Microvesicles Released from Human Red Blood Cells: Properties and Potential Applications

Microvesicles (MVs) are small spherical fragments of plasma membrane between 50 and 1000 nm in diameter. MVs arise through direct outward budding and fission of the plasma membrane. As almost all cells, human red blood cells (RBCs) are able to release MVs into extracellular environment under stimulating or storage conditions. Recently, it has been known that MVs not only play a role in homeostasis but also have diverse functions in cell-cell interactions and in the pathogenesis of diseases. In this chapter, the formation and release of MVs from human RBCs have been described. In addition, MVs have demonstrated to be potential vehicle for transport of nucleic acid and other molecules to the target cells. Although RBC-derived MVs are potential material for the development of delivery systems, it is still a great challenge to the clinical application. Future research should pay more attention to MVs as biological targets for diagnosis and practical therapeutics of cancer and other diseases

Intracellular Ca2+ Concentration and Phosphatidylserine Exposure in Healthy Human Erythrocytes in Dependence on in vivo Cell Age

After about 120 days of circulation in the blood stream, erythrocytes are cleared by macrophages in the spleen and the liver. The "eat me" signal of this event is thought to be the translocation of phosphatidylserine from the inner to the outer membrane leaflet due to activation of the scramblase, while the flippase is inactivated. Both processes are triggered by an increased intracellular Ca2+ concentration. Although this is not the only mechanism involved in erythrocyte clearance, in this minireview, we focus on the following questions: Is the intracellular-free Ca2+ concentration and hence phosphatidylserine exposure dependent on the erythrocyte age, i.e. is the Ca2+ concentration, progressively raising during the erythrocyte aging in vivo? Can putative differences in intracellular Ca2+ and exposure of phosphatidylserine to the outer membrane leaflet be measured in age separated cell populations? Literature research revealed less than dozen of such publications with vastly contradicting results for the Ca2+ concentrations but consistency for a lack of change for the phosphatidylserine exposure. Additionally, we performed reanalysis of published data resulting in an ostensive illustration of the situation described above. Relating these results to erythrocyte physiology and biochemistry, we can conclude that the variation of the intracellular free Ca2+ concentration is limited with 10 μM as the upper level of the concentration. Furthermore, we propose the hypothesis that variations in measured Ca2+ concentrations may to a large extent depend on the experimental conditions applied but reflect a putatively changed Ca2+ susceptibility of erythrocytes in dependence of in vivo cell age

Red Blood Cells Actively Contribute to Blood Coagulation and Thrombus Formation

The chapter describes the likely molecular mechanisms leading to the aggregation of human red blood cells (RBCs) under conditions of physiological coagulation when prostaglandin E2 (PGE2) or lysophosphatidic acid (LPA) is released from activated platelets and under pathophysiological conditions, in particular thrombi formation in sickle cell disease when patients are in a vaso-occlusive crisis. In both scenarios cation channels are activated. This leads to an increase of the free intracellular Ca2+ concentration resulting in an activation of the lipid scramblase, which in turn mediates a movement of phosphatidylserine (PS) from the inner to the outer membrane leaflet. In addition, the increased Ca2+ concentration leads to the activation of the Gardos channel. Experiments suggesting this mechanism have been performed with fluorescence microscopy, flow cytometry as well as single-cell force spectroscopy. The Ca2+-triggered RBC aggregation force has been identified to be close to 100 pN, a value large enough to play a significant role during thrombus formation or in pathological situations

Lhx9 and lhx9alpha: differential biochemical properties and effects on neuronal differentiation

The Lhx9 LIM-homeodomain transcription factor and its truncated isoform Lhx9alpha are generated by alternative splicing of the Lhx9 gene. Here we investigated the differential functional properties of these two isoforms. Lhx9alpha, which lacks parts of the homeodomain, was unable to bind DNA in EMSA experiments, but was able to associate with CLIM cofactors in GST pull-down assays. In transfection experiments in PC12 cells, Lhx9alpha fusion constructs systematically showed a nuclear localization, as opposed to Lhx9 fusion constructs, which also localized to the cytoplasm. Moreover, Lhx9 increased NGF-induced neuronal differentiation of PC12 cells. Lhx9alpha, on the other hand, did not significantly increase neuronal differentiation but had an effect on the morphology of PC12 cells. Finally, as tested by RT-PCR experiments on transfected PC12 cells, Lhx9 was not able to induce the transcription of Lhx9alpha. Our results show significantly different functional properties for Lhx9 and Lhx9alpha, and suggest that Lhx9alpha can compete away limiting amounts of nuclear CLIM cofactors. Thus, Lhx9 and Lhx9alpha isoforms could be implicated in regulating various aspects of neuronal differentiation

