Cytokines such as erythropoietin (EPO) and thrombopoitein (TPO) and so on, which stimulate hematopoiesis, can regulate self-renewal, proliferation, differentiation, maturation and programmed cell death of hematopoietic cells through specifically binding to surface receptors. Recently random phage display peptide libraries and other screening methods have been used to isolate mimetic including small peptides and non-peptides molecules, which can mimic the same effects as cytokines, such as EPO and TPO, and demonstrate the similar potency and activity as EPO and TPO in a panel of in vitro biological assays and in animal experiments. These approaches are critical to further research of interactive mechanisms between cytokine and receptor, receptor activation and rational design of other desired cytokine mimetic. This review concisely introduced recent advances in research on mimetic of EPO, TPO and other cytokines and future directions.
Animals ; Cytokines ; pharmacology ; Erythropoietin ; pharmacology ; Hematopoiesis ; drug effects ; physiology ; Humans ; Peptide Library ; Peptides ; pharmacology ; Thrombopoietin ; pharmacology

Erythropoietin (EPO) is an acidic glycoprotein that was first detected as a hematopoietic factor and its synthesis is triggered in response to cellular hypoxia-sensing. EPO binds to type I cytokine receptors, which associate with the non-receptor tyrosine kinase Jak2, and thereby activate Stat 5a/5b, Ras/MAPK, and PI3-K/Akt signaling pathways. The recent discovery shows that there is a specific EPO/EPO-receptor system in the central nervous system (CNS), independently of the haematopoietic system. Hypoxia and anemia can up-regulate EPO/EPOR expressions in the CNS. Further studies demonstrate that EPO has substantial neuro-protective effects and acts as a neurotrophic factor on central cholinergic neurons, influencing their differentiation and regeneration. EPO also exerts neuro-protective activities in different models of brain damage in vivo and in vitro, such as hypoxia, cerebral ischaemia and sub-arachnoid haemorrhage. EPO may also be involved in synaptic plasticity via the inhibition or stimulation of various neurotransmitters. Therefore, human recombinant EPO that activate its receptors in the central nervous system might be utilized in the future clinical practice involving neuroprotection and brain repair.
Animals ; Brain ; metabolism ; Cell Differentiation ; drug effects ; Erythropoietin ; metabolism ; pharmacology ; Humans ; Neurons ; cytology ; drug effects ; Neuroprotective Agents ; metabolism ; pharmacology ; Receptors, Erythropoietin ; metabolism

Erythropoietin ; pharmacology ; Folic Acid ; pharmacology ; therapeutic use ; Humans ; Infant, Newborn ; Infant, Premature ; Platelet Count ; Vitamin B 12 ; pharmacology ; therapeutic use

OBJECTIVEFunctionally, erythropoietin (EPO) can promote the proliferation and growth of erythroid progenitor cells, and it is widely used in the treatment of anemia in chronic diseases caused by tumor and inflammation. However, it is unclear whether EPO has any effect on tumor cell iron metabolism and tumor cell proliferation. The purpose of this study was to explore the effects of recombinant human EPO (rhEPO) on the expression of transferrin receptor (TfR, CD(71) antigen) of leukemic cell K562 and its relation to cell cycle.

METHODSIn vitro culture of K562 cell was performed with additions of various concentrations of rhEPO and Fe. Treatments were terminated at 24 h and 72 h, respectively. Then each group of cells was incubated with FITC-IgG antibody to CD(71) or PI, a kind of DNA dye. And TfR expression and DNA synthesis status were analyzed by flow-cytometry.

RESULTS(1) The expression of TfR by K562 cells increased significantly when incubated for 72 h with different concentrations of rhEPO. The measurement values of 5 U/ml, 10 U/ml and 20 U/ml groups were 12.2 +/- 1.40, 10.7 +/- 0.99 and 11.1 +/- 0.90, respectively. They were markedly increased when compared with that of control group (6.27 +/- 0.11, P < 0.05). (2) When incubated with rhEPO (5 u/ml) alone or combined with FeCl(3) (100 micro mol/L), the percentages of cells in S phase were 51.1% and 59.6%, respectively. They significantly increased when compared with that of control group (42.9%, P < 0.05).

CONCLUSIONSIron is very important for the proliferation of both normal cells and leukemic cells. It is essential to the activity of ribonucleotide reductase (RR). The authors hypothesized that rhEPO would increase the expression of TfR and intracellular iron content of leukemic cells, which would enhance the DNA synthesis and cell proliferation. Therefore, the clinical application of rhEPO to promote erythropoiesis of cancer patients should be cautious.

