Eritropoietina – un antioxidant inert pentru organele genitale interne feminine la șobolan

Introduction

Bailey et al. claimed⁽¹⁾ that erythropoietin (Epo) may act as a potent antioxidant, particularly through its cytoprotective and antiapoptotic mechanisms that combat oxidative stress. Beyond red blood cell production stimulation, Epo scavenges directly free radicalsand acts indirectly by inducing antioxidant enzymes (e.g., glutathione peroxidase) and reducing the iron-dependent oxidative damage. In the present study, four histologic variables of ischemia reperfusion (IR) uteri⁽²⁾, fallopian tubes⁽³⁾ and ovaries⁽⁴⁾ (internal genitalia; GIR) were examined for this purpose. These variables for every organ were: epithelium edema (E), epithelium karyorrhexis (K), congestion (C) and inflammation (I) – EKCI. Furthermore, the cytokine (TNF-a) and the oxidative marker malondialdehyde (MDA) were also calculated. This review analyses the effect of Epo on rat genitalia pathology totally, associated by the two redox markers and a biochemical access⁽⁵⁾.

Materials and method

Since this is a review study, the reader is called to backdate at referenced publications^(2-5) for technical details settings, such as the two ethics committee approvals under the 3693/12-11-2010 and 14/10-1-2012 numbers fully complied with the tenants of the Declaration of Helsinki; the granting company; the experiment location and also the pathology department and the peri-experimental Albino female Wistar rats management. The rats were 16-18 weeks old. Similar details are found in the works of Tsompos et al. (2019; 2022; 2024; 2017)^(2-5); the randomization assignment to four groups consisted in N=10; the introductory common stage of 45-minute ischemia for all four groups. Group A: placebo intravenous reperfusion of 60 minutes; Group B: placebo intravenous reperfusion of 120 minutes; Group C: immediate Epo intravenous reperfusion of 60 minutes; Group D: immediate Epo intravenous reperfusion of 120 minutes. The dose height assessment was described at preliminary studies as 10 mg/kg body mass.

Also, perioperational details about ischemic laparotomic clamping of inferior aorta over renal arteries with forceps for 45 minutes; its restoration removal for blood patency and reperfusion; as well the iterated Epo administration at the time of reperfusion; through inferior vena cava catheter for each experimental group, can be found in the same works^(2-5). The EKCI variables, TNF-a, MDA and the blood values were determined at the 60^th minute of reperfusion (for A and C groups) and at the 120^th minute of reperfusion (for B and D groups). The increased relations proven between animals’ mass with either each EKCI, or marker or serum variables, were included using corrected values. The pathologic score grading was maintained widely the same: 0-0.499 – lesions-free; 0.5-1.499 – mild lesions; 1.5-2.499 – moderate lesions; 2.5-3 – serious lesions damage. It is worth noting that the functional statistical test used in the preliminary studies freed the final findings from strict timepoints and endpoints, so that the Table 1 is applicable to any time point within the first two hours. The fourth preliminary study simultaneously examined the effect of Epo on 35 blood and plasma substances from the central circulatory system of rats using exactly the same IR methodology in the inferior vena cava, also with the same exact timepoints⁽⁵⁾ (Table 2). The aim was to obtain a more accurate and reliable numerical estimate of the direction and severity of the general metabolic disorder caused by this particular antioxidant agent in the body, with the ultimate goal of using it as a basis for comparison with the pathologic deviations of the internal genitalia (Table 3).

Discussion

It is observed that researchers are less interested in using Epo in tissue IR experiments, being reserved only for biotechnology experiments in recent literature. Indeed, Nuchpramool et al. revealed⁽⁶⁾ the myomatous erythrocytosis syndrome (MES), a rare syndrome, cha­rac­terized by erythrocytosis, myomatous uterus, blood normalization after hysterectomy or myomectomy and high risk of thromboembolic events, in a premenopausal nulliparous 50-year-old woman. Her largest myoma was measuring 19.5 cm, along with a right ovarian hemorrhagic cyst without abnormal uterine bleeding, erythrocytosis with normal tumor markers and serum Epo. Postoperatively, her blood levels were normalized, confirming MES. The exact mechanism remains unclear, but it may involve somewhen normal Epo levels often linked to myomas. Nong et al. also described⁽⁷⁾ MES, the same secondary erythrocytosis associated with fibroids, presumably resulted from ectopic Epo production by the fibroids, which activated the Janus kinase 2 (JAK-2) pathway and increased red blood cell counts, in a 33-year-old woman. Preoperative hemoglobin level was 19.7 g/dL; hematocrit 60.4% with a 25×20×12 cm fibromatous uterus under low-molecular-weight heparin thromboprophylaxis and intravenous hydration. After abdominal myomectomy, the hemoglobin level was normalized. This case highlights the importance of required accurate Epo-related therapeutic targets for MES.

