Survival and senescence of human young red cells in vitro.

Background: A number of experimental investigations in vivo suggest that in humans a decrease of circulating erythrocyte number ensues whenever erythropoietin (EPO) plasma level decreases. Since the process seems to selectively eliminate young red cells (neocytes), it has been named neocytolysis. The experimental models in vivo have revealed and documented multiple forms of neocytolysis but have not fully elucidated the specificity of the target red cells and the relation with EPO level changes. In an attempt to better characterize the neocytolytic process, we have undertaken an in vitro investigation on age-ranked human red cells. Methods: By centrifugation on Percoll density gradient we separated the red cells population into three subsets, neocytes, middle-aged and old. Then we comparatively investigated the kinetics of survival of the subsets cultured under different conditions: with medium alone, with 10% autologous plasma, with EPO, alone or in combination with autologous monocytes. Results: Neocytes showed a viability and a survival rate lower than the other red cells when cultured in medium or with 10% plasma. EPO at physiological doses increased their survival rate, but not that of the other subsets. This effect was enhanced by co-culture with monocytes. Conclusion: Likely neocytes are more sensitive than the other RBCs subsets to presence or absence of survival signals, such as EPO or plasma or monocytes derived factors. These observations could provide an insight into the link between the decrease in EPO plasma level and the reduction of circulating red cells mass and account for the specificity of neocytes clearance

Immortalized HEK 293 Kidney Cell Lines as Models of Renal Cells: Friends or Foes?

The immortalized cell lines derived from human embryonic kidney, named HEK 293, are extensively used as models of human renal cells in in vitro studies. Nevertheless, ample evidence in the literature shows that HEK 293 cells display genotypic and phenotypic characteristics that differ substantially from primary kidney cells, with potential detrimental effects on the quality of the experimental results. Among the differences documented between HEK 293 and renal cells, there is an altered pattern of expression of many proteins involved in the development and physiological functions of the kidney. Methionine sulfoxide reductase (Msr) enzymes are ubiquitous components of the cellular machinery, evolved to counteract the damages inflicted to methionine residues by oxidative stress, particularly intense in kidney tissues. In this article, we have compared the levels of expression of several different Msr enzymes in human kidney and in a HEK 293 strain and have observed significant differences between the two cell types

Rap2, but not Rap1 GTPase is expressed in human red blood cells and is involved in vesiculation

AbstractRecent studies have suggested that Rap1 and Rap2 small GTP-binding proteins are both expressed in human red blood cells (RBCs). In this work, we carefully examined the expression of Rap proteins in leukocytes- and platelets-depleted RBCs, whose purity was established on the basis of the selective expression of the β2 subunit of the Na+/K+-ATPase, as verified according to the recently proposed “β-profiling test” [J.F. Hoffman, A. Wickrema, O. Potapova, M. Milanick, D.R. Yingst, Na pump isoforms in human erythroid progenitor cells and mature erythrocytes, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 14572-14577]. In pure RBCs preparations, Rap2, but not Rap1 was detected immunologically. RT-PCR analysis of mRNA extracted from highly purified reticulocytes confirmed the expression of Rap2b, but not Rap2a, Rap2c, Rap1a or Rap1b. In RBCs, Rap2 was membrane-associated and was rapidly activated upon treatment with Ca2+/Ca2+-ionophore. In addition, Rap2 segregated and was selectively enriched into microvesicles released by Ca2+-activated RBCs, suggesting a possible role for this GTPase in membrane shedding

"So is science …"1 : No evidence for neocytolysis on descending the mountains (Response to Rice and Gunga).

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Membrane Rearrangements in the Maturation of Circulating Human Reticulocytes

Red blood cells (RBCs) begin their circulatory life as reticulocytes (Retics) after their egress from the bone marrow where, as R1 Retics, they undergo significant rearrangements in their membrane and intracellular components, via autophagic, proteolytic, and vesicle-based mechanisms. Circulating, R2 Retics must complete this maturational process, which involves additional loss of significant amounts of membrane and selected membrane proteins. Little is known about the mechanism(s) at the basis of this terminal differentiation in the circulation, which culminates with the production of a stable biconcave discocyte. The membrane of R1 Retics undergoes a selective remodeling through the release of exosomes that are enriched in transferrin receptor and membrane raft proteins and lipids, but are devoid of Band 3, glycophorin A, and membrane skeletal proteins. We wondered whether a similar selective remodeling occurred also in the maturation of R2 Retics. Peripheral blood R2 Retics, isolated by an immunomagnetic method, were compared with mature circulating RBCs from the same donor and their membrane protein and lipid content was analyzed. Results show that both Band 3 and spectrin decrease from R2 Retics to RBCs on a “per cell” basis. Looking at membrane proteins that are considered as markers of membrane rafts, flotillin-2 appears to decrease in a disproportionate manner with respect to Band 3. Stomatin also decreases but in a more proportionate manner with respect to Band 3, hinting at a heterogeneous nature of membrane rafts. High resolution lipidomics analysis, on the contrary, revealed that those lipids that are typically representative of the membrane raft phase, sphingomyelin and cholesterol, are enriched in mature RBCs with respct to Retics, relative to total cell lipids, strongly arguing in favor of the selective retention of at least certain subclasses of membrane rafts in RBCs as they mature from Retics. Our hypothesis that rafts serve as additional anchoring sites for the lipid bilayer to the underlying membrane-skeleton is corroborated by the present results. It is becoming ever more clear that a proper lipid composition of the reticulocyte is necessary for the production of a normal mature RBC

