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Am J Physiol Cell Physiol 293: C960-C966, 2007. First published May 9, 2007; doi:10.1152/ajpcell.00530.2006
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RECEPTORS AND SIGNAL TRANSDUCTION

Reduced sickle erythrocyte dehydration in vivo by endothelin-1 receptor antagonists

Alicia Rivera

Department of Laboratory Medicine, Children's Hospital Boston, and Department of Pathology, Harvard Medical School, Boston, Massachusetts

Submitted 13 October 2006 ; accepted in final form 6 May 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Elevated plasma levels of cytokines such as endothelin-1 (ET-1) have been shown to be associated with sickle cell disease (SCD). However, the role of ET-1 in the pathophysiology of SCD is not entirely clear. I now show that treatment of SAD mice, a transgenic mouse model of SCD, with BQ-788 (0.33 mg·kg–1·day–1 intraperitoneally for 14 days), an ET-1 receptor B (ETB) antagonist, induced a significant decrease in Gardos channel activity (1.7 ± 0.1 to 1.0 ± 0.4 mmol·1013 cell–1·h–1, n = 3, P = 0.019) and reduced the erythrocyte density profile by decreasing the mean density (D50; n = 4, P = 0.012). These effects were not observed in mice treated with BQ-123, an ET-1 receptor A (ETA) antagonist. A mixture of both antagonists induced a similar change in density profile as with BQ-788 alone that was associated with an increase in mean cellular volume and a decrease in corpuscular hemoglobin concentration mean. I also observed in vitro effects of ET-1 on human sickle erythrocyte dehydration that was blocked by BQ-788 and a mixture of ETB/ETA antagonists but not by ETA antagonist alone. These results show that erythrocyte hydration status in vivo is mediated via activation of the ETB receptor, leading to Gardos channel modulation in SCD.

cellular dehydration; Gardos channel; transgenic sickle mice

ACUTE CHEST SYNDROME AND STROKE are the leading causes of morbidity and mortality among sickle cell disease (SCD) patients. One major clinical manifestation of this disease is the onset of recurrent episodes of severe pain called vaso-occlusive crises. These crises are caused by the occlusion within blood vessels of dehydrated sickle erythrocytes and the ischemia that follows (18). Epidemiological data indicate that 5% of the patients with SCD experience 3–10 crises a year (24, 25). It is believed that crises are initiated by the interaction among the endothelium, sickle cells, and plasma factors (33).

Patients with SCD have been shown to have elevated levels of cytokines such as ET-1 (16, 31), transforming growth factor-beta, stem cell factor, soluble transferrin receptor (7), IL-8 (11), and platelet-activating factor (PAF) (20). These vasoactive molecules control a variety of physiological processes, which are mediated by cell surface receptors, reactive oxygen species, H2O2, and nitric oxide. However, the mechanisms for their interaction with the vasoactive molecules and their pathophysiological roles in SCD are not completely understood. Nonetheless, it is believed that they play an important role in the adhesion of sickle erythrocytes to the endothelium and in the pathogenesis of vaso-occlusive crises (2, 7).

Sickle erythrocytes have been shown to interact with vascular endothelial cells, stimulating the release of ET-1 and regulating the expression of the ET-1 gene in cultured endothelial cells (17). My coworkers and I have recently shown that the Gardos channel is coupled to ET-1, C-X-C (cytokines), and C-C (chemokines) receptors in both human (27) and mouse sickle erythrocytes (28). ET-1 acts via two major receptor isoforms, ET-1 receptor A (ETA) and B (ETB). The ETA receptors are predominantly present in vascular smooth muscle cells mediating vasoconstriction, whereas ETB receptors are mostly located in endothelial cells mediating vasodilatation. Both normal and sickle erythrocytes express ETB receptors (27). In addition, my group has shown that ET-1 enhances the formation of dense cells in vitro. However, it is not known whether ET-1 can modulate Gardos channel activity and modulate erythrocyte cellular volume in vivo. It was hypothesized that blockade of ET-1 receptors in vivo would reduce formation of dehydrated sickle erythrocytes by decreasing Gardos channel activity in SCD.

In this study the in vivo role of ET-1 in sickle cell dehydration in a transgenic sickle mouse model was investigated using ET-1 receptor antagonists. It was observed that ET-1 receptor antagonists in vivo significantly decreased corpuscular hemoglobin concentration mean (CHCM) and the percentage of dense cells while increasing mean cellular volume (MCV) and decreasing Gardos channel activity. These results provide evidence that ET-1 receptor antagonists represent a novel target for the development of new therapeutic strategies to ameliorate the formation of sickle erythrocytes in SCD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Drugs and Chemicals Charybdotoxin (ChTX), ET-1, calphostin C, BQ-788 (selective ETB receptor antagonist), and BQ-123 (selective ETA receptor antagonist) were purchased from Sigma Chemical (St. Louis, MO). All peptides were prepared as indicated by the manufacturer and stored at –20°C for less than 2 mo. The A-23187 ionophore was purchased from Calbiochem (La Jolla, CA). 86Rb was purchased from PerkinElmer Life Sciences (Boston, MA). All other reagents were purchased from Sigma Chemical.

