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

Figures

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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.

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  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.