Kojima et al.⁽⁸⁾ studied genetically manipulated chi­cken oviducts that express human Epo (hEpo) under the control of a lysozyme promoter-enhancer in order to examine the regulatory mechanisms of chicken lysozyme gene expression in vivo. They found that a -1.9 kb DNase I hypersensitive site (DHS) was essential for oviduct-specific expression by using several deletion mutants of the promoter-flanking region. These will enable target genes to produce proteinaceous materials into egg white by transgenic chickens. The concentration of hEpo in egg white was 14-75 µg/ml, suggesting that the chicken lysozyme promoter containing -1.9 kb DHS is sufficient for the production of pharmaceuticals using transgenic chickens.

Chen et al.⁽⁹⁾ demonstrated a rapid workflow for Epo capture, quantitation and glycan profiling in Chinese hamster ovary (CHO) cell supernatant. Glycans at the three N-glycosylation sites strongly influence Epo’s stability and bioactivity, so accurate quantitation of Epo and analysis of its glycosylation patterns are critical during production and subsequent processing. Newly developed porous membranes containing affinity peptides selectively capture Epo from cell culture supernatants. Captured Epo was labeled with a fluorescent antibody to enable quantitation in a 10-minute assay with an average coefficient of variation approximately 12% in a 96-well-plate format. The rapid 2-cm glass-fiber membrane combined with tryptic in-membrane digestion yielded glycan profiles comparable to those obtained from overnight in-solution digestion. Thus, the membrane-based assays provide a novel approach for rapid Epo quantitation and facilitating glycan identification.

Tajbakhsh et al.⁽¹⁰⁾ compared two natural hydrogel composites containing alginate (Alg), Wharton’s Jelly (WJ/Alg) and collagen (Col/Alg) as models for human artificial ovary (AO), a bioengineered approach aimed at increasing fertility potential, especially in women and prepubertal girls with a history of cancer. In each experimental group from the six groups, 40 isolated human ovarian follicles were seeded in 10 µl of the desired hydrogel and xenotransplanted in ovariectomized NMRI mice for one week. FSH (7.5 IU) and Epo (200 IU/kg as an angiogenic factor) were injected every other day. In contrast, most of follicles in Col/Alg remained at the primordial stage. Although FSH injection helped granulosa cells differentiation, especially in the WJ/Alg group (2.84-fold), and Epo injection increased blood vessels in both groups (p<0.0001), the follicles could not be preserved in the experimental groups. The WJ/Alg-base artificial ovary provided better support for follicle growth and development after one week than other groups. In addition, adjusting FSH and Epo dosage may improve follicle survival and growth in future studies.

Gan et al.⁽¹¹⁾ fused recombinant hEpo (rhEpo) with human immunoglobulin G (IgG) Fc fragment (rhEPO-Fc) as a novel erythropoiesis-stimulating agent designed to extend plasma half-life and enhance biological activity in hemodialysis patients with chronic kidney disease (CKD)-related anemia. This phase 3 trial enrolled patients randomized (2:1) to receive either rhEpo-Fc or rhEpo (Chinese hamster ovary cell-derived) for 28 weeks. The rhEpo-Fc responders were eligible for a 24-week extension period. The primary endpoint was assessed between weeks 21 and 28. Patients receiving rhEpo-Fc demonstrated non-inferiority Hb maintenance than ones with rhEpo. The inter-group least square mean (LSM) differences were approximately 3.96 g/L (p<0.001) and approximately 2.27 g/L (p=0.008), respectively, for rhEPO-Fc and rhEpo. Dose adjustments due to treatment-emergent were both adverse events significantly lower (0% versus 2.2%; p<0.05) and reduced deaths unrelated with both drugs, for the rhEpo-Fc branch. These findings indicated that rhEpo-Fc effectively maintained Hb levels in patients with CKD anemia undergoing hemodialysis, showing comparable efficacy to rhEpo, with reduced dosing frequency and a similar safety profile.

Catalán-Tatjer et al.⁽¹²⁾ employed the recombinase-mediated cassette exchange strategy to develop isogenic cell lines expressing one copy of Epo, as a model protein product and various antiapoptotic genes: bcl-2 and bcl-xL from Chinese hamster ovary (CHO) and human origin, mcl-1 and bhrf-1. Programmed induced cell death by sodium butyrate is a critical factor in all CHO cultures, dictating the duration until harvest in fed-batch cultures and viable cell density in perfusion. The observed phenotype varied significantly, depending on the overexpressed gene; therefore, the metabolic differences were further characterized. They showed that overexpressing bcl-2 from the CHO origin significantly improved productivity, established a methodology to successfully test candidate genes via targeted integration and would enable future metabolic engineering strategies.

Tanaka et al.⁽¹³⁾ established a novel specific and sensitive anti-human EphA1 monoclonal antibodies (mAb), clone Ea1Mab-30 (mouse IgG1, kappa), by the cell-based immunization and screening (CBIS) method. Ea1Mab-30 demonstrated reactivity with an EphA1-overexpressed Chinese hamster ovary-K1 cell line (CHO/EphA1) among other carcinoma tissues, in flow cytometry. Epo-producing hepatocellular receptor A1 (EphA1) is one of the Eph receptor family members, the largest group of receptor tyrosine kinases. EphA1 is expressed in various tissues, and regulates cellular homeostasis by interacting with its membrane-bound ephrin ligands and other receptors. EphA1 critically correlates with the pathogenesis in several disorders, including Alzheimer’s disease and cancers. Furthermore, Ea1Mab-30 detected EphA1 protein in CHO/EphA1 and 5637 lysates. Ea1Mab-30 also clearly stained EphA1 of formalin-fixed paraffin-embedded CHO/EphA1. Ea1Mab-30, established by CBIS method, could help analyze the EphA1-contributed cellular functions, having potential applications in basic research, pathological diagnosis and treatment with specificity and high affinity for EphA1-expressing cells.