Animals SAD1 mice (8–10 mo old) were kindly provided by Dr. Seth Alper (Beth Israel Hospital and Harvard Medical School, Boston, MA). These studies were part of the research protocol A04-09-131R, which was submitted to and approved by the Children's Hospital Animal Care and Use Committee. Animals were fed standard mouse chow and given water ad libitum during the treatment. After treatment, animals were disposed of according to the animal handling protocols and regulations of the Children's Hospital Boston and Harvard Medical School.

Treatment of Mice With ET-1 Antagonists Transgenic SAD sickle cell mice have been widely used by various independent groups for ion transport and cellular dehydration studies (8, 10). Transgenic SAD sickle cell mice were placed on a regime for 14 days with ET-1 antagonist as follows: mice were divided into four groups of four animals per group and intraperitoneally injected for 14 consecutive days. ET-1 antagonists were diluted in sterile normal saline. SAD mice in the group A received sterile saline alone (0.1 ml). SAD mice in group B received 0.1 ml of a 0.1 mg/ml stock solution of the ETA antagonist BQ-123 (0.33 mg/kg). SAD mice in group C received the ETB antagonist BQ-788 (0.1 ml of 0.1 mg/ml stock). The mice in group D received 0.1 ml of mixed BQ-123 (0.2 mg/ml) and BQ-788 (0.2 mg/ml) dissolved into 1 ml of saline. At day 15, mice were killed, and whole blood was immediately collected into heparinized tubes for further experimentation.

ADVIA Hematological Parameters Cell blood counts were determined using the ADVIA automated hematology analyzer (Bayer Diagnostics, Tarrytown, NY). Freshly isolated whole blood was collected in heparin-containing tubes, and an aliquot of 250 µl was used to perform the erythrocyte and reticulocyte counts and white blood differential for each sample by using a software program specific for mouse blood.

Measurement of Whole Blood Gardos Channel Activity Freshly isolated blood collected from treated animals was used to determine the Gardos channel activity in whole blood as previously described in detail (28). Briefly, the whole blood sample was mixed with Tris-MOPS, pH 8.0, at 25°C (final concentration, 20 mM), 1 mM ouabain, and 10 µM bumetanide in three separate Eppendorf microtubes. A-23187 was added at a final concentration of 6 µmol per liter of cells, and the cell suspension was incubated for 1 h at room temperature. 86Rb (10 µCi/ml) was then added to each tube at time 0. A sample of 100 µl was removed at 1, 3, and 5 min in duplicate and transferred to a 1.5-ml Eppendorf tube containing 0.3 ml of phthalate oil and 0.8 ml of normal saline with 5 mM EGTA. Samples were immediately spun down, and the cell pellet was counted for radioactivity. The suspension remaining after 3 min was spun down, and the supernatant was used to determine total specific activity.

Phthalate Density Profile Density distribution curves were obtained using phthalate esters in microhematocrit tubes as previously described in detail by Kurantsin-Mills et al. (19). Briefly, phthalate solutions were prepared to give a range of densities between 1.08 and 1.11 g/ml. The hematocrit tubes were filled with 30 µl of whole blood or cell suspension and 10 µl of different phthalate solutions. Tubes were centrifuged at 12,200 rpm for 10 min at room temperature in a temperature-controlled microcentrifuge. The amount of dense cells was calculated from the total cell content below the oil layer (lower layer) divided by the total amount of cells and expressed as a percentage as shown previously by my group (27). The data are presented as percentages of dense cells versus phthalate oil densities and the connecting data points unless otherwise stated. The best-fit sigmoidal curve analysis using SigmaPlot 9.0 graphic software for Windows was done in each individual experimental curve for each condition to assess the statistical difference between treatments. The phthalate oil density at 50% (D50) is the phthalate oil density that divides the cell population into two equal parts. This value is used to determine alterations in the cellular density profile of the entire red cell population (27).

Cyclic Deoxygenation-Oxygenation Experiments In Vitro Blood was collected into tubes containing heparin and prepared as described in detail previously (27). Briefly, freshly isolated human erythrocytes were incubated in a plasmalike buffer containing (mM) 145 NaCl, 2 KCl, 25 NaHCO3, 10 glucose, 0.06 adenosine, 0.04 inosine, 0.15 MgCl2, and 2 CaCl2 for 3 h (30% hematocrit) under a 10-min oxygenation-deoxygenation cycle. Each cycle provided 3 min of 15% O2-5% CO2 balanced with N2 and 7 min of 5% CO2 balanced with N2 gas. The gases were humidified by bubbling in a column that contained an isotonic saline solution at 37°C. The cell suspension was then transferred to an ice bath. Aliquots were obtained at different time points to evaluate the changes in gas levels during the 10-min cycles. Gases were measured using a Co-oximeter (model 845; Ciba-Corning, Medfield, MA).

