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. 2015 Apr;11(4):2387-96.
doi: 10.3892/mmr.2014.3025. Epub 2014 Dec 1.

Therapeutic effect of human umbilical cord mesenchymal stem cells modified by angiotensin-converting enzyme 2 gene on bleomycin-induced lung fibrosis injury

Fang Min  1 , Fengying Gao  2 , Qian Li  3 , Zhenwei Liu  4
Affiliations

Therapeutic effect of human umbilical cord mesenchymal stem cells modified by angiotensin-converting enzyme 2 gene on bleomycin-induced lung fibrosis injury

Fang Min et al. Mol Med Rep. 2015 Apr.

Abstract

The aim of the present study was to evaluate the therapeutic effects of human umbilical cord mesenchymal stem cells (uMSCs) in the presence of angiotensin‑converting enzyme 2 gene (ACE2; ACE2‑uMSCs) on bleomycin (BLM)‑induced lung injury and pulmonary fibrosis in mice. A total of 100 male C57BL/6 mice were divided at random into five groups (n=20) as follows: Control group, BLM group, ACE2 group, uMSC group and ACE2‑uMSC group. At 7, 14 and 28 days post‑treatment, the following parameters were evaluated in lung tissue: Oxidation indexes [malondialedehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) and oxidized glutathione (GSSG)]; fibrosis factors [tumor necrosis factor (TNF)‑α, interferon (IFN)‑γ and transforming growth factor (TGF)‑β]; inflammatory cytokines [Interleukin (IL)‑1, IL‑2, IL‑6 and IL‑10]; ACE2 gene expression; hydroxyproline and collagen type 1 messenger RNA (mRNA) concentration; as well as matrix metalloproteinase (MMPs; 2 and 9) and tissue inhibitor of metalloproteinase (TIMP)1‑4 expression. ACE2‑uMSC injection following bleomycin pretreatment significantly alleviated lung injury in mice. In addition, treatment with ACE2‑uMSCs demonstrated a stronger therapeutic effect than ACE2‑ or uMSC treatment alone, indicated by decreased expression of MDA, GSSG, TNF‑α, IFN‑γ, TGF‑β, IL‑1, IL‑2, IL‑6, collagen type 1 mRNA, MMPs and TIMPs as well as hydroxyproline concentration, and upregulation of SOD, GSH and ACE2 and IL‑10. In conclusion, the results of the present study demonstrated that ACE2 and uMSCs had a synergistic therapeutic effect on bleomycin‑induced acute lung injury.

