Role of MiRNA in the Regulation of Blood Group Expression.

Background: MicroRNAs (miRNAs) are small, endogenous non-coding RNA molecules that inhibit gene expression through either destabilization of the target mRNA or translational repression. MiRNAs recognize target sites, most commonly found in the 3'-untranslated regions of cognate mRNAs. This review aims to provide a state-of-the-art overview of the role of miRNAs in the regulation of major blood group antigens such as ABH as well as cancer-specific glycans. Summary: Besides their known roles in the control of developmental processes, proliferation, apoptosis, and carcinogenesis, miRNAs have recently been identified to play a regulatory role during erythropoiesis and blood group antigen expression. Since only little is known about the function of the red cell membrane proteins carrying blood group antigens, it is of great interest to shed light on the regulatory mechanisms of blood group gene expression. Some carrier proteins of blood group antigens are not restricted to red blood cells and are widely expressed in other bodily fluids and tissues and quite a few play a crucial role in tumor cells, as either tumor suppressors or promoters. Key Message: All available data point at a tremendous physiological as well as pathophysiological relevance of miRNAs in context of blood group regulation. Furthermore, miRNAs are involved in the regulation of pleiotropic genetic pathways such as hematopoiesis and tumorigenesis and thus have to be studied in future research on this subject.

Characterization of immortalized bone marrow erythroid progenitor adult (imBMEP-A)-The first inducible immortalized red blood cell progenitor cell line derived from bone marrow CD71-positive cells.

BACKGROUND AIMS: Ex vivo production of red blood cells (RBCs) represents a promising alternative for transfusion medicine. Several strategies have been described to generate erythroid cell lines from different sources, including embryonic, induced pluripotent, and hematopoietic stem cells. All these approaches have in common that they require elaborate differentiation cultures whereas the yield of enucleated RBCs is inefficient. METHODS: We generated a human immortalized adult erythroid progenitor cell line derived from bone marrow CD71-positive erythroid progenitor cells (immortalized bone marrow erythroid progenitor adult, or imBMEP-A) by an inducible expression system, to shorten differentiation culture necessary for terminal erythroid differentiation. It is the first erythroid cell line that is generated from direct reticulocyte progenitors and demonstrates robust hemoglobin production in the immortalized state. RESULTS: Morphologic analysis of the immortalized cells showed that the preferred cell type of the imBMEP-A line corresponds to hemoglobin-producing basophilic erythroblasts. In addition, we were able to generate a stable cell line from a single cell clone with the triple knockout of RhAG, RhDCE and KELL. After removal of doxycycline, part of the cells differentiated into normoblasts and reticulocytes within 5-7 days. CONCLUSIONS: Our results demonstrate that the imBMEP-A cell line can serve as a stable and straightforward modifiable platform for RBC engineering in the future.

Long-COVID is Associated with Impaired Red Blood Cell Function.

COVID-19 disease, caused by the severe acute respiratory syndrome virus 2 (SARS-CoV-2), induces a broad spectrum of clinical symptoms ranging from asymptomatic cases to fatal outcomes. About 10-35% of all COVID-19 patients, even those with mild COVID-19 symptoms, continue to show symptoms, i. e., fatigue, shortness of breath, cough, and cognitive dysfunction, after initial recovery. Previously, we and others identified red blood cell precursors as a direct target of SARS-CoV-2 and suggested that SARS-CoV-2 induces dysregulation in hemoglobin- and iron-metabolism contributing to the severe systemic course of COVID-19. Here, we put particular emphasis on differences in parameters of clinical blood gas analysis and hematological parameters of more than 20 healthy and Long-COVID patients, respectively. Long-COVID patients showed impaired oxygen binding to hemoglobin with concomitant increase in carbon monoxide binding. Hand in hand with decreased plasma iron concentration and transferrin saturation, mean corpuscular hemoglobin was elevated in Long-COVID patients compared to healthy donors suggesting a potential compensatory mechanism. Although blood pH was within the physiological range in both groups, base excess- and bicarbonate values were significantly lower in Long-COVID patients. Furthermore, Long-COVID patients displayed reduced lymphocyte levels. The clinical relevance of these findings, e. g., as a cause of chronic immunodeficiency, remains to be investigated in future studies. In conclusion, our data suggest impaired erythrocyte functionality in Long-COVID patients, leading to diminished oxygen supply. This in turn could be an explanation for the CFS, dyspnea and anemia. Further investigations are necessary to identify the underlying pathomechanisms.

