Secretory phospholipase A 2 (sPLA 2 ) type IIa, elevated in inflammation, breaks down membrane phospholipids and generates arachidonic acid. We hypothesized that sPLA 2 will hydrolyze red blood cells that expose phosphatidylserine (PS) and generate lysophosphatidic acid (LPA) from phosphatidic acid that is elevated in PS-exposing red blood cells. In turn, LPA, a powerful lipid mediator, could affect vascular endothelial cell function. Although normal red blood cells were not affected by sPLA 2 , at levels of sPLA 2 observed under inflammatory conditions (100 ng/ml) PS-exposing red blood cells hemolyzed and generated LPA (1.2 nM/10 8 RBC). When endothelial cell monolayers were incubated in vitro with LPA, a loss of confluence was noted. Moreover, a dose-dependent increase in hydraulic conductivity was identified in rat mesenteric venules in vivo with 5 M LPA, and the combination of PS-exposing red blood cells with PLA 2 caused a similar increase in permeability. In the presence of N-palmitoyl L-serine phosphoric acid, a competitive inhibitor for the endothelial LPA receptor, loss of confluence in vitro and the hydraulic permeability caused by 5 M LPA in vivo were abolished. The present study demonstrates that increased sPLA 2 activity in inflammation in the presence of cells that have lost their membrane phospholipid asymmetry can lead to LPA-mediated endothelial dysfunction and loss of vascular integrity.Secretory phospholipase A 2 (sPLA 2 ) 2 type IIa is a low molecular weight, ubiquitous enzyme that is elevated in inflammation. The enzyme generates arachidonic acid from phospholipids for the generation of thromboxanes and leukotrienes and, as such, acts as an essential mediator in the inflammatory pathways (1). The specific membranes targeted by the enzyme for the generation of arachidonic acid during inflammation have not been clearly defined. Secretory PLA 2 has a strong preference for phospholipids that are negatively charged at physiologic pH: phosphatidylserine (PS) and phosphatidylethanolamine (PE) (2). In normal mammalian cells, these phospholipids are mainly (PE) or exclusively (PS) confined to the inner layer of the plasma membrane (1-3). Loss of this asymmetric distribution and exposure of PS on the external surface generates a thrombogenic surface and signals macrophages to remove cells by phagocytosis (1-5). Although normal mammalian cells do not seem to act as targets for sPLA 2 , loss of phospholipid asymmetry in plasma membranes and the exposure of PS have been shown to render them vulnerable to phospholipid hydrolysis (1). In addition, it has been well established that bacterial membranes are excellent substrates for this enzyme (6). Together, membranes with altered phospholipid packing are potential targets for sPLA 2 type IIa.We have previously shown that sPLA 2 is elevated after injury, predicts hypoxemia, and is related to multiorgan failure (7,8). In addition, elevated levels of sPLA 2 predict the onset of the acute chest syndrome in sickle cell disease (9 -11), the severe lung damage that...

Iron-mediated oxidative stress plays an important role in the pathophysiology of thalassemia. Oxidative stress can cause lesions in DNA, including double-strand breaks. DNA damage, which is a cause of cancer (although not the only one), is recognized as deleterious. Unlike cancer, DNA damage can be assayed easily and relatively inexpensively in humans. In this study, a sensitive micronucleus assay was used to measure the frequency of chromosomal breaks in patients with alpha- and beta-thalassemia. The micronucleus test is based on the observation that a secondary nucleus (micronucleus) is formed around a chromosomal fragment, outside the main nucleus of a dividing cell. Micronuclei are readily apparent in red blood cells (RBCs), which otherwise lack DNA. We combined an immunomagnetic separation technique with single-laser flow cytometry to isolate and analyze reticulocytes in peripheral blood for the presence of micronuclei before these cells are removed by the spleen. Blood samples were obtained from patients with thalassemia and healthy volunteers. After immunomagnetic enrichment of CD71-positive reticulocytes, the cells were stained for micronuclei using the DNA dye 7-aminoactinomycin D (7-AAD) and evaluated by flow cytometry. Our findings indicate that higher levels of micronuclei frequencies are present in thalassemic RBCs.

