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Degradation of Lung Protective Angiotensin Converting Enzyme-2 by Meconium in Human Alveolar Epithelial Cells: A Potential Pathogenic Mechanism in Meconium Aspiration Syndrome

  • Chintan K. Gandhi
  • Romel Holmes
  • Ira H. Gewolb
  • Bruce D. UhalEmail author



Pancreatic digestive enzymes present in meconium might be responsible for meconium-induced lung injury. The local Renin Angiotensin System plays an important role in lung injury and inflammation. Particularly, angiotensin converting enzyme-2 (ACE-2) has been identified as a protective lung enzyme against the insult. ACE-2 converts pro-apoptotic Angiotensin II to anti-apoptotic Angiotensin 1–7. However, the effect of meconium on ACE-2 has never been studied before.


To study the effect of meconium on ACE-2, and whether inhibition of proteolytic enzymes present in the meconium reverses its effects on ACE-2.


Alveolar epithelial A549 cells were exposed to F-12 medium, 2.5% meconium, meconium + a protease inhibitor cocktail (PIc) and PIc alone for 16 h. At the end of incubation, apoptosis was measured with a nuclear fragmentation assay and cell lysates were collected for ACE-2 immunoblotting and enzyme activity.


Meconium caused a fourfold increase in apoptotic nuclei (p < 0.001). The pro-apoptotic effect of meconium can be reversed by PIc. Meconium reduced ACE-2 enzyme activity by cleaving ACE-2 into a fragment detected at ~ 37 kDa by immunoblot. PIc prevented the degradation of ACE-2 and restored 50% of ACE-2 activity (p < 0.05).


These data suggest that meconium causes degradation of lung protective ACE-2 by proteolytic enzymes present in meconium, since the effects of meconium can be reversed by PIc.


Meconium aspiration syndrome Angiotensin-converting enzyme 2 Renin-angiotensin system Neonatal lung injury 



We thank Dr. Amal T. Abdul-Hafez for helping in designing and executing the experiments. We also thank Sparrow Hospital (Lansing, MI) Newborn Unit nursing staff for helping in collecting the meconium for the study.


This work was supported by a grant from the Fellowship Research Fund of Sparrow Hospital, Lansing, MI (to C.G).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Approval

The study protocol was approved by the Sparrow Hospital and Michigan State University institutional review board.


