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L-citrulline Prevents Alveolar and Vascular Derangement in a Rat Model of Moderate Hyperoxia-induced Lung Injury

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Abstract

Background

Moderate normobaric hyperoxia causes alveolar and vascular lung derangement in the newborn rat. Endogenous nitric oxide (NO), which promotes lung growth, is produced from the metabolism of l-arginine to l-citrulline in endothelial cells. We investigated whether administering l-citrulline by raising the serum levels of l-arginine and enhancing NO endogenous synthesis attenuates moderate hyperoxia-induced lung injury.

Methods

Newborn rats were exposed to FiO2 = 0.6 or room air for 14 days to induce lung derangement and then were administered l-citrulline or a vehicle (sham). Lung histopathology was studied with morphometric features. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected for analysis. Lung vascular endothelial growth factor (VEGF), nitric oxide synthase (eNOS), and matrix metalloproteinase 2 (MMP2) gene and protein expressions were assessed.

Results

Serum l-arginine rose in the L-citr + hyperoxia group (p = 0.05), as well as the Von Willebrand factor stained vessels count (p = 0.0008). Lung VEGF immune staining, localized on endothelial cells, was weaker in the sections under hyperoxia than the l-citr + hyperoxia and room air groups. This pattern was comparable with the VEGF gene and protein expression profiles. Mean alveolar size increased in the untreated hyperoxia and sham-treated groups compared with the groups reared in room air or treated with l-citrulline under exposure to hyperoxia (p = 0.0001). Lung VEGF and eNOS increased in the l-citrulline-treated rats, though this treatment did not change MMP2 gene expression but regulated the MMP2 active protein, which rose in BALF (p = 0.003).

Conclusions

We conclude that administering l-citrulline proved effective in improving alveolar and vascular growth in a model of oxygen-induced pulmonary damage, suggesting better lung growth and matrix regulation than in untreated groups.

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Abbreviations

BPD:

Bronchopulmonary dysplasia

MMP:

Matrix metalloproteinase

BALF:

Bronchoalveolar lavage fluid

l-citr:

l-Citrulline

References

  1. Filippone M, Sartor M, Zacchello F, Baraldi E (2003) Flow limitation in infants with bronchopulmonary dysplasia and respiratory function at school age. Lancet 361:753–754

    Article  PubMed  Google Scholar 

  2. Balasubramaniam V, Mervis CF, Maxey AM, Markham NE, Abman SH (2007) Hyperoxia reduces bone marrow, circulating, and lung endothelial progenitor cells in the developing lung: implications for the pathogenesis of bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol 292:L1073–L1084

    Article  PubMed  CAS  Google Scholar 

  3. Balasubramaniam V, Ingram DA (2009) Endothelial progenitors in the risk of developing bronchopulmonary dysplasia: can we include endothelial progenitor cells in BPD risk assessment? Am J Respir Crit Care Med 180:488–490

    Article  PubMed  Google Scholar 

  4. Abman SH, Baker C, Balasubramaniam V (2008) Growth and development of the lung circulation: mechanisms and clinical implications. In: Bancalari E (ed) The newborn lung. Saunders Elsevier, Philadelphia, pp 50–72

    Chapter  Google Scholar 

  5. Jakkula M, Le Cras TD, Gebb S, Hirth KP, Tuder RM, Voelkel NF, Abman SH (2000) Inhibition of angiogenesis decreases alveolarization in the developing rat lung. Am J Physiol Lung Cell Mol Physiol 279:L600–L607

    PubMed  CAS  Google Scholar 

  6. Tang K, Rossiter HB, Wagner PD, Breen EC (2004) Lung-targeted VEGF inactivation leads to an emphysema phenotype in mice. J Appl Physiol 97:1559–1566

    Article  PubMed  CAS  Google Scholar 

  7. Kallapur SG, Bachurski CJ, Le Cras TD, Joshi SN, Ikegami M, Jobe AH (2004) Vascular changes after intra-amniotic endotoxin in preterm lamb lungs. Am J Physiol Lung Cell Mol Physiol 287:L1178–L1185

