Incubation Of Red Blood Cells Research Articles

Procoagulant and proinflammatory effects of red blood cells on lipopolysaccharide‐stimulated monocytes

We aimed to evaluate the mechanisms underlying the effects of red blood cells (RBCs) on the reactivity of monocytes to lipopolysaccharide (LPS) stimulation. Measurements of tissue factor (TF) antigen and activity were performed on freshly isolated white blood cells (WBCs)/platelets resuspended in heparinized plasma, as well as cultured monocytic cells. In a dose-dependent manner, RBCs significantly enhanced LPS-induced TF activity and antigen levels in blood monocytes; potentiation of TF activity by both human and murine RBCs did not require the presence of neutrophils and/or platelets. We also measured the levels of monocyte chemotactic protein-1 (MCP-1), the key proinflammatory chemokine that binds to duffy antigen receptor for chemokines (DARC) on RBC surface, in plasma and RBC lysates after the incubation of RBCs with WBC/platelets; at the concentrations corresponding to normal blood counts, RBCs exerted a significant influence on the free plasma levels of MCP-1, with about two-thirds of detectable MCP-1 post-LPS stimulation being associated with RBCs. Critically, DARC-deficient murine RBCs failed to enhance LPS-induced TF activity, confirming the mechanistic significance of RBC-DARC. Our study reports a novel mechanism by which RBCs promote procoagulant and proinflammatory sequelae of WBC exposure to LPS, likely mediated by RBC-DARC in the microenvironment(s) that bring monocytes and RBCs in close proximity.

Renitrosylation of banked human red blood cells improves deformability and reduces adhesivity.

Transfusion of red blood cells (RBCs) is a frequent health care practice. However, unfavorable consequences may occur from transfusions of stored RBCs and are associated with RBC changes during storage. Loss of S-nitrosohemoglobin (SNO-Hb) and other S-nitrosothiols (SNOs) during storage is implicated as a detriment to transfusion efficacy. It was hypothesized that restoring SNOs within banked RBCs would improve RBC functions relevant to successful transfusion outcomes, namely, increased deformability and decreased adhesivity. Stored human RBCs were incubated with nitric oxide (NO) donors PROLI/NO and DEA/NO (disodium 1-[2-(carboxylato)-pyrrolidin-1-yl]diazen-1-ium-1,2-diolate and diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate) under varying experimental conditions (e.g., aerobic/anaerobic incubation, NO donor to RBC ratio). SNO restoration was evaluated in vitro and in vivo as a means to improve RBC function after storage. Incubation of RBCs with the NO donors resulted in 10-fold greater levels of SNO-Hb versus untreated control or sham RBCs, with significantly higher Hb-bound NO yields from an NO dose delivered by DEA/NO. RBC incubation with DEA/NO at a stoichiometry of 1:62.5 NO:Hb significantly increased RBC deformabilty and reduced adhesion to cultured endothelial cells. RBC incubation with DEA/NO also increased S-nitrosylation of RBC cytoskeletal and membrane proteins, including the β-spectrin chain. Renitrosylation attenuated both RBC sequestration in the lung and the mild blood oxygen saturation impairments seen with banked RBCs in a mouse model of transfusion. RBC renitrosylation using NO donors has promise for correcting deficient properties (e.g., adhesivity, rigidity, and SNO loss) of banked RBCs and in turn improving transfusion outcomes.

Amyloid beta peptide (1-42)-mediated antioxidant imbalance is associated with activation of protein kinase C in red blood cells.

Glycolysis and pentose phosphate pathway (PPP) in red blood cell (RBC) are modulated by the cell oxygenation state. This metabolic modulation is connected to variations in intracellular nicotinamide adenine dinucleotide phosphate-reduced form (NADPH) and adenosine triphosphate (ATP) levels as a function of the oxygenation state of the cell, and, consequently, it should have physiologic relevance. In the present study, we analysed the effects of amyloid beta peptide (1-42) (Abeta) on RBC metabolism and its relationship with the activity of protein kinase C (PKC). Our results showed that metabolic response to Abeta depended on the degree of cell oxygenation. In particular, under high O2 pressure, in Abeta-treated RBC, glucose metabolized through PPP approached that metabolized by RBC under low O2 pressure, differently to that observed in untreated cells. The effect of Abeta on RBC metabolism was paralleled by increase in PKC enzyme activity, but cytosolic Ca2+ concentration does not seem to be involved in this mechanism. Incubation of Abeta-treated RBC with a specific inhibitor of PKC partially restores PPP flux. A possible rationalization of the different metabolic behaviours shown by RBC following Abeta treatment is proposed. It takes into account the known post-translational modifications to cytoskeleton proteins induced by PKC. The reduction in PPP flux may lead to a weakened defence system of antioxidant reserve in RBC, becoming a source of reactive species, and, consequently, its typical, structural and functional features are lost. Therefore, oxidative stress may outflow from the RBC and trigger damage events in adjacent cells and tissue, thus contributing to vascular damage.

