The hereditary blood disorder sickle cell disease (SCD) results from a single nucleotide mutation in the β-globin gene, which leads to the loss of red blood cell deformability. Diseased red blood cells are susceptible to changes of their shape (sickling), which can be precipitated by hypoxia, stress, and hypothermia. The relatively stiff cells can no longer undergo the required shape change necessary to transit the microvasculature, resulting in vessel occlusion, ischemia, and pain crises. During the past several decades, our understanding of the complex pathophysiology of vaso-occlusion, which is the hallmark of SCD, has improved dramatically. Originally, it was believed that stiff (sickled) red blood cells resulting from polymerization of hemoglobin S were the primary contributor to the clinical pathophysiology of SCD. Research in human subjects and in animal models, however, has identified dysregulated vascular pathways, known as sickle vasculopathy, which features severe hemolytic anemia leading to scavenging of nitric oxide (NO).1 This diminished NO bioavailability in turn leads to pulmonary hypertension, cutaneous leg ulceration, priapism, ischemic stroke, as well as renal insufficiency, systolic hypertension, and overall endothelial dysfunction.2–4 NO has been described as a mediator of SCD, and it has been demonstrated previously that the disruption of endothelial regulation of vasoreactivity is a likely contributor to sickle vasculopathy and pulmonary hypertension.5 Moreover, NO was shown to inhibit the adherence of sickle cells (SCs) to the vascular endothelium in SCD.6 Thus, in principle, restoring NO bioavailability should therefore result in a marked improvement in vascular function in SCD. It is therefore critical to either verify or refute the role of NO bioavailability and its pharmacological manipulation in SCD in preclinical models, which will in future lay the foundation to develop specific therapeutic agents that impact pathways affecting NO resistance in SCD.
Arginase, an enzyme that is released from red blood cells, competes with NO synthase (NOS) for their common substrate, L-arginine. Increased arginase activity consequently results in decreased L-arginine availability for endothelial NOS (eNOS).7 Increased arginase activity in patients with SCD has been suggested by previous studies. Morris et al.8 showed that a lower L-arginine to ornithine ratio (which corresponds to greater arginase activity) was associated with more severe pulmonary hypertension and a greater mortality in human subjects. To determine whether L-arginine supplementation can recover eNOS activity, Little et al.9 conducted a phase I/II clinical trial in which exogenous L-arginine was administered to patients with SCD; however, L-arginine supplementation resulted in increased plasma arginine and ornithine levels without changing citrulline levels, suggesting that most of the arginine administered is metabolized by arginase rather than NOS. Furthermore, chronic L-arginine exposure has been demonstrated to increase endothelial cell arginase, essentially “adding fuel to the fire.”10
There is increasing evidence that arginase reciprocally regulates eNOS function in the vasculature. We and others have shown that arginase activation contributes to an age-related decrease in endothelial NO and subsequent vascular endothelial dysfunction and stiffness. Moreover, in the absence of sufficient substrate availability (L-arginine), eNOS produces superoxide instead of NO.11 Arginase thereby contributes to eNOS uncoupling in cellular and animal models of atherosclerosis by limiting substrate availability for eNOS.7,12,13 Importantly, we have shown that arginase inhibition improves vascular endothelial NO bioavailability, decreases reactive oxygen species (ROS) production, and enhances endothelial-dependent vasodilation.14 Importantly, there are distinct intracellular pools of L-arginine, of which only some can be accessed by NOS.15,16 Thus, failure in previous trials to improve vascular mechanics by L-arginine supplementation might be either because of the inaccessibility of L-arginine pools to NOS or because of L-arginine consumption by arginase. One approach to improve L-arginine bioavailability to NOS would be to limit L-arginine consumption by arginase. We therefore examined whether arginase inhibition can improve global NO bioavailability, and thereby attenuate vascular endothelial dysfunction, pulmonary pressures, and vascular stiffness in transgenic mice with SCD.
