Int J Stem Cells.  2016 May;9(1):145-151. 10.15283/ijsc.2016.9.1.145.

The Effect of Bone Marrow Mononuclear Cells on Lung Regeneration and Apoptosis in a Simple Model of Pulmonary Emphysema

• El-Badrawy MK
• Shalabi NM
• Mohamed MA
• Ragab A
• Abdelwahab HW
• Anber N
• Sobh MA
• Khater Y
• Abdel Hamid AA

• KMID: 2164171
• DOI: http://doi.org/10.15283/ijsc.2016.9.1.145

Abstract

BACKGROUND
In severe chronic stages of emphysema the only treatment is lung transplantation. SO, an urgent need exists for the development of effective treatments. Stem cells therapy arises as a new therapeutic approach. AIM OF THE WORK: To investigate whether bone marrow mononuclar cells (BMMNCs) can promote lung regeneration and decrease apoptosis in lipopolysaccharide (LPS) induced pulmonary emphysema in C57Bl/6 mice. MATERIAL AND METHODS: 14 weeks old female mice (C57Bl/6), weighing around 25 g were used in this study. The mice were divided into 4 groups (10 in each group): group A: mice received no treatment, group B: mice received intranasal instillation of LPS with no further treatment, group C: mice received intranasal instillation of LPS then given a dose of BMMNCs and evaluated 21 days later and group D: the mice that received intranasal instillation of LPS then given a dose of Dulbecco's Modified Eagle's Medium (DMEM) and evaluated 21 days later. Imaging analysis was done using imagej program. To measure apoptotic index, Anti-caspase 3 polyclonal antibody staining was done.
RESULTS
Analysis of the mean of airspace equivalent diameters (D0) and its statistical distribution (D1) for the different groups allowed to observe that group treated with BMMNCs (group C) showed the significant improvement in D0 and D1 than the group received LPS only (group B). Analysis of apoptotic index showed significant difference between BMMNCs treated group (group C) and that received LPS only (group B).
CONCLUSIONS
BMMNCs effectively promote lung regeneration and reduction of apoptosis in pulmonary emphysema.

Keyword

Emphysema; Stem cells; Mononuclear cells; Apoptosis; Lipopolysaccharide

MeSH Terms

Animals
Apoptosis*
Bone Marrow*
Emphysema
Female
Humans
Lung Transplantation
Lung*
Mice
Pulmonary Emphysema*
Regeneration*
Stem Cells

Figure

• 

  Fig. 1 Cluster bar graph represents means and standard deviations of the mean of airspace equivalent diameters (D0) and its statistical distribution (D1) in studied groups. Group A: (the mice that received no treatment), group B (the mice received intranasal instillation of LPS), group C (the mice received BMMNCs after LPS instillation) and group D (the mice received DMEM media after LPS instillation).

• 

  Fig. 2 Lung parenchyma stained with hematoxylin-eosin, (A) represents group A (the mouse that received no treatment), (B) represents group B (the mouse that received LPS) with an overall increase in air space size compared to group A, (C) represents group C (the mouse received BMMNCs after LPS) with decrease in air space size compared to group B and (D) represents group D (the mouse received DMEM media after LPS instillation) with an overall increase in air space size similar to group B.

• 

  Fig. 3 Lung sections stained with immunohistochemistry for activated caspase3; (A) showing increased number of caspase3 positive cells in the alveolar septa (arrows) of mouse received LPS only (group B); (B) BMMNCs treated mouse lung (group C), showing decreased number of caspase3 positive cells in the alveolar septa in the lung (arrow); (C) 40× magnification to show caspase-3 positive cells.

Reference

References

1. Snider GL. Distinguishing among asthma, chronic bronchitis, and emphysema. Chest. 1985; 87(1 Suppl):35S–39S. DOI: 10.1378/chest.87.1.35S. PMID: 3964740.

2. Miravitlles M, Calle M, Soler-Cataluña JJ. Clinical phenotypes of COPD: identification, definition and implications for guidelines. Arch Bronconeumol. 2012; 48:86–98. DOI: 10.1016/j.arbres.2011.10.007.