Rapid Reversal of Red Blood Cell Sickling Promoted By Pegylated Carboxyhemoglobin Bovine Gas Transfer Properties

Background: Red blood cell (RBC) sickling is the hallmark phenotype of Sickle Cell Disease (SCD) resulting from an inherited hemoglobin (HbS) point mutation genotype. Sickling is a direct consequence of HbS polymerization that occurs as RBC encounter low oxygen environments. Sickled RBCs have increased rigidity restricting their passage through the microvasculature that can lead to a vaso-occlusive crisis (VOC). VOCs are painful, debilitating and can be associated with more severe co morbidities including acute chest syndrome, leg ulcers and stroke. Treatments for VOC are limited and include IV hydration and opioid for pain control. Extensive VOC events often require hospitalization, supplemental oxygen and transfusions. Improved therapies that can be administered early during VOC that directly target the causative RBC sickling events are warranted.PEGylated carboxyhemoglobin bovine (Sanguinate SG) is currently in clinical development being evaluated for the ability to treat the comorbidities of SCD including VOC. SG carries oxygen (O2) and/or carbon monoxide (CO) in the mammalian vasculature for delivery to tissues to synergistically treat inflammation and vasoconstriction and other comorbidities characterized by tissue hypoxia. Early intervention of VOC with SG treatment could limit the crisis event and reduce pain severity while providing a timely crisis resolution thereby reducing the likelihood of more severe VOC clinically related complications.Methods: SG treatment effects were evaluated under controlled environmental conditions to determine its capacity for gas exchange with RBCs obtained with IRB consent from healthy and SCD volunteers. Carboxyhemoglobin and oxyhemoglobin levels were monitored to determine dose and time effects as well as the repetitive capacity of SG to facilitate gas transfer processes. RBC treated samples were also analyzed by light microscopy and image capture flow cytometry to visualize and quantify the effects of SG treatment on reversing sickled SCD RBC. A PEGylated bovine serum albumin (PBSA) product was also produced and analyzed in parallel as a control agent.Results: SG addition to normal oxygenated RBC resulted in CO and O2 exchange between RBC and SG that followed mass balance and reached equilibrium in closed system. Kinetic analysis revealed SG rapidly transferred its CO component to oxygenated RBC with concomitant O2 loading of SG. Using experimentally loaded RBC with CO and SG with O2 produced similar reciprocal gas exchange results. Additionally the primary RBC/ SG reaction products were isolated and cycled demonstrating the ability of SG to continually facilitate gas transfer through multiple exposure events.Similar studies using SG, oxygenated SG (produced by an RBC exchange reaction) or PBSA control were conducted with SCD RBC. Sickling was induced by incubation of RBC in a hypoxic chamber for 3 hours prior to SG or control treatments. After 2 hours of treatment, cells were fixed by addition of glutaraldehyde. Photomicroscopy showed a marked reduction in the sickled RBC population with both SG or oxygenated SG treatments but not with the PBSA control (Fig. 1). Results from imaging flow cytometry further supported the microscopy findings and revealed a significant quantitative reduction in the percentage of sickled RBC levels.Summary: RBC rheology and lifespan are negatively impacted by repetitive HbS polymerization/de-polymerization cycling and treatments that reverse RBC sickling during a VOC event could be expected to provide broad clinical benefits through reduction of SCD comorbidities. SG was designed to promote CO and oxygen transfer in concentration dependent manners providing physiological supplementation of O2 transport/delivery in conditions of hemolytic or ischemic anemia.These ex vivo data demonstrated for the first time that under controlled conditions, a therapeutic agent could be observed serving as an active gas transport agent providing either CO or O2 to sickled RBCs, prompting rapid unsickling. Furthermore, the use of image capture flow cytometry provided a quantitative measurement of sickled RBC fraction decrease. This mechanism may provide a useful biomarker test in future clinical studies to monitor SG treatment effects on SCD patients. [Display omitted] DisclosuresJubin:Prolong Pharmaceuticals: Employment. Buontempo:Prolong Pharmaceuticals: Employment. Yglesias:Prolong Pharmaceuticals: Employment. Abuchowski:Prolong Pharmaceuticals: Employment. Chen:Prolong Pharmaceuticals: Employment. Kazo:Prolong Pharmaceuticals: Employment. Kazo:Prolong Pharmaceuticals: Employment.