KAT-Independent Gene Regulation by Tip60 Promotes ESC Self-Renewal but Not Pluripotency

Although histone-modifying enzymes are generally assumed to function in a manner dependent on their enzymatic activities, this assumption remains untested for many factors. Here, we show that the Tip60 (Kat5) lysine acetyltransferase (KAT), which is essential for embryonic stem cell (ESC) self-renewal and pre-implantation development, performs these functions independently of its KAT activity. Unlike ESCs depleted of Tip60, KAT-deficient ESCs exhibited minimal alterations in gene expression, chromatin accessibility at Tip60 binding sites, and self-renewal, thus demonstrating a critical KAT-independent role of Tip60 in ESC maintenance. In contrast, KAT-deficient ESCs exhibited impaired differentiation into mesoderm and endoderm, demonstrating a KAT-dependent function in differentiation. Consistent with this phenotype, KAT-deficient mouse embryos exhibited post-implantation developmental defects. These findings establish separable KAT-dependent and KAT-independent functions of Tip60 in ESCs and during differentiation, revealing a complex repertoire of regulatory functions for this essential chromatin remodeling complex

Characterization of microvesicles released from human red blood cells

Background/Aims: Extracellular vesicles (EVs) are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs) and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs) under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. Methods: Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. Results: Treatment of RBCs with 4-bromo-A23187 (positive control), lysophosphatidic acid (LPA), or phorbol-12 myristate-13 acetate (PMA) in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS) and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 mV depended on the solutions and buffers used. Conclusion: An increase of intracellular Ca2+ or an activation of protein kinase C leads to the formation and release of MVs in human RBCs

Chondrolectin mediates growth cone interactions of motor axons with an intermediate target

The C-type lectin chondrolectin (chodl) represents one of the major gene products dysregulated in spinal muscular atrophy models in mice. However, to date, no function has been determined for the gene. We have identified chodl and other novel genes potentially involved in motor axon differentiation, by expression profiling of transgenically labeled motor neurons in embryonic zebrafish. To enrich the profile for genes involved in differentiation of peripheral motor axons, we inhibited the function of LIM-HDs (LIM homeodomain factors) by overexpression of a dominant-negative cofactor, thereby rendering labeled axons unable to grow out of the spinal cord. Importantly, labeled cells still exhibited axon growth and most cells retained markers of motor neuron identity. Functional tests of chodl, by overexpression and knockdown, confirm crucial functions of this gene for motor axon growth in vivo. Indeed, knockdown of chodl induces arrest or stalling of motor axon growth at the horizontal myoseptum, an intermediate target and navigational choice point, and reduced muscle innervation at later developmental stages. This phenotype is rescued by chodl overexpression, suggesting that correct expression levels of chodl are important for interactions of growth cones of motor axons with the horizontal myoseptum. Combined, these results identify upstream regulators and downstream functions of chodl during motor axon growth

Roles of the Rlim-Rex1 axis during X chromosome inactivation in mice

In female mice, the gene dosage from X chromosomes is adjusted by a process called X chromosome inactivation (XCI) that occurs in two steps. An imprinted form of XCI (iXCI) that silences the paternally inherited X chromosome (Xp) is initiated at the 2-to 4-cell stages. As extraembryonic cells including trophoblasts keep the Xp silenced, epiblast cells that give rise to the embryo proper reactivate the Xp and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI require the lncRNA Xist, which is expressed from the X to be inactivated. The X-linked E3 ubiquitin ligase Rlim (Rnf12) in conjunction with its target protein Rex1 (Zfp42), a critical repressor of Xist, have emerged as major regulators of iXCI. However, their roles in rXCI remain controversial. Investigating early mouse development, we show that the Rlim-Rex1 axis is active in pre-implantation embryos. Upon implantation Rex1 levels are downregulated independently of Rlim specifically in epiblast cells. These results provide a conceptual framework of how the functional dynamics between Rlim and Rex1 ensures regulation of iXCI but not rXCI in female mice.</p