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Increasing nitric oxide bioavailability fails to improve collateral vessel formation in humanized sickle cell mice

Abstract

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.

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Fig. 1: L-Arginine supplementation fails to improve recovery from hindlimb ischemia.
Fig. 2: Analysis of arginine pathways in sickle cell mice.
Fig. 3: Nitric oxide (NO)-producing hydrogel PA-YK-NO worsens response to hindlimb ischemia in sickle cell mice.
Fig. 4: L-NG-nitro arginine methyl ester (L-NAME) worsens response to hindlimb ischemia but protects against aortic inflammation in sickle cell mice.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

References

  1. 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).

    PubMed  Article  Google Scholar 

  2. Francis, R. B. Large-vessel occlusion in sickle cell disease: pathogenesis, clinical consequences, and therapeutic implications. Med. Hypotheses. 35, 88–95 (1991).

    CAS  PubMed  Article  Google Scholar 

  3. Miller, A. C. & Gladwin, M. T. Pulmonary complications of sickle cell disease. Am. J. Respir. Crit. Care. Med. 185, 1154–1165 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. Scheinman, J. I. Sickle cell disease and the kidney. Nat. Clin. Pract. Nephrol. 5, 78–88 (2009).

    PubMed  Article  Google Scholar 

  5. Okwan-Duodu, D. et al. Impaired collateral vessel formation in sickle cell disease. Arterioscler Thromb. Vasc. Biol. 38, 1125–1133 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. 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).

    CAS  PubMed  Article  Google Scholar 

  9. Moens, A. L. & Kass, D. A. Tetrahydrobiopterin and cardiovascular disease. Arterioscler Thromb. Vasc. Biol. 26, 2439–2444 (2006).

    CAS  PubMed  Article  Google Scholar 

  10. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Morris, C. R. et al. Dysregulated arginine metabolism, hemolysis-associated pulmonary hypertension, and mortality in sickle cell disease. Jama. 294, 81–90 (2005).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. 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).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  13. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 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).

    CAS  PubMed  Article  Google Scholar 

  15. Reiter, C. D. et al. Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease. Nat. Med. 8, 1383–1389 (2002).

    CAS  PubMed  Article  Google Scholar 

  16. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. 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).

    CAS  PubMed  Article  Google Scholar 

  19. Benites, B. D. & Olalla-Saad, S. T. An update on arginine in sickle cell disease. Expert Rev. Hematol. 12, 235–244 (2019).

    CAS  PubMed  Article  Google Scholar 

  20. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. 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).

    CAS  PubMed  Article  Google Scholar 

  22. 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).

    CAS  PubMed  Article  Google Scholar 

  23. 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).

    CAS  PubMed  Google Scholar 

  24. 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).

    Article  Google Scholar 

  25. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. 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).

    CAS  PubMed  Article  Google Scholar 

  27. 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).

    PubMed  PubMed Central  Article  Google Scholar 

  28. 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).

    PubMed  Article  Google Scholar 

  29. Gkaliagkousi, E. & Ferro, A. Nitric oxide signalling in the regulation of cardiovascular and platelet function. Front. Biosci. (Landmark Ed) 16, 1873–1897 (2011).

    CAS  Article  Google Scholar 

  30. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 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).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  34. 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).

    CAS  PubMed  Article  Google Scholar 

  35. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 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).

    CAS  PubMed  Article  Google Scholar 

  37. 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).

    CAS  PubMed  Article  Google Scholar 

  38. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. 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).

    CAS  PubMed  Article  Google Scholar 

  40. 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).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  41. Schnog, J.-J. B. et al. Evidence for a metabolic shift of arginine metabolism in sickle cell disease. Ann. Hematol. 83, 371–375 (2004).

    CAS  PubMed  Article  Google Scholar 

  42. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Minniti, C. P. & Kato, G. J. Critical reviews: How we treat sickle cell patients with leg ulcers. Am. J. Hematol. 91, 22–30 (2016).

    PubMed  Article  Google Scholar 

  44. 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).

    Article  Google Scholar 

  45. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. 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).

    CAS  PubMed Central  Article  Google Scholar 

  47. 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).

    PubMed  PubMed Central  Article  Google Scholar 

  48. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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Funding

This work was funded by an NIH R01 Grant: NHLBI R01 HL131414.

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C.V.L. and W.R.T. performed study concept and design and writing and revision of paper; H.W.J., L.A.B., and D.R.A. performed development of methodology; C.V.L., L.A.B., H.S., L.H., G.J., and J.H. provided acquisition, analysis and interpretation of data; D.R.A., L.A.B. and H.W.J. provided technical support and all authors contributed to review and revision of the paper.

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Correspondence to W. Robert Taylor.

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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

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