Plasmodium falciparum, but not P. vivax, can induce erythrocytic apoptosis.

BackgroundApoptosis can occur in red blood cells (RBC) and seems to be involved in hematologic disorders related to many diseases. In malaria it is known that parasitized RBC (pRBC) is involved in the development of anemia and thrombosis; however, non-parasitized RBC (nRBC) apoptosis could amplify these malaria-associated hematologic events. In fact, in experimental malaria, increased levels of apoptosis were observed in nRBC during lethal Plasmodium yoelii 17XL infection, but in human malaria erythrocytic apoptosis has never been studied. The present study was performed to investigate if nRBC apoptosis also occurs in P. vivax and P. falciparum infections.FindingsApoptosis of nRBC was evaluated in blood samples of P. vivax malaria patients and clinically healthly individuals living in Manaus, Brazil, both ex vivo and after incubation of RBC for 24 h. Additionally, the capacity of plasma from P. vivax or P. falciparum patients was tested for induction of in vitro apoptosis of normal RBC from a clinically healthy individual living in a non-endemic malaria region. Apoptosis was detected by flow cytometry using annexin V staining. In contrast to experimental malaria that significantly increased the levels of apoptotic nRBC both ex-vivo and after 24 h of incubation, no significant alteration on apoptotic nRBC rates was detected in P. vivax infected patients when compared with non-infected control individuals. Similar results were observed when plasma of these P. vivax patients was incubated with normal RBC. Conversely, plasma from P. falciparum-infected subjects induced significant apoptosis of these cells.ConclusionsApoptosis of normal RBC can be induced by plasma from individuals with P. falciparum (but not with P. vivax) malaria. This finding could reflect the existence of erythrocytic apoptosis during infection that could contribute to the pathogenesis of hematological and vascular complications associated with falciparum malaria.

Complement inhibition significantly decreases red blood cell lysis in a rat model of acute intravascular hemolysis.

Prevention of acute hemolytic transfusion reactions is a worldwide concern. The objective of this study was to develop a simple rat model of complement-mediated acute intravascular hemolysis. Human AB red blood cells (RBCs) were incubated with complement-sufficient or complement-deficient Wistar rat serum (WRS) in the presence and absence of human RBC antibody in vitro to elucidate the mechanism of hemolysis. To study the role of complement in acute intravascular hemolysis in vivo, Wistar rats were treated either with or without cobra venom factor (CVF) to deplete complement activity. Human AB RBCs were then injected into both groups of rats, followed by serial blood draws up to 2 hours. Venous blood clearance and lysis of transfused RBCs at each time point were measured by flow cytometry and spectrophotometry. RBC sequestration was determined in the liver, spleen, and kidney by immunohistochemistry. In vitro incubation of human RBCs with WRS demonstrated that RBC lysis was mediated via the classical complement pathway and that hemolysis was antibody dependent. Transfusion of human RBCs into rats showed significantly less hemolysis in the CVF group versus untreated group. RBC sequestration in the spleen and liver 2 hours posttransfusion were not quantitatively different between the two groups. Given the much higher degree of similarity for rat and human complement compared to mice, this simple rat model is ideal for testing novel inhibitors of classical pathway activation for the prevention and treatment of acute intravascular hemolysis.

Could Na,K-ATPase play a role in potassium leakage from irradiated erythrocytes?

BackgroundIn developing countries, the access to red blood cell (RBC) irradiators is restricted. Thus, it is a common practice in blood banks to stock irradiated RBC units until they expire. The aim of this work is to elucidate the involvement of Na,K-ATPase in potassium leakage from prophylactically irradiated RBCs. MethodsWhole blood was collected from healthy donors, and blood concentrates were irradiated with 25Gy of γ-radiation within 24h of collection. At days 3, 5, 7, 9, 11, 14 and 28 post-irradiation, fractions were removed and centrifuged and Na,K-ATPase activity from ghost membranes was determined. ResultsThe inhibition of Na,K-ATPase activity in RBCs reached 12.6% by day 7 of storage and up to 50% by day 14 of storage. The addition of vitamin C prevented the irradiation-induced loss of Na,K-ATPase activity. The irradiation of RBCs provoked an increase in potassium plasma levels and a decrease in sodium plasma levels. The incubation of RBCs with ouabain did not change the sodium or potassium levels in the plasma, and the addition of vitamin C only partially prevented a decrease in sodium levels caused by irradiation. ConclusionBecause the inhibition of Na,K-ATPase by ouabain did not cause potassium accumulation in the plasma, we conclude that the irradiation-induced inhibition of the pump is not a key factor driving this effect.

