Yonsei Med J.  2017 Jan;58(1):206-216. 10.3349/ymj.2017.58.1.206.

Angiopoietin-1 Modified Human Umbilical Cord Mesenchymal Stem Cell Therapy for Endotoxin-Induced Acute Lung Injury in Rats

• Huang ZW
• Liu N
• Li D
• Zhang HY
• Wang Y
• Liu Y
• Zhang LL
• Ju XL

• KMID: 2374208
• DOI: http://doi.org/10.3349/ymj.2017.58.1.206

Abstract

PURPOSE
Angiopoietin-1 (Ang1) is a critical factor for vascular stabilization and endothelial survival via inhibition of endothelial permeability and leukocyte- endothelium interactions. Hence, we hypothesized that treatment with umbilical cord mesenchymal stem cells (UCMSCs) carrying the Ang1 gene (UCMSCs-Ang1) might be a potential approach for acute lung injury (ALI) induced by lipopolysaccharide (LPS).
MATERIALS AND METHODS
UCMSCs with or without transfection with the human Ang1 gene were delivered intravenously into rats one hour after intra-abdominal instillation of LPS to induce ALI. After the rats were sacrificed at 6 hours, 24 hours, 48 hours, 8 days, and 15 days post-injection of LPS, the serum, the lung tissues, and bronchoalveolar lavage fluid (BALF) were harvested for analysis, respectively.
RESULTS
Administration of fluorescence microscope confirmed the increased presence of UCMSCs in the injured lungs. The evaluation of UCMSCs and UCMSCs-Ang1 actions revealed that Ang1 overexpression further decreased the levels of the pro-inflammatory cytokines TNF-α, TGF-β1, and IL-6 and increased the expression of the anti-inflammatory cytokine IL-10 in the injured lungs. This synergy caused a substantial decrease in lung airspace inflammation and vascular leakage, characterized by significant reductions in wet/dry ratio, differential neutrophil counts, myeloperoxidase activity, and BALF. The rats treated by UCMSCs-Ang1 showed improved survival and lower ALI scores.
CONCLUSION
UCMSCs-Ang1 could improve both systemic inflammation and alveolar permeability in ALI. UC-derived MSCs-based Ang1 gene therapy may be developed as a potential novel strategy for the treatment of ALI.

Keyword

Angiopoietin-1; human umbilical cord mesenchymal stem cell; acute lung injury

MeSH Terms

Acute Lung Injury/chemically induced/*therapy
Angiopoietin-1/*genetics
Animals
Bronchoalveolar Lavage Fluid
Cytokines/metabolism
Endotoxins
Genetic Therapy
Interleukin-10/metabolism
Interleukin-6/metabolism
Leukocyte Count
Lipopolysaccharides
Lung/metabolism
Male
*Mesenchymal Stem Cell Transplantation
Mesenchymal Stromal Cells/metabolism
Neutrophils/metabolism
Rats
Transforming Growth Factor beta1/metabolism
Tumor Necrosis Factor-alpha/metabolism
Umbilical Cord/*cytology
Angiopoietin-1
Cytokines
Endotoxins
Interleukin-10
Interleukin-6
Lipopolysaccharides
Transforming Growth Factor beta1
Tumor Necrosis Factor-alpha

Figure

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  Fig. 1 The morphology and immunophenotype identification of UCMSCs. (A and B) Respectively by the inverted microscope and HE staining observation of MSCs (×100). (C) Immunophenotype of UC-derived MSCs. HE, hematoxylin and eosin; UCMSCs, umbilical cord-derived mesenchy mal stem cells.

• 

  Fig. 2 Differentiation and identification of UCMSCs. (A) The formation of mineralized matrix shown by alizarin red staining (×200). (B) Alkaline phosphatase expression (×200). (C) Differentiation into adipocytes by the inverted microscope (×200). (D) Positive oil-red O staining (×200). (E) Immunohistochemical detection of cartilage specific type II collagen expression (×200). (F) Toluidine blue staining (×200). UCMSCs, umbilical cord-derived mesenchy mal stem cells.

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  Fig. 3 (A) The information of vector GV287. Ubi-MCS-3FLAG-SV40-EGFP At a MOI=8, transduction of Ang1 into MSCs using a lentivirus vector. Viability of MSCs and high transduction efficiency (>95%) of GFP at 72 h after MSCs-Ang1 tranduction, ×200. (B) Fluorescence microscopy. (C) Flow cytometry. (D) The expression of Ang1 protein detection of transfected MSCs-Ang1 in western blotting. CON, control group with no transfection; NC (OE), negative control group transfected with GFP lentiviral vectors LV-GV287; OE, Ang1 overexpression group transfected with GFP and LV-Ang1. MOI, multiplicity of infection; GFP, green fluorescent protein.

