Necrotizing enterocolitis (NEC) is an idiopathic, inflammatory bowel necrosis of premature infants. Clinical studies have linked NEC with antecedent red blood cell (RBC) transfusions, but the ...underlying mechanisms are unclear. Here we report a neonatal murine model to investigate this association. C57BL/6 mouse pups rendered anemic by timed phlebotomy and then given RBC transfusions develop NEC-like intestinal injury with prominent necrosis, inflammation, and submucosal edema/separation of the lamina propria in the ileocecal region and colon within 12-24 h. The anemic intestine is infiltrated by inflammatory macrophages, which are activated in situ by RBC transfusions via a Toll-like receptor (TLR)-4-mediated mechanism and cause bowel injury. Chelation of RBC degradation products with haptoglobin, absence of TLR4, macrophage depletion, and inhibition of macrophage activation is protective. Intestinal injury worsens with increasing severity and the duration of anemia prior to transfusion, indicating a need for the re-evaluation of current transfusion guidelines for premature infants.
Background
The formation of extracellular vesicles (EVs) occurs during cold storage of RBCs. Transfusion of EVs may contribute to adverse responses in recipients receiving RBCs. However, EVs are ...poorly characterized with limited data on whether distinct vesicles are formed, their composition, and potential biological effects.
Study Design and Methods
Stored RBC‐derived EVs were purified using protocols that separate larger microvesicle‐like EVs (LEVs) from smaller exosome‐like vesicles (SEVs). Vesicles were analyzed by electron microscopy, content of hemoglobin, heme, and proteins (by mass spectrometry), and the potential to mediate lipid peroxidation and endothelial cell permeability in vitro.
Results
SEVs were characterized by having an electron‐dense double membrane whereas LEVs had more uniform electron density across the particles. No differences in hemoglobin nor heme levels per particle were observed, however, due to smaller volumes, SEVs had higher concentrations of oxyHb and heme. Both particles contained antioxidant proteins peroxiredoxin‐2 and copper/zinc superoxide dismutase, these were present in higher molecular weight fractions in SEVs suggesting either oxidized proteins are preferentially packaged into smaller vesicles and/or that the environment associated with SEVs is more pro‐oxidative. Furthermore, total glutathione (GSH + GSSG) levels were lower in SEVs. Both EVs mediated oxidation of liposomes that were prevented by hemopexin, identifying heme as the pro‐oxidant effector. Addition of SEVs, but not LEVs, induced endothelial permeability in a process also prevented by hemopexin.
Conclusion
These data show that distinct EVs are formed during cold storage of RBCs with smaller particles being more likely to mediate pro‐oxidant and inflammatory effects associated with heme.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Peroxiredoxin‐2 (Prx‐2) is a critical antioxidant protein in red blood cells (RBC). Prx‐2 is oxidized to a disulfide covalently‐bound dimer by H2O2, and then reduced back by the NADPH‐dependent ...thioredoxin—thioredoxin reductase system. The reduction of oxidized Prx‐2 is relatively slow in RBCs. Since Prx‐2 is highly abundant, Prx‐2s' peroxidase catalytic cycle is not considered to be limiting under normal conditions. However, whether Prx‐2 recycling becomes limiting when RBCs are exposed to stress is not known. Using three different model systems characterized by increased oxidative damage to RBCs spanning the physiologic (endogenous RBCs of different ages), therapeutic (cold‐stored RBCs in blood banks) and pathologic (RBCs from sickle cell disease (SCD) patients and humanized SCD mice) spectrum, basal levels of Prx‐2 oxidation and Prx‐2 recycling kinetics after addition of H2O2 were determined. The reduction of oxidized Prx‐2 was significantly slower in older versuin older versus younger RBCs, in RBCs stored for 4–5 weeks compared to 1 week, and in RBC from pediatric SCD patients compared to RBCs from control non‐SCD patients. Similarly, the rate of Prx‐2 recycling was slower in humanized SCD mice compared to WT mice. Treatment of RBC with carbon monoxide (CO) to limit heme‐peroxidase activity had no effect on Prx‐2 recycling kinetics. Treatment with glucose attenuated slowed Prx‐2 recycling in older RBCs and SCD RBCs, but not stored RBCs. In conclusion, the reduction of oxidized Prx‐2 can be further slowed in RBCs, which may limit the protection afforded by this antioxidant protein in settings associated with erythrocyte stress.