Endothelium-derived nitric oxide production is increased by ATP released from red blood cells incubated with hydroxyurea

Red blood cells (RBCs) release adenosine triphosphate (ATP) in response to a variety of stimuli, including flow-induced deformation. Hydroxyurea (HU), a proven therapy for individuals with sickle cell disease (SCD), is known to improve blood flow. However, the exact mechanism leading to the improved blood flow is incomplete. Here, we report that the incubation of human RBCs with HU enhances ATP release from these cells and that this ATP is capable of stimulating nitric oxide (NO) production in an endothelium. RBCs incubated with HU were pumped through micron-size flow channels in a microfluidic device. The release of ATP from the RBCs was measured using the luciferin–luciferase assay in detection wells on the device that were separated from the flow channels by a porous polycarbonate membrane. NO released from a layer of bovine artery endothelial cells (bPAECs) cultured on the polycarbonate membrane was also measured using the extracellular NO probe DAF-FM. ATP release from human RBCs incubated with 100μM HU was observed to be 2.06±0.37-fold larger than control samples without HU (p<0.05, N⩾3). When HU-incubated RBCs were flowed under a layer of bPAECs, NO released from the bPAEC layer was measured to be 1.34±0.10-fold higher than controls. An antagonist of the P2Y receptor established that this extra 30% increase in NO release is ATP mediated. Furthermore, when RBCs were incubated with L-NAME, a significant decrease in endothelium-derived NO production was observed. Control experiments suggest that RBC-generated NO indirectly affects endothelial NO production via its effects on RBC-derived ATP release.

An effect of glycoprotein IIb/IIIa inhibitors on the kinetics of red blood cells aggregation

The reversible aggregation of red blood cells (RBCs) continues to be of the basic science and clinical interest. Recently it has been reported about a specific binding between fibrinogen and unknown erythrocyte glycoprotein receptors. The aim of this study was to investigate whether the red blood cell aggregation (RBCA) include the cell-cell interaction using the membrane receptors that bind such ligands as fibrinogen or fibronectin. To test this hypothesis the RBCs were incubated with monafram - the drug of the monoclonal antibodies against glycoprotein (GP) IIb/IIIa, with the GPIIb-IIIa receptor antagonist tirofiban, epifibatide and with the fibrinogen inhibiting peptide. It has been found that the RBC incubation with monafram resulted in a marked RBCA decrease mainly in persons with high level of aggregation. Another research session has shown that RBC incubation with fibronectin was accompanied by a significant RBCA rise. The monafram addition to red cell incubation medium resulted in a significant RBCA lowering. The cell incubation with tirofiban and epifibatide issued in RBCA decrease. The similar results were obtained when RBCs were incubated with the fibrinogen inhibiting peptide. Although monafram, tirofiban, eptifibatide and the fibrinogen inhibiting peptide were related to fibrinogen function they didn't inhibit RBCA completely. Therefore, under moderate and low red blood cell aggregation the cell binding is probably related to nonspecific mode. It seems evident that the specific and nonspecific modes of red blood cell aggregate formation could co-exist. Additional theoretical and experimental investigations in this area are needed.

A star polymer based on a polyethylene glycol with a porphyrinic core as a photosensitizing agent for application in photodynamic therapy: tests in vitro on human erythrocytes

In this study, human erythrocytes were used to assess the cytotoxicity of an uncharged, water-soluble and bio-compatible porphyrin system [the 5,10,15,20-tetrakis-p-(ω-methoxypolyethyleneoxyphenyl)porphyrin, H2–P], which has the potential to act as a photosensitizing agent in photodynamic therapy (PDT). By investigating the cytotoxicity effects under controlled conditions, at different porphyrin concentrations and upon increasing irradiation times, the data in the absence and in the presence of H2–P were compared. It was shown that the H2–P system, without the assistance of any additional carrier, is easily internalized into cells, unlike the process which occurs for charged porphyrins. Incubation of red blood cells with H2–P resulted in non-cytotoxicity in control conditions but, under irradiation, the cytotoxicity increased significantly at 20 μM of porphyrin; it further increased at 40 μM, reaching total cell death at 80 μM. These results suggest that, as already observed for other cellular species, PDT-mediated oxidative stress occurs.

