening in generation of cells coming from all the three embryonic sheets, ectoderm, mesoderm and en-doderm. Their use could be crucial in the treatment of single-cell deficiency, as in the case of liver damage due to alcohol or Diabetes mellitus type I or in the course of cardiac or neu-rological diseases [67].

Even the study of mesenchymal stem cells allowed to subvert in a drastic way the very concept of stem cell as it had been considered for years, destroying the strict vision of a progressive differentiation of immature precursors to a

Table 3.

Study Population and Methods Results

Leyland-Jones et al. J Clin Oncol 2005

Patients with breast cancer

Epoetin alfa versus placebo

• Terminated early because of significantly higher mortality rate in epoetin alpha group

• Greater incidence of breast cancer progression than placebo

• Higher incidence of fatal thrombotic and vascular events

Henke et al. Lancet 2003

Patients with head and neck cancer

Epoetin beta versus placebo

• Poorer Locoregional progression-free survival in epoetin beta than placebo

• Higher incidence of thromboembolic events in epoetin beta than placebo

Smith et al. J Clin Oncol 2008

Patients with active cancer and anaemia not receiv-

ing chemotherapy or radiotherapy

Darbepoetin alpha versus placebo

• Trial terminated early

• Increased Locoregional failure rate in darbepoetin group

• Overall survival favoured placebo group but was not statistically significant

Regenerative Medicine Current Pharmaceutical Design, 2009, Vol. 15, No. 17 2035

precise cellular line. The mesenchymal cells seem able to migrate in sites other than those of origin, participate in re-generative phenomena and have differentiative and prolifera-tive properties similar to those of the blastocyst in the first two weeks of implantation. These cells are also predominant in the bone marrow, action domain of Erythropoietin [68], and may differ under appropriate and still largely unknown stimulus, in adipocytes, osteoblasts, chondrocytes and mus-cle cells [69].

Erythropoietin could after all play a key role on the so-called mesenchymal epithelial transition of mesenchymal stem cells and on its mutual, as already highlighted for other substances with a autocrine or paracrine abilities like TGF- , EGF, HGF and PDGF [70].

This phenomenon, which accompanies stages of embryo-genesis, morphogenesis and organogenesis [71], allows in fact the bidirectional transition between cellular phenotypes extremely different, the epithelial and the mesenchymal one, inside of which there are intermediate phenotypes that pro-gress towards one of two poles, depending on the environ-mental stimuli [72].

The experimental evidence of angiogenic, regenerative stem-induced, anti-apoptotic hormones like erythropoietin finally poses many questions: will it be possible in the future to use in humans certain residues of the regenerator mecha-nisms, which are admirably present in some amphibians?

What role will be played by the phenomena of dediffer-entiation and regeneration, such as the ones present in fish?

Will we be able to mobilise any pre-existent multipotent stem cells?

What will be the contribution of stem cells cultivated both embryonic or adult?

Why must humans be satisfied with the limited regenera-tive action of Erythropoietin, having lost the phenomenal ability of many metazoaria and certain vertebrates to rebuild entire portions of their own injured body?

These are some of the questions posed by Regenerative Medicine on the table of experimentation. In this, erythro-poietin, in physiology, pathophysiology, pharmacology has already mapped out a tenuous furrow.

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Received: March 11, 2009 Accepted: March 13, 2009