Issidora S. Papassideri

An update on red blood cell storage lesions, as gleaned through biochemistry and omics technologies

Red blood cell (RBC) aging in the blood bank is characterized by the accumulation of a significant number of biochemical and morphologic alterations. Recent mass spectrometry and electron microscopy studies have provided novel insights into the molecular changes underpinning the accumulation of storage lesions to RBCs in the blood bank. Biochemical lesions include altered cation homeostasis, reprogrammed energy, and redox metabolism, which result in the impairment of enzymatic activity and progressive depletion of high‐energy phosphate compounds. These factors contribute to the progressive accumulation of oxidative stress, which in turn promotes oxidative lesions to proteins (carbonylation, fragmentation, hemoglobin glycation) and lipids (peroxidation). Biochemical lesions negatively affect RBC morphology, which is marked by progressive membrane blebbing and vesiculation. These storage lesions contribute to the altered physiology of long‐stored RBCs and promote the rapid clearance of up to one‐fourth of long‐stored RBCs from the recipients bloodstream after 24 hours from administration. While prospective clinical evidence is accumulating, from the present review it emerges that biochemical, morphologic, and omics profiles of stored RBCs have observable changes after approximately 14 days of storage. Future studies will assess whether these in vitro observations might have clinically meaningful effects.

RBC‐derived vesicles during storage: ultrastructure, protein composition, oxidation, and signaling components

BACKGROUND: Red cells (RBCs) lose membrane in vivo, under certain conditions in vitro, and during the ex vivo storage of whole blood, by releasing vesicles. The vesiculation of the RBCs is a part of the storage lesion. The protein composition of the vesicles generated during storage of banked RBCs has not been studied in detail.

Stage-specific apoptotic patterns during Drosophila oogenesis

In the present study we demonstrate the existence of two apoptotic patterns in Drosophila nurse cells during oogenesis. One is developmentally regulated and normally occurs at stage 12 and the other is stage-specific and is sporadically observed at stages 7 and 8 of abnormally developed follicles. The apoptotic manifestation of the first pattern begins at stage 11 and is marked by a perinuclear rearrangement of the actin cytoskeleton and the development of extensive lobes and engulfments of the nurse cell nuclei located proximal to the oocyte. Consequently, at late stage 12 (12C), half of the nurse cell nuclei exhibit condensed chromatin, while at late stage 13 all the nuclei have fragmented DNA, as it is clearly shown by TUNEL assay. Finally, the apoptotic vesicles that are formed during stage 13, are phagocytosed by the neighboring follicle cells and at stage 14 the nurse cell nuclear remnants can be easily detected within the adjacent follicle cell phagosomes. In the second sporadic apoptotic pattern, all the nurse cell nuclei are highly condensed with fragmented DNA, accompanied by a completely disorganized actin cytoskeleton. When we induced apoptosis in Drosophila follicles through an etoposide and staurosporine in vitro treatment, we observed a similar pattern of stage-specific cell death at stages 7 and 8. These observations suggest a possible protective mechanism throughout Drosophila oogenesis that results in apoptosis of abnormal, damaged or spontaneously mutated follicles before they reach maturity.

Anti-ageing and rejuvenating effects of quercetin.

Homeostasis is a key feature of the cellular lifespan. Its maintenance influences the rate of ageing and it is determined by several factors, including efficient proteolysis. The proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. Alterations of proteasome function have been recorded in various biological phenomena including ageing and replicative senescence. Proteasome activities and function are decreased upon replicative senescence, whereas proteasome activation confers enhanced survival against oxidative stress, lifespan extension and maintenance of the young morphology longer in human primary fibroblasts. Several natural compounds possess anti-ageing/anti-oxidant properties. In this study, we have identified quercetin (QUER) and its derivative, namely quercetin caprylate (QU-CAP) as a proteasome activator with anti-oxidant properties that consequently influence cellular lifespan, survival and viability of HFL-1 primary human fibroblasts. Moreover, when these compounds are supplemented to already senescent fibroblasts, a rejuvenating effect is observed. Finally, we show that these compounds promote physiological alterations when applied to cells (i.e. whitening effect). In summary, these data demonstrate the existence of naturally occurring anti-ageing products that can be effectively used through topical application.

Intracellular Clusterin Inhibits Mitochondrial Apoptosis by Suppressing p53-Activating Stress Signals and Stabilizing the Cytosolic Ku70-Bax Protein Complex

Purpose: Secretory clusterin (sCLU)/apolipoprotein J is an extracellular chaperone that has been functionally implicated in DNA repair, cell cycle regulation, apoptotic cell death, and tumorigenesis. It exerts a prosurvival function against most therapeutic treatments for cancer and is currently an antisense target in clinical trials for tumor therapy. However, the molecular mechanisms underlying its function remained largely unknown. Experimental Design: The molecular effects of small interfering RNA-mediated sCLU depletion in nonstressed human cancer cells were examined by focusing entirely on the endogenously expressed sCLU protein molecules and combining molecular, biochemical, and microscopic approaches. Results: We report here that sCLU depletion in nonstressed human cancer cells signals stress that induces p53-dependent growth retardation and high rates of endogenous apoptosis. We discovered that increased apoptosis in sCLU-depleted cells correlates to altered ratios of proapoptotic to antiapoptotic Bcl-2 protein family members, is amplified by p53, and is executed by mitochondrial dysfunction. sCLU depletion-related stress signals originate from several sites, because sCLU is an integral component of not only the secretory pathway but also the nucleocytosolic continuum and mitochondria. In the cytoplasm, sCLU depletion disrupts the Ku70-Bax complex and triggers Bax activation and relocation to mitochondria. We show that sCLU binds and thereby stabilizes the Ku70-Bax protein complex serving as a cytosol retention factor for Bax. Conclusions: We suggest that elevated sCLU levels may enhance tumorigenesis by interfering with Bax proapoptotic activities and contribute to one of the major characteristics of cancer cells, that is, resistance to apoptosis.