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  Fig. 4 (A) Histological analysis indicated LPS injection caused capillary vessel engorgement, congestion, abundant inflammatory infiltration in lung stroma (×100). Administration of MSCs or MSCs-Ang1 improved the lung injury at all time points. (B) Lung injury score decreased significantly in the MSCs group and MSCs-Ang1 group at 6 hours, 24 hours, 48 hours, 8 days, and 15 days after LPS injection. (*p<0.05 compared with LPS controls; †p<0.05 comparing MSCs-Ang1 with MSCs). Fibroblast injections have no effect on improvement of both pathological morphology and lung injury score. NS, normal saline; LPS, lipopolysaccharide; MSCs, mesenchymal stem cells.

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  Fig. 5 BALF neutrophil counts (A) and lung MPO activity (B). Neutrophil counts and lung tissue MPO activity were significantly higher in the LPS group, compared to the control group. Treatment with UCMSCs or UCMSCs-Ang1 significantly reduced LPS-induced increases in BALF neutrophil counts and lung MPO activity after 24 hours of acute lung injury (*p<0.05 compared with LPS controls; †p<0.05 comparing UCMSCs-Ang1 with UCMSCs). NS, normal saline; LPS, lipopolysaccharide; MSCs, mesenchymal stem cells; BALF, bronchoalveolar lavage fluid; MPO, myeloperoxidase; UCMSCs, umbilical cord-derived mesenchy mal stem cells.

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  Fig. 6 Injection of UCMSCs-Ang1 reduced pulmonary edema in ALI. Pulmonary edema was measured as wet-dry ratio (*p<0.05 vs. the LPS group; †p<0.05 comparing UCMSCs-Ang1 with UCMSCs). NS, normal saline; LPS, lipopolysaccharide; UCMSCs, umbilical cord-derived mesenchy mal stem cells; ALI, acute lung injury.

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  Fig. 7 Levels of proinflammatory and antiinflammatory cytokines and chemokines in serum by ELISA. Increased levels of the pro-inflammatory cytokines TNF-α, IL-6, and TGF-β1 were down-regulated significantly comparing UCMSCs-Ang1 with UCMSCs group, which suggested Ang1 assistance anti-inflammatory effect in the early stage of inflammation (A, B, and C). In addition, the response of antiinflammatory IL-10 of UCMSCs-Ang1 group peaked at 48 hours after injection of LPS and decreased gradually at the following time points (D). The LPS-induced IL-10 increase appeared to be no different among the LPS and fibroblast groups (*p<0.05 vs. the LPS group; †p<0.05 comparing UCMSCs-Ang1 with UCMSCs). NS, normal saline; LPS, lipopolysaccharide; MSCs, mesenchymal stem cells; ELISA, enzyme-linked immunosorbent assay; TNF, tumor necrosis factor; IL, interleukin; TGF, transforming growth factor; UCMSCs, umbilical cord-derived mesenchy mal stem cells.

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  Fig. 8 The survival rate of the MSCs-Ang1 group over 15 days is significantly higher than that of the MSCs group (70% vs. 50%; *p=0.0141). MSCs-Ang1 treatment improved survival rates at all time-points. NS, normal saline; LPS, lipopolysaccharide; MSCs, mesenchymal stem cells.

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  Fig. 9 Ang1 expressing UCMSCs labeled with the GFP were observed in lung sections from LPS-injured animals killed at 6 hours after acute lung injury. (A) UCMSCs-treated lungs. (B) UCMSCs-Ang1-treated recipient lungs. (C) UCMSCs-Ang1-treated recipient lungs at 8 days. RT-PCR results demonstrated that expression of GFP mRNA in MSCs-Ang1 group was higher than that in the MSCs group at 8 days and 15 days after ALI. (D) GFP mRNA relative quantitative analysized by RT-PCR at the designated time points. (E) At 8 days, UCMSCs-Ang1 expressed a greater quantity of GFP mRNAs than UCMSCs. 1–3 were respectively UCMSCs group, LPS group, UCMSCs-Ang1 group. GAPDH as the internal control (*p<0.05 compared with PBS control). GFP, green fluorescent protein; UCMSCs, umbilical cord-derived mesenchy mal stem cells; LPS, lipopolysaccharide.

Reference

1. Mei SH, McCarter SD, Deng Y, Parker CH, Liles WC, Stewart DJ. Prevention of LPS-induced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin 1. PLoS Med. 2007; 4:e269.