Cell Cycle ; drug effects ; Erythropoietin ; pharmacology ; Flow Cytometry ; Humans ; K562 Cells ; Receptors, Transferrin ; metabolism ; Recombinant Proteins

The effects of erythropoietin and recombinant cytokines (G-CSF, SCF, IL-3 and GM-CSF) on colony formation and self-renewal by erythroid burst-forming units (BFU-E) and granulocyte-macrophage progenitors (CFU-GM) from mobilized peripheral blood progenitor cells (PBPCs) of the normal donors and patients were investigated. To better understand how combinations of the cytokines may be appropriate for stem cell manipulation, a model involving replating of individual BFU-E and CFU-GM colonies has been used to study the effects of cytokines on colony formation and self-renewal. Based on granulocyte colony-stimulating factor (G-CSF) and erythropoietin(EPO) alone serve as a baseline with which to compare the effects of the combinations with stem cell factor (SCF), interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). The results revealed that: 1. BFU-E derived from PBPCs produced a significantly great numbers of subcolonies in EPO plus IL-3 and EPO+SCF+IL-3, and a difference in EPO+SCF compared with EPO alone. 2. Compared patients to normal donors the self-renewal ability of BFU-E was not influenced after EPO plus SCF or IL-3. 3. There was a significantly increased frequency of CFU-GM in the presence of SCF, IL-3, and GM-CSF used in conjunction with G-CSF. Comparing the frequencies of CFU-GM in patients and donors, patients' PBPCs were more sensitive to G-CSF+SCF and GMix (G-CSF+SCF+IL-3+GM-CSF), especially to G-CSF alone than donors. 4. There was a significant increase in AUC (area under the curve of subcolony distribution) in the presence of G-CSF combined with other cytokines. GMix was identified as the optimal combination of cytokines for both the expansion of CFU-GM as well as the expansion of clonogenic progenitor cells. 5. Donors have a high AUC than patients, especially there was a significant increase AUC in G-CSF alone (P = 0.0067).
Cytokines ; pharmacology ; Erythroid Precursor Cells ; drug effects ; Erythropoietin ; pharmacology ; Humans ; Leukocytes, Mononuclear ; drug effects ; physiology ; Recombinant Proteins ; pharmacology ; Stem Cells ; drug effects

OBJECTIVETo investigate the in vitro effect of erythropoietin (EPO) on hepcidin of monocytes and its molecular mechanisms.

METHODSHepcidin and signaling molecules including C/EBPalpha, Smad1/5/8, p-Smad1/5/8 and p-STAT3 were detected by real time PCR and Western blot. THP-1 monocytes were stimulated by interleukin-6 (IL-6) or lipopolysaccharide (LPS). EPO receptor (EPOR) antibody was added to observe its antagonistic effect on EPO and impact on the signaling proteins.

RESULTSEPO suppressed mRNA expression of THP-1 hepcidin of monocytes induced by 20 ng/ml IL-6 or 1 microg/ml LPS in both dose and time dependent manner. The most decrease of hepcidin expression was observed at 2 IU/ml EPO for 6 hours. EPO also down-regulated hepcidin protein induced by 20 ng/ml IL-6. At 2 IU/ml EPO for 6 hours hepcidin protein was down-regulated, as was C/EBPalpha, p-Smad1/5/8 and p-STAT3. Antibody to EPOR antagonized the down-regulation of EPO on hepcidin and signaling proteins.

CONCLUSIONSMonocytes hepcidin can be reduced by EPO when stimulated by IL-6 or LPS. The mechanism of which may be at least in part, via suppression of C/EBPalpha, p-Smad1/5/8 and p-STAT3 signaling.

Antimicrobial Cationic Peptides ; metabolism ; Cells, Cultured ; Erythropoietin ; pharmacology ; Hepcidins ; Humans ; Interleukin-6 ; pharmacology ; Lipopolysaccharides ; pharmacology ; Monocytes ; drug effects ; metabolism ; Signal Transduction

This study was aimed to investigate the effect of rhIL-6 and rhEPO on hepcidin mRNA expression in HepG2 cells and human primary hepatocytes, and mechanism of rhEPO in treatment of anemia of chronic disease (ACD). The HepG2 cells and human primary hepatocytes were cultured with medium containing different concentrations of rhIL-6 and rhEPO for a certain time, then mRNA was isolated and its RT-PCR was performed, the bands were photographed and analyzed by UVI band, the hepcidin and G3PDH mRNA ratio were semi-quantitatively analyzed. The expression levels of hepcidin in GepG2 cells and human primary hepatocytes at different conditions were compared. The results showed that the hepcidin mRNA expression in HepG2 cells and human primary hepatocytes could be enhanced by rhIL-6, the rhEPO could inhibit rhIL6-induced hepcidin mRAN expression. The rhEPO alone basically did not influence hepcidin mRNA expression in HepG2 cells. It is concluded that Hepcidin mRNA expression in HepG2 cells and human primary hepatocytes can be elevated by rhIL-6 with concentration- and time-dependent manner in certain range. rhEPO can inhibit this effect of rhIL-6.
Antimicrobial Cationic Peptides ; genetics ; metabolism ; Erythropoietin ; pharmacology ; Hep G2 Cells ; Hepatocytes ; drug effects ; metabolism ; Hepcidins ; Humans ; Interleukin-6 ; pharmacology ; RNA, Messenger ; genetics ; Recombinant Proteins ; pharmacology

OBJECTIVETo evaluate the effect of early application of recombinant human erythropoietin (rhEPO) on white matter development in preterm infants using fractional anisotropy (FA) of magnetic resonance diffusion tensor imaging (DTI).