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The process of lipid peroxidation is widespread in biology and is mediated through both enzymatic and non-enzymatic pathways. A significant proportion of the oxidized lipid products are electrophilic ...in nature, the RLS (reactive lipid species), and react with cellular nucleophiles such as the amino acids cysteine, lysine and histidine. Cell signalling by electrophiles appears to be limited to the modification of cysteine residues in proteins, whereas non-specific toxic effects involve modification of other nucleophiles. RLS have been found to participate in several physiological pathways including resolution of inflammation, cell death and induction of cellular antioxidants through the modification of specific signalling proteins. The covalent modification of proteins endows some unique features to this signalling mechanism which we have termed the 'covalent advantage'. For example, covalent modification of signalling proteins allows for the accumulation of a signal over time. The activation of cell signalling pathways by electrophiles is hierarchical and depends on a complex interaction of factors such as the intrinsic chemical reactivity of the electrophile, the intracellular domain to which it is exposed and steric factors. This introduces the concept of electrophilic signalling domains in which the production of the lipid electrophile is in close proximity to the thiol-containing signalling protein. In addition, we propose that the role of glutathione and associated enzymes is to insulate the signalling domain from uncontrolled electrophilic stress. The persistence of the signal is in turn regulated by the proteasomal pathway which may itself be subject to redox regulation by RLS. Cell death mediated by RLS is associated with bioenergetic dysfunction, and the damaged proteins are probably removed by the lysosome-autophagy pathway.
There are a wide variety of reactive species which can affect cell function, including reactive oxygen, nitrogen, and lipid species. Some are formed endogenously through enzymatic or non-enzymatic ...pathways, and others are introduced through diet or environmental exposure. Many of these reactive species can interact with biomolecules and can result in oxidative post-translational modification of proteins. It is well documented that some oxidative modifications cause macromolecular damage and cell death. However, a growing body of evidence suggests that certain classes of reactive species initiate cell signaling by reacting with specific side chains of peptide residues without causing cell death. This process is generally termed "redox signaling," and its role in physiological and pathological processes is a subject of active investigation. This review will give an overview of oxidative protein modification as a mechanism of redox signaling, including types of reactive species and how they modify proteins, examples of modified proteins, and a discussion about the current concepts in this area.
Toxicity mediated by free heme has emerged as an important element of end organ injuries and adverse outcomes in critically ill disease states. Free heme is thought to be derived from oxidative ...denaturation of free hemoglobin, secondary to red cell hemolysis. In this study, we evaluated the ability of oxidants (H2O2, nitrite, peroxynitrite and hypochlorous acid) formed during inflammation to cause heme release from purified hemoglobin and hemolysates, at pH 7.4 and 6.8. Supraphysiological concentrations of nitrite, peroxynitrite or hypochlorous acid were required to cause appreciable heme release from either free hemoglobin or hemolysates. However, H2O2 administered as a bolus or generated in situ, was more potent at promoting free heme release with free hemoglobin. With hemolysates, only in situ H2O2 formation resulted in significant free heme release. In all cases, free heme release was higher at lower pH and required oxidation of ferrous heme, but was not dependent on ferrylHb formation. Moreover, ligating ferric heme with cyanide or blocking the β93Cys did not prevent, but in fact increased free heme release. The salient observations from this study are that free heme release is likely mediated by continuous generation of H2O2 versus other heme oxidants, and facilitated at low pH.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Trauma is the leading cause of death and disability in patients aged 1-46 y. Severely injured patients experience considerable blood loss and hemorrhagic shock requiring treatment with massive ...transfusion of red blood cells (RBCs). Preclinical and retrospective human studies in trauma patients have suggested that poorer therapeutic efficacy, increased severity of organ injury, and increased bacterial infection are associated with transfusion of large volumes of stored RBCs, although the mechanisms are not fully understood.