    Article  PubMed  CAS  Google Scholar 

  8. Lin YJ, Markham NE, Balasubramaniam V, Tang JR, Maxey A, Kinsella JP, Abman SH (2005) Inhaled nitric oxide enhances distal lung growth after exposure to hyperoxia in neonatal rats. Pediatr Res 58:22–29

    Article  PubMed  CAS  Google Scholar 

  9. McCurnin DC, Pierce RA, Chang LY, Gibson LL, Osborne-Lawrence S, Yoder BA, Kerecman JD, Albertine KH, Winter VT, Coalson JJ, Crapo JD, Grubb PH, Shaul PW (2005) Inhaled NO improves early pulmonary function and modifies lung growth and elastin deposition in a baboon model of neonatal chronic lung disease. Am J Physiol Lung Cell Mol Physiol 288:L450–L459

    Article  PubMed  CAS  Google Scholar 

  10. ter Horst SA, Walther FJ, Poorthuis BJ, Hiemstra PS, Wagenaar GT (2007) Inhaled nitric oxide attenuates pulmonary inflammation and fibrin deposition and prolongs survival in neonatal hyperoxic lung injury. Am J Physiol Lung Cell Mol Physiol 293:L35–L44

    Article  PubMed  CAS  Google Scholar 

  11. Bode-Böger SM, Muke J, Surdacki A, Brabant G, Böger RH, Frölich JC (2003) Oral 1-arginine improves endothelial function in healthy individuals older than 70 years. Vasc Med 8:77–81

    Article  PubMed  Google Scholar 

  12. Piatti PM, Monti LD, Valsecchi G, Magni F, Setola E, Marchesi F, Galli-Kienle M, Pozza G, Alberti KG (2001) Long-term oral l-arginine administration improves peripheral and hepatic insulin sensitivity in type 2 diabetic patients. Diabetes Care 24:875–880

    Article  PubMed  CAS  Google Scholar 

  13. Walker HA, McGing E, Fisher I, Böger RH, Bode-Böger SM, Jackson G, Ritter JM, Chowienczyk PJ (2001) Endothelium-dependent vasodilation is independent of the plasma l-arginine/ADMA ratio in men with stable angina: lack of effect of oral l-arginine on endothelial function, oxidative stress and exercise performance. J Am Coll Cardiol 38:499–505

    Article  PubMed  CAS  Google Scholar 

  14. Morris SM Jr (2004) Enzymes of arginine metabolism. J Nutr 134:2743S–2747S

    PubMed  CAS  Google Scholar 

  15. Curis E, Nicolis I, Moinard C, Osowska S, Zerrouk N, Benazeth S, Cynober L (2005) Almost all about l-citrulline in mammals. Amino Acids 29:177–205

    Article  PubMed  CAS  Google Scholar 

  16. Waugh WH, Daeschner CW, Files BA, McConnell ME, Strandjord SE (2001) Oral l-citrulline as arginine precursor may be beneficial in sickle cell disease: early phase two results. J Natl Med Assoc 93:363–371

    PubMed  CAS  Google Scholar 

  17. Schwedhelm E, Maas R, Freese R, Jung D, Lukacs Z, Jambrecina A, Spickler W, Schulze F, Böger RH (2008) Pharmacokinetic and pharmacodynamic properties of oral l-citrulline and l-arginine: impact on nitric oxide metabolism. Br J Clin Pharmacol 65:51–59

    Article  PubMed  CAS  Google Scholar 

  18. Solomonson LP, Flam BR, Pendleton LC, Goodwin BL, Eichler DC (2003) The caveolar nitric oxide synthase/arginine regeneration system for NO production in endothelial cells. J Exp Biol 206:2083–2087

    Article  PubMed  CAS  Google Scholar 

  19. Greenlee KJ, Werb Z, Kheradmand F (2007) Matrix metalloproteinases in lung: multiple, multifarious, and multifaceted. Physiol Rev 87:69–98

    Article  PubMed  CAS  Google Scholar 

  20. Chetty C, Lakka SS, Bhoopathi P, Rao JS (2010) MMP-2 alters VEGF expression via alphaVbeta3 integrin-mediated PI3 K/AKT signaling in A549 lung cancer cells. Int J Cancer 127:1081–1095

