Sickle cell disease (SCD) is associated with repeated bouts of vascular insufficiency leading to organ dysfunction. Deficits in revascularization following vascular injury are evident in SCD patients and animal models. We aimed to elucidate whether enhancing nitric oxide bioavailability in SCD mice improves outcomes in a model of vascular insufficiency. Townes AA (wild type) and SS (sickle cell) mice were treated with either L-Arginine (5% in drinking water), L-NAME (N(ω)-nitro-L-arginine methyl ester; 1 g/L in drinking water) or NO-generating hydrogel (PA-YK-NO), then subjected to hindlimb ischemia via femoral artery ligation and excision. Perfusion recovery was monitored over 28 days via LASER Doppler perfusion imaging. Consistent with previous findings, perfusion was impaired in SS mice (63 ± 4% of non-ischemic limb perfusion in AA vs 33 ± 3% in SS; day 28; P < 0.001; n = 5–7) and associated with increased necrosis. L-Arginine treatment had no significant effect on perfusion recovery or necrosis (n = 5–7). PA-YK-NO treatment led to worsened perfusion recovery (19 ± 3 vs. 32 ± 3 in vehicle-treated mice; day 7; P < 0.05; n = 4–5), increased necrosis score (P < 0.05, n = 4–5) and a 46% increase in hindlimb peroxynitrite (P = 0.055, n = 4–5). Interestingly, L-NAME worsened outcomes in SS mice with decreased in vivo lectin staining following ischemia (7 ± 2% area in untreated vs 4 ± 2% in treated mice, P < 0.05, n = 5). Our findings demonstrate that L-arginine and direct NO delivery both fail to improve postischemic neovascularization in SCD. Addition of NO to the inflammatory, oxidative environment in SCD may result in further oxidative stress and limit recovery.
Your institute does not have access to this article
Subscribe to Journal
Get full journal access for 1 year
We are sorry, but there is no personal subscription option available for your country.
Get time limited or full article access on ReadCube.
All prices are NET prices.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Choudhury, N. A. et al. Intracranial vasculopathy and infarct recurrence in children with sickle cell anaemia, silent cerebral infarcts and normal transcranial Doppler velocities. Br. J. Haematol. 183, 324–326 (2018).
Francis, R. B. Large-vessel occlusion in sickle cell disease: pathogenesis, clinical consequences, and therapeutic implications. Med. Hypotheses. 35, 88–95 (1991).
Miller, A. C. & Gladwin, M. T. Pulmonary complications of sickle cell disease. Am. J. Respir. Crit. Care. Med. 185, 1154–1165 (2012).
Scheinman, J. I. Sickle cell disease and the kidney. Nat. Clin. Pract. Nephrol. 5, 78–88 (2009).
Okwan-Duodu, D. et al. Impaired collateral vessel formation in sickle cell disease. Arterioscler Thromb. Vasc. Biol. 38, 1125–1133 (2018).
Dai, X. & Faber, J. E. Endothelial nitric oxide synthase deficiency causes collateral vessel rarefaction and impairs activation of a cell cycle gene network during arteriogenesis. Circ. Res. 106, 1870–1881 (2010).
Yu, J. et al. Endothelial nitric oxide synthase is critical for ischemic remodeling, mural cell recruitment, and blood flow reserve. Proc. Natl. Acad. Sci. USA. 102, 10999–11004 (2005).
Landburg, P. P. et al. Plasma asymmetric dimethylarginine concentrations in sickle cell disease are related to the hemolytic phenotype. Blood Cells Mol. Dis. 44, 229–232 (2010).
Moens, A. L. & Kass, D. A. Tetrahydrobiopterin and cardiovascular disease. Arterioscler Thromb. Vasc. Biol. 26, 2439–2444 (2006).
Vilas-Boas, W. et al. Arginase levels and their association with Th17-related cytokines, soluble adhesion molecules (sICAM-1 and sVCAM-1) and hemolysis markers among steady-state sickle cell anemia patients. Ann. Hematol. 89, 877–882 (2010).