Signaling pathways regulating red blood cell aggregation.

The exposure of red blood cells (RBC) to some hormones (epinephrine, insulin and glucagon) and agonists of α- and β-adrenergic receptors (phenylephrine, clonidine and isoproterenol) may modify RBC aggregation (RBCA). Prostaglandin E1 (PGE1) significantly decreased RBCA, and PGE2 had a similar but lesser effect. Adenylyl cyclase (AC) stimulator forskolin added to RBC suspension, caused a decrease of RBCA. More marked lowering of RBCA occurred after RBC treatment by dB-cAMP. Phosphodiesterase (PDE) inhibitors markedly reduced RBCA. Ca2+ influx stimulated by A23187 was accompanied by an increase of RBCA. The blocking of Ca2+ entry into the RBC by verapamil or the chelation of Ca2+ by EGTA led to a significant RBCA decrease. Lesser changes of aggregation were found after RBC incubation with protein kinase C stimulator phorbol 12-myristate 13-acetate (PMA). A significant inhibitory effect of tyrosine protein kinase (TPK) activator cisplatin on RBCA was revealed, while selective TPK inhibitor, lavendustin, eliminated the above mentioned effect. Taken together, the data demonstrate that changes in RBCA are connected with activation of different intracellular signaling pathways. We suggest that alterations in RBCA are mainly associated with the crosstalk between the adenylyl cyclase-cAMP system and Ca2+ control mechanisms.

Plasmodium falciparum , but not P. vivax , can induce erythrocytic apoptosis

Apoptosis can occur in red blood cells (RBC) and seems to be involved in hematologic disorders related to many diseases. In malaria it is known that parasitized RBC (pRBC) is involved in the development of anemia and thrombosis; however, non-parasitized RBC (nRBC) apoptosis could amplify these malaria-associated hematologic events. In fact, in experimental malaria, increased levels of apoptosis were observed in nRBC during lethal Plasmodium yoelii 17XL infection, but in human malaria erythrocytic apoptosis has never been studied. The present study was performed to investigate if nRBC apoptosis also occurs in P. vivax and P. falciparum infections. Apoptosis of nRBC was evaluated in blood samples of P. vivax malaria patients and clinically healthly individuals living in Manaus, Brazil, both ex vivo and after incubation of RBC for 24 h. Additionally, the capacity of plasma from P. vivax or P. falciparum patients was tested for induction of in vitro apoptosis of normal RBC from a clinically healthy individual living in a non-endemic malaria region. Apoptosis was detected by flow cytometry using annexin V staining. In contrast to experimental malaria that significantly increased the levels of apoptotic nRBC both ex-vivo and after 24 h of incubation, no significant alteration on apoptotic nRBC rates was detected in P. vivax infected patients when compared with non-infected control individuals. Similar results were observed when plasma of these P. vivax patients was incubated with normal RBC. Conversely, plasma from P. falciparum-infected subjects induced significant apoptosis of these cells. Apoptosis of normal RBC can be induced by plasma from individuals with P. falciparum (but not with P. vivax) malaria. This finding could reflect the existence of erythrocytic apoptosis during infection that could contribute to the pathogenesis of hematological and vascular complications associated with falciparum malaria.

In vitro effect of pentoxifylline and lisofylline on deformability and aggregation of red blood cells from healthy subjects and patients with chronic venous disease.

Purpose. The aim of the study was to assess the in vitro potency of pentoxifylline (PTX) and one of its most active metabolites lisofylline (LSF) to improve rheological properties of red blood cells (RBC) from healthy individuals and patients with chronic venous disease (CVD). Additionally, the study aimed to compare the effects of PTX and LSF on RBC deformability and aggregation. Methods. Blood samples were collected from healthy volunteers (antecubital vein) and from CVD patients (varicose and antecubital vein). Deformability and aggregation of RBC were assessed using Laser-assisted Optical Rotational Cell Analyser (LORCA). Results. PTX and LSF increased RBC elongation significantly. Additionally, RBC incubation with PTX resulted in a marked decrease in RBC aggregation. PTX reduced the tendency towards the formation of RBC aggregates and of their stability. The beneficial effect of PTX on RBC aggregation was most apparent for those cells whose aggregation tendency and aggregate stability was the greatest. Conclusions. In vitro addition of PTX or LSF effectively increased deformability of RBC from healthy donors and patients with CVD. Thus, LSF may contribute to the in vivo hemorheological effects of pentoxifylline. On the other hand, there was no significant effect of LSF on aggregation of RBC in vitro. Hence, LSF has no contribution to this particular effect of PTX. Additionally, the present study demonstrated the use of RBC with impaired deformability and aggregation for the evaluation of in vitro rheological activity of xenobiotics.

