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, 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).
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.
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.
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.
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