Erythrocyte Fractionation Freshly isolated erythrocytes were washed three times with normal saline and suspended at 10% in the same solution. Isotonic Stractan (arabinogalactan) solutions at different densities were prepared as described by Corash (6). Densities were prepared between 1.077 and 1.101 g/dl and carefully layered (1.0 ml of each density) in a 12-ml polypropylene tube. Cell suspensions (1 ml) were layered on top of the gradient and placed in an ultracentrifuge (Sorvall RC-28S) for 45 min at 72,000 g (8°C) in a swinging bucket rotor. Cells were divided into three fractions (top, middle, and bottom). The CHCM was measured using an aliquot of each cell fraction in an ADVIA 120 hematology analyzer.

Statistical Analysis All values are means ± SE. When applicable, nonpaired t-test was used to calculate P values.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In Vivo Studies Effects of ET-1 receptor antagonists on mouse hematological parameters. The in vivo effects of ET-1 receptor antagonists were studied in SAD transgenic mice. The hematological parameters after 14 days of administration of ET-1 receptor antagonists are shown in Table 1. MCV was significantly increased after treatment with a mixture of the ET-1 receptor antagonists BQ-123 and BQ-788. This change was accompanied by a decrease in CHCM and an increase in the percentage of hypochromic cells. In reticulocytes, similar effects were observed in MCV and CHCM values. These results suggest that ET receptor activation may play an important role in the regulation of cellular volume and hemoglobin concentration in sickle mice.


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  		Table 1. In vivo effects of 14 days of treatment with ET-1 receptor antagonists on hematological parameters in SAD mice

 
ET-1 receptor antagonist affects erythrocyte dehydration in vivo in sickle erythrocytes. Treatment with the ETA receptor antagonist BQ-123 did not significantly affect the density of the sickle erythrocytes (data not shown). However, BQ-788, an ETB receptor antagonist, induced a significant change in the density profile (D50 from 1.1052 ± 0.0007 to 1.1033 ± 0.0006, n = 4, P = 0.012), especially among the younger cell population (density <1.101 g/ml, Fig. 1). These results strongly support the role of ETB receptors in sickle cell dehydration. However, no significant differences in MCV and CHCM in whole blood from animals treated with the antagonist for ETB receptor alone were observed. This suggests that the effects of the antagonist might be directed at the young cell population, which was masked in whole cell measurements (Table 1). A mixture of both antagonists (BQ-123/BQ-788) in equal amounts induced a significant reduction of dense cell formation, changing the D50 from 1.1058 ± 0.0007 to 1.1047 ± 0.0002 (P = 0.034, n = 4) in vivo (Fig. 1). These data suggest that by blocking both receptors, a reduction in cellular dehydration can be induced in vivo. These results are in agreement with the MCV and CHCM changes observed under similar conditions (Table 1).


Figure 1
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  		Fig. 1. Effects of ET-1 receptor antagonists in the erythrocyte density profile in vivo. Transgenic SAD sickle cell mice were injected intraperitoneally with saline (bullet), BQ-788 ({triangleup}), or a mixture of BQ-123/BQ-788 receptor antagonists ({circ}) for 14 days. Mice were killed at day 15, and an erythrocyte density profile was determined using the phthalate method in heparinized whole blood. Data are means ± SE of 4 experimental determinations (n = 4 mice).

 
Gardos channel activity reduced by ET-1 receptor antagonists in vivo. The channel activity was measured in ex vivo heparinized whole blood to preserve the conditions in circulation of SAD mouse erythrocytes. Figure 2 shows that in SAD mice, treatment with BQ-123 antagonist did not significantly change the channel activity, which is in agreement with the lack of an effect on cellular dehydration. However, BQ-788 antagonist treatment significantly reduced the activity of the channel (from 1.7 ± 0.1 to 1.0 ± 0.4 mmol·1013 cells–1·min–1, n = 4, P = 0.035). Treatment with a mixture of both antagonists produced an even greater inhibition on the channel activity, which was consistent with the changes observed in the density profile (Fig. 1). Together these results strongly support the hypothesis that the ET-1 receptor antagonists are changing cellular volume via Gardos channel inhibition in vivo.


Figure 2
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  		Fig. 2. Effects of ET-1 receptor antagonists on the charybdotoxin (ChTX)-sensitive K+ influx (Gardos channel activity) in sickle erythrocytes in vivo. Whole blood was collected after 14 days of treatment with and without BQ-123, BQ-788, or a mixture of both in heparin-containing tubes. Gardos channel activity was measured in the presence or absence of 50 nM ChTX as described in MATERIALS AND METHODS. *P = 0.035; **P = 0.019.

