Dietary supplementation with docosahexanoic acid (DHA) increases red blood cell membrane flexibility in mice with sickle cell disease.

Humans and mice with sickle cell disease (SCD) have rigid red blood cells (RBCs). Omega-3 fatty acids, such as docosahexanoic acid (DHA), may influence RBC deformability via incorporation into the RBC membrane. In this study, sickle cell (SS) mice were fed natural ingredient rodent diets supplemented with 3% DHA (DHA diet) or a control diet matched in total fat (CTRL diet). After 8weeks of feeding, we examined the RBCs for: 1) stiffness, as measured by atomic force microscopy; 2) deformability, as measured by ektacytometry; and 3) percent irreversibly sickled RBCs on peripheral blood smears. Using atomic force microscopy, it is found that stiffness is increased and deformability decreased in RBCs from SS mice fed CTRL diet compared to wild-type mice. In contrast, RBCs from SS mice fed DHA diet had markedly decreased stiffness and increased deformability compared to RBCs from SS mice fed CTRL diet. Furthermore, examination of peripheral blood smears revealed less irreversibly sickled RBCs in SS mice fed DHA diet as compared to CTRL diet. In summary, our findings indicate that DHA supplementation improves RBC flexibility and reduces irreversibly sickled cells by 40% in SS mice. These results point to potential therapeutic benefits of dietary omega-3 fatty acids in SCD.

Sickle cell disease increases high mobility group box 1: a novel mechanism of inflammation.

High mobility group box 1 (HMGB1) is a chromatin-binding protein that maintains DNA structure. On cellular activation or injury, HMGB1 is released from activated immune cells or necrotic tissues and acts as a damage-associated molecular pattern to activate Toll-like receptor 4 (TLR4). Little is known concerning HMGB1 release and TLR4 activity and their role in the pathology of inflammation of sickle cell disease (SCD). Circulating HMGB1 levels were increased in both humans and mice with SCD compared with controls. Furthermore, sickle plasma increased HMGB1-dependent TLR4 activity compared with control plasma. HMGB1 levels were further increased during acute sickling events (vasoocclusive crises in humans or hypoxia/reoxygenation injury in mice). Anti-HMGB1 neutralizing antibodies reduced the majority of sickle plasma-induced TLR4 activity both in vitro and in vivo. These findings show that HMGB1 is the major TLR4 ligand in SCD and likely plays a critical role in SCD-mediated inflammation.

A novel hemoglobin-binding peptide reduces cell-free hemoglobin in murine hemolytic anemia.

Hemolysis can saturate the hemoglobin (Hb)/heme scavenging system, resulting in increased circulating cell-free Hb (CF-Hb) in hereditary and acquired hemolytic disease. While recent studies have suggested a central role for intravascular hemolysis and CF-Hb in the development of vascular dysfunction, this concept has stimulated considerable debate. This highlights the importance of determining the contribution of CF-Hb to vascular complications associated with hemolysis. Therefore, a novel Hb-binding peptide was synthesized and linked to a small fragment of apolipoprotein E (amino acids 141-150) to facilitate endocytic clearance. Plasma clearance of hE-Hb-b10 displayed a rapid phase t(1/2) of 16 min and slow phase t(1/2) of 10 h, trafficking primarily through the liver. Peptide hE-Hb-B10 decreased CF-Hb in mice treated with phenylhydrazine, a model of acute hemolysis. Administration of hE-Hb-B10 also attenuated CF-Hb in two models of chronic hemolysis: Berkeley sickle cell disease (SS) mice and mice with severe hereditary spherocytosis (HS). The hemolytic rate was unaltered in either chronic hemolysis model, supporting the conclusion that hE-Hb-B10 promotes CF-Hb clearance without affecting erythrocyte lysis. Interestingly, hE-Hb-B10 also decreased plasma ALT activity in SS and HS mice. Although acetylcholine-mediated facialis artery vasodilation was not improved by hE-Hb-B10 treatment, the peptide shifted vascular response in favor of NO-dependent vasodilation in SS mice. Taken together, these data demonstrate that hE-Hb-B10 decreases CF-Hb with a concomitant reduction in liver injury and changes in vascular response. Therefore, hE-Hb-B10 can be used to investigate the different roles of CF-Hb in hemolytic pathology and may have therapeutic benefit in the treatment of CF-Hb-mediated tissue damage.

