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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 13, 2005 as doi:10.1096/fj.04-2450fje. |
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Departments of
* Drug Research and Evaluation and
Cell Biology and Neurosciences, Istituto Superiore di Sanita’, Rome, Italy
2Correspondence: Department of Drug Research and Evaluation, Section of Cell Aging and Degeneration, Istituto Superiore di Sanita’, Viale Regina Elena 299, Rome 00161, Italy. E-mail: malorni{at}iss.it
SPECIFIC AIMS
Erythrocytes are "simplified" cells that, having undergone oxidative imbalance, are committed to senescence and finally to their own destruction. Red blood cells (RBC) from patients with different redox-associated pathologies have been analyzed from functional, structural, or biochemical points of view. The aim was to correlate oxidative changes occurring in RBC with the pathogenesis of certain human diseases and eventually identify specific cellular markers of degeneration. The importance of redox-induced changes in RBC integrity and function is recognized as a pathogenic mechanism of some acquired and genetic diseases, especially surface molecules associated with the clearance of senescent RBC. Glycophorin A (GA) and apoptosis (i.e., phosphatidylserine, or PS) have been studied. Expression of the first (GA) was described to be altered in some pathological conditions (some respiratory diseases). PS, originally detected in nucleated cells by annexin V (AV) binding, was recently described as an apoptotic marker of RBC that can be significantly increased in some pathologic conditions (sickle cell disease). Apoptosis of RBC, since it occurred in a cell void of mitochondria and nucleus, was considered a paradoxical type of programmed cell death. Although lacking the mitochondrial regulators that play a pivotal role in nucleated cell apoptosis (caspase-9, Apaf-1, and cytochrome c), erythrocytes represent a unique cell model system with which to investigate plasma membrane-dependent programmed cell death pathways. We performed a series of experiments using human RBC to analyze subcellular effects of the product generated by the reaction of superoxide and nitric oxide radicals (peroxynitrite, or ONOO–). This term refers to the anion oxoperoxynitrate (1–), ONOO–, and its conjugate acid hydrogen oxoperoxynitrate, ONOOH. About 50% of peroxynitrite generated in the blood crosses RBC membrane and is inactivated inside the cell. By its potent oxidant and nitrating ability, peroxynitrite has been proposed as an important mediator of inflammation-induced tissue injury and dysfunction, and is considered the most efficient nitrating species of biological relevance so far identified.
PRINCIPAL FINDINGS
1. Oxidative effects of peroxynitrite
Treatments with 50 µM ONOO– lead to a significant increase (+26±3%) of methemoglobin (Met-Hb) in RBC. Two antioxidants were considered: N-acetyl-cysteine (NAC), a thiol supplier, and quercetin (Quer), a flavonoid ubiquitously present in fruits and vegetables. We found that Met-Hb increase was fully counteracted by the presence of both antioxidants NAC or Quer (100% oxy-Hb). Measurements of thiol content were performed. A significant GSH depletion and total thiol decrease was detected after ONOO– exposure. We found that NAC, but not Quer, was able to significantly counteract thiol depletion exerted by ONOO–. Evaluation of thiol content is essentially referred to as cytoskeletal thiol groups. Accordingly, an analysis of the main component of RBC membranoskeleton (i.e., spectrin molecule) was carried out. Static cytometry analyses clearly indicated that ONOO– exposure leads to spectrin aggregation and patching and that these changes were fully counteracted by NAC pretreatments and only partially by Quer.
2. Peroxynitrite induces structural alterations of RBC
In consideration of the key role played by the cytoskeleton in cell shape maintenance, ultrastructural and morphometrical analyses were performed by means of scanning electron microscopy (SEM). Peroxynitrite treatment induced the formation of several acanthocytes together with several blebbing cells and few leptocytic forms (Fig. 1
B; control cells are shown in Fig. 1A
). At variance, SEM observation of RBC samples pretreated with NAC (5 mM) or Quer (50 µM) before peroxynitrite administration clearly indicated that, although some rare shrunken leptocytic form could be detected, the normal discoid shape was generally maintained (Fig. 1C, D
). These qualitative results were confirmed by morphometric analyses clearly indicating a significant increase of shrunken RBC after ONOO– administration, which was significantly counteracted by pre-exposure to antioxidant drugs (Fig. 1E
). On these bases, RBC osmotic fragility was assessed. These evaluations indicated that ONOO– treatment increased RBC vulnerability; this was significantly counteracted by both NAC and Quer (Fig. 1F
). Parallel analyses carried out on the cytoskeleton component instructing RBC shape maintenance (i.e., spectrin molecule) clearly indicated a peroxynitrite-induced rearrangement and patching of this molecule, which was counteracted by NAC antioxidant.
