Tuberc Respir Dis.  2015 Jul;78(3):239-245. 10.4046/trd.2015.78.3.239.

The Therapeutic Effects of Optimal Dose of Mesenchymal Stem Cells in a Murine Model of an Elastase Induced-Emphysema

• Kim YS
• Kim JY
• Huh JW
• Lee SW
• Choi SJ
• Oh YM

• KMID: 2320649
• DOI: http://doi.org/10.4046/trd.2015.78.3.239

Abstract

BACKGROUND
Chronic obstructive pulmonary disease is characterized by emphysema, chronic bronchitis, and small airway remodeling. The alveolar destruction associated with emphysema cannot be repaired by current clinical practices. Stem cell therapy has been successfully used in animal models of cigarette smoke- and elastase-induced emphysema. However, the optimal dose of mesenchymal stem cells (MSCs) for the most effective therapy has not yet been determined. It is vital to determine the optimal dose of MSCs for clinical application in emphysema cases.
METHODS
In the present study, we evaluated the therapeutic effects of various doses of MSCs on elastase-induced emphysema in mice. When 3 different doses of MSCs were intravenously injected into mice treated with elastase, only 5x10(4) MSCs showed a significant effect on the emphysematous mouse lung. We also identified action mechanisms of MSCs based on apoptosis, lung regeneration, and protease/antiprotease imbalance.
RESULTS
The MSCs were not related with caspase-3/7 dependent apoptosis. But activity of matrix metalloproteinase 9 increased by emphysematous lung was decreased by intravenously injected MSCs. Vascular endothelial growth factor were also increased in lung from MSC injected mice, as compared to un-injected mice.
CONCLUSION
This is the first study on the optimal dose of MSCs as a therapeutic candidate. This data may provide important basic data for determining dosage in clinical application of MSCs in emphysema patients.

Keyword

Emphysema; Mesenchymal Stromal Cells; Therapy

MeSH Terms

Airway Remodeling
Animals
Apoptosis
Bronchitis, Chronic
Emphysema
Humans
Lung
Matrix Metalloproteinase 9
Mesenchymal Stromal Cells*
Methods
Mice
Models, Animal
Pancreatic Elastase*
Pulmonary Disease, Chronic Obstructive
Regeneration
Stem Cells
Tobacco Products
Vascular Endothelial Growth Factor A
Matrix Metalloproteinase 9
Pancreatic Elastase
Vascular Endothelial Growth Factor A

Figure

• 

  Figure 1 Optimal dose of mesenchymal stem cells (MSCs) to repair elastase induced emphysema in mice. C57BL/6J mice were administered 0.4 U of elastase on day 0 by intratracheal application and then intravenously injected with MSCs on day 7. Data are measures of the histological staining with hematoxylin and eosin in lung section on day 14. (A) Control (n=5), Ela (elastase only, n=15), Ela+1×104 (elastase+1×104 MSC, n=10), Ela+2.5×104 (elastase+2.5×104 MSC, n=10), Ela+5×104 (elastase+5×104 MSC, n=16), and Ela+1×105 (elastase+1×105 MSC, n=6) (×10). Scale bars=1.0 mm. (B) Morphometic analysis of the mean linear intercept. Values are presented as the mean±SEM.

• 

  Figure 2 The anti-apoptotic effects of mesenchymal stem cell (MSC) in elastase induced emphysema. C57BL/6J mice were intratracheally applied with 0.4 U of elastase on day 0 and then intravenously injected with MSCs on day 7. Lung tissue were collected on day 14 (n=6-9 per group). Caspase 3/7 activity was measured with fluorimetric emzymatic assay and normalized by protein concentration in lung homogenates. Ela: elastase only; Ela+5×104: elastase+5×104 MSC.