2. Gotts JE, Matthay MA. Mesenchymal stem cells and acute lung injury. Crit Care Clin. 2011; 27:719–733.

3. Gharib SA, Liles WC, Matute-Bello G, Glenny RW, Martin TR, Altemeier WA. Computational identification of key biological modules and transcription factors in acute lung injury. Am J Respir Crit Care Med. 2006; 173:653–658.

4. Slutsky AS, Hudson LD. PEEP or no PEEP--lung recruitment may be the solution. N Engl J Med. 2006; 354:1839–1841.

5. Harting MT, Jimenez F, Xue H, Fischer UM, Baumgartner J, Dash PK, et al. Intravenous mesenchymal stem cell therapy for traumatic brain injury. J Neurosurg. 2009; 110:1189–1197.

6. Li J, Li D, Liu X, Tang S, Wei F. Human umbilical cord mesenchymal stem cells reduce systemic inflammation and attenuate LPS-induced acute lung injury in rats. J Inflamm (Lond). 2012; 9:33.

7. Paul A, Nayan M, Khan AA, Shum-Tim D, Prakash S. Angiopoietin-1-expressing adipose stem cells genetically modified with baculovirus nanocomplex: investigation in rat heart with acute infarction. Int J Nanomedicine. 2012; 7:663–682.

8. Xu J, Qu J, Cao L, Sai Y, Chen C, He L, et al. Mesenchymal stem cell-based angiopoietin-1 gene therapy for acute lung injury induced by lipopolysaccharide in mice. J Pathol. 2008; 214:472–481.

9. Gupta N, Su X, Popov B, Lee JW, Serikov V, Matthay MA. Intrapulmonary delivery of bone marrow-derived mesenchymal stem cells improves survival and attenuates endotoxin-induced acute lung injury in mice. J Immunol. 2007; 179:1855–1863.

10. Davies MJ. Myeloperoxidase-derived oxidation: mechanisms of biological damage and its prevention. J Clin Biochem Nutr. 2011; 48:8–19.

11. Bustos ML, Huleihel L, Kapetanaki MG, Lino-Cardenas CL, Mroz L, Ellis BM, et al. Aging mesenchymal stem cells fail to protect because of impaired migration and antiinflammatory response. Am J Respir Crit Care Med. 2014; 189:787–798.

12. Fang X, Neyrinck AP, Matthay MA, Lee JW. Allogeneic human mesenchymal stem cells restore epithelial protein permeability in cultured human alveolar type II cells by secretion of angiopoietin-1. J Biol Chem. 2010; 285:26211–26222.

13. Kanki-Horimoto S, Horimoto H, Mieno S, Kishida K, Watanabe F, Furuya E, et al. Implantation of mesenchymal stem cells overexpressing endothelial nitric oxide synthase improves right ventricular impairments caused by pulmonary hypertension. Circulation. 2006; 114:1 Suppl. I181–I185.

14. Yamada M, Kubo H, Kobayashi S, Ishizawa K, Numasaki M, Ueda S, et al. Bone marrow-derived progenitor cells are important for lung repair after lipopolysaccharide-induced lung injury. J Immunol. 2004; 172:1266–1272.

15. Thurston G, Rudge JS, Ioffe E, Papadopoulos N, Daly C, Vuthoori S, et al. The anti-inflammatory actions of angiopoietin-1. EXS. 2005; (94):233–245.

16. Gamble JR, Drew J, Trezise L, Underwood A, Parsons M, Kasminkas L, et al. Angiopoietin-1 is an antipermeability and anti-inflammatory agent in vitro and targets cell junctions. Circ Res. 2000; 87:603–607.

17. Piao W, Wang H, Inoue M, Hasegawa M, Hamada H, Huang J. Transplantation of Sendai viral angiopoietin-1-modified mesenchymal stem cells for ischemic limb disease. Angiogenesis. 2010; 13:203–210.

18. McCarter SD, Mei SH, Lai PF, Zhang QW, Parker CH, Suen RS, et al. Cell-based angiopoietin-1 gene therapy for acute lung injury. Am J Respir Crit Care Med. 2007; 175:1014–1026.

19. Bhandari V, Choo-Wing R, Lee CG, Zhu Z, Nedrelow JH, Chupp GL, et al. Hyperoxia causes angiopoietin 2-mediated acute lung injury and necrotic cell death. Nat Med. 2006; 12:1286–1293.

20. Johnson ER, Matthay MA. Acute lung injury: epidemiology, pathogenesis, and treatment. J Aerosol Med Pulm Drug Deliv. 2010; 23:243–252.