    Article  PubMed  CAS  Google Scholar 

  21. Guidolin D, Albertin G, Spinazzi R, Sorato E, Mascarin A, Cavallo D, Antonello M, Ribatti D (2008) Adrenomedullin stimulates angiogenic response in cultured human vascular endothelial cells: involvement of the vascular endothelial growth factor receptor 2. Peptides 29:2013–2023

    Article  PubMed  CAS  Google Scholar 

  22. Kubista M, Andrade JM, Bengtsson M, Forootan A, Jonak J, Lind K, Sindelka R, Sjoback R, Sjogreen B, Strombom L, Stahlberg A, Zoric N (2006) The real-time polymerase chain reaction. Mol Aspects Med 27:95–125

    Article  PubMed  CAS  Google Scholar 

  23. Onisto M, Slongo ML, Gregnanin L, Gastaldi T, Carli M, Rosolen A (2005) Expression and activity of vascular endothelial growth factor and metalloproteinases in alveolar and embryonal rhabdomyosarcoma cell lines. Int J Oncol 27:791–798

    PubMed  CAS  Google Scholar 

  24. Thurlbeck WM (1975) Postnatal growth and development of the lung. Am Rev Respir Dis 111:803–844

    PubMed  CAS  Google Scholar 

  25. Yi M, Jankov RP, Belcastro R, Humes D, Copland I, Shek S, Sweezey NB, Post M, Albertine KH, Auten RL, Tanswell AK (2004) Opposing effects of 60% oxygen and neutrophil influx on alveologenesis in the neonatal rat. Am J Respir Crit Care Med 170:1188–1196

    Article  PubMed  Google Scholar 

  26. Masood A, Yi M, Lau M, Belcastro R, Shek S, Pan J, Kantores C, McNamara PJ, Kavanagh BP, Belik J, Jankov RP, Tanswell AK (2009) Therapeutic effects of hypercapnia on chronic lung injury and vascular remodeling in neonatal rats. Am J Physiol Lung Cell Mol Physiol 297:L920–L930

    Article  PubMed  CAS  Google Scholar 

  27. Donohue PK, Gilmore MM, Cristofalo E, Wilson RF, Weiner JZ, Lau BD, Robinson KA, Allen MC (2011) Inhaled nitric oxide in preterm infants: a systematic review. Pediatrics 127:e414–e422

    Article  PubMed  Google Scholar 

  28. Vadivel A, Aschner JL, Rey-Parra GJ, Magarik J, Zeng H, Summar M, Eaton F, Thébaud B (2010) L-citrulline attenuates arrested alveolar growth and pulmonary hypertension in oxygen-induced lung injury in newborn rats. Pediatr Res 68:519–525

    Article  PubMed  CAS  Google Scholar 

  29. Husain AN, Siddiqui NH, Stocker JT (1998) Pathology of arrested acinar development in postsurfactant bronchopulmonary dysplasia. Hum Pathol 29:710–717

    Article  PubMed  CAS  Google Scholar 

  30. Jobe AH, Bancalari E (2001) Bronchopulmonary dysplasia. Am J Respir Crit Care Med 163:1723–1729

    PubMed  CAS  Google Scholar 

  31. Duda DG, Fukumura D, Jain RK (2004) Role of eNOS in neovascularization: NO for endothelial progenitor cells. Trends Mol Med 10:143–145

    Article  PubMed  CAS  Google Scholar 

  32. Ziche M, Morbidelli L, Choudhuri R, Zhang HT, Donnini S, Granger HJ, Bicknell R (1997) Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J Clin Invest 99:2625–2634

    Article  PubMed  CAS  Google Scholar 

  33. Dulak J, Józkowicz A (2003) Regulation of vascular endothelial growth factor synthesis by nitric oxide: facts and controversies. Antioxid Redox Signal 5:123–132

    Article  PubMed  CAS  Google Scholar 

  34. Ananthakrishnan M, Barr FE, Summar ML, Smith HA, Kaplowitz M, Cunningham G, Magarik J, Zhang Y, Fike CD (2009) L-citrulline ameliorates chronic hypoxia-induced pulmonary hypertension in newborn piglets. Am J Physiol Lung Cell Mol Physiol 297:L506–L511