 
In Vitro Studies Cellular dehydration via ET-1 receptor activation. To investigate the pathways by which activation of the ET-1 receptors induces cellular dehydration, freshly isolated sickle erythrocytes were exposed to deoxygenation-oxygenation cycles for 3 h. This procedure has been well described by my group (27) and others (17a, 32a) to induce significant sickle erythrocyte dehydration. Aliquots were removed and gases were determined. Figure 3 shows that during the 10-min cycle, the pH and PCO2 gas levels did not significantly change. Variation of PO2 was observed as expected when the system was switched from O2 to N2. A concomitant increase in the PO2 and O2 saturation (SO2) was observed during those 3 min of oxygenation. After 3 min, PO2 and SO2 started to decrease as the O2 gas was switched off, demonstrating the cycling pattern during the deoxygenation-oxygenation cycle protocol. Under these experimental conditions, erythrocytes from SCD patients were incubated with 500 nM ET-1 in the presence or absence of 1 µM of either ETB (BQ-788) or ETA antagonist (BQ-123) during deoxygenation-oxygenation cycles. The presence of 1 µM BQ-788 induced a leftward shift of the red cell density profile in the presence of 500 nM ET-1 (D50 from 1.1027 to 1.098; data not shown). Figure 4 shows that ET-1-induced cellular dehydration was blocked by BQ-788 and a mixture of ETB and ETA antagonists but not by ETA antagonist alone. These data support the contention that ETB receptors are involved in cellular dehydration in sickle erythrocytes and are consistent with the in vivo findings in mice.


Figure 3
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  		Fig. 3. Cyclic deoxygenation-oxygenation pattern in erythrocytes. Freshly isolated human erythrocytes were incubated in plasmalike buffer containing 60 µM adenine, 40 µM inosine, and 1.5 mM CaCl2 for 3 h at 37°C. Aliquots were removed at time points indicated, and pH, PO2, O2 saturation (SO2), and PCO2 were immediately measured using a co-oximeter gas blood analyzer. Oxygenated phase (3 min): 15% O2, 5% CO2, 80% N2; deoxygenated phase (7 min): 95% N2, 5% CO2. Graph shows the values for a representative experiment that was repeated in 3 different samples.

 

Figure 4
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  		Fig. 4. Effects of ET-1 antagonists on sickle cell dehydration during deoxygenation-oxygenation conditions. Freshly isolated human sickle erythrocytes were incubated with and without 500 nM ET-1 and 1 µM ET-1 receptor antagonists for B (BQ-788) or A (BQ-123) subtype. The suspension was incubated for 3 h in deoxygenation-oxygenation cycles as described in MATERIALS AND METHODS. The density profile was measured using the phthalate method (oil density 1.092 g/ml) in whole blood as described in MATERIALS AND METHODS. Data are means ± SE of 3 separate experiments. **P < 0.02 compared with control. {alpha}P < 0.024 compared with ET-1 alone.

 
To study the effect of deoxygenation-oxygenation-stimulated ET-1 cellular dehydration on Gardos channel activity, sickle erythrocytes were incubated with ET-1 in the presence or absence of 10 nM ChTX or 10 µM clotrimazole (CTL) during 3 h of deoxy-oxygenation cycles (Fig. 5). Both ChTX and CLT are well-described inhibitors of the Gardos channel. ET-1-induced cellular dehydration was inhibited in the presence of either ChTX or CLT, indicating that ET-1-induced cellular dehydration is mediated by the activation of the Gardos channel under deoxygenation-oxygenation conditions. These results support the hypothesis that in vivo, the Gardos channel plays an important role in cellular dehydration.


Figure 5
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  		Fig. 5. Effect of Gardos channel blockers on ET-1-stimulated cell dehydration in sickle erythrocytes. Phthalate oil density profile (oil density 1.092 g/ml) was performed after incubation of sickle erythrocytes in the presence or absence of 500 nM ET-1 and 10 nM ChTX or 10 µM clotrimazole (CLT). The suspensions were kept for 3 h in deoxygenation-oxygenation conditions as detailed in MATERIALS AND METHODS. Data are means ± SE of 3 similar experiments. B, baseline; C, control. **P < 0.031 compared with control. {alpha},betaP < 0.021 compared with ET-1 alone.