  1. 1.
    Gandhi CK (2018) Management of meconium-stained newborns in the delivery room. Neonatal Netw NN 37(3):141–148. Google Scholar
  2. 2.
    Tyler DC, Murphy J, Cheney FW (1978) Mechanical and chemical damage to lung tissue caused by meconium aspiration. Pediatrics 62(4):454–459Google Scholar
  3. 3.
    Zagariya A, Bhat R, Chari G, Uhal B, Navale S, Vidyasagar D (2005) Apoptosis of airway epithelial cells in response to meconium. Life Sci 76(16):1849–1858. Google Scholar
  4. 4.
    Gandhi C, Uhal BD (2016) Roles of the angiotensin system in neonatal lung injury and disease. JSM Atheroscler 1(3):1014PubMedCentralGoogle Scholar
  5. 5.
    Sparks MA, Crowley SD, Gurley SB, Mirotsou M, Coffman TM (2014) Classical renin-angiotensin system in kidney physiology. Compr Physiol 4(3):1201–1228. PubMedCentralGoogle Scholar
  6. 6.
    Yoshiji H, Kuriyama S, Yoshii J, Ikenaka Y, Noguchi R, Nakatani T, Tsujinoue H, Fukui H (2001) Angiotensin-II type 1 receptor interaction is a major regulator for liver fibrosis development in rats. Hepatology 34(4 Pt 1):745–750. Google Scholar
  7. 7.
    Paul M, Poyan Mehr A, Kreutz R (2006) Physiology of local renin-angiotensin systems. Physiol Rev 86(3):747–803. Google Scholar
  8. 8.
    Chen LN, Yang XH, Nissen DH, Chen YY, Wang LJ, Wang JH, Gao JL, Zhang LY (2013) Dysregulated renin-angiotensin system contributes to acute lung injury caused by hind-limb ischemia-reperfusion in mice. Shock (Augusta GA) 40(5):420–429. Google Scholar
  9. 9.
    Liu L, Qiu HB, Yang Y, Wang L, Ding HM, Li HP (2009) Losartan, an antagonist of AT1 receptor for angiotensin II, attenuates lipopolysaccharide-induced acute lung injury in rat. Arch Biochem Biophys 481(1):131–136. Google Scholar
  10. 10.
    Marshall RP (2003) The pulmonary renin-angiotensin system. Curr Pharm Des 9(9):715–722Google Scholar
  11. 11.
    Zhang H, Sun GY (2005) LPS induces permeability injury in lung microvascular endothelium via AT(1) receptor. Arch Biochem Biophys 441(1):75–83. Google Scholar
  12. 12.
    Wang R, Alam G, Zagariya A, Gidea C, Pinillos H, Lalude O, Choudhary G, Oezatalay D, Uhal BD (2000) Apoptosis of lung epithelial cells in response to TNF-alpha requires angiotensin II generation de novo. J Cell Physiol 185 (2):253–259.;2-%23 Google Scholar
  13. 13.
    Li X, Molina-Molina M, Abdul-Hafez A, Uhal V, Xaubet A, Uhal BD (2008) Angiotensin converting enzyme-2 is protective but downregulated in human and experimental lung fibrosis. Am J Physiol Lung Cell Mol Physiol 295(1):L178–L185. PubMedCentralGoogle Scholar
  14. 14.
    Antonowicz I, Shwachman H (1979) Meconium in health and in disease. Adv Pediatr 26:275–310Google Scholar
  15. 15.
    Ivanov VA, Gewolb IH, Uhal BD (2010) A new look at the pathogenesis of the meconium aspiration syndrome: a role for fetal pancreatic proteolytic enzymes in epithelial cell detachment. Pediatr Res 68(3):221–224. Google Scholar
  16. 16.
    Uhal BD, Dang M, Dang V, Llatos R, Cano E, Abdul-Hafez A, Markey J, Piasecki CC, Molina-Molina M (2013) Cell cycle dependence of ACE-2 explains downregulation in idiopathic pulmonary fibrosis. Eur Respir J 42(1):198–210. Google Scholar
  17. 17.
    Xiao F, Burns KD (2017) Measurement of angiotensin converting enzyme 2 activity in biological fluid (ACE2). Methods Mol Biol (Clifton NJ) 1527:101–115. Google Scholar
  18. 18.
    Li X, Zhang H, Soledad-Conrad V, Zhuang J, Uhal BD (2003) Bleomycin-induced apoptosis of alveolar epithelial cells requires angiotensin synthesis de novo. Am J Physiol Lung Cell Mol Physiol 284(3):L501–L507. Google Scholar
  19. 19.
    Uhal BD, Joshi I, Hughes WF, Ramos C, Pardo A, Selman M (1998) Alveolar epithelial cell death adjacent to underlying myofibroblasts in advanced fibrotic human lung. Am J Physiol 275(6 Pt 1):L1192–L1199Google Scholar
  20. 20.
    Wosten-van Asperen RM, Lutter R, Specht PA, Moll GN, van Woensel JB, van der Loos CM, van Goor H, Kamilic J, Florquin S, Bos AP (2011) Acute respiratory distress syndrome leads to reduced ratio of ACE/ACE2 activities and is prevented by angiotensin-(1–7) or an angiotensin II receptor antagonist. J Pathol 225(4):618–627. Google Scholar
  21. 21.
    Garg M, Angus PW, Burrell LM, Herath C, Gibson PR, Lubel JS (2012) Review article: the pathophysiological roles of the renin-angiotensin system in the gastrointestinal tract. Aliment Pharmacol Ther 35(4):414–428. Google Scholar