Morris, C. R. et al. Dysregulated arginine metabolism, hemolysis-associated pulmonary hypertension, and mortality in sickle cell disease. Jama. 294, 81–90 (2005).
Steppan, J. et al. Arginase Inhibition Reverses Endothelial Dysfunction, Pulmonary Hypertension, and Vascular Stiffness in Transgenic Sickle Cell Mice. Anesth. Analg. 123, 652–658 (2016).
Taylor, C. M. et al. Hydroxyurea improves nitric oxide bioavailability in humanized sickle cell mice. Am. J. Physiol. Regul. Integr. Comp. Physiol. 320, R630–R640 (2021).
Eberhardt, R. T. et al. Sickle cell anemia is associated with reduced nitric oxide bioactivity in peripheral conduit and resistance vessels. Am. J. Hematol. 74, 104–111 (2003).
Reiter, C. D. et al. Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease. Nat. Med. 8, 1383–1389 (2002).
Hsu, L. L. et al. Hemolysis in sickle cell mice causes pulmonary hypertension due to global impairment in nitric oxide bioavailability. Blood 109, 3088–3098 (2007).
Gladwin, M. T. et al. Nitric oxide for inhalation in the acute treatment of sickle cell pain crisis: a randomized controlled trial. Jama 305, 893–902 (2011).
Maitre, B. et al. Inhaled nitric oxide for acute chest syndrome in adult sickle cell patients: a randomized controlled study. Intensive Care Med. 41, 2121–2129 (2015).
Benites, B. D. & Olalla-Saad, S. T. An update on arginine in sickle cell disease. Expert Rev. Hematol. 12, 235–244 (2019).
Dasgupta, T., Hebbel, R. P. & Kaul, D. K. Protective effect of arginine on oxidative stress in transgenic sickle mouse models. Free Radic. Biol. Med. 41, 1771–1780 (2006).
Jaja, S. I., Ogungbemi, S. O., Kehinde, M. O. & Anigbogu, C. N. Supplementation with l-arginine stabilizes plasma arginine and nitric oxide metabolites, suppresses elevated liver enzymes and peroxidation in sickle cell anaemia. Pathophysiology 23, 81–85 (2016).
Kehinde, M. O., Ogungbemi, S. I., Anigbogu, C. N. & Jaja, S. I. l-Arginine supplementation enhances antioxidant activity and erythrocyte integrity in sickle cell anaemia subjects. Pathophysiology 22, 137–142 (2015).
Morris, C. R. et al. Arginine therapy: A novel strategy to induce nitric oxide production in sickle cell disease. Br. J. Haematol. 111, 498–500 (2000).
Styles, L. et al. Arginine therapy does not benefit children with sickle cell anemia — results of the CSCC clinical trial consortium multi-institutional study. Blood 110, 2252–2252 (2007).
Morris, C. R. et al. A randomized, placebo-controlled trial of arginine therapy for the treatment of children with sickle cell disease hospitalized with vaso-occlusive pain episodes. Haematologica 98, 1375–1382 (2013).
Onalo, R. et al. Randomized control trial of oral arginine therapy for children with sickle cell anemia hospitalized for pain in Nigeria. Am. J. Hematol. 96, 89–97 (2021).
Morris, C. R. et al. Impact of arginine therapy on mitochondrial function in children with sickle cell disease during vaso-occlusive pain. Blood 136, 1402–1406 (2020).
Ghimire, K., Altmann, H. M., Straub, A. C. & Isenberg, J. S. Nitric oxide: what’s new to NO? Am. J. Physiol. Cell Physiol. 312, C254–C262 (2017).
Gkaliagkousi, E. & Ferro, A. Nitric oxide signalling in the regulation of cardiovascular and platelet function. Front. Biosci. (Landmark Ed) 16, 1873–1897 (2011).
Peng, X. et al. Gender differences affect blood flow recovery in a mouse model of hindlimb ischemia. Am. J. Physiol. Heart Circ. Physiol. 300, H2027–H2034 (2011).
