Transplantation of human matrix metalloproteinase-1 gene-modified bone marrow-derived mesenchymal stem cell attenuates CCL4-induced liver fibrosis in rats

Abstract

It has been reported that bone marrow-derived mesenchymal stem cells (BMSCs) alleviated liver fibrosis. We investigated whether BMSCs transfected with human matrix metalloproteinase 1 (BMSCs/MMP1) would improve their therapeutic effect in liver fibrosis induced by carbon tetrachloride (CCl4) in rats. BMSCs were transfected with an adenovirus carrying enhanced green fluorescence protein (GFP) and human MMP1 gene. BMSCs or BMSCs/MMP1 were directly injected into fibrotic rats via the tail vein. GFP-labeled cells appeared in the fibrotic liver after BMSC transplantation. The expression of BMSCs/MMP1 elevated levels of MMP1 in vitro. Although BMSC administration reduced liver fibrosis, transplantation of BMSCs/MMP1 enhanced the reduction of liver fibrosis to a higher level. Treatment with BMSCs/MMP1 not only decreased collagen content but also suppressed activation of hepatic stellate cells (HSCs) in fibrotic liver, which led to subsequent improvement of both liver injury and fibrosis. Treatment with BMSCs/MMP1 resulted in an improved therapeutic effect compared with BMSCs alone, which is probably because of the sustainably expressed MMP1 level in the liver. BMSCs/MMP1 transplantation not only improved biochemical parameters but also attenuated progression of liver fibrosis, suggesting that BMSCs may be a potential cell source in preventing liver fibrosis and MMP1 gene may enhance the anti-fibrotic effect of BMSCs.

Figure 1

Characteristics of bone marrow-derived mesenchymal stem cells (BMSCs) and BMSCs/matrix metalloproteinase 1 (MMP1). (A and B) BMSCs (passage 16) observed by light microscopy (×100). (C) Green fluorescence protein (GFP)-expressing BMSCs shown by fluorescence microscopy (×100). (D and E) GFP expression rate in gene transfected BMSCs detected by flow cytometry 72 h later at MOI 50, 100, 200 and 300 pfu/cell.

Figure 2

Expression of molecules on cells by flow cytometry analysis. Cells were positive on (A) CD90, and (B) CD105, while negative on (C) CD14, (D) CD34, (E) CD45 and (F) CD79a, indicating that isolated cells were bone marrow-derived mesenchymal stem cells (BMSCs).

Figure 3

Effect of bone marrow-derived mesenchymal stem cells (BMSCs) on CCl₄-induced liver fibrosis. (A) Tracing of green fluorescence protein (GFP) gene-modified BMSCs in liver. (B) Liver histology after BMSCs/matrix metalloproteinase 1 (MMP1) administration by H&E staining. (C) Collagen content in fibrotic liver by Masson staining. (D) Fibrosis area. (E) Hepatic hydroxyproline content. (F and G) Hepatic α-smooth muscle actin by western blotting. (×100). *P<0.05 and ^**P<0.01.

Figure 3

Effect of bone marrow-derived mesenchymal stem cells (BMSCs) on CCl₄-induced liver fibrosis. (A) Tracing of green fluorescence protein (GFP) gene-modified BMSCs in liver. (B) Liver histology after BMSCs/matrix metalloproteinase 1 (MMP1) administration by H&E staining. (C) Collagen content in fibrotic liver by Masson staining. (D) Fibrosis area. (E) Hepatic hydroxyproline content. (F and G) Hepatic α-smooth muscle actin by western blotting. (×100). *P<0.05 and ^**P<0.01.

Figure 4

Bone marrow-derived mesenchymal stem cells (BMSCs)/matrix metalloproteinase 1 (MMP1) attenuated CCl₄-induced liver injury. Serum (A) alanine aminotransferase (ALT), (B) aspartate aminotransferase (AST), (C) prothrombin time (PT) and (D) albumin (ALB) levels in normal, CCl₄-treated, CCl₄-treated transfused with BMSCs, CCl₄-treated transfused with BMSCs/green fluorescence protein (GFP), and CCl₄-treated transfused with BMSCs/MMP1 animals. ^*P<0.05 and ^**P<0.01.

Figure 5

Expression of matrix metalloproteinase 1 (MMP1) and tissue inhibitor of metalloproteinase 1 (TIMP1) from bone marrow-derived mesenchymal stem cells (BMSCs)/MMP1 and liver. (A) Secretion of MMP1 from the BMSCs/MMP1 by enzyme-linked immunosorbent assay (ELISA). (B) Hepatic MMP1 and (C) TIMP1 expression after transplantation of BMSCs/MMP1. (D) Enzyme activity of MMP1 in vitro and (E) in liver. ^*P<0.05 and ^**P<0.01.