We developed a murine model of trauma hemorrhage (TH) followed by resuscitation with plasma and leukoreduced RBCs (in a 1:1 ratio) that were banked for 0 (fresh) or 14 (stored) days. Two days later, lungs were infected with Pseudomonas aeruginosa K-strain (PAK). Resuscitation with stored RBCs significantly increased the severity of lung injury caused by P. aeruginosa, as demonstrated by higher mortality (median survival 35 h for fresh RBC group and 8 h for stored RBC group; p < 0.001), increased pulmonary edema (mean 95% CI 106.4 μl 88.5-124.3 for fresh RBCs and 192.5 μl 140.9-244.0 for stored RBCs; p = 0.003), and higher bacterial numbers in the lung (mean 95% CI 1.2 × 10(7) -1.0 × 10(7) to 2.5 × 10(7) for fresh RBCs and 3.6 × 10(7) 2.5 × 10(7) to 4.7 × 10(7) for stored RBCs; p = 0.014). The mechanism underlying this increased infection susceptibility and severity was free-heme-dependent, as recombinant hemopexin or pharmacological inhibition or genetic deletion of toll-like receptor 4 (TLR4) during TH and resuscitation completely prevented P. aeruginosa-induced mortality after stored RBC transfusion (p < 0.001 for all groups relative to stored RBC group). Evidence from studies transfusing fresh and stored RBCs mixed with stored and fresh RBC supernatants, respectively, indicated that heme arising both during storage and from RBC hemolysis post-resuscitation plays a role in increased mortality after PAK (p < 0.001). Heme also increased endothelial permeability and inhibited macrophage-dependent phagocytosis in cultured cells. Stored RBCs also increased circulating high mobility group box 1 (HMGB1; mean 95% CI 15.4 ng/ml 6.7-24.0 for fresh RBCs and 50.3 ng/ml 12.3-88.2 for stored RBCs), and anti-HMGB1 blocking antibody protected against PAK-induced mortality in vivo (p = 0.001) and restored macrophage-dependent phagocytosis of P. aeruginosa in vitro. Finally, we showed that TH patients, admitted to the University of Alabama at Birmingham ER between 1 January 2015 and 30 April 2016 (n = 50), received high micromolar-millimolar levels of heme proportional to the number of units transfused, sufficient to overwhelm endogenous hemopexin levels early after TH and resuscitation. Limitations of the study include lack of assessment of temporal changes in different products of hemolysis after resuscitation and the small sample size precluding testing of associations between heme levels and adverse outcomes in resuscitated TH patients.
We provide evidence that large volume resuscitation with stored blood, compared to fresh blood, in mice increases mortality from subsequent pneumonia, which occurs via mechanisms sensitive to hemopexin and TLR4 and HMGB1 inhibition.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A method to acquire the Raman spectra of sub-surface components using diffusely focused radiation in a microscope sampling configuration is described. This procedure generates Raman scattering at ...various sample depths by producing a converging beam at the back aperture of the objective lens. This method requires illumination of the sample with a defocused laser, while simultaneously increasing the number of CCD pixels that are binned along the spatial axis of the detector. We applied this diffuse sampling method to the analysis of stored red blood cells (RBCs). During storage, biochemical changes to RBCs occur (the "storage lesion"). However, there are no existing non-invasive methods to assess this. We evaluated the instrumental parameters needed to maximize the diffusely scattered signal, including pixel binning, slit width, and bandwidth. We demonstrated the effectiveness of this diffuse resonance Raman spectroscopy (DRRS) method by detecting RBCs through a blood bag segment (1 mm wall thickness). We directly compared the DRRS method to the more common stand-off Raman spectroscopy (SORS) method using both 633 nm and 785 nm excitation. Time-dependent DRRS spectra were used in a multivariate model for classification of RBCs in polymer segments by storage age. Young (6-8 day) RBCs were differentiated from old (35-40) RBCs with 100% sensitivity and 98.5% selectivity. These data indicated that DRRS is a promising, non-invasive technique for acquiring the spectra of sub-surface components, and is particularly applicable when the underlying sample can be resonantly enhanced.