    Article  PubMed  CAS  Google Scholar 

  35. Crosby LM, Waters CM (2010) Epithelial repair mechanisms in the lung. Am J Physiol Lung Cell Mol Physiol 298:L715–L731

    Article  PubMed  CAS  Google Scholar 

  36. van Hinsbergh VW, Engelse MA, Quax PH (2006) Pericellular proteases in angiogenesis and vasculogenesis. Arterioscler Thromb Vasc Biol 26:716–728

    Article  PubMed  Google Scholar 

  37. Walter A, Etienne-Selloum N, Sarr M, Kane MO, Beretz A, Schini-Kerth VB (2008) Angiotensin II induces the vascular expression of VEGF and MMP-2 in vivo: preventive effect of red wine polyphenols. J Vasc Res 45:386–394

    Article  PubMed  CAS  Google Scholar 

  38. Le NT, Xue M, Castelnoble LA, Jackson CJ (2007) The dual personalities of matrix metalloproteinases in inflammation. Front Biosci 12:1475–1487

    Article  PubMed  CAS  Google Scholar 

  39. Kheradmand F, Rishi K, Werb Z (2002) Signaling through the EGF receptor controls lung morphogenesis in part by regulating MT1-MMP-mediated activation of gelatinase A/MMP2. J Cell Sci 115:839–848

    PubMed  CAS  Google Scholar 

  40. Danan C, Jarreau PH, Franco ML, Dassieu G, Grillon C, Abd Alsamad I, Lafuma C, Harf A, Delacourt C (2002) Gelatinase activities in the airways of premature infants and development of bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol 283:L1086–L1093

    PubMed  CAS  Google Scholar 

  41. Schulz CG, Sawicki G, Lemke RP, Roeten BM, Schulz R, Cheung PY (2004) MMP-2 and MMP-9 and their tissue inhibitors in the plasma of preterm and term neonates. Pediatr Res 55:794–801

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors are indebted to Professor Franco Zacchello, who strongly encouraged the creation of the Neonatal Developmental Biology Group, and to Professor A. S. Belloni for her kind advice. The authors also thank the personnel at the Department of Anatomy and Physiology (G. Sarasin, A. Rambaldo, and D. Guidolin), at the Pathology Section of the Department of Oncological and Surgical Sciences, and at the NICU, and the Associazione Pulcino. This work was funded by the Italian Ministry of Health grant CPDA081132/08, University of Padova, Padova, Italy.

Conflicts of interest

The authors have no conflict of interests or financial ties to disclose.

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Authors and Affiliations

  1. Neonatal Intensive Care Unit, Department of Pediatrics, University of Padova, Padova, Italy

    Davide Grisafi, Arben Dedja, Anna Milan, Michela Alfiero Bordigato, Lino Chiandetti, Marco Filippone & Patrizia Zaramella

  2. Department of Experimental Biomedical Sciences, University of Padova, Padova, Italy

    Evelyne Tassone, Valentina Masola & Maurizio Onisto

  3. Department of Anatomy and Physiology, University of Padova, Padova, Italy

    Barbara Oselladore, Andrea Porzionato, Giovanna Albertin, Veronica Macchi & Raffaele De Caro

  4. Section of Pathology, Department of Oncological and Surgical Sciences, University of Padova, Padova, Italy

    Vincenza Guzzardo, Roberto Salmaso & Ambrogio Fassina

  5. Department of Laboratory Medicine, University-Hospital, Padova, Italy

    Carlo Artusi & Martina Zaninotto

  6. Department of Pediatrics, Neonatal Intensive Care Unit, University-Hospital, Padova, via Giustiniani, 3, 35128, Padova, Italy

    Patrizia Zaramella

Authors
  1. Davide Grisafi
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  2. Evelyne Tassone
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Corresponding author

Correspondence to Patrizia Zaramella.

Additional information

D. Grisafi and E. Tassone contributed equally to this work.

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Grisafi, D., Tassone, E., Dedja, A. et al. L-citrulline Prevents Alveolar and Vascular Derangement in a Rat Model of Moderate Hyperoxia-induced Lung Injury. Lung 190, 419–430 (2012). https://doi.org/10.1007/s00408-012-9382-z

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  • DOI: https://doi.org/10.1007/s00408-012-9382-z

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