• 

  Figure 3 The effects on protease and anti-protease in elastase induced emphysema with or without mesenchymal stem cells (MSCs). C57BL/6J mice were intratracheally applied with 0.4 U of elastase on day 0 and then intravenously injected with MSCs on day 7. Lung tissues were collected on day 14 (n=6-9 per group). (A) Matrix metalloproteinase (MMP) 2, MMP9, and MMP12 mRNA expression were measured with quantitative polymerase chain reaction (PCR) using SYBR Green and normalized by β-actin expression and then displayed with ratio to control group. Values are presented as the mean±SEM. *p<0.05, as compared with control group. (B) Activity of MMP2 and 9 were measured with gelatin zymography. (C) The mRNA expression of SLPI (left) and tissue inhibitor of metalloproteinases-1 (TIMP1) (right) were measured with quantitative PCR using SYBR Green and normalized by β-actin expression and then displayed with ratio to control group. PBS: phosphate buffered saline; Ela: elastase only; Ela+5×104: elastase+5×104 MSC.

• 

  Figure 4 The production of growth factors in elastase induced emphysema with or without mesenchymal stem cells (MSCs). C57BL/6J mice were intratracheally applied with 0.4 U of elastase on day 0 and then intravenously injected with MSCs on day 7. Lung tissue were collected on day14 (n=5-9 per group). The growth factors were measured with enzyme-linked immunosorbent assay for hepatocyte growth factor (HGF) (A), fibroblast growth factor 2 (FGF2) (B), and vascular endothelial growth factor (VEGF) (C). Values are presented as the mean±SEM. *p<0.05 was statistical significance of the comparison between 2 groups. Ela: elastase only; Ela+5×104: elastase+5×104 MSC.

Reference

1. Senior RM, Anthonisen NR. Chronic obstructive pulmonary disease (COPD). Am J Respir Crit Care Med. 1998; 157(4 Pt 2):S139–S147.

2. Sutherland ER, Cherniack RM. Management of chronic obstructive pulmonary disease. N Engl J Med. 2004; 350:2689–2697.

3. Churg A, Cosio M, Wright JL. Mechanisms of cigarette smoke-induced COPD: insights from animal models. Am J Physiol Lung Cell Mol Physiol. 2008; 294:L612–L631.

4. Segura-Valdez L, Pardo A, Gaxiola M, Uhal BD, Becerril C, Selman M. Upregulation of gelatinases A and B, collagenases 1 and 2, and increased parenchymal cell death in COPD. Chest. 2000; 117:684–694.

5. Yokohori N, Aoshiba K, Nagai A. Respiratory Failure Research Group in Japan. Increased levels of cell death and proliferation in alveolar wall cells in patients with pulmonary emphysema. Chest. 2004; 125:626–632.

6. Aaron SD, Vandemheen KL, Fergusson D, Maltais F, Bourbeau J, Goldstein R, et al. Tiotropium in combination with placebo, salmeterol, or fluticasone-salmeterol for treatment of chronic obstructive pulmonary disease: a randomized trial. Ann Intern Med. 2007; 146:545–555.

7. Calverley PM, Anderson JA, Celli B, Ferguson GT, Jenkins C, Jones PW, et al. Salmeterol and fluticasone propionate and survival in chronic obstructive pulmonary disease. N Engl J Med. 2007; 356:775–789.

8. Huh JW, Kim SY, Lee JH, Lee JS, Van Ta Q, Kim M, et al. Bone marrow cells repair cigarette smoke-induced emphysema in rats. Am J Physiol Lung Cell Mol Physiol. 2011; 301:L255–L266.

9. Longhini-Dos-Santos N, Barbosa-de-Oliveira VA, Kozma RH, Faria CA, Stessuk T, Frei F, et al. Cell therapy with bone marrow mononuclear cells in elastase-induced pulmonary emphysema. Stem Cell Rev. 2013; 9:210–218.

10. Schweitzer KS, Johnstone BH, Garrison J, Rush NI, Cooper S, Traktuev DO, et al. Adipose stem cell treatment in mice attenuates lung and systemic injury induced by cigarette smoking. Am J Respir Crit Care Med. 2011; 183:215–225.

11. Kern S, Eichler H, Stoeve J, Kluter H, Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells. 2006; 24:1294–1301.

12. Wexler SA, Donaldson C, Denning-Kendall P, Rice C, Bradley B, Hows JM. Adult bone marrow is a rich source of human mesenchymal 'stem' cells but umbilical cord and mobilized adult blood are not. Br J Haematol. 2003; 121:368–374.