METHODS
Animals
Transgenic SC mice were obtained from Lawrence Berkeley National Laboratory.17 These mice express exclusively human sickle hemoglobin, and all mouse hemoglobin genes are deleted. Age-matched C57Bl/6 mice were used as wild-type (WT) controls. Forty SC animals (4- to 5-month-old males) were randomized into 2 groups with 20 animals per group: untreated SC mice (SC) and SC mice treated with 2(S)-amino-6-boronohexanoic acid (SC + ABH). WT animals were used as controls. Noninvasive pulse wave velocity (PWV) measurements were acquired in all these mice. At the end of the experimental period, the 20 animals from each cohort were randomized for end-point measures as follows: 6 animals were used for biochemistry, 6 for vasoreactivity (organ bath), and 8 for cardiac catheterization. Each animal in the treatment group consumed approximately 400 μg of ABH (Calbiochem, San Diego, CA) daily in their drinking water for 4 weeks. All mice were maintained on standard rodent diet, and all studies were approved by the Animal Care and Use Committees of the Johns Hopkins Medical Institute.
Arginase Activity
Mouse aorta was frozen in liquid nitrogen and pulverized. Resulting tissue powder was combined 1:4 (wt:vol) with ice-cold lysis buffer (1× radioimmunoprecipitation assay [RIPA] containing protease inhibitors [phenylmethane sulfonyl fluoride, leupeptin, and aprotinin]) and homogenized. The homogenate was centrifuged at 12,000g for 10 minutes and the supernatant recovered for an arginase activity assay as described previously.18
Production of ROS and NO
ROS were measured ex vivo as described previously, with the use of dihydroethidium (DHE) fluorescence (Pierce Protein Biology Products; Thermo Scientific, Rockford, IL).19 NO production was measured ex vivo with 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF FM-DA) (Molecular Probes, Eugene, OR), a NO-sensitive fluorescent dye, as described previously.20
Vasoreactivity Studies
Aortic rings were prepared and suspended in organ chambers, as previously described.14 Vessels were preconstricted with phenylephrine (10−6 mol/L; Sigma-Aldrich, St. Louis, MO) or PGF2α (10−8 mol/L; Sigma-Aldrich). Endothelium-dependent and endothelium-independent responses were determined with acetylcholine (ACh, 10−9 to 10−5 mol/L) and sodium nitroprusside (10−9 to 10−5 mol/L; Sigma), respectively.
Cardiac Catheterization
The procedure was conducted as described previously.21 In brief, all animals were anesthetized with 0.08 to 0.15 mL of 300 mg/mL urethane, 0.05 mL of 2 mg/mL etomidate, and 0.02 μL of 1.5 g/mL morphine and placed on a heating pad. Rectal temperature was measured throughout the experiment and maintained at baseline. The duration of the experiments was 45 to 60 minutes, which is well below the duration of the anesthetics used (90 minutes), and therefore, we did not adjust the depth of anesthesia during the experiments. A tracheotomy tube was placed and connected to a 845 Mouse Minivent (Hugo Sachs Elektronik, March, Germany) set at a respiratory rate between 110 and 120 breaths per minutes at 300 μL of air per breath. As described previously, the apex of the heart was accessed through the diaphragm via a lateral incision below the xyphoid process.22,23 Using a 27½ gauge needle, the tip of a 1.4-F SPR-839 Millar conductance catheter (Millar, Inc, Houston, TX) was introduced into the right ventricle via a retrograde approach and then into the left ventricle via a similar approach. Data were recorded using a Millar Aria 1 PV Conductance System and Chart 5 from AD Instrument PowerLab (AD instruments, Colorado Springs, CO). To obtain pressure volume loops and chamber-specific hemodynamic variables, manual occlusion of the isolated inferior vena cava was used to initiate a transient reduction in preload.
Pulse Wave Velocity
PWV was measured noninvasively using high-frequency Doppler (Indus Instruments, Webster, TX) as previously described.24 PWV is measured at 2 points along the aorta with 1 point in the upper thoracic cavity and 1 point in the lower abdominal cavity. PWV is calculated as the separation distance divided by the pulse transit time between the 2 points.
Statistical Analysis
Data are reported as mean ± SD. Arginase activity, NO activity, ROS production, cardiac catheterization, and PWV were analyzed with 1-way analysis of variance (ANOVA) followed by the Tukey test to obtain P values for 1-way ANOVA and multiplicity-adjusted P values for each comparison. For the vasoreactivity experiments, a 2-way-ANOVA followed by Bonferroni test for multiple comparisons was used, with a total of 27 effective groups. A P value of <0.01 was used as the criterion for statistical significance. The P values of the ANOVA are presented in the Results section. The post hoc P values for all direct comparisons are presented in the figure legends.