A method to acquire the Raman spectra of sub-surface components using diffusely focused radiation in a microscope sampling configuration is described.
Primary mitral regurgitation (PMR) is associated with oxidative and inflammatory myocardial damage. We reported greater exosome hemoglobin (Hb) in pericardial fluid (PCF) versus plasma, suggesting a ...cardiac source of Hb.
Test the hypothesis that Hb is produced in the PMR heart and is associated with increased inflammation.
Hb gene expression for subunits alpha (HBA) and beta (HBB) was assessed in right atria (RA), left atria (LA) and left ventricular (LV) tissue from donor hearts (n = 10) and PMR patient biopsies at surgery (n = 11). PMR patients (n = 22) had PCF and blood collected for macrophage markers, pro-inflammatory cytokines, and matrix metalloproteinases (MMPs). In-situ hybridization for HBA mRNA and immunohistochemistry for Hb-alpha (Hbα) and Hb-beta (Hbβ) protein was performed on PMR tissue.
HBA and HBB genes are significantly increased (>4-fold) in RA, LA, and LV in PMR vs. normal hearts. In PMR tissue, HBA mRNA is expressed in both LV cardiomyocytes and interstitial cells by in-situ hybridization; however, Hbα and Hbβ protein is only expressed in interstitial cells by immunohistochemistry. PCF oxyHb is significantly increased over plasma along with low ratios (<1.0) of haptoglobin:oxyHb and hemopexin:heme supporting a highly oxidative environment. Macrophage chemotactic protein-1, tumor necrosis factor-α, interleukin-6, and MMPs are significantly higher in PCF vs. plasma.
There is increased Hb production in the PMR heart coupled with the inflammatory state of the heart, suggests a myocardial vulnerability of further Hb delivery and/or production during cardiac surgery that could adversely affect LV functional recovery.
Display omitted
•Hemoglobin is produced by cardiomyocytes and interstitial cells in the PMR heart.•Cell-free hemoglobin is elevated and haptoglobin levels are depleted in PCF vs. plasma.•PCF has a greater oxidative environment vs. plasma.•A gradient of pro-inflammatory cytokines, chemoattractants, and MMPs are higher in PCF vs. plasma supporting a myocardial source.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Hemolysis, characterized by formation of free hemoglobin (Hb), occurs in patients undergoing cardiopulmonary bypass (CPB). However, there is no study of the dynamic changes in red blood cell ...(RBC)-derived exosomes (Exos) released during CPB, nor whether these particles mediate acute kidney injury (AKI).
This study is a comprehensive time–course analysis, at baseline, 30 minutes, to 24 hours post-crossclamp release (XCR) to determine (1) Exos Hb content; (2) free Hb/heme, haptoglobin, hemopexin; and (3) urinary markers of AKI over the same time period. In addition, we developed a model system in Sprague–Dawley rats to test for AKI after intravenous injection of Exos Hb released during CPB.
In 30 patients undergoing CPB, there is a significant increase in plasma Hb-positive Exos but not microvesicles 30 minutes post-XCR versus other time points, with a simultaneous decrease in the haptoglobin/Hb ratio. These changes presage a significant increase in urine neutrophil gelatinase–associated lipocalin and kidney injury molecule-1 at 24 hours. Intravenous injection of plasma Exos (109-10 particles obtained 30 minutes post-XCR) into rats causes AKI at 72 hours, manifested by multifocal degeneration of proximal tubular epithelium. At 21 days, there is persistent tubular injury and interstitial fibrosis. Intravenous injection of Exos from 35-day-old stored RBCs into rats results in glomerular–tubular injury, increased kidney ferritin and hemoxygenase-1 expression, and significant elevation of kidney injury molecule-1 and proteinuria at 72 hours.
These combined studies raise the potential for RBC-derived Exos, released during CPB, to target the kidney and mediate AKI.
Display omitted
PDF