 
Protein kinase C (PKC) has been reported by my group (28) and others (14) to activate the Gardos channel in erythrocytes. Furthermore, activation of PKC activity is seen upon activation of ET-1 receptors. To test whether PKC plays a role in ET-1-induced cellular dehydration, red blood cells were incubated with ET-1 in the presence or absence of 1 µM calphostin C (CC) under deoxygenation-oxygenation conditions for 3 h (Fig. 6). Similar experiments were performed with 1 µM chelerythrine, a structurally distinct PKC inhibitor. Inhibition of PKC activity completely blocked the ET-1-induced cell dehydration in sickle erythrocytes, suggesting that PKC activity is involved in cellular dehydration and dense cell formation in deoxygenation-oxygenation conditions.


Figure 6
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  		Fig. 6. Effects of PKC blocker on ET-1-induced sickle cell dehydration in vitro. Freshly isolated sickle erythrocytes were incubated in a plasmalike buffer in the presence or absence 500 nM ET-1 with and without 1 µM calphostin C (CC) for 3 h in oxygenation-deoxygenation conditions. Density profile was performed immediately afterward as described in detail in MATERIALS AND METHODS. Graph represents 1 of 3 similar experiments (n = 3).

 
ET-1 receptor activation is hyperresponsive in discocytes from sickle cell anemia subjects. It has been described that a subpopulation of erythrocytes, discocytes, have the tendency to dehydrate at a higher rate than other subpopulations, leading to increased rates of hemoglobin S polymerization in SCD patients (13). It seems that this fraction of cells is responsible for a fast rate of dense erythrocyte formation in sickle patients. To investigate whether these populations of cells are more sensitive to the presence of elevated concentrations of ET-1, dense cell formation was measured in three subsets of erythrocytes separated by their hemoglobin concentration and migration in a Stractan gradient as described in MATERIALS AND METHODS. Figure 7 shows ET-1-induced dense cell formation in the top and middle fractions but not in the bottom fraction. Since the top fractions contain light and young cells, these results suggest that the fast-dehydrated erythrocyte fraction may be responsible for the dense cell formation in the presence of ET-1 in vivo.


Figure 7
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  		Fig. 7. Effect of ET-1 on cell dehydration at different sickle erythrocyte cell fractions under deoxygenation-oxygenation cycles. Freshly isolated red cells were fractionated according to their buoyant density as described in MATERIALS AND METHODS. Isolated fractions were incubated in the presence or absence of 500 nM ET-1 for 3 h in deoxygenation-oxygenation cycles. Cell density was measured using the phthalate method at a density of 1.090 with a baseline of 45.8%. Values are means ± SE of 3 determinations. *P < 0.035; **P < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The physiological role of chemokine and cytokine receptors in erythrocyte function is unclear. A role of chemokine receptors in the clearance of chemokines from the circulation has been proposed (35). This receptor is identical to the Duffy antigen, which also functions as a receptor for Plasmodium vivax (21). My coworkers and I (27) recently reported that the Duffy antigen is involved in erythrocyte volume regulation, since RANTES (regulated on activation normal T-expressed and presumably secreted) and PAF significantly modulated the kinetic parameters of the Ca2+-activated K+ channel (Gardos channel) activity. In this study, the in vivo role of the cytokine receptor for ET-1 in promoting cellular dehydration was evaluated in a transgenic mouse model of SCD. Reduction of cellular dehydration and dense cell formation was shown in sickle cell mice treated with ET-1 receptor antagonists. These results support and extend previous studies showing increased channel activity in vitro in the presence of exogenous ET-1 (27). In addition, these in vitro studies now show that the mechanism for this reduction in cellular dehydration is via activation of the Gardos channel through ETB receptor stimulation and a PKC-dependent mechanism. Together, these results suggest an in vivo coupling of sickle cell formation and ETB receptor activation.

A role for ET-1 levels in SCD pathophysiology has been previously suggested. It is possible that suppression of ET-1 expression may lead to changes in erythrocyte volume and, hence, hemoglobin S polymerization in vivo. Consistent with this hypothesis, ET-1 levels, erythrocyte dehydration, and Gardos channel activity are decreased when sickle transgenic animals are fed a diet supplemented with arginine (29). These data suggest that arginine diet supplementation interferes with ET-1 expression and/or release in sickle cell transgenic mice and support the hypothesis that modulation of ET-1 levels is associated with cellular hydration status.

ET-1-induced Gardos channel activity was significantly inhibited when cells were incubated in the presence of the ETB receptor antagonist but not the ETA antagonist (27). However, a mixture of both ETA and ETB antagonist in vivo showed a further decrease in the activity of the Gardos channel from that observed with ETB alone, suggesting that both receptors are involved in the regulation of dense erythrocyte formation. It is possible that the presence of ETA antagonist may enhance the specific effects of the ETB antagonist. This proposal is supported by previous observations showing that a PCR product corresponding to ETB and not ETA was identified in human erythroid precursor cDNA with the use of primers for ETB (27). Alternatively, the presence of ETA receptor antagonists may induce a decrease in the vessel tone that reduces sheer stress of the sickle erythrocytes, leading to reduced local production of endogenous ET-1 or/and activation of the endothelium. This effect may lead to the reduction in the overall levels of ET-1 and/or other cytokines in the circulation.

