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, 2019, 5310202
eCollection

The Effect of Vascular Endothelial Growth Factor on Bone Marrow Mesenchymal Stem Cell Engraftment in Rat Fibrotic Liver Upon Transplantation

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The Effect of Vascular Endothelial Growth Factor on Bone Marrow Mesenchymal Stem Cell Engraftment in Rat Fibrotic Liver Upon Transplantation

Ke Yuan et al. Stem Cells Int.

Abstract

Background: According to existing related experiments and research reports, stem cell transplantation therapy has been shown to have a positive effect on the recovery of liver fibrosis/cirrhosis, but for some reason, this therapy still cannot be widely used in clinical work. One of the reasons that cannot be ignored is the low quantity of exogenous stem cells transplanted into the liver in vivo. Thus, we investigated whether the use of the vascular endothelial growth factor (VEGF) can increase the number of stem cell transplants and improve the efficacy of stem cell transplantation therapy.

Methods: Using a Sprague-Dawley rat liver fibrosis model, we transplanted into fibrosis liver allograft bone marrow mesenchymal stem cells (BMSCs) which were labelled with chlormethylbenzamido-1,1-dioctadecyl-3,3,3'3'-tetramethylin-docarbocyamine (CM-DiI) or injected VEGF adenovirus solution through the tail vein or conducted the above two operations simultaneously. The cell surface receptor profile of BMSC was examined by flow cytometry and immunofluorescence staining. Hepatic sinusoidal vascular leakage was measured with Evan's blue dye assay. Paraffin section staining, immunofluorescent staining, RT-qPCR (quantitative reverse transcription polymerase chain reaction), and Western blot were used to evaluate hepatic pathological changes and physiology function.

Result: The in vivo study indicated that, comparing with other groups of rats, the rats with combined treatment of BMSC transplantation and VEGF injection exhibited obvious reduction in liver fibrosis. Evan's blue dye assay suggests that after injecting with VEGF adenovirus solution, the rat's hepatic sinusoidal permeability would be increased. We confirmed the expression of very late antigen-4 (VLA4, integrin α 4 β 1) on rat BMSCs and the elevated expression of vascular adhesion molecule-1 (VCAM-1) in the hepatic sinusoidal endothelial cells. In addition, the analysis of CM-DiI-labeled BMSCs showed that the BMSC+VEGF group exhibited better cell engraftment and that the engrafted cells were mainly distributed in the hepatic parenchyma. Furthermore, compared with the other situation, it is best to reconstitute the liver secretion and regeneration function of rats after combined application of VEGF and BMSC.

Conclusion: We showed that VEGF promotes the engraftment of BMSCs in liver fibrosis, enhances liver regeneration, and improves liver function. These outcomes may be related to the increasing hepatic sinusoidal endothelium permeability and VCAM-1-increased expression.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Masson staining of the papillary process in four groups of liver fibrosis model rats. The blue stained area in the images depicts the hepatic collagen fibrous band.
Figure 2
Figure 2
Morphology and identification of BMSCs. (a) Morphology of the BMSCs. Phenotypic characterization of the cultured BMSCs. (b) Immunofluorescent staining of BMSC surface markers CD29, CD34, and CD45. (c) Flow cytometry analysis of the cell surface markers CD45, CD29, and CD90 on cultured BMSCs at the third passage. (d) Flow cytometry analysis of VLA4 expression on cultured BMSCs at the third passage.
Figure 3
Figure 3
Assessment of liver fibrosis in rats. (a) Three different pathological sections with staining of the liver papillary lobe in four groups (Masson's trichrome stain, Sirius red stain, and H&E stain, n = 3/group; original magnification, 40x). (b) Evaluation of the distribution of α-smooth muscle actin (α-SMA; red) and collagen III (red) in four groups examined by immunofluorescent staining. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 100 μm. (c) Protein levels of liver fibrotic markers: TGFβ (transforming growth factor beta), α-SMA, and collagen III in four groups of rats examined by Western blot with β-actin used as an internal control. (d) RT-qPCR was used to compare the expression of COL3A1 (collagen type III alpha 1) and IL-6 (interleukin 6) in four groups. (e) EMT-related parameters (vimentin, Twist1, Snail, Slug, E-cadherin, and occludin) were evaluated by RT-qPCR, and GADPH was used as a housekeeping gene. Results are shown as fold changes compared to the model group. p < 0.05 compared to the model group; #p < 0.05 between the BMSC+VEGF group and the BMSC group, n = 3.
Figure 4
Figure 4
The impact of VEGF on rat liver fibrosis after BMSC transplant. (a) Immunofluorescent staining of VEGF and VCAM-1 (both in red) with nuclei shown in blue. (b) VEGF and VCAM-1 expression in the liver was evaluated by Western blot with β-actin used as an internal reference. (c) VEGF and VCAM-1 expression in the liver was evaluated by RT-qPCR, and GADPH was used as a housekeeping gene. Results are shown as fold changes compared to the model group. (d) Immunofluorescent findings 28 days after transplantation of BMSC. DiI-labeled BMSC engraftment in the liver (in red). (e) Residual amount of Evan's blue dye in the liver reflecting hepatic sinusoidal endothelium permeability. p < 0.05 compared to the model group; #p < 0.05 between the BMSC+VEGF group and the BMSC group, n = 3.
Figure 5
Figure 5
Assessment of liver function. (a) Immunofluorescent staining of Ki67 and PCNA (both in red), with nuclei shown in blue. (b) The expression of CYP3A1 and the expression of ALB in the liver was evaluated by Western blot, and β-actin was used as an internal reference. (c) ALB and G6pase expression in the liver was evaluated by RT-qPCR, and GADPH was used as a housekeeping gene. Results are shown as fold changes compared to the model group. p < 0.05 when compared to the model group, n = 3.
Figure 6
Figure 6
Immunostaining of the BMSC group and the BMSC+adenovirus vector group. Representative histopathological changes observed in Masson's trichrome-, Sirius red-, and H&E-stained liver tissues in the BMSC group and the BMSC+adenovirus vector group; original magnification, 40x.

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