Investigation of mesalazine as an antifibrotic drug following myocardial infarction in male mice.

OBJECTIVES: Myocardial infarction (MI) initiates a complex reparative response during which damaged cardiac muscle is replaced by connective tissue. While the initial repair is essential for survival, excessive fibrosis post-MI is a primary contributor to progressive cardiac dysfunction, and ultimately heart failure. Currently, there are no approved drugs for the prevention or the reversal of cardiac fibrosis. Therefore, we tested the therapeutic potential of repurposed mesalazine as a post-MI therapy, as distinct antifibrotic effects have recently been demonstrated. METHODS: At 8 weeks of age, MI was induced in male C57BL/6J mice by LAD ligation. Mesalazine was administered orally at a dose of 100 µg/g body weight in drinking water. Fluid intake, weight development, and cardiac function were monitored for 28 days post intervention. Fibrosis parameters were assessed histologically and via qPCR. RESULTS: Compared to controls, mesalazine treatment offered no survival benefit. However, no adverse effects on heart and kidney function and weight development were observed, either. While total cardiac fibrosis remained largely unaffected by mesalazine treatment, we found a distinct reduction of perivascular fibrosis alongside reduced cardiac collagen expression. CONCLUSIONS: Our findings warrant further studies on mesalazine as a potential add-on therapy post-MI, as perivascular fibrosis development was successfully prevented.

Novel evidence that the ABO blood group shapes erythropoiesis and results in higher hematocrit for blood group B carriers.

The ABO blood group (BG) system is of great importance for blood transfusion and organ transplantation. Since the same transcription factors (TFs) and microRNAs (miRNAs) govern the expression of ABO BG antigens and regulate erythropoiesis, we hypothesized functional connections between both processes. We found significantly higher hemoglobin and hematocrit values in BG B blood donors compared to BG A. Furthermore, we observed that erythropoiesis in BG B hematopoietic stem/progenitor cells (HSPCs) was accelerated compared to BG A HSPCs. Specifically, BG B HSPCs yielded more lineage-specific progenitors in a shorter time (B: 31.3 ± 2.2% vs. A: 22.5 ± 3.0%). Moreover, non-BG A individuals exhibited more terminally differentiated RBCs with higher enucleation rates containing more hemoglobin compared to BG A. Additionally, we detected increased levels of miRNA-215-5p and -182-5p and decreased expression of their target TFs RUNX1 and HES-1 mRNAs in erythroid BG B precursor cells compared to BG A. This highlights the important roles of these factors for the disappearance of differentiation-specific glycan antigens and the appearance of cancer-specific glycan antigens. Our work contributes to a deeper understanding of erythropoiesis gene regulatory networks and identifies its interference with BG-specific gene expression regulations particularly in diseases, where ABO BGs determine treatment susceptibility and disease progression.

K562 erythroleukemia line as a possible reticulocyte source to culture Plasmodium vivax and its surrogates.

Establishing an in vitro "red blood cell matrix" that would allow uninterrupted access to a stable, homogeneous reticulocyte population would facilitate the establishment of continuous, long-term in vitro Plasmodium vivax blood stage cultures. In this study, we have explored the suitability of the erythroleukemia K562 cell line as a continuous source of such reticulocytes and have investigated regulatory factors behind the terminal differentiation (and enucleation, in particular) of this cell line that can be used to drive the reticulocyte production process. The Duffy blood group antigen receptor (Fy), essential for P. vivax invasion, was stably introduced into K562 cells by lentiviral gene transfer. miRNA-26a-5p and miRNA-30a-5p were downregulated to promote erythroid differentiation and enucleation, resulting in a tenfold increase in the production of reticulocytes after stimulation with an induction cocktail compared with controls. Our results suggest an interplay in the mechanisms of action of miRNA-26a-5p and miRNA-30a-5p, which makes it necessary to downregulate both miRNAs to achieve a stable enucleation rate and Fy receptor expression. In the context of establishing P. vivax-permissive, stable, and reproducible reticulocytes, a higher enucleation rate may be desirable, which may be achieved by the targeting of further regulatory mechanisms in Fy-K562 cells; promoting the shift in hemoglobin production from fetal to adult may also be necessary. Despite the fact that K562 erythroleukemia cell lines are of neoplastic origin, this cell line offers a versatile model system to research the regulatory mechanisms underlying erythropoiesis.