3. Agustí AG, Noguera A, Sauleda J, Sala E, Pons J, Busquets X. Systemic effects of chronic obstructive pulmonary disease. Eur Respir J. 2003; 21:347–360. DOI: 10.1183/09031936.03.00405703. PMID: 12608452.

4. Chen ZH, Kim HP, Ryter SW, Choi AM. Identifying targets for COPD treatment through gene expression analyses. Int J Chron Obstruct Pulmon Dis. 2008; 3:359–370. PMID: 18990963. PMCID: 2629979.

5. Vidal D, Fortunato G, Klein W, Cortizo L, Vasconcelos J, Ribeiro-Dos-Santos R, Soares M, Macambira S. Alterations in pulmonary structure by elastase administration in a model of emphysema in mice is associated with functional disturbances. Rev Port Pneumol. 2012; 18:128–136. DOI: 10.1016/j.rppneu.2011.12.007. PMID: 22425364.

6. Hackett TL, Knight DA, Sin DD. Potential role of stem cells in management of COPD. Int J Chron Obstruct Pulmon Dis. 2010; 5:81–88. PMID: 20463889. PMCID: 2865028.

7. Cruz FF, Antunes MA, Abreu SC, Fujisaki LC, Silva JD, Xisto DG, Maron-Gutierrez T, Ornellas DS, Sá VK, Rocha NN, Capelozzi VL, Morales MM, Rocco PR. Protective effects of bone marrow mononuclear cell therapy on lung and heart in an elastase-induced emphysema model. Respir Physiol Neurobiol. 2012; 182:26–36. DOI: 10.1016/j.resp.2012.01.002. PMID: 22266352.

8. Parameswaran H, Majumdar A, Ito S, Alencar AM, Suki B. Quantitative characterization of airspace enlargement in emphysema. J Appl Physiol (1985). 2006; 100:186–193. DOI: 10.1152/japplphysiol.00424.2005.

9. Liu H, Ma L, Wu J, Wang K, Chen X. Apoptosis of alveolar wall cells in chronic obstructive pulmonary disease patients with pulmonary emphysema is involved in emphysematous changes. J Huazhong Univ Sci Technolog Med Sci. 2009; 29:466–469. DOI: 10.1007/s11596-009-0415-7. PMID: 19662364.

10. Dawkins PA, Stockley RA. Animal models of chronic obstructive pulmonary disease. Thorax. 2001; 56:972–977. DOI: 10.1136/thorax.56.12.972. PMID: 11713362. PMCID: 1745992.

11. Obot C, Lee K, Fuciarelli A, Renne R, McKinney W. Characterization of mainstream cigarette smoke-induced bio-marker responses in ICR and C57Bl/6 mice. Inhal Toxicol. 2004; 16:701–719. DOI: 10.1080/08958370490476604. PMID: 15371058.

12. Phillips B, Veljkovic E, Peck MJ, Buettner A, Elamin A, Guedj E, Vuillaume G, Ivanov NV, Martin F, Boué S, Schlage WK, Schneider T, Titz B, Talikka M, Vanscheeuwijck P, Hoeng J, Peitsch MC. A 7-month cigarette smoke inhalation study in C57BL/6 mice demonstrates reduced lung inflammation and emphysema following smoking cessation or aerosol exposure from a prototypic modified risk tobacco product. Food Chem Toxicol. 2015; 80:328–345. DOI: 10.1016/j.fct.2015.03.009. PMID: 25843363.

13. Massaro D, Massaro GD. Developmental alveologenesis: longer, differential regulation and perhaps more danger. Am J Physiol Lung Cell Mol Physiol. 2007; 293:L568–L569. DOI: 10.1152/ajplung.00258.2007. PMID: 17631611.

14. Tibboel J, Reiss I, de Jongste JC, Post M. Ceramides: a potential therapeutic target in pulmonary emphysema. Respir Res. 2013; 14:96. DOI: 10.1186/1465-9921-14-96. PMID: 24083966. PMCID: 3851206.