Methaemalbumin formation in sickle cell disease: effect on oxidative protein modification and HO-1 induction.

Normally, cell free haemoglobin is bound by haptoglobin and efficiently cleared. However, the chronic haemolysis in sickle cell disease (SCD) overwhelms haptoglobin binding capacity and protein turnover, resulting in elevated cell free haemoglobin. Cell free haemoglobin acts as both a scavenger of vasoactive nitric oxide and a pro-oxidant. In addition, methaemoglobin (metHb) releases the haem moiety, which can bind to albumin to form methaemalbumin (metHSA). This study used electron paramagnetic resonance to detect metHSA in SCD plasma and demonstrated that haptoglobin prevents haem transfer from metHb to HSA. MetHSA may either provide a second line of defence against haemoglobin/haem-mediated oxidation or contribute to the pro-oxidant environment of SCD plasma. We demonstrated that HSA inhibited oxidative protein modification induced by metHb. Additionally, we showed that while metHb induced haem oxygenase 1 (HO-1), an indicator of oxidative stress, HSA attenuated metHb induction of this enzyme, thereby limiting the potential benefits of HO-1. Furthermore, HO-1 induction by metHSA was less than HO-1 induction by equimolar metHb not bound to albumin. Our findings confirm the presence of metHSA in SCD and suggest that haem transfer from metHb to HSA reduces the oxidative effects of free haemoglobin/haem on endothelium with both beneficial (reduced protein oxidation) and potentially harmful (reduced HO-1 induction) outcomes.

Regulation of cAMP by phosphodiesterases in erythrocytes.

The erythrocyte, a cell responsible for carrying and delivering oxygen in the body, has often been regarded as simply a vehicle for the circulation of hemoglobin. However, it has become evident that this cell also participates in the regulation of vascular caliber in the microcirculation via release of the potent vasodilator, adenosine triphosphate (ATP). The regulated release of ATP from erythrocytes occurs via a defined signaling pathway and requires increases in cyclic 3',5'- adenosine monophosphate (cAMP). It is well recognized that cAMP is a critical second messenger in diverse signaling pathways. In all cells increases in cAMP are localized and regulated by the activity of phosphodiesterases (PDEs). In erythrocytes activation of either beta adrenergic receptors (beta(2)AR) or the prostacyclin receptor (IPR) results in increases in cAMP and ATP release. Receptor-mediated increases in cAMP are tightly regulated by distinct PDEs associated with each signaling pathway as shown by the finding that selective inhibitors of the PDEs localized to each pathway potentiate both increases in cAMP and ATP release. Here we review the profile of PDEs identified in erythrocytes, their association with specific signaling pathways and their role in the regulation of ATP release from these cells. Understanding the contribution of PDEs to the control of ATP release from erythrocytes identifies this cell as a potential target for the development of drugs for the treatment of vascular disease.

Insulin inhibits human erythrocyte cAMP accumulation and ATP release: role of phosphodiesterase 3 and phosphoinositide 3-kinase.

In non-erythroid cells, insulin stimulates a signal transduction pathway that results in the activation of phosphoinositide 3-kinase (PI3K) and subsequent phosphorylation of phosphodiesterase 3 (PDE3). Erythrocytes possess insulin receptors, PI3K and PDE3B. These cells release adenosine triphosphate (ATP) when exposed to reduced O(2) tension via a signaling pathway that requires activation of the G protein, Gi, as well as increases in cAMP. Although insulin inhibits ATP release from human erythrocytes in response to Gi activation by mastoparan 7 (Mas 7), no effect on cAMP was described. Here, we investigated the hypothesis that insulin activates PDE3 in human erythrocytes via a PI3K-mediated mechanism resulting in cAMP hydrolysis and inhibition of ATP release. Incubation of human erythrocytes with Mas 7 resulted in a 62 +/- 7% increase in cAMP (n = 9, P < 0.05) and a 306 +/- 69% increase in ATP release (n = 9, P < 0.05), both of which were attenuated by pre-treatment with insulin. Selective inhibitors of PDE3 (cilostazol) or PI3K (LY294002) rescued these effects of insulin. These results support the hypothesis that insulin activates PDE3 in erythrocytes via a PI3K-dependent mechanism. Once activated, PDE3 limits Mas 7-induced increases in intracellular cAMP. This effect of insulin leads, ultimately, to decreased ATP release in response to Mas 7. Activation of Gi is required for reduced O(2) tension-induced ATP release from erythrocytes and this ATP release has been shown to participate in the matching of O(2) supply with demand in skeletal muscle. Thus, pathological increases in circulating insulin could, via activation of PDE3 in erythrocytes, inhibit ATP release from these cells, depriving the peripheral circulation of one mechanism that could aid in the regulation of the delivery of O(2) to meet tissue metabolic need.