The role of hypoxia in SCD has been well established, but the contribution of cytokines to hypoxia-mediated cellular dehydration is not clear. Hypoxia has been shown to increase ET-1 levels in vivo (23). ET-1 induces an increase in cellular dehydration that leads to enhanced dense cell formation under hypoxic conditions in vitro. It is possible that in sickle transgenic mice, hypoxia-induced dense cell formation in vivo may be blocked by treatment with these antagonists. This is strongly supported by the effects of these antagonists on hypoxia-induced cellular dehydration in sickle erythrocytes (Fig. 4). Reduction of hypoxia-induced sickle erythrocyte dehydration in vivo by clotrimazole or Mg2+ supplementation, both blockers of K+ transport, has been shown in a mouse model of SCD (9). However, the effect of these blockers on cytokine and chemokine release has not been investigated.

ET-1 receptor antagonists have been used as a potential therapeutic approach to pulmonary hypertension and asthma. Both conditions have been shown to be associated with increased plasma levels of ET-1 (12, 15, 22) as observed in SCD. In patients with pulmonary hypertension, a pilot study has shown therapeutic efficacy of selective antagonists to ET-1 receptors (1). In addition, in a mouse model of pulmonary hypertension, this therapy showed benefits in both prevention and reversal of hypoxia-induced pulmonary vascular remodeling. Furthermore, treatment with bosentan, a nonselective blocker of ET-1 receptors, has been reported to reduce the levels of proinflammatory molecules that are associated with the development of asthma (15). A clinical trial is currently underway to assess the effect of bosentan on pulmonary vascular resistance and exercise capacity in SCD patients (http://clinicaltrials.gov/ct/show/NCT00313196) (5).

Briehl and Christoph (3) found that a 15% change in hemoglobin concentration increased hemoglobin polymerization rate fivefold. Inhibition of the Gardos channel by clotrimazole proved to be effective in the reduction of cellular dehydration in only a subset of sickle cell patients (4, 10). In the present study, only 14 days of ET-1 receptor antagonist treatment changed the CHCM by 3% in sickle cell mice (Table 1). These changes are smaller, albeit significant, compared with what has been observed with clotrimazole, which specifically targets Gardos channel activity, for 14 days in human (4%) or sickle mice (12%) (4, 10). However, it is possible that increasing either the exposure or dose of the ET receptor antagonists will lead to similar effects on CHCM as observed with clotrimazole. Nonetheless, these observations suggest that suppression of specific endothelial cytokine signaling not only mediates reduction of hemoglobin S polymerization but also may interfere with ET-1 effects on the endothelium in vivo. Consistent with my original observations (26), a preliminary report now suggests that treatment with bosentan reduces hypoxic-induced kidney injury in SAD mice (32). Although the mechanism(s) for this effect were not described, they provide further evidence of the role of ET-1 in the pathophysiology of SCD that supports the observations presented in the present study.

A search for novel therapies based on regulation of signaling pathways that affect both K+ loss and volume regulation continues to provide valuable insight into the progression of SCD. Consistent with this approach, the new Gardos channel blocker ICA-17043, a clotrimazole analog (34) that is currently in phase III clinical trials, and arginine diet supplementation (30), which is in phase II clinical trials, have been shown to modulate red cell volume in vivo in sickle cell animal models via reduction of Gardos channel activity. The present results suggest an important interplay between the endothelium via endothelium-derived cytokine/chemokines and erythrocyte volume regulation in vivo. Most importantly, these studies demonstrate a role for ET-1 in the pathophysiology of SCD. Thus these results add to the arsenal of potential therapies that are designed to reduce dense cell formation in SCD and suggest that a combination of therapies such as ICA-17043 together with arginine diet supplementation and ET-1 receptor antagonist may have synergistic effects on erythrocyte volume regulation and as such warrant further investigation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants HL067699 and DK069388 (to A. Rivera).


   ACKNOWLEDGMENTS
 
The expert technical assistance of Lin-Chie Pong and Lawrence Chien is acknowledged and greatly appreciated. A. Rivera is a Fellow of the Center of Excellence in Minority Health and Health Disparities at Harvard Medical School.