15. Vernooy JH, Dentener MA, van Suylen RJ, Buurman WA, Wouters EF. Long-term intratracheal lipopolysaccharide exposure in mice results in chronic lung inflammation and persistent pathology. Am J Respir Cell Mol Biol. 2002; 26:152–159. DOI: 10.1165/ajrcmb.26.1.4652.

16. Brass DM, Hollingsworth JW, Cinque M, Li Z, Potts E, Toloza E, Foster WM, Schwartz DA. Chronic LPS inhalation causes emphysema-like changes in mouse lung that are associated with apoptosis. Am J Respir Cell Mol Biol. 2008; 39:584–590. DOI: 10.1165/rcmb.2007-0448OC. PMID: 18539952. PMCID: 2574529.

17. van Houwelingen AH, Weathington NM, Verweij V, Blalock JE, Nijkamp FP, Folkerts G. Induction of lung emphysema is prevented by L-arginine-threonine-arginine. FASEB J. 2008; 22:3403–3408. DOI: 10.1096/fj.07-096230. PMID: 18556462. PMCID: 2518250.

18. Pera T, Zuidhof A, Valadas J, Smit M, Schoemaker RG, Gosens R, Maarsingh H, Zaagsma J, Meurs H. Tiotropium inhibits pulmonary inflammation and remodelling in a guinea pig model of COPD. Eur Respir J. 2011; 38:789–796. DOI: 10.1183/09031936.00146610. PMID: 21349917.

19. Muñoz-Barrutia A, Ceresa M, Artaechevarria X, Montuenga LM, Ortiz-de-Solorzano C. Quantification of lung damage in an elastase-induced mouse model of emphysema. Int J Biomed Imaging. 2012; 2012:734734. DOI: 10.1155/2012/734734. PMID: 23197972. PMCID: 3503307.

20. Weibel ER, Hsia CC, Ochs M. How much is there really? Why stereology is essential in lung morphometry. J Appl Physiol (1985). 2007; 102:459–467. DOI: 10.1152/japplphysiol.00808.2006.

21. Alvarez-Viejo M, Menendez-Menendez Y, Blanco-Gelaz MA, Ferrero-Gutierrez A, Fernandez-Rodriguez MA, Gala J, Otero-Hernandez J. Quantifying mesenchymal stem cells in the mononuclear cell fraction of bone marrow samples obtained for cell therapy. Transplant Proc. 2013; 45:434–439. DOI: 10.1016/j.transproceed.2012.05.091. PMID: 23375334.

22. Longhini-Dos-Santos N, Barbosa-de-Oliveira VA, Kozma RH, Faria CA, Stessuk T, Frei F, Ribeiro-Paes JT. Cell therapy with bone marrow mononuclear cells in elastase-induced pulmonary emphysema. Stem Cell Rev. 2013; 9:210–218. DOI: 10.1007/s12015-012-9419-y.

23. Zarogoulidis P, Hohenforst-Schmidt W, Huang H, Sahpatzidou D, Freitag L, Sakkas L, Rapti A, Kioumis I, Pitsiou G, Kouzi-Koliakos K, Papamichail A, Papaiwannou A, Tsiouda T, Tsakiridis K, Porpodis K, Lampaki S, Organtzis J, Gschwendtner A, Zarogoulidis K. A gene therapy induced emphysema model and the protective role of stem cells. Diagn Pathol. 2014; 9:195. DOI: 10.1186/s13000-014-0195-7. PMID: 25394479. PMCID: 4243373.

24. Yuhgetsu H, Ohno Y, Funaguchi N, Asai T, Sawada M, Takemura G, Minatoguchi S, Fujiwara H, Fujiwara T. Beneficial effects of autologous bone marrow mononuclear cell transplantation against elastase-induced emphysema in rabbits. Exp Lung Res. 2006; 32:413–426. DOI: 10.1080/01902140601047633. PMID: 17162649.

25. Rojas M, Xu J, Woods CR, Mora AL, Spears W, Roman J, Brigham KL. Bone marrow-derived mesenchymal stem cells in repair of the injured lung. Am J Respir Cell Mol Biol. 2005; 33:145–152. DOI: 10.1165/rcmb.2004-0330OC. PMID: 15891110. PMCID: 2715309.