RESULTS
Arginase Activity
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Figure 1.: Arginase activity in mouse aortic homogenates: arginase activity is greater in sickle cell mice (SC; n = 5) compared with wild-type controls (WT; n = 4; P = 0.0009). Arginase inhibition (2(S)-amino-6-boronohexanoic acid [ABH]; n = 5) in SC mice decreases arginase activity, to a level comparable with WTs (P = 0.69 compared with WT and P = 0.002 compared with SC [*post hoc P value <0.01]).
Arginase activity was significantly greater in the aortic homogenates of untreated SC mice compared with WT controls. Treatment with ABH resulted in a marked reduction in arginase activity in the SC mice compared with the untreated SC cohort (SC versus WT versus SC + ABH: 341 ± 69.3 vs 90.1 ± 17.3 vs 120 ± 17.3 pmol urea/mg protein/min; P = 0.0007 by 1-way ANOVA; n = 4–5 animals per group; Figure 1).
NO and Reactive Oxygen Species
Figure 2.: Nitric oxide levels and reactive oxygen species: (A) nitric oxide production is decreased in sickle cell (SC) aorta compared with wild types (WT; P = 0.009). Arginase inhibition with 2(S)-amino-6-boronohexanoic acid (ABH) increases nitric oxide production (n = 3 animals per group; P = 0.39 compared with WT and P = 0.05 compared with SC). B, Rates of reactive oxygen species production are greater in SC mice compared with WT (P = 0.03). Treatment with ABH decreases this in SC mice (n = 5 animals per group; P = 0.87 compared with WT and P = 0.07 compared with SC; *post hoc P value <0.01).
Aortic strips from untreated SC mice showed significantly decreased NO production compared with WT controls. Arginase inhibition with ABH resulted in increased NO production in SC mice compared with the untreated SC cohort (rate of NO production SC: 0.76 ± 0.14; WT: 1.34 ± 0.17; SC + ABH: 1.16 ± 0.16 relative florescence units [RFU]/s; n = 3 animals per group; Figure 2A; P = 0.0006 by 1-way ANOVA). Conversely, the rate of ROS production was greater in the aortic strips from untreated SC mice compared with age-matched WT controls. Arginase inhibition decreased ROS production in SC mice compared with the untreated SC group (rate of ROS production: SC: 3.96 ± 1.70; WT: 1.63 ± 1.20; SC + ABH: 2.02 ± 0.45 RFU/s; n = 5 per group; Figure 2B; P = 0.02 by 1-way ANOVA).
Vasoreactivity Studies and Endothelial Function
Figure 3.: Vasoreactivity and endothelial function: (A) sickle cell mice (SC) have a reduced maximal endothelial-dependent vasodilatory response to acetylcholine (ACh) compared with wild types (WT, P = 0.018), which becomes apparent at an ACh dose of 10−5 M. Inhibition of arginase restores endothelial-dependent vasodilation (P > 0.99 compared with WT). B, There is no difference in the endothelial-independent response (sodium nitroprusside) between the groups (n = 6 animals per group, P > 0.99 in all comparisons). ABH = 2(S)-amino-6-boronohexanoic acid. SNP indicates sodium nitroprusside.
Vasodilation in response to increasing concentrations of ACh was similar in all groups. Only the maximal endothelial-dependent vasodilatory response to ACh (10−5 M) decreased in SC mice when compared with WT controls (P = 0.02 by 2-way ANOVA with the Bonferroni multiple comparisons test). Treatment with ABH restored maximal endothelial-dependent vasodilation in SC mice to that of WT mice (Figure 3A). The endothelial-independent response to the NO donor sodium nitroprusside was similar in all 3 groups (half maximal effective concentration in SC: 1.1 × 10−8, WT: 8.5 × 10−9, and SC + ABH: 1.3 × 10−8; maximal relaxation in SC: 98% ± 5.8%, SC + ABH: 95% ± 2.6%, and WT: 95% ± 5.9%; n = 6; Figure 3B).
Right Heart Catheterization
Figure 4.: Right heart catheterization: (A) pulmonary vascular resistance index (PVRI) and (B) right ventricular end-systolic pressure (RVESP) were elevated in sickle cell (SC) mice compared with wild types (WT). C, Right ventricular cardiac output index (RVCOI) and (D) right ventricular end-systolic elastance (RVEes) were similar in SC mice compared with WT. Treatment with ABH for 4 weeks restored PVRI, RVESP, RVCOI, and RVEes (n = 8 animals per group, post hoc P values: PVRI: WT versus SC P = 0.004, WT versus SC + 2(S)-amino-6-boronohexanoic acid (ABH) P = 0.76, SC versus SC + ABH P = 0.019; RVESP: WT versus SC P = 0.03, WT versus SC + ABH P = 0.70, SC versus SC + ABH P = 0.13; RVCOI: WT versus SC P = 0.10, WT versus SC + ABH P = 0.80, SC versus SC + ABH P = 0.03; RVEes: WT versus SC P = 0.18, WT versus SC + ABH P = 0.83, SC versus SC + ABH P = 0.44; *P < 0.01).