A preliminary report of these findings was presented in abstract form (Blood 102: 264a, 2003) at the 45th Annual Meeting of the American Society of Hematology, in San Diego, CA.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Rivera, Dept. of Laboratory Medicine, Bader 7, Children's Hospital Boston, 300 Longwood Ave., Boston, MA 02115 (e-mail: alicia.rivera{at}childrens.harvard.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Ambalavanan N, Bulger A, Murphy-Ullrich J, Oparil S, Chen YF. Endothelin-A receptor blockade prevents and partially reverses neonatal hypoxic pulmonary vascular remodeling. Pediatr Res 57: 631–636, 2005.[CrossRef][Web of Science][Medline]

2. Bourantas KL, Dalekos GN, Makis A, Chaidos A, Tsiara S, Mavridis A. Acute phase proteins and interleukins in steady state sickle cell disease. Eur J Haematol 61: 49–54, 1998.[Web of Science][Medline]

3. Briehl RW, Christoph GW. Exponential progress curves and shear in the gelation of hemoglobin S. Prog Clin Biol Res 240: 129–149, 1987.[Medline]

4. Brugnara C, Gee B, Armsby CC, Kurth S, Sakamoto M, Rifai N, Alper SL, Platt OS. Therapy with oral clotrimazole induces inhibition of the Gardos channel and reduction of erythrocyte dehydration in patients with sickle cell disease. J Clin Invest 97: 1227–1234, 1996.[Web of Science][Medline]

5. Castro O, Gladwin MT. Pulmonary hypertension in sickle cell disease: mechanisms, diagnosis, and management. Hematol Oncol Clin North Am 19: 881–896, 2005.[CrossRef][Web of Science][Medline]

6. Corash L. Density dependent red cell separation. In: Red Cell Metabolism. Methods in Haematology, edited by Beutler E. New York: Churchill Livingston, 1986, p. 90.

7. Croizat H, Nagel RL. Circulating cytokines response and the level of erythropoiesis in sickle cell anemia. Am J Hematol 60: 105–115, 1999.[CrossRef][Web of Science][Medline]

8. De Franceschi L, Beuzard Y, Jouault H, Brugnara C. Modulation of erythrocyte potassium chloride cotransport, potassium content, and density by dietary magnesium intake in transgenic SAD mouse. Blood 88: 2738–2744, 1996.[Abstract/Free Full Text]

9. De Franceschi L, Brugnara C, Rouyer-Fessard P, Jouault H, Beuzard Y. Formation of dense erythrocytes in SAD mice exposed to chronic hypoxia: evaluation of different therapeutic regimens and of a combination of oral clotrimazole and magnesium therapies. Blood 94: 4307–4313, 1999.[Abstract/Free Full Text]

10. De Franceschi L, Saadane N, Trudel M, Alper SL, Brugnara C, Beuzard Y. Treatment with oral clotrimazole blocks Ca2+-activated K+ transport and reverses erythrocyte dehydration in transgenic SAD mice. A model for therapy of sickle cell disease. J Clin Invest 93: 1670–1676, 1994.[Web of Science][Medline]

11. Duits AJ, Schnog JB, Lard LR, Saleh AW, Rojer RA. Elevated IL-8 levels during sickle cell crisis. Eur J Haematol 61: 302–305, 1998.[Web of Science][Medline]

12. Eddahibi S, Raffestin B, Clozel M, Levame M, Adnot S. Protection from pulmonary hypertension with an orally active endothelin receptor antagonist in hypoxic rats. Am J Physiol Heart Circ Physiol 268: H828–H835, 1995.[Abstract/Free Full Text]

13. Fabry ME, Romero JR, Buchanan ID, Suzuka SM, Stamatoyannopoulos G, Nagel RL, Canessa M. Rapid increase in red blood cell density driven by K:Cl cotransport in a subset of sickle cell anemia reticulocytes and discocytes. Blood 78: 217–225, 1991.[Abstract/Free Full Text]

14. Fathallah H, Sauvage M, Romero JR, Canessa M, Giraud F. Effects of PKC{alpha} activation on Ca2+ pump and KCa channel in deoxygenated sickle cells. Am J Physiol Cell Physiol 273: C1206–C1214, 1997.[Abstract/Free Full Text]

15. Finsnes F, Lyberg T, Christensen G, Skjonsberg OH. Effect of endothelin antagonism on the production of cytokines in eosinophilic airway inflammation. Am J Physiol Lung Cell Mol Physiol 280: L659–L665, 2001.[Abstract/Free Full Text]

16. Graido-Gonzalez E, Doherty JC, Bergreen EW, Organ G, Telfer M, McMillen MA. Plasma endothelin-1, cytokine, and prostaglandin E2 levels in sickle cell disease and acute vaso-occlusive sickle crisis. Blood 92: 2551–2555, 1998.[Abstract/Free Full Text]

17. Hammerman SI, Kourembanas S, Conca TJ, Tucci M, Brauer M, Farber HW. Endothelin-1 production during the acute chest syndrome in sickle cell disease. Am J Respir Crit Care Med 156: 280–285, 1997.[Abstract/Free Full Text]

17a. Holtzclaw JD, Jiang M, Yasin Z, Joiner CH, Franco RS. Rehydration of high-density sickle erythrocytes in vitro. Blood 100: 3017–3025, 2002.[Abstract/Free Full Text]