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3. Effects of peroxynitrite on cell surface molecules
To investigate whether band 3 as an anion channel molecule was affected by peroxynitrite, static and flow cytometry analyses were conducted. They clearly showed a band 3 rearrangement and quantitative reduction in ONOO–-treated RBC. NAC was able to fully counteract these changes whereas Quer provided a less efficient protection. We evaluated both distribution and quantitative expression of GA on the RBC surface. Results obtained clearly indicated a significant decrease of GA expression in 
40% of peroxynitrite-exposed RBC, which was prevented by NAC and, although to a lesser extent, by Quer pretreatments.
4. Peroxynitrite induces activation of cathepsin E (CathE) and µ-calpain
Two further markers of RBC degeneration were taken into consideration: CathE and µ-calpain. Molecular antibodies recognizing active forms of these proteases were used. In a subset of RBC exposed to ONOO–, we observed by static or flow cytometry an increase of the active form of these proteases. The amount of RBC in which CathE was activated was represented by a small percentage of cells (7%). In the same vein, only a small percentage of ONOO–-treated RBC (7.3%) expressed active µ-calpain. These values differed from that described above that indicated a higher rate of altered cells.
5. Peroxynitrite induces phosphatidylserine (PS) externalization
The loss of plasma membrane asymmetry and exposure of PS was reported to be associated with cell death by apoptosis in different cell types. We decided to evaluate PS externalization by using annexin V (AV) binding assay in the experimental model system described herein. We found that: 1) a significant increase of AV-positive cells was detectable after ONOO– exposure (7%), which was 2) significantly counteracted by Quer or NAC pretreatments (Fig. 2
A). In line with the above findings and the hypothesized role of cathepsins and calpains in apoptosis, a significant (P<0.01) reduction of AV-positive cells in the presence of cathepsin and µ-calpain inhibitors was detected (Fig. 2B
).
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6. Peroxynitrite and caspase activity.
The activity of apoptosis-related specific proteases (i.e., caspases) was then considered by measuring their specific substrate cleavage. We found that peroxynitrite treatment leads to the activation of caspase 2 and caspase 3 whereas caspase 8 activity was undetectable. The specificity of these results was demonstrated by using selective inhibitors. ONOO–-induced activation of caspase 2 and caspase 3 was significantly (P<0.01) hindered by their specific inhibitors (VDAVD and DEVD, respectively) as well as by the pan-caspase inhibitor (ZVAD), whereas the caspase 8 inhibitor (IETD) was ineffective (Fig. 2C
).
CONCLUSIONS AND SIGNIFICANCE
Treatments with peroxynitrite induced a series of subcellular alterations: 1) oxidation of oxyhemoglobin to methemoglobin; 2) cytoskeletal and ultrastructural alterations; and 3) changes in senescence expression markers (decreased expression of GA), in a significant percentage (40%) of RBC. In a small percentage of erythrocytes (6–8%), peroxynitrite induced an increased expression of the active form of CathE and µ-calpain as well as PS externalization, a well known early marker of apoptosis. Accordingly, caspase 2 and caspase 3 were found activated. The presence of antioxidants inhibited peroxynitrite-induced senescence and, partially, apoptosis. Inhibition of aspartyl-proteases cathepsin and calpain as well as inhibitors of caspases resulted in a significant impairment of senescence-associated changes (cell shrinking and down-regulation of glycophorin A) as well as of apoptosis-associated changes (i.e., PS externalization). Collectively, these results indicate that peroxynitrite, a milestone of redox-mediated damage in human pathology, can hijack human red blood cells toward senescence and/or apoptosis by a mechanism involving both cysteinyl and aspartyl proteases. These results could be of importance in consideration of some points that recently emerged from literature indicating that redox changes induced by peroxynitrite can be considered as a paradigmatic source of oxidative imbalance. Peroxynitrite, generated by the reaction of superoxide and nitric oxide radicals, was recognized as a key pro-oxidant species implicated in cell pathology and involved in the pathogenetic mechanisms underlying several human diseases, including respiratory and degenerative diseases. For instance, an apparent correlation between the oxidative damage and the loss of PS asymmetry has been reported in RBC from patients with important human diseases, including sickle cell anemia, thalassemia, glucose-6-phosphate dehydrogenase deficiency, and diabetes. PS externalization, a well-defined sign of apoptosis, contributes to the shortened life span of defective erythrocytes as well as to important changes occurring in RBC function, such as hypercoagulability. This, in turn, can contribute to microclot generation that is of importance in the pathogenesis of respiratory diseases (chronic obstructive pulmonary disease). Finally, ONOO–-mediated RBC oxidative senescence could be proposed as a representative model system to not only evaluate oxidative imbalance-mediated senescence, but also the so-called erythroptosis (Fig. 3
). In a mummified cell such as the erythrocyte (a cell devoid of organelles) a sort of apoptosis seems to occur via a mechanism that brings into play all the protease cascades, aspartyl, and cysteinyl proteases, still offered by the cytosol of the "mummy."
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2450fje;
1 These authors contributed equally to this work. 
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