Figure 5.: Right ventricular (RV) pressure volume loops: representative traces of RV pressure volume loops in (A) wild-type, (B) sickle cell (SC), and (C) SC mice after treatment with 2(S)-amino-6-boronohexanoic acid (ABH).
Pulmonary vascular resistance index (PVRI) and right ventricular end-systolic pressure (RVESP) may be significantly elevated in SC mice compared with WT animals (PVRI: 5.5 ± 1.9 vs 2.9 ± 0.28 mm Hg × min/μL/100 g; P = 0.004; RVESP: 23.1 ± 4.0 vs 18.9 ± 1.13 mm Hg; P = 0.03; Figure 4). Right ventricular cardiac output index (RVCOI) and right ventricular end-systolic elastance (RVEes) in SC mice were similar to WT animals (4.60 ± 0.51 vs 2.9 ± 0.85 mL/min/100 g; P = 0.10; and 0.58 ± 0.11 vs 0.89 ± 0.48 mm Hg/μL; P = 0.18; Figures 4 and 5). Treatment with ABH for 4 weeks restored PVRI and RVESP to levels similar to WT animals (PVRI: 3.4 ± 1.4 mm Hg × min/μL/100 g; RVESP: 20.1 ± 3.1 mm Hg; Figures 4 and 5). Treatment with ABH may elevate RVCOI in the SC animals compared with untreated SC animals, with no effect on RVEes (RVCOI: 5.1 ± 2.5 mL/min/100 g, P = 0.03 versus SC; RVEes: 0.79 ± 0.31 mm Hg/μL; Figures 4 and 5).
PWV and Arterial Stiffness
Figure 6.: Pulse wave velocity (PWV): PWV is greater in sickle cell (SC) mice than in wild types (WT, P = 0.09). SC mice treated with 2(S)-amino-6-boronohexanoic acid (ABH) have significantly reduced PWV to a level that is not statistically different from WT (n = 20 animals per group; P = 0.73 compared with WT and P = 0.001 compared with SC; *P < 0.01).
PWV, a measure of vascular stiffness, was greater in untreated SC mice than in WT controls. SC mice treated with ABH for 4 weeks had significantly reduced PWVs compared with untreated SC mice and were no different from WT controls (PWV in m/s: SC: 3.74 ± 0.54; WT: 3.25 ± 0.21 m/s; SC + ABH: 3.06 ± 0.31; P = 0.0009 by 1-way ANOVA; n = 20; Figure 6).
DISCUSSION
Loss of global NO and overall endothelial dysfunction is now well established in SCD. In this study, we show that arginase activity is significantly increased in SC mouse aorta compared with WT, whereas NO production is reduced. More importantly, we believe this is the first demonstration that arginase inhibition improves vascular function and reverses the vascular phenotype of SCD. These findings are in accordance with previous studies demonstrating not only arginase upregulation but more specifically upregulation of arginase II, as well as ornithine decarboxylase antizyme mRNA in platelets of SC patients.25,26 Moreover, we demonstrated an attenuated endothelial-dependent vasorelaxation response to ACh in SC mice.