18. Kaul DK, Fabry ME, Nagel RL. The pathophysiology of vascular obstruction in the sickle syndromes. Blood Rev 10: 29–44, 1996.[CrossRef][Web of Science][Medline]

19. Kurantsin-Mills J, Jacobs HM, Lessin LS. Assessment of the hydration state of sickle cells by phthalate ester density distribution. J Lab Clin Med 109: 486–494, 1987.[Web of Science][Medline]

20. Longenecker GL, Beyers BJ, Mankad VN. Platelet regulatory prostanoids and platelet release products in sickle cell disease. Am J Hematol 40: 12–19, 1992.[CrossRef][Web of Science][Medline]

21. Miller CL, Mishell RI. Differential regulatory effects of accessory cells on the generation of cell-mediated immune reactions. J Immunol 114: 692–695, 1975.[Abstract/Free Full Text]

22. Pearl JM, Wellmann SA, McNamara JL, Lombardi JP, Wagner CJ, Raake JL, Nelson DP. Bosentan prevents hypoxia-reoxygenation-induced pulmonary hypertension and improves pulmonary function. Ann Thorac Surg 68: 1714–1721, 1999.[Abstract/Free Full Text]

23. Phelan M, Perrine SP, Brauer M, Faller DV. Sickle erythrocytes, after sickling, regulate the expression of the endothelin-1 gene and protein in human endothelial cells in culture. J Clin Invest 96: 1145–1151, 1995.[Web of Science][Medline]

24. Platt OS, Brambilla DJ, Rosse WF, Milner PF, Castro O, Steinberg MH, Klug PP. Mortality in sickle cell disease. Life expectancy and risk factors for early death. N Engl J Med 330: 1639–1644, 1994.[Abstract/Free Full Text]

25. Platt OS, Thorington BD, Brambilla DJ, Milner PF, Rosse WF, Vichinsky E, Kinney TR. Pain in sickle cell disease. Rates and risk factors. N Engl J Med 325: 11–16, 1991.[Abstract]

26. Rivera A. In vivo regulation of cellular dehydration by endothelin-1 receptor B antagonist in sickle cell mouse model. Blood 102: 934a, 2003.[CrossRef]

27. Rivera A, Jarolim P, Brugnara C. Modulation of Gardos channel activity by cytokines in sickle erythrocytes. Blood 99: 357–603, 2002.[Abstract/Free Full Text]

28. Rivera A, Rotter MA, Brugnara C. Endothelins activate Ca2+-gated K+ channels via endothelin B receptors in CD-1 mouse erythrocytes. Am J Physiol Cell Physiol 277: C746–C754, 1999.[Abstract/Free Full Text]

29. Romero JR, Rivera A, Suzuka S, Nagel RL, Fabry ME. Effect of arginine diet for sickle transgenic mice o plasma cytokine levels and the mechanism of reduction of Gardos channel activity. Blood 102: 260a, 2003.

30. Romero JR, Suzuka SM, Nagel RL, Fabry ME. Arginine supplementation of sickle transgenic mice reduces red cell density and Gardos channel activity. Blood 99: 1103–1108, 2002.[Abstract/Free Full Text]

31. Rybicki AC, Benjamin LJ. Increased levels of endothelin-1 in plasma of sickle cell anemia patients. Blood 92: 2594–2596, 1998.[Free Full Text]

32. Sabaa N, De Franceschi L, Bonnin P, Malpeli G, Germain S, Vandermeersch S, Mater-Redelsperger M, Scarpa A, Janin A, Levy B, Girot B, Beuzard Y, Leboeuf C, Henri A, Dussaule JC, Tharaux PL. The dual endothelin receptor antagonist bosentan prevents the acute sickle cell-related hypoxic lung and kidney injury in transgenic SAD mice. Blood 108: 687a, 2006.

32a. Schwartz RS, Musto S, Fabry ME, Nagel RL. Two distinct pathways mediate the formation of intermediate density cells and hyperdense cells from normal density sickle red blood cells. Blood 92: 4844, 1998.[Abstract/Free Full Text]

33. Steinberg MH. Management of sickle cell disease. N Engl J Med 340: 1021–1030, 1999.[Free Full Text]

34. Stocker JW, De FL, Naughton-Smith GA, Corrocher R, Beuzard Y, Brugnara C. ICA-17043, a novel Gardos channel blocker, prevents sickled red blood cell dehydration in vitro and in vivo in SAD mice. Blood 101: 2412–2418, 2003.[Abstract/Free Full Text]

35. Szabo MC, Soo KS, Zlotnik A, Schall TJ. Chemokine class differences in binding to the Duffy antigen-erythrocyte chemokine receptor. J Biol Chem 270: 25348–25351, 1995.[Abstract/Free Full Text]




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