Lyle, A. N. et al. Reactive oxygen species regulate osteopontin expression in a murine model of postischemic neovascularization. Arterioscler. Thromb. Vasc. Biol. 32, 1383–1391 (2012).
El-Ferzli, G. T. et al. A Nitric Oxide-Releasing Self-Assembled Peptide Amphiphile Nanomatrix for Improving the Biocompatibility of Microporous Hollow Fibers. Asaio. J. 61, 589–595 (2015).
Moon, C. Y. et al. Effects of the nitric oxide releasing biomimetic nanomatrix gel on pulp-dentin regeneration: Pilot study. PLoS One. 13, e0205534 (2018).
Kushwaha, M. et al. A nitric oxide releasing, self assembled peptide amphiphile matrix that mimics native endothelium for coating implantable cardiovascular devices. Biomaterials 31, 1502–1508 (2010).
Kaul, D. K., Zhang, X., Dasgupta, T. & Fabry, M. E. Arginine therapy of transgenic-knockout sickle mice improves microvascular function by reducing non-nitric oxide vasodilators, hemolysis, and oxidative stress. Am. J. Physiol. Heart Circ. Physiol. 295, H39–H47 (2008).
Wood, K. C., Hebbel, R. P., Lefer, D. J. & Granger, D. N. Critical role of endothelial cell-derived nitric oxide synthase in sickle cell disease-induced microvascular dysfunction. Free Radic. Biol. Med. 40, 1443–1453 (2006).
Morris, C. R., Kuypers, F. A., Larkin, S., Vichinsky, E. P. & Styles, L. A. Patterns of arginine and nitric oxide in patients with sickle cell disease with vaso-occlusive crisis and acute chest syndrome. J. Pediatr. Hematol. Oncol. 22, 515–520 (2000).
Antwi-Boasiako, C. & Campbell, A. D. Low nitric oxide level is implicated in sickle cell disease and its complications in Ghana. Vasc. Health Risk Manag. 14, 199–204 (2018).
Almeida, L. E. F. et al. Sickle cell disease subjects and mouse models have elevated nitrite and cGMP levels in blood compartments. Nitric Oxide 94, 79–91 (2020).
Eleutério, R. M. N. et al. Double-Blind Clinical Trial of Arginine Supplementation in the Treatment of Adult Patients with Sickle Cell Anaemia. Adv. Hematol. 2019, 4397150 (2019).
Schnog, J.-J. B. et al. Evidence for a metabolic shift of arginine metabolism in sickle cell disease. Ann. Hematol. 83, 371–375 (2004).
Bakshi, N. & Morris, C. R. The role of the arginine metabolome in pain: implications for sickle cell disease. J. Pain Res. 9, 167–175 (2016).
Minniti, C. P. & Kato, G. J. Critical reviews: How we treat sickle cell patients with leg ulcers. Am. J. Hematol. 91, 22–30 (2016).
Powars, D. R., Chan, L. S., Hiti, A., Ramicone, E. & Johnson, C. Outcome of sickle cell anemia: a 4-decade observational study of 1056 patients. Medicine (Baltimore) 84, 363–376 (2005).
Lopes, F. C. et al. Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea. Haematologica 100, 730–739 (2015).
Zhang, J., et al. HIF-1α and HIF-2α redundantly promote retinal neovascularization in patients with ischemic retinal disease. J. Clin. Invest. 131, e139202 (2021).
Kauv, P. et al. Characteristics of moyamoya syndrome in sickle-cell disease by magnetic resonance angiography: An adult-cohort study. Front. Neurol. 10, 15 (2019).
Potoka, K. P. et al. Nitric oxide-independent soluble guanylate cyclase activation improves vascular function and cardiac remodeling in sickle cell disease. Am. J. Respir. Cell Mol. Biol. 58, 636–647 (2018).
This work was funded by an NIH R01 Grant: NHLBI R01 HL131414.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Lewis, C.V., Sellak, H., Hansen, L. et al. Increasing nitric oxide bioavailability fails to improve collateral vessel formation in humanized sickle cell mice. Lab Invest 102, 805–813 (2022). https://doi.org/10.1038/s41374-022-00780-0