Continuous Percoll Gradient Centrifugation of Erythrocytes—Explanation of Cellular Bands and Compromised Age Separation

(1) Background: When red blood cells are centrifuged in a continuous Percoll-based density gradient, they form discrete bands. While this is a popular approach for red blood cell age separation, the mechanisms involved in banding were unknown. (2) Methods: Percoll centrifugations of red blood cells were performed under various experimental conditions and the resulting distributions analyzed. The age of the red blood cells was measured by determining the protein band 4.1a to 4.1b ratio based on western blots. Red blood cell aggregates, so-called rouleaux, were monitored microscopically. A mathematical model for the centrifugation process was developed. (3) Results: The red blood cell band pattern is reproducible but re-centrifugation of sub-bands reveals a new set of bands. This is caused by red blood cell aggregation. Based on the aggregation, our mathematical model predicts the band formation. Suppression of red blood cell aggregation reduces the band formation. (4) Conclusions: The red blood cell band formation in continuous Percoll density gradients could be explained physically by red blood cell aggregate formation. This aggregate formation distorts the density-based red blood cell age separation. Suppressing aggregation by osmotic swelling has a more severe effect on compromising the RBC age separation to a higher degree

Continuous Percoll Gradient Centrifugation of Erythrocytes—Explanation of Cellular Bands and Compromised Age Separation

(1) Background: When red blood cells are centrifuged in a continuous Percoll-based density gradient, they form discrete bands. While this is a popular approach for red blood cell age separation, the mechanisms involved in banding were unknown. (2) Methods: Percoll centrifugations of red blood cells were performed under various experimental conditions and the resulting distributions analyzed. The age of the red blood cells was measured by determining the protein band 4.1a to 4.1b ratio based on western blots. Red blood cell aggregates, so-called rouleaux, were monitored microscopically. A mathematical model for the centrifugation process was developed. (3) Results: The red blood cell band pattern is reproducible but re-centrifugation of sub-bands reveals a new set of bands. This is caused by red blood cell aggregation. Based on the aggregation, our mathematical model predicts the band formation. Suppression of red blood cell aggregation reduces the band formation. (4) Conclusions: The red blood cell band formation in continuous Percoll density gradients could be explained physically by red blood cell aggregate formation. This aggregate formation distorts the density-based red blood cell age separation. Suppressing aggregation by osmotic swelling has a more severe effect on compromising the RBC age separation to a higher degree

In Vitro Erythropoiesis at Different pO2 Induces Adaptations That Are Independent of Prior Systemic Exposure to Hypoxia

Hypoxia is associated with increased erythropoietin (EPO) release to drive erythropoiesis. At high altitude, EPO levels first increase and then decrease, although erythropoiesis remains elevated at a stable level. The roles of hypoxia and related EPO adjustments are not fully understood, which has contributed to the formulation of the theory of neocytolysis. We aimed to evaluate the role of oxygen exclusively on erythropoiesis, comparing in vitro erythroid differentiation performed at atmospheric oxygen, a lower oxygen concentration (three percent oxygen) and with cultures of erythroid precursors isolated from peripheral blood after a 19-day sojourn at high altitude (3450 m). Results highlight an accelerated erythroid maturation at low oxygen and more concave morphology of reticulocytes. No differences in deformability were observed in the formed reticulocytes in the tested conditions. Moreover, hematopoietic stem and progenitor cells isolated from blood affected by hypoxia at high altitude did not result in different erythroid development, suggesting no retention of a high-altitude signature but rather an immediate adaptation to oxygen concentration. This adaptation was observed during in vitro erythropoiesis at three percent oxygen by a significantly increased glycolytic metabolic profile. These hypoxia-induced effects on in vitro erythropoiesis fail to provide an intrinsic explanation of the concept of neocytolysis