Storage-dependent remodeling of the red blood cell membrane is associated with increased immunoglobulin G binding, lipid raft rearrangement, and caspase activation

BACKGROUND: The elucidation of the storage lesion is important for the improvement of red blood cell (RBC) storage. Ex vivo storage is also a model system for studying cell‐signaling events in the senescence and programmed cell death of RBCs. The membrane hosts critical steps in these mechanisms and undergoes widespread remodeling over the storage period.

Cell death during Drosophila melanogaster early oogenesis is mediated through autophagy.

Autophagy is a physiological and evolutionarily conserved process maintaining homeostatic functions, such as protein degradation and organelle turnover. Accumulating data provide evidence that autophagy also contributes to cell death under certain circumstances, but how this is achieved is not well known. Herein, we report that autophagy occurs during developmentally-induced cell death in the female germline, observed in the germarium and during middle developmental stages of oogenesis in Drosophila melanogaster. Degenerating germline cells exhibit caspase activation, chromatin condensation, DNA fragmentation and punctate staining of mCherry-DrAtg8a, a novel marker for monitoring autophagy in Drosophila. Genetic inhibition of autophagy, by removing atg1 or atg7 function, results in significant reduction of DNA fragmentation, suggesting that autophagy acts genetically upstream of DNA fragmentation in this tissue. This study provides new insights into the mechanisms that regulate cell death in vivo during development.

Red blood cell aging markers during storage in citrate‐phosphate‐dextrose–saline‐adenine‐glucose‐mannitol

BACKGROUND: It has been suggested that red blood cell (RBC) senescence is accelerated under blood bank conditions, although neither protein profile of RBC aging nor the impact of additive solutions on it have been studied in detail.

Dynamics of apoptosis in the ovarian follicle cells during the late stages of Drosophila oogenesis

Abstract. In the present study, we demonstrate the apoptotic events of the ovarian follicle cells during the late stages of oogenesis in Drosophila melanogaster. Follicle cell morphology appears normal from stage 10 up to stage 14, exhibiting a euchromatic nucleus and a well-organized cytoplasm. First signs of apoptosis appear at the anterior pole of the egg chamber at stage 14A. They are characterized by loss of microvilli at the apical cell membrane, alterations in nuclear morphology, such as chromatin condensation and convolution of the nuclear membrane, and also by condensation and vacuolization of the cytoplasm. During the following stage 14B, the follicle cell nuclei contain fragmented DNA as is demonstrated by acridine orange staining and TUNEL (TdT-mediated dUTP nick end-labeling) assay. Finally, the apoptotic follicle cells seem to detach from the eggshell when the mature egg chamber exits the ovariole. The detached follicle cells exhibit condensed nuclear chromatin, a disorganized cytoplasm with crowded organelles and are surrounded by epithelial cells. The above results seem to be associated with the abundant phagocytosis that we observed at the entry of the lateral oviducts, where the epithelial cells contain apoptotic cell bodies. Additionally, we tested the effect of etoposide treatment in the follicular epithelium and found that it induces apoptosis in a stage- and site-specific manner. These observations suggest a possible method of absorption of the apoptotic follicle cells that prevents the blockage of the ovarioles and helps the regular production of mature eggs.

Aging and death signalling in mature red cells: from basic science to transfusion practice

The red blood cells (RBCs) aging process is considered as an issue of special scientific and clinical interest. It represents a total of unidirectional, time-dependent but not-necessarily linear series of molecular events that finally lead to cell clearance1. Under normal circumstances, all human RBCs live approximately 120±4 days in blood circulation, implying the existence of tightly regulated molecular mechanism(s), responsible for the programming of the lifespan and the nonrandom removal of senescent RBCs2,3. Although the RBCs have already been used as a model for aging study1, the molecular participants, as well as the signalling pathways involved, are not yet completely clarified. RBCs storage under blood bank conditions is far from being considered analogous to the physiologic in vivo aging process. The putative implicated in vivo signalling pathways are expected to be more-or-less preserved under in vitro conditions, nevertheless slightly modulated, in response to a totally different environment. A storage period of up to 35–42 days at 4 °C probably is not a “congenial interlude” of the physiological maturity process and definitely does not represent an ignorable time period, compared to the RBCs lifespan. Stored RBCs age without the normal adjacency of other cells or plasma, which continuously provide them with survival factors and signals and, moreover, they are obligated to share their living space with their own and other cells’ wastes. Since no clearance mechanisms seem to function, senescent RBCs are probably sentenced to “survive” for a longer period than they were probably programmed for. Although there is evidence suggesting that storage disturbs the physiological RBC aging process4–7, the mechanistic basis of the aging progress inside the blood unit and the functional reactivity of the modified RBCs in vivo, remain still elusive. Given the fundamental need for safe and efficient transfusions, the clinical impact of stored blood, as a function of the storage parameters, has attracted considerable attention. Clinical trials that focus on the potential adverse clinical consequences of transfusing older storage-age RBCs units vs. younger ones have already been reported8. Apart from the skepticism around their design and execution9, these studies indicate the necessity of thorough examination of the storage effect on packed RBCs, before analyzing their potential impact on the clinical outcome. This review focuses on the current knowledge on aging and death signalling pathways operating in both in vivo systems and stored RBCs and suggests future directions in the preservation science, helpful for addressing what seem to be the current critical questions in transfusion medicine.