Role molecular signaling pathways in changes of red blood cell deformability

This study was designed to investigate the dependency of the red blood cell deformability upon activation of extra- and intracellular signaling pathways. Exposures of red blood cells (RBCs) to catecholamines and to insulin led to positive change in the RBC deformability. When forskolin, a stimulator of adenylyl cyclase (AC), was added to RBC suspension, the RBC deformability was increased. Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP. The inhibitors of phosphodiesterase (PDE) activity increased red cell deformability. These results revealed a considerable role of the AC-cAMP signaling system in the regulation of red blood cell deformability. The rise of the red blood cell Ca(2+) influx, stimulated by mechanical loading or A23187 was accompanied by a marked lowering of RBC deformability. At the same time blocking of Ca(2+) entry into RBC by verapamil or Ca(2+) chelating by EGTA led to significant deformability rise. The comparison of the effect of the different protein kinases on the red blood cell deformability showed that it was altered more considerable under PKA activation by forskolin or dB-cAMP than by other protein kinases. There was a lesser but quite statistically significant effect of tyrosine protein kinase (TPK) on RBC microrheology. Whereas the microrheological effect of PKC was not so considerable. The problem of the short-term regulation of red blood cell microrheology is examined. The latter includes: the modes of activation of extra- and intracellular molecular signaling pathways, ligand - receptor interaction, second messengers, membrane protein phosphorylation. On the whole the total data clearly show that the red cell deformability changes are connected with activation of different extra - and intracellular signaling pathways. It seems reasonable to suppose that red blood cell deformability changes were mainly associated with activation of the AC-cAMP-PKA pathway, and with decrease of Ca(2+) entry into cells.

Abstract 19046: RBC-Macrophage Inflammatory Axis: Potential Role in Atherosclerosis

Background: Red blood cells (RBC) modulate vasoreactivity, serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC), and contribute to the pathology of atherosclerotic plaque rupture, suggesting a broad role in vascular pathophysiology. High fat diet (HFD) is known to negatively impact vascular function and modify RBC membrane composition, but how this affects RBC chemokine binding, and interaction with macrophages and blood vessels, is unknown. Methods and Results: RBC from C57BL/6J mice fed chow diet (chow RBC) or 60% HFD (HFD RBC) for 16 weeks were collected and studied in vitro and in vivo. Reactive oxygen species (DCFH fluorescence) and phosphatidylserine externalization (annexin V binding), a potential signal for RBC phagocytosis, were increased ~1.5 fold (p&lt;0.05) in HFD RBC compared to chow RBC. HFD RBC released ~1.5 fold more monocyte chemoattractant protein-1 (MCP-1) (p&lt;0.05) compared to chow RBC upon addition of heparin to disrupt DARC binding. In a transwell system, HFD RBC, as compared with chow RBC, produced a significant increase in the migration of thioglycollate-elicited macrophages through an endothelial monolayer (p&lt;0.05). Incubation of HFD RBC with murine macrophages demonstrated a ~1.5 fold (p&lt;0.05) increase in phagocytosis compared to chow RBC; upon their internalization by macrophages, HFD RBC more potently upregulated transcription of major pro-inflammatory chemokines Il-1b, Ccl3, and Cxcl2. As compared with chow RBC, exposure of murine aortic segments to HFD RBC under orbital shear conditions led to enhanced endothelial activation as evidenced by increased adhesion of labeled murine macrophages to the aortic endothelium (p&lt;0.05). Finally, when labeled and injected in vivo, HFD RBC exhibited a ~3 fold (p&lt;0.05) increase in splenic phagocytosis as compared with chow RBC. Conclusion: HFD profoundly impact RBC biology, inducing oxidative stress, altering membrane phospholipid display, and increasing chemokine release, phagocytosis by macrophages, and macrophage-blood vessel interactions. These novel findings suggest that RBC may play an important role in transducing vascular inflammation, and thus contributing to atherosclerosis, during HFD.