Insulin inhibits low oxygen-induced ATP release from human erythrocytes: implication for vascular control.

OBJECTIVE: ATP released from human erythrocytes in response to reduced oxygen tension (pO(2)) participates in the matching of oxygen (O(2)) supply with need in skeletal muscle by stimulating increases in blood flow to areas with increased O(2) demand. Here, we investigated the hypothesis that hyperinsulinemia inhibits ATP release from erythrocytes and impairs their ability to stimulate dilation of isolated arterioles exposed to decreased extraluminal pO(2). MATERIALS AND METHODS: Erythrocyte ATP release was stimulated pharmacologically (mastoparan 7) and physiologically (reduced pO(2)) in the absence or presence of insulin. We also examined the ability of isolated skeletal muscle arterioles perfused with buffer containing erythrocytes treated with insulin or its vehicle (saline) to dilate in response to decreased extraluminal pO(2). RESULTS: Insulin significantly attenuated mastoparan 7- and reduced pO(2)-induced ATP release. In vessels perfused with untreated erythrocytes, low extraluminal pO(2) resulted in an increase in vessel diameter. In contrast, when erythrocytes were treated with insulin, no vasodilation occurred. CONCLUSIONS: These studies demonstrate that insulin inhibits ATP release from erythrocytes in response to reduced pO(2) and impairs their ability to stimulate dilation of skeletal muscle arterioles. These results suggest that hyperinsulinemia could hinder the matching of O(2) supply with need in skeletal muscle.

Iloprost- and isoproterenol-induced increases in cAMP are regulated by different phosphodiesterases in erythrocytes of both rabbits and humans.

Activation of the G protein G(s) results in increases in cAMP, a necessary step in the pathway for ATP release from rabbit and human erythrocytes. In all cells, the level of cAMP is the product of its synthesis by adenylyl cyclase and its hydrolysis by phosphodiesterases (PDEs). Both iloprost (Ilo), a PGI(2) analog, and isoproterenol (Iso), a beta-agonist, stimulate receptor-mediated increases in cAMP in rabbit and human erythrocytes. However, the specific PDEs associated with each of these signaling pathways in the erythrocyte have not been fully characterized. Previously, we reported that PDE3B is present in rabbit and human erythrocyte membranes and that PDE3 inhibitors potentiate Ilo-induced increases in cAMP. Here we report that inhibitors of either PDE2 or PDE4, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and rolipram, respectively, potentiate Iso-induced increases in cAMP in rabbit and human erythrocytes. Importantly, these inhibitors had no effect on cAMP increases associated with the incubation of erythrocytes with Ilo. In addition, we establish, for the first time, the presence of PDE2A protein in rabbit and human erythrocyte membranes. Finally, we determined that preincubation of human erythrocytes with EHNA and rolipram together potentiate Iso-induced ATP release, whereas preincubation with cilostazol enhances Ilo-induced release of ATP. These results are consistent with the hypothesis that, in rabbit and human erythrocytes, Ilo-induced increases in cAMP and ATP release are regulated by PDE3, whereas those associated with Iso are regulated by the activities of both PDE2 and PDE4. These studies demonstrate that PDE activity in these cells is localized to specific signaling pathways.

Rabbit erythrocytes release ATP and dilate skeletal muscle arterioles in the presence of reduced oxygen tension.