Léon-Félix et al.⁽¹⁴⁾ aimed to examine the effects of simvastatin and Epo on follicular survival and revascularization after subcutaneous autotransplantation of cryopreserved ovarian tissue in domestic cats as a model for endangered felids: control, simvastatin (SIM) and Epo. The SIM group received a single oral dose of 5 mg/kg four hours prior to ovariohysterectomy, while the Epo group received subcutaneous injections of 500 IU/kg for seven consecutive days prior to grafting. Frozen/thawed ovarian fragments were removed each week after grafting in queens. Treatment with SIM and Epo resulted in generally smaller areas of inflammation in the tissue. The SIM group presented six Ki67-positive, morphologically normal growing follicles on days 49 and 63, significantly more than the control and Epo groups. The Epo group showed a significantly larger number of follicle-like structures that were composed mainly of proliferative granulosa cells with no oocyte. These results suggest that Epo selectively supports somatic cell survival, and SIM may promote the long-term viability and growth of residual follicles. Future optimized efforts may harness these beneficial effects to improve ovarian tissue cryopreservation and transplantation outcomes in felids.

Hasheminejad et al.⁽¹⁵⁾ showed that incorporating a ubiquitous chromatin opening elements (UCOE) reduces position-dependent gene silencing and increases recombinant mRNA and protein expression on Epo production in CHO DG44 cell pools. Transcriptional gene silencing remains a major limitation in mammalian expression systems and often reduces protein yield. Incorporation of UCOEs into expression cassettes has emerged as a promising strategy to mitigate position effects and enhance transgene expression. A codon-optimized Epo expression cassette was introduced into CHO DG44 cells using either a UCOE-containing vector or a conventional non-UCOE control one, following random genomic integration. The UCOE-containing cell pool exhibited a significant enhancement in recombinant Epo expression, with approximately a 3.8-fold increase in mRNA levels and a seven-fold increase in secreted protein levels than the non-UCOE control cell pool. This strategy supports improved efficiency during the early stages of cell line development for recombinant protein production.

Rahimi et al.⁽¹⁶⁾ suggested that the downregulation of the BAX and caspase-3 genes, using the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9)-mediated knockdown of BAX and caspase-3 genes (CRISPR method), inhibits apoptosis, extends cell lifespan, enhances the yield of recombinant Epo (approximately 1.63-fold), exhibits a higher cell density (approximately 1475±5%) even in the presence of an apoptosis inducer. Additionally, reduction of caspase-3 expression was proven to be more effective than BAX in extending the lifespan of cells and producing heterologous recombinant proteins in rCHO cells. BAX and caspase-3 are known essential genes in the apoptotic pathway of cells, promoting the apoptotic cascade through different mechanisms. Inhibition of these genes can increase the longevity of cells in cell culture.

Ter Wee et al.⁽¹⁷⁾ evaluated the impact of (hyper)glycosylation in CHO cell produced erythropoiesis-stimulating agents (ESAs) on in vivo efficacy, after epoetin glycoforms fractionation and characterization for a better comprehension of the relationship of epoetin glycoforms’ structure to function and the range of in vivo potency response. The results demonstrate a strong link only between sialylation or antennary structure, and confirmed an inverse relationship for the potency of darbepoetin between in vivo and in vitro methods.

The convergence of statistically nonsignificant findings regarding the internal genitalia, the oxidative markers and the overall effect of Epo on serum levels supported by the literature – which points to future, more thorough studies on erythropoietin – leads to the conclusion that Epo is an oxidatively inert drug, at least within the dimensions of the present experiment we conducted.

Conclusions

Erythropoietin appears not to be a promising factor, since both other authors and we leave the investigation of Epo’s efficacy to more robust future studies, regarding the production of pharmaceuticals using transgenic chickens, the rapid Epo quantitation and easy glycan identification, the blood vessels increase in human bioengineered artificial ovary, the hamster ovary cell recombinant human Epo production, the isogenic cell lines development expressing Epo copies, the pathogenetic Epo-producing hepatocellular receptor A1 study, the ovarian tissue cryopreservation and transplantation improvement, the cell line development for recombinant protein production efficiency improvement, the longevity increase of cells in cultures, and the sialylation or antennary structure linkage in epoetin glycoforms.

Acknowledgement. Acknowledged in preliminary studies. 

Autor corespondent:   Constantinos Tsompos E-mail: constantinos1tsompos@gmail.com

CONFLICT OF INTEREST: none declared.

FINANCIAL SUPPORT: none declared.

This work is permanently accessible online free of charge and published under the CC-BY.