Novel Role for Bioactive Lipids in Mobilization of Bone Marrow Stem Cells During Myocardial Ischemia: Sphingosine-1 Phosphate (S1P) As Potential Therapeutic Target

Abstract 1911 Background:Bone marrow (BM) contains a variety of stem cells, including mobile pool of hematopoietic stem cells and non-hematopoietic stem cells and acute myocardial infarction (AMI) triggers mobilization of BM-derived stem cells (BMSCs) through poorly understood processes. Recently we have postulated a major role for bioactive lipids such as sphingosine-1 phosphate (S1P) in G-CSF- and AMD3100-induced mobilization of hematopoietic stem cells (HSCs) into peripheral blood (PB) (Leukemia 2010;24:976–85). Hypothesis:We hypothesized that S1P could also play a role in mobilization of BMSCs in patients during tissue and organ injury as seen for example in patients with AMI. Methods:Peripheral blood (PB) samples from matched controls and patients presenting with ST-elevation myocardial infarction (STEMI) were examined i) by employing FACS for a number of circulating in PB lineage negative (Lin-)/CD45-/CD34+ and CD133+ and CXCR4+ BMSCs, ii) PB level of S1P by employing mass spectrometry, iii) SDF-1 level by ELISA and finally iv) BMSCs were tested in Transwell migration assays for their chemotactic responsiveness to S1P gradient in plasma from STEMI patients. Results:Plasma S1P levels were highest within 6 hours of AMI presentation 0.31 ± 0.02 μM and declined to 0.14 ± 0.02 μM in controls (P < 0.05 for 6 hours vs. controls). The elevation of plasma S1P corresponded with complement cascade (CC) activation and higher levels of C5b-C9 membrane attack complex (MAC) complex in the plasma. The largest reservoir of S1P in whole blood was red blood cells (RBCs) and incubation of RBCs with activated complement initiated the release of S1P. Elevated plasma S1P levels was paralleled by early significant increase in circulating lineage negative (Lin-)/CD45-/CD34+, CD133+ and CXCR4+ BMSCs (P < 0.01 vs. controls). Plasma obtained from STEMI patients in the early phases following acute injury stimulated the migration of human BM-derived Lin-/CXCR4+ and Lin-/CD34+ BMSCs in chemotaxis assays (7–8-fold increase vs. vehicle, P < 0.05), this effect was blunted by charcoal stripping of the plasma (CSP) and was further inhibited by the specific S1P receptor type 1 (S1P1) antagonists W146 (10 μM) and VPC20139 (10 μM). At the same time to our surprise we did not observed significant chemotactic relevant changes in SDF-1 level in PB plasma. Conclusion:This is the first human study to suggest that elevated S1P level in PB early in the course of AMI mobilizes BMSCs. Furthermore, we demonstrate for the first time that the release of hematopoietic and non-hematopoietic BMSCs into PB correlates in AMI patients with activation of CC and generation of MAC, that may release S1P from erythrocytes and platelets. We postulate that based on these results future studies examining the therapeutic manipulation of S1P and its receptors for myocardial regeneration are warranted. Disclosures:No relevant conflicts of interest to declare.

Role of protein kinases of human red cell membrane in deformability and aggregation changes

The proteomic analysis has showed that red cell membrane contains several kinases and phosphatases. Therefore the aim of this study was to investigate the role of protein kinases of human red cell membrane in deformability and aggregation changes. Exposure of red blood cells (RBCs) to some chemical compounds led to change in the RBC microrheological properties. When forskolin (10 microM), an adenylyl cyclase (AC) and a protein kinase A (PKA) stimulator was added to RBC suspension, the RBC deformability (RBCD) was increased by 20% (p < 0.05). Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP (by 26%; p < 0.01). Red cell aggregation (RBCA) was significantly decreased under these conditions (p < 0.01). Markedly less changes of deformability was found after RBC incubation with protein kinase stimulator C (PKC)--phorbol 12-myristate 13-acetate (PMA). This drug reduced red cell aggregation only slightly. It was inhibited red cell tyrosine phosphotase activity by N-vanadat and was obtained a significant RBCD rise and RBCA lowering. The similar effect was found when cells were incubated with cisplatin as a tyrosine protein kinase (TPK) activator. It is important to note that a selective TPK inhibitor--lavendustin eliminated the above mention effects. On the whole the total data clearly show that the red cell aggregation and deformation changes were connected with an activation of the different intracellular signaling pathways.