In skeletal muscle, oxygen (O(2)) delivery to appropriately meet metabolic need requires mechanisms for detection of the magnitude of O(2) demand and the regulation of O(2) delivery. Erythrocytes, when exposed to a decrease in O(2) tension, release both O(2) and the vasodilator adenosine triphosphate (ATP). The aims of this study were to establish that erythrocytes release ATP in response to reduced O(2) tension and determine if erythrocytes are necessary for the dilation of isolated skeletal muscle arterioles exposed to reduced extraluminal O(2) tension. Rabbit erythrocytes exposed to reduced O(2) tension in a tonometer (n = 5, pO(2) = 27 +/- 3, p < 0.01) released ATP in response to reduced O(2) tension. ATP release increased in proportion to the decrease in O(2) tension. The contribution of erythrocytes to the response of skeletal muscle arterioles to reduced extraluminal O(2) tension was determined using isolated hamster cheek pouch retractor muscle arterioles perfused with buffer (n = 11, mean diameter 52 +/- 3 mum) in the absence and presence of rabbit erythrocytes. Without erythrocytes, arterioles did not dilate when exposed to reduced extraluminal O(2) tension (pO(2) = 32 +/- 4 mmHg). In contrast, when rabbit erythrocytes were present in the perfusate (hematocrit 15%), the same decrease in O(2) tension resulted in a 20 +/- 4% dilation (p < 0.01). These results provide support for the hypothesis that erythrocytes, via their ability to release O(2) along with ATP in response to exposure to reduced O(2) tension, can participate in the matching of O(2) delivery with metabolic need in skeletal muscle.

Phosphodiesterase 3 is present in rabbit and human erythrocytes and its inhibition potentiates iloprost-induced increases in cAMP.

Increases in the second messenger cAMP are associated with receptor-mediated ATP release from erythrocytes. In other signaling pathways, cAMP-specific phosphodiesterases (PDEs) hydrolyze this second messenger and thereby limit its biological actions. Although rabbit and human erythrocytes possess adenylyl cyclase and synthesize cAMP, their PDE activity is poorly characterized. It was reported previously that the prostacyclin analog iloprost stimulated receptor-mediated increases in cAMP in rabbit and human erythrocytes. However, the PDEs that hydrolyze erythrocyte cAMP synthesized in response to iloprost were not identified. PDE3 inhibitors were reported to augment increases in cAMP stimulated by prostacyclin analogs in platelets and pulmonary artery smooth muscle cells. Additionally, PDE3 activity was identified in embryonic avian erythrocytes, but the presence of this PDE in mammalian erythrocytes has not been investigated. Here, using Western blot analysis, we determined that PDE3B is a component of rabbit and human erythrocyte membranes. In addition, we report that the preincubation of rabbit and human erythrocytes with the PDE3 inhibitors milrinone and cilostazol potentiates iloprost-induced increases in cAMP. In addition, cilostamide, the parent compound of cilostazol, potentiated iloprost-induced increases in cAMP in human erythrocytes. These findings demonstrate that PDE3B is present in rabbit and human erythrocytes and are consistent with the hypothesis that PDE3 activity regulates cAMP levels associated with a signaling pathway activated by iloprost in these cells.

Prostacyclin analogs stimulate receptor-mediated cAMP synthesis and ATP release from rabbit and human erythrocytes.

OBJECTIVES: The purpose of this study was to establish that the prostacyclin (PGI(2)) receptor (IP receptor) is present on rabbit and human erythrocytes and that its activation stimulates cyclic adenosine monophosphate (cAMP) synthesis and adenosine triphosphate (ATP) release. METHODS: The effect of incubation of erythrocytes with the active PGI(2) analogs, iloprost or UT-15C, on cAMP levels and ATP release was determined in the absence and presence of the IP receptor antagonist, CAY10441. Western analysis was used to determine the presence of the IP receptor on isolated membranes. To establish that effects of PGI(2) analogs were not due to prostaglandin E(2)(PGE(2)) receptor activation, the effect of PGE(2) on cAMP levels and ATP release was determined. RESULTS: Rabbit and human erythrocytes possess IP receptors. Iloprost and UT-15C stimulated increases in cAMP and ATP release that were prevented by the IP receptor antagonist, CAY10441. PGE(2) did not stimulate cAMP accumulation or ATP release and did not inhibit iloprost-induced increases in cAMP. CONCLUSIONS: This study establishes that the IP receptor is present on rabbit and human erythrocytes and that its activation results in increases in cAMP and ATP release. These results suggest a novel mechanism by which PGI(2) and its active analogs, when administered pharmacologically, could produce vasodilation.