Physiological changes in SC vasculopathy include increased pulmonary resistance, increased right ventricular systolic pressures, and greater PWV, suggesting stiffer vessels. These changes are recapitulated well in the SC mouse. One way to improve NO signaling downstream of NO in human subjects is through phosphodiesterase (PDE) inhibition. Interestingly, modest increases in plasma cyclic guanosine monophosphate and citrulline levels and improved pulmonary pressures, as well as 6-minute walk test distance, were observed in patients receiving sildenafil.9 A subsequent double-blind study in patients with pulmonary hypertension and decreased exercise tolerance that compared placebo with sildenafil was terminated after enrollment of 74 patients because there were more hospitalizations for pain in the sildenafil group, no difference in the 6-minute walk distance, and a trend to increased pulmonary hypertension in the sildenafil group.27 This is likely because the PDE5 inhibitor does not address the issue of eNOS uncoupling, and the high oxidative stress and low NO bioavailability in the vasculature remain unchanged. Indeed, if PDE5 inhibitors are to work, at least some bioavailable NO needs to be present to activate soluble guanylate cyclase. However, because these studies were performed in human subjects, only plasma levels of arginase were evaluated and endothelial arginase was not examined.8
In this study, treatment of SC mice with the arginase inhibitor ABH for 4 weeks decreased arginase activity and restored pulmonary pressures, resistance, and PWV to a level comparable with age-matched WT animals. Vasodilation in response to increasing concentrations of ACh in aortic rings was similar in all cohorts. This could, in part, have been because of the age of the mice used in this study, with older mice demonstrating impaired vasoreactivity. In addition, the increased PWV in these mice suggests that the in vivo environment with its fully functional mechanical and biochemical signaling mechanisms along with sickling cells may be required to observe alterations in vasoreactivity; a feature that is lost in the in vitro study. Finally, endothelial dysfunction may be dependent on the vascular bed. Indeed, to our knowledge, the only observation to date of endothelial dysfunction in this SC mouse model has been in the pulmonary circulation in vivo.28
Overall, these findings support previous studies that have proposed that arginase may be an important target in SCD-related vasculopathy and provide evidence for the use of arginase inhibitors for the treatment of SC vasculopathy.28,29 Although there are currently no specific arginase inhibitors available for human use, in 2007 it was demonstrated that chloroquine, a drug used as an antimalarial or antirheumatic medication, inhibits arginase in a dose-dependent manner including in sickle erythrocytes.30 Continued efforts to identify and characterize specific and selective arginase inhibitors are needed to further elucidate the therapeutic index of arginase in human subjects.
It is widely accepted that hemolytic rates and release of free hemoglobin into the circulation closely correlate with scavenging of NO. Two mechanisms can account for this loss of NO: first, cell-free hemoglobin can rapidly react with NO, and second, the released arginase 1 can consume L-arginine to further compromise substrate availability for NOS.29 However, given that arginase 2 also is upregulated in the endothelium of SC mice, there appears to be an endothelial contribution to SC vasculopathy that is independent from red blood cell hemolysis. Future studies will focus on separating the contribution of each arginase isoform (arginase 1 in the red blood cells versus arginase 2 in the endothelium) to the SC phenotype.
Limitations
This study was performed in a rodent model with some experiments conducted ex vivo. The physiological experiments were performed with mice under general anesthesia with the chest cavity exposed to ambient pressure and air, and the lungs ventilated with positive pressure. This could have resulted in a change of pressures, flows, and volumes measurements and might not have been identical to awake and intact animals. Furthermore, placement of the catheter is difficult in small rodents and was done by visual recognition of pressure waveforms by a single provider without verification by other imaging modalities. Because methodologic variations may limit the generalizability of our results, we used a more stringent post hoc P value <0.01 as statistically significant. In the present study, it was not possible to delineate whether the detrimental effects of arginase in SCD are because of intracellular arginase 2 of the affected cells or because of the amount of arginase 1 liberated from the erythrocytes by hemolysis. These mechanisms can be distinguished by measuring endothelial and plasma NO and arginase in SC mice and their WT controls using pharmacological inhibition of each isoform.
CONCLUSIONS
Two important findings of this study are (1) increased arginase activity contributes to endothelial dysfunction, pulmonary hypertension, and vascular stiffness in transgenic SC mice; and (2) chronic treatment with an arginase inhibitor, ABH, attenuates systemic and pulmonary vascular dysfunction in transgenic SC mice. Thus, arginase is a potential novel therapeutic target in SC vasculopathy.
DISCLOSURES
Name: Jochen Steppan, MD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Name: Huong T. Tran, PhD.
Contribution: This author helped design the study, conduct the study, and analyze the data.
Name: Valeriani R. Bead, MD.
Contribution: This author helped design the study, conduct the study, and analyze the data.
Name: Young Jun Oh, MD.
Contribution: This author helped conduct the study.
Name: Gautam Sikka, MD.
Contribution: This author helped conduct the study.
Name: Trinity J. Bivalacqua, MD, PhD.
Contribution: This author helped design the study.
Name: Arthur L. Burnett, MD.
Contribution: This author helped design the study.
Name: Dan E. Berkowitz, MD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Name: Lakshmi Santhanam, PhD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
This manuscript was handled by: Avery Tung, MD.
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