  22. 22.
    Mullinger M, Palasi M (1966) Tryptic and chymotryptic activity of stools of newborn infants. Pediatrics 38(4):657–659Google Scholar
  23. 23.
    Wyllie AH (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284(5756):555–556Google Scholar
  24. 24.
    Riccardi C, Nicoletti I (2006) Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protoc 1(3):1458–1461. Google Scholar
  25. 25.
    Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139(2):271–279Google Scholar
  26. 26.
    Dini L, Coppola S, Ruzittu MT, Ghibelli L (1996) Multiple pathways for apoptotic nuclear fragmentation. Exp Cell Res 223(2):340–347. Google Scholar
  27. 27.
    Oberhammer F, Fritsch G, Schmied M, Pavelka M, Printz D, Purchio T, Lassmann H, Schulte-Hermann R (1993) Condensation of the chromatin at the membrane of an apoptotic nucleus is not associated with activation of an endonuclease. J Cell Sci 104(Pt 2):317–326Google Scholar
  28. 28.
    Ghibelli L, Maresca V, Coppola S, Gualandi G (1995) Protease inhibitors block apoptosis at intermediate stages: a compared analysis of DNA fragmentation and apoptotic nuclear morphology. FEBS Lett 377(1):9–14Google Scholar
  29. 29.
    Gopallawa I, Uhal BD (2016) Angiotensin-(1–7)/mas inhibits apoptosis in alveolar epithelial cells through upregulation of MAP kinase phosphatase-2. Am J Physiol Lung Cell Mol Physiol 310(3):L240–L248. Google Scholar
  30. 30.
    Zagariya A, Bhat R, Uhal B, Navale S, Freidine M, Vidyasagar D (2000) Cell death and lung cell histology in meconium aspirated newborn rabbit lung. Eur J Pediatr 159(11):819–826Google Scholar
  31. 31.
    Rosenfeld CR, Zagariya AM, Liu XT, Willis BC, Fluharty S, Vidyasagar D (2008) Meconium increases type 1 angiotensin II receptor expression and alveolar cell death. Pediatr Res 63(3):251–256. Google Scholar
  32. 32.
    Huang L, Sexton DJ, Skogerson K, Devlin M, Smith R, Sanyal I, Parry T, Kent R, Enright J, Wu QL, Conley G, DeOliveira D, Morganelli L, Ducar M, Wescott CR, Ladner RC (2003) Novel peptide inhibitors of angiotensin-converting enzyme 2. J Biol Chem 278(18):15532–15540. Google Scholar
  33. 33.
    Kuba K, Imai Y, Rao S, Gao H, Guo F, Guan B, Huan Y, Yang P, Zhang Y, Deng W, Bao L, Zhang B, Liu G, Wang Z, Chappell M, Liu Y, Zheng D, Leibbrandt A, Wada T, Slutsky AS, Liu D, Qin C, Jiang C, Penninger JM (2005) A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury. Nat Med 11(8):875–879. Google Scholar
  34. 34.
    Imai Y, Kuba K, Rao S, Huan Y, Guo F, Guan B, Yang P, Sarao R, Wada T, Leong-Poi H, Crackower MA, Fukamizu A, Hui CC, Hein L, Uhlig S, Slutsky AS, Jiang C, Penninger JM (2005) Angiotensin-converting enzyme 2 protects from severe acute lung failure. Nature 436(7047):112–116. Google Scholar
  35. 35.
    Wiener RS, Cao YX, Hinds A, Ramirez MI, Williams MC (2007) Angiotensin converting enzyme 2 is primarily epithelial and is developmentally regulated in the mouse lung. J Cell Biochem 101(5):1278–1291. Google Scholar
  36. 36.
    Oarhe CI, Dang V, Dang M, Nguyen H, Gopallawa I, Gewolb IH, Uhal BD (2015) Hyperoxia downregulates angiotensin-converting enzyme-2 in human fetal lung fibroblasts. Pediatr Res 77(5):656–662. PubMedCentralGoogle Scholar
  37. 37.
    Mohamed TL, Nguyen HT, Abdul-Hafez A, Dang VX, Dang MT, Gewolb IH, Uhal BD (2016) Prior hypoxia prevents downregulation of ACE-2 by hyperoxia in fetal human lung fibroblasts. Exp Lung Res 42(3):121–130. PubMedCentralGoogle Scholar
  38. 38.
    Khan A, Benthin C, Zeno B, Albertson TE, Boyd J, Christie JD, Hall R, Poirier G, Ronco JJ, Tidswell M, Hardes K, Powley WM, Wright TJ, Siederer SK, Fairman DA, Lipson DA, Bayliffe AI, Lazaar AL (2017) A pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome. Critic Care (Lond Engl) 21(1):234. Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Chintan K. Gandhi
    • 1
    • 2
  • Romel Holmes
    • 3
  • Ira H. Gewolb
    • 1
  • Bruce D. Uhal
    • 4
    Email author
  1. 1.Division of Neonatology, Department of Pediatrics & Human DevelopmentMichigan State UniversityEast LansingUSA
  2. 2.Division of Neonatal Perinatal MedicinePennsylvania State Health Children’s HospitalHersheyUSA
  3. 3.BiologyTuskegee UniversityTuskegeeUSA
  4. 4.Department of PhysiologyMichigan State UniversityEast LansingUSA

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