Hemoglobin E (β26 Glu→Lys) is the most common abnormal hemoglobin (Hb) variant in the world. Homozygotes for HbE have a benign clinical picture (microcytic and mildly anemic) with rare clinical symptoms. However, when HbE coexists with β0-thalassemia, it is the most common severe hemoglobinopathy world-wide. National research priorities in the United States now include HbE related diseases because of the growing number of immigrants with these diseases, as well as the significant world impact of this life-threatening condition. In efforts to understand these diseases, a transgenic mouse model of homozygous HbE Disease was generated (Chen et al. Blood Cells Mol. Dis., 2004). Subsequent breeding has yielded knockout transgenic mice expressing exclusively human HbE: RBC smears show hypochromia, target cells, and stress reticulocytes. Measurement of PS exposure with Annexin V does not show a significantly increased population of PS exposing cells in the blood of these mice. Density gradients showing broader RBC density distributions correlate with increased RDW. RBC parameters determined by the ADVIA show low MCV (31.6±1.5fl vs. 46.9±2.1fl for C57) and MCH (9.8±0.1pg vs. 14.8±0.7pg for C57); and normal MCHC (32.0±2.0g/dl vs. 31.2±1.7 g/dl for C57) [n=5]. These RBC parameters mirror human HbE homozygotes. Increased levels of zinc protoporphyrin (ZPP), free protoporphyrin (PPIX), and fluorescent heme degradation products (FHDP) are found. To see if these porphyrins and FHDP arise from alterations in heme synthesis, real-time PCR technology methods were used. A comparison of the gene expression of enzymes involved in heme synthesis was made from RNA extracted from the spleens (the mouse hematopoietic organ). Upregulation in the rate-limiting enzyme erythroid delta-aminolevulinate synthase (ALAS) and other heme synthesis enzymes is suggested. These observations may be explained by increased erythropoiesis, compatible with the increase in % reticulocytes (7.2%±2.5, n=5); and/or elevated RBC porphyrins observed in these mice. This result is consistent with the effect of elevated porphyrins on ALAS in peripheral blood of erythropoietic protoporphyria-chemically induced mice (Inafuku et al., J Dermatol Sci, 1999). Considering the above, we conclude that the HbE full knockout mice are an excellent model for the benign human homozygous EE disease, and will serve as an important control for the proposed transgenic studies to generate HbE/β-thalassemia mice.

Double-stranded DNA breaks are serious lesions that contribute to the pathogenesis of cancer and chronic illnesses. Free radical injury is an important mechanism for the production of DNA modifications that eventually lead to chromosomal breaks. The generation of free radicals is increased in sickle cell disease (SCD) owing to the presence of the relatively unstable HbS and recurrent ischemia-reperfusion events. In the present study, a sensitive reticulocyte micronucleus assay was used to determine the frequency of chromosomal breaks in patients with SCD (Offer et al. FASEB J. 2005;19:485). A micronucleus is a piece of a chromosome left behind in a reticulocyte as a consequence of DNA double-stranded break after the nucleus is extruded. Blood samples were obtained from patients with SCD (n=33) and healthy volunteers (n=23). After immunomagnetic enrichment of CD71+ reticulocytes (Trf-Ret), RNA was removed with RNAse, and the cells were stained for miconuclei using the DNA dye 7-aminoactinomycin D (7-AAD) followed by flow cytometric evaluation. The appearance of reactive oxygen species in cytosol and membrane lipid oxidation in RBCs were measured by flow cytometry using the fluorescent markers 2, 7-dichlorofluorescin diacetate (DCF) and C11-BODIPY, respectively. The frequency of micronucleated Trf-Ret was significantly higher (p<.0001) in patients with SCD (mean 3.061/10,000 Trf-Ret, range 0.256 to 9.779) as compared with normal controls (mean 0.750/10,000 Trf-Ret, range 0.043 to 2.659). Mean DCF (125.1%) and C11-BODIPY (117.6%) fluorescence intensities were significantly greater than the mean control value (100%, p 0.0001). Our data indicate that patients with sickle cell disease have increased double stranded DNA-breaks compared with healthy controls, and suggest that elevated production of oxidants could contribute to the development of genetic damage in these patients.

Phosphatidylserine (PS) exposure contributes to recognition and removal of aged erythrocytes. PS exposure can only arise in cells in which the aminophospholipid translocase, or flippase, is defunct, or otherwise PS would be transported back into the inner cell monolayer. To provide evidence that flippase activity is reduced in old erythrocytes, we studied mouse erythrocytes that were biotinylated in vivo. This way we were able to distinguish the aging, biotinylated, RBC population from the newly formed, unbiotinylated RBC. Detection of the biotinylation with fluorescent streptavidin was combined with a flow cytometric assay for flippase activity using NBD-PS. We found that flippase activity decreased over time during the life-span of the RBC, as shown by a decrease in flippase activity of the biotinylated RBC compared to the newly formed RBC. A particularly high flippase activity was found in CD71-positive reticulocytes. Murine sickle cells show a much higher erythrocyte turnover than normal erythrocytes, with a 25–50% newly formed population each day and complete elimination of all biotinylated cells in 10 days. As in control mice, flippase activity decreased during the (short) life-span of sickle RBC and at all times, a subpopulation of sickle cells had a markedly reduced flippase activity. High flippase activity was only found in newly released erythrocytes. Our data indicate that the flippase activity decreases throughout the life-span of erythrocytes and can be markedly different in subpopulations of RBC in vivo. This decrease reduces the cell’s potential to maintain its phospholipid asymmetry, which results in PS exposure as a removal signal for the damaged cell. We conclude that the inability of sickle RBC to maintain flippase activity is related to the decreased survival time of those cells.