13. Murphy MB, Moncivais K, Caplan AI. Mesenchymal stem cells: environmentally responsive therapeutics for regenerative medicine. Exp Mol Med. 2013; 45:e54.

14. Saether EE, Chamberlain CS, Leiferman EM, Kondratko-Mittnacht JR, Li WJ, Brickson SL, et al. Enhanced medial collateral ligament healing using mesenchymal stem cells: dosage effects on cellular response and cytokine profile. Stem Cell Rev. 2014; 10:86–96.

15. Richardson JD, Bertaso AG, Psaltis PJ, Frost L, Carbone A, Paton S, et al. Impact of timing and dose of mesenchymal stromal cell therapy in a preclinical model of acute myocardial infarction. J Card Fail. 2013; 19:342–353.

16. Cataldo D, Munaut C, Noel A, Frankenne F, Bartsch P, Foidart JM, et al. MMP-2- and MMP-9-linked gelatinolytic activity in the sputum from patients with asthma and chronic obstructive pulmonary disease. Int Arch Allergy Immunol. 2000; 123:259–267.

17. Chang YS, Ahn SY, Yoo HS, Sung SI, Choi SJ, Oh WI, et al. Mesenchymal stem cells for bronchopulmonary dysplasia: phase 1 dose-escalation clinical trial. J Pediatr. 2014; 164:966–972.e6.

18. Hare JM, Traverse JH, Henry TD, Dib N, Strumpf RK, Schulman SP, et al. A randomized, double-blind, placebo-controlled, dose-escalation study of intravenous adult human mesenchymal stem cells (prochymal) after acute myocardial infarction. J Am Coll Cardiol. 2009; 54:2277–2286.

19. Liang J, Zhang H, Hua B, Wang H, Lu L, Shi S, et al. Allogenic mesenchymal stem cells transplantation in refractory systemic lupus erythematosus: a pilot clinical study. Ann Rheum Dis. 2010; 69:1423–1429.

20. Haynesworth SE, Baber MA, Caplan AI. Cytokine expression by human marrow-derived mesenchymal progenitor cells in vitro: effects of dexamethasone and IL-1 alpha. J Cell Physiol. 1996; 166:585–592.

21. Hegab AE, Kubo H, Yamaya M, Asada M, He M, Fujino N, et al. Intranasal HGF administration ameliorates the physiologic and morphologic changes in lung emphysema. Mol Ther. 2008; 16:1417–1426.

22. Katsha AM, Ohkouchi S, Xin H, Kanehira M, Sun R, Nukiwa T, et al. Paracrine factors of multipotent stromal cells ameliorate lung injury in an elastase-induced emphysema model. Mol Ther. 2011; 19:196–203.

23. Muyal JP, Muyal V, Kotnala S, Kumar D, Bhardwaj H. Therapeutic potential of growth factors in pulmonary emphysematous condition. Lung. 2013; 191:147–163.

24. Kasahara Y, Tuder RM, Taraseviciene-Stewart L, Le Cras TD, Abman S, Hirth PK, et al. Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J Clin Invest. 2000; 106:1311–1319.

25. Guan XJ, Song L, Han FF, Cui ZL, Chen X, Guo XJ, et al. Mesenchymal stem cells protect cigarette smoke-damaged lung and pulmonary function partly via VEGF-VEGF receptors. J Cell Biochem. 2013; 114:323–335.

26. Weiss DJ, Casaburi R, Flannery R, LeRoux-Williams M, Tashkin DP. A placebo-controlled, randomized trial of mesenchymal stem cells in COPD. Chest. 2013; 143:1590–1598.

27. Koc ON, Day J, Nieder M, Gerson SL, Lazarus HM, Krivit W. Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH). Bone Marrow Transplant. 2002; 30:215–222.

28. Horwitz EM, Prockop DJ, Fitzpatrick LA, Koo WW, Gordon PL, Neel M, et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med. 1999; 5:309–313.