METHODSA total of 81 preterm infants with gestational age ≤32 weeks, birth weight <1 500 g, and hospitalization within 24 hours after birth were randomly divided into rhEPO group (42 infants) and control group (39 infants). The infants in the rhEPO group were administered rhEPO, while those in the control group were given the same volume of normal saline. The preterm infants of both groups took examinations of head magnetic resonance imaging, diffusion-weighted imaging, and DTI at the corrected gestational age of 35-37 weeks. FA was calculated for the regions of interest in both groups.

RESULTSThere was no significant difference in the incidence of intracranial hemorrhage, periventricular leukomalacia, focal cerebral white matter damage (CWMD), and extensive CWMD between rhEPO and control groups (P>0.05). Compared with the control group, the rhEPO group showed higher FA values at the posterior limb of the internal capsule, the splenium of the corpus callosum, frontal white matter, and occipital white matter (P<0.05). There was no significant difference in FA values at the parietal white matter, thalamus, lenticular nucleus, and caudate nucleus between the two groups (P>0.05).

CONCLUSIONSEarly application of rhEPO has a neuroprotective effect on white matter development in preterm infants.

Diffusion Tensor Imaging ; Erythropoietin ; pharmacology ; Female ; Humans ; Infant, Newborn ; Infant, Premature ; Male ; Neuroprotective Agents ; pharmacology ; Recombinant Proteins ; pharmacology ; White Matter ; drug effects ; growth & development

BACKGROUND/AIMS: Darbepoetin alfa (DPO) exhibits comparable renoprotective effects to erythropoietin (EPO) in several animal models of acute renal injury. We examined whether DPO also attenuated renal injury in a rat model of cisplatin nephrotoxicity. METHODS: Male Spague-Dawley rats were divided into four groups: untreated, DPO-treated, cisplatin-injected, and DPO-treated cisplatin-injected. DPO pretreatment was conducted 24 hours after and just before cisplatin administration. Ninety-six hours after cisplatin administration, animals in all experimental groups were sacrificed. We examined serology; real-time reverse transcription polymerase chain reaction (RT-PCR) for TNF-alpha, Bcl-2, and MCP-1 gene expression; and Western blots for caspase-3. We also conducted terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and light microscopy. RESULTS: Pretreatment with DPO significantly reduced the levels of blood urea nitrogen and serum creatinine, the magnitude of renal tubular epithelial damage, and renal gene expression of TNF-alpha, Fas, and MCP-1 in kidneys injured by cisplatin. Pretreatment with DPO significantly increased Bcl-2 mRNA levels in kidneys injured by cisplatin, and significantly reduced activated caspase-3 and TUNEL-positive cells. CONCLUSIONS: DPO exhibits a renoprotective effect in experimental cisplatin-induced renal injury, the mechanism of which may involve DPO antiinflammatory and antiapoptotic effects.
Animals ; Anti-Inflammatory Agents/pharmacology ; Antineoplastic Agents/*toxicity ; Apoptosis/drug effects ; Cisplatin/*toxicity ; Erythropoietin/*analogs & derivatives/pharmacology ; Hematocrit ; Kidney/*drug effects/pathology ; Male ; Rats ; Rats, Sprague-Dawley

To study whether recombinant human erythropoietin (rhEPO) reduces neuronal apoptosis through inhibiting over-expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in nucleus induced by brain ischemia/reperfusion in rats, 48 adult Sprague-Dawley rats were randomly divided into 3 groups: sham, saline and EPO groups. Animal models of brain ischemia/reperfusion were established by middle cerebral artery occlusion in rats. The effects of EPO on the sizes of ischemia tissue were observed by TTC staining. The over-expression of GAPDH in nucleus was detected by Hoechst-33258 and anti-GAPDH antibody double staining. The neuronal apoptosis in penumbral was detected by Nissl's staining and Hoechst-33258 immunofluorescence, respectively. The results showed that rhEPO treatment (3 000 U/kg, three times daily, i.p.) apparently reduced the sizes of infarct brain tissue in ischemia/reperfusion rats. rhEPO inhibited over-expression of GAPDH in nucleus of apoptotic neurons. In the meantime rhEPO decreased the number of apoptotic neurons in ischemia/reperfusion rats. These results suggest that rhEPO may induced reduction of neuronal apoptosis in penumbra may be through inhibiting over-expression of GAPDH in nucleus of apoptotic neurons induced by ischemia/reperfusion. Reduction of GAPDH over-expression in nucleus may play a pivotal role in EPO inhibiting neuronal apoptosis in cerebral ischemia/reperfusion rats, providing experimental evidence for EPO neuro-protecting effects against ischemia/reperfusion.
Animals ; Apoptosis ; Brain ; enzymology ; pathology ; Brain Ischemia ; pathology ; Erythropoietin ; pharmacology ; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) ; metabolism ; Humans ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; pharmacology ; Reperfusion Injury ; pathology