The role of red blood cell adenylyl cyclase activation in changes of erythrocyte membrane microrheological properties

The ability of red blood cells (RBCs) for the reversible change of their shape under passing through capillaries in microcirculation mainly depends on membrane elasticity of these cells. Phosphorylation of some membrane proteins can result in the changes of microrheological red blood cell properties. Here we show a significant increase in RBC deformability (RBCD) after incubation with isoproterenol (10−6 M). Red blood cell aggregation (RBCA) decreased under these conditions only slightly. When forskolin (10 μM), an adenylyl cyclase (AC) stimulator, was added to the RBC suspension, RBCD increased significantly (p < 0.05). Some more changes of deformability were found after incubation of RBC with stable penetrating analog of cyclic adenosine phosphate (cAMP), dibutyryl-cAMP, (dB-cAMP, 50 μM) and after phosphodiesterase (PDE) activity inhibition with Vinpocetine, Rolipram, or IBMX. It was found that Gs-proteins inhibitor Clonidine and specific Gi-protein stimulator Mastaparan 7 increased both RBCD and RBCA. On the whole, the data clearly show that the RBC aggregation and deformation changes are related with activation of the different intracellular signaling pathways. We suppose that RBCD increase was mainly associated with activation of the adenylyl-cyclase-cAMP system.

Abstract 4448: Rational design of a novel nanocarrier for doxorubicin delivery in cancer treatment

Abstract Doxorubicin is a very important anti-cancer drug and is currently being used in the treatment of many hematologic malignancies and solid tumors. There is a great need to develope a more effective nanoformulation of doxorubicin that can overcome cardiotoxicity of doxorubicin, as well as the problems associated with Doxil (PEGylated liposomal doxorubicin): large particle size, therefore unable to penetrate deep into the tumor, foot and hand syndrome, and myelotoxicity. We have recently developed a novel biocompatible telodendrimer (PEG block dendritic oligomer of cholic acids), which can self-assemble into stable micelles (∼50 nm) in water when mixed with a hydrophobic drug, such as paclitaxel, with high loading capacity. However, the doxorubicin delivery using this formulation suffers from lower drug loading capacity, less stability and series hemolytic properties. We have optimized the structure of the telodendrimer via introducing the specific building blocks, which has strong interactions with doxorubicin through TT- TT interaction and the hydrogen bonding. The engineered telodendrimer showed better doxorubicin drug loading capacity (4 mg/mL) and greater physical stability (18 nm, stable &amp;gt;3 months) compared with the original formulation. Nanocarrier is nontoxic in cell culture even at &amp;gt;1 mg/mL concentration. The doxorubicin-loaded formulation exhibited the similar potency as the free doxorubicin and Doxil in killing of cancer cells in the cell culture. This engineered formulation after loaded with doxorubicin can completely avoid the hemolysis in the in vitro red blood cell incubation. The in vivo biodistribution and the in vivo antitumor efficacy of this novel DOX-nanotherapeutics will be further studied in the animal models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4448. doi:10.1158/1538-7445.AM2011-4448

Hydroxyurea stimulates the release of ATP from rabbit erythrocytes through an increase in calcium and nitric oxide production

Hydroxyurea, a proven therapy for sickle cell disease, is known to improve blood flow and reduce vaso-occlusive crises, although its exact mechanism of action is not clear. The objective of this study was to determine if hydroxyurea results in an increase of ATP release from the red blood cell (RBC) via the drug's ability to stimulate nitric oxide (NO) production in these cells. A system enabling the flow of RBCs through microbore tubing was used to investigate ATP release from the RBC. Incubation of rabbit RBCs (7% hct) with 50 μM hydroxyurea resulted in a significant increase in the release of ATP from these cells. This level of ATP release was not detected in the absence of flow. Studies also showed that increments in hydroxyurea and NO (from spermine NONOate) resulted in an initial increase in ATP release, followed by a decrease in this release at higher concentrations of hydroxyurea and the NO donor. Incubation with L-NAME abolished the effect of the hydroxyurea, suggesting that NO production by the RBC was involved. Indeed, in the presence of 50 μM hydroxyurea, the amount of total Ca 2+ measured (by atomic absorption spectroscopy) in a 7% solution of RBCs increased from 363 ± 47 ng/ml and 530 ± 52 ng/ml. Finally, EPR studies suggest that an increase in nitrosylated Hb in the RBC is only measured for those studies involving hydroxyurea and a Ca 2+-containing buffer.