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Figures

Figure 1
Figure 1
Flow cytometric analysis of cell surface antigens confirms the uMSC phenotype of cells. At passage three of the logarithmic phase, adherent fibroblast-like cells were analyzed for expression of cell surface antigens. Cells expressed the typical mesenchymal pattern of markers: CD29(+), CD44(+), CD105(+), CD34(−), CD45(−), CD86(−). uMSC, human umbilical mesenchymal stem cell.
Figure 2
Figure 2
Examination of the differentiation and transfection efficiency of uMSCs. Following transfection with ACE2, uMSCs were stained with (A) Alizarin Red for calcium deposition (orange) to indicate osteoblastic activity and bone formation and (B) oil red O for intracellular lipid droplets (red) to show adipogenic differentiation. Immunofluoresence staining of chondrocytes to reveal expression of collagen type II in the (C) uMSCs only group; (D) ACE2-uMSC group at 14 days and (E) 28 days; and (F) negative control group. Green fluorescent protein-labelled ACE2-uMSCs detected using (G) fluorescence analysis and (H) light microscopy (magnification, ×200). uMSC, human umbilical cord mesenchymal stem cells; ACE2, angiotensin-converting enzyme 2 gene; ACE2-uMSCs, uMSCs transfected with ACE2.
Figure 3
Figure 3
Hematoxylin-eosin staining of lung tissue. Lung tissue sections from each group as follows: ACE2-uMSC group at (A) 7, (B) 14 and (C) 28 days; (D) control group; bleomycin group at (E) 7, (F) 14 and (G) 28 days; ACE2 group at (H) 7, (I) 14 and (J) 28 days; and uMSC group at (K) 7, (L) 14 and (M) 28 days (magnification, ×200). uMSCs, group injected with human umbilical cord mesenchymal stem cells; ACE2, group injected with angiotensin-converting enzyme 2 gene; ACE2-uMSCs, group injected with uMSCs transfected with ACE2; BLM, group with bleomycin-induced lung injury only.
Figure 4
Figure 4
Evaluation of pulmonary fibrosis in the different groups. (A) Ashcroft score of fibrosis for each group at 7, 14 and 28 days following bleomycin-induced lung injury and the control group. (B) Protein expression levels, as determined by ELISA, of profibrotic and proinflammatory factors for each group 14 days following treatment. *P<0.05 and **P<0.01 compared with BLM group. uMSCs, group injected with human umbilical cord mesenchymal stem cells; ACE2, group injected with angiotensin-converting enzyme 2 gene; ACE2-uMSCs, group injected with uMSCs transfected with ACE2; BLM, group with bleomycin-induced lung injury only; IL, interleukin; TGF, transforming growth factor; TNF, tumor necrosis factor; IFN, interferon.
Figure 5
Figure 5
ACE2 expression in lung tissue is significantly increased in the ACE2 and ACE2-uMSC groups. (A) Western blot and (B) densitometric analysis of protein expression levels of ACE2 in lung tissue from each group at 7, 14 and 28 days following injection treatment. β-actin served as control. *P<0.05 and **P<0.01 compared with BLM group. uMSCs, group injected with human umbilical cord mesenchymal stem cells; ACE2, group injected with angiotensin-converting enzyme 2 gene; ACE2-uMSCs, group injected with uMSCs transfected with ACE2; BLM, group with bleomycin-induced lung injury only.
Figure 6
Figure 6
Collagen deposition was significantly reduced by ACE2 and uMSC treatments individually and in combination. Determination of (A) hydroxyproline levels and (B) collagen type 1 messenger RNA in different lung tissue at 7, 14 and 28 days following treatment. *P<0.05 compared with BLM group; #P<0.05 compared with ACE2 group; &P<0.05 compared with uMSC group. uMSCs, group injected with human umbilical cord mesenchymal stem cells; ACE2, group injected with angiotensin-converting enzyme 2 gene; ACE2-uMSCs, group injected with uMSCs transfected with ACE2; BLM, group with bleomycin-induced lung injury only.
Figure 7
Figure 7
The expression of MMPs (2 and 9) and TIMPs (–4) in each group at 28 days. (A) Western blot analysis of MMPs (2 and 9) and TIMPs (1 and 2) in each group. Densitometric analysis of (B) MMP-2 and (C) MMP-9 protein expression levels. (D) Quantitative polymerase chain reaction analysis of TIMPs (–4) mRNA expression. β-actin served as the control. (E) Immunohistochemical analysis of MMP-2 and MMP-9 expression in each group (magnification, ×200). *P<0.05 and **P<0.01 compared with BLM group. MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; uMSCs, group injected with human umbilical cord mesenchymal stem cells; ACE2, group injected with angiotensin-converting enzyme 2 gene; ACE2-uMSCs, group injected with uMSCs transfected with ACE2; BLM, group with bleomycin-induced lung injury only.
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References

    1. Bakowitz M, Bruns B, McCunn M. Acute lung injury and the acute respiratory distress syndrome in the injured patient. Scand J Trauma Resusc Emerg Med. 2012;20:54. doi: 10.1186/1757-7241-20-54. - DOI - PMC - PubMed
    1. Wada T, Jesmin S, Gando S, et al. The role of angiogenic factors and their soluble receptors in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) associated with critical illness. J Inflamm (Lond) 2013;10:6. doi: 10.1186/1476-9255-10-6. - DOI - PMC - PubMed
    1. Hayes M, Curley G, Ansari B, Laffey JG. Clinical review: Stem cell therapies for acute lung injury/acute respiratory distress syndrome - hope or hype? Crit Care. 2012;16:205. doi: 10.1186/cc10570. - DOI - PMC - PubMed
    1. Walkey AJ, Summer R, Ho V, Alkana P. Acute respiratory distress syndrome: epidemiology and management approaches. Clin Epidemiol. 2012;4:159–169. doi: 10.2147/CLEP.S28800. - DOI - PMC - PubMed
    1. Kumar P, Goldstraw P, Yamada K, et al. Pulmonary fibrosis and lung cancer: risk and benefit analysis of pulmonary resection. J Thorac Cardiovasc Surq. 2003;125:1231–1237. - PubMed

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