Effects of Fibrotic Tissue on Liver-targeted Hydrodynamic Gene Delivery

Abstract

Hydrodynamic gene delivery is a common method for gene transfer to the liver of small animals, and its clinical applicability in large animals has been demonstrated. Previous studies focused on functional analyses of therapeutic genes in animals with normal livers and little, however, is known regarding its effectiveness and safety in animals with liver fibrosis. Therefore, this study aimed to examine the effects of liver fibrosis on hydrodynamic gene delivery efficiency using a rat liver fibrosis model. We demonstrated for the first time, using pCMV-Luc plasmid, that this procedure is safe and that the amount of fibrotic tissue in the liver decreases gene delivery efficiency, resulting in decrease in luciferase activity depending on the volume of fibrotic tissue in the liver and the number of hepatocytes that are immunohistochemically stained positive for transgene product. We further demonstrate that antifibrotic gene therapy with matrix metalloproteinase-13 gene reduces liver fibrosis and improves efficiency of hydrodynamic gene delivery. These results demonstrate the negative effects of fibrotic tissue on hydrodynamic gene delivery and its recovery by appropriate antifibrotic therapy.

Figure 1

Effect of fibrosis on the efficiency of hydrodynamic gene delivery. (a) The standard Sirius red staining was performed to evaluate the fibrotic liver tissues in normal rats and LF rats 9 weeks after bile duct ligation. A quantitative analysis of fibrotic tissue positively stained was performed using ImageJ software (version 1.6.0_20, National Institutes of Health) as previously reported (a total of 75 sections from three rats each were analyzed). (b) The distribution of the fibrotic area (%) in each lobe (15 sections from all lobes). The values represent mean ± SD. (c) Each group of three normal rats and LF rats were hydrodynamically injected with pCMV-Luc, and liver samples were collected 4 hours after the transfection followed by the standard luciferase assay. The values represent mean ± SD (five samples from every liver lobe per rat from all three rats in each group, yielding 15 samples per group). (d) Luciferase activity in each lobe of the rat groups (three tissue samples from each lobe). *P < 0.05,** P < 0.01, *** P < 0.001, and N.S., no statistical significance. t-test or one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test. LF, liver fibrosis group; RL, right lateral lobe; RM, right medial lobe; LM, left medial lobe; LL, left lateral lobe; and CL, caudate lobe.

Figure 2

Correlation between liver fibrosis and efficiency of liver-targeted HGD (a–d) representative images of Sirius red staining; (a) ~5% of fibrotic tissue, (b) for 10%, (c) for 15%, and (d) for 20%. (e) Correlation analysis between area of fibrotic liver tissue and luciferase activity (n = 120, *** for P < 0.001); Scale bar represents 200 µm (100 ×). r, correlation coefficient.

Figure 3

Experimental protocol for antifibrotic treatment. The rat liver fibrosis (LF) model was developed by the bile duct ligation (BDL) (n = 40). Two groups of LF ( n = 20) and LF-MMP13, BDL simultaneously followed by hydrodynamic delivery of pBGI-MMP13 (n = 20) were developed. Each model was analyzed for the severity of the LF at appropriate time points using a serum marker of hyaluronic acid, type 4 collagen, albumin, and hepatobiliary enzymes. The liver-targeted hydrodynamic gene delivery (HGD) of pCMV-Luc was performed on 5 rats in each group at the time points of 0 (n = 10), 5 (n = 10), 8 (n = 10), and 10 (n = 10) weeks after BDL followed by euthanasia and the collection of tissue samples.

Figure 4

Effect of antifibrotic gene therapy on gene delivery efficiency. (a) Time-dependent change in fibrotic liver tissue. The Sirius red staining was followed by the quantitative analysis of fibrotic tissue using ImageJ software (version 1.6.0_20, National Institutes of Health). The values represent mean ± SD (five sections from five lobes of five rats each in the group, yielding 125 sections for each value from 40 rats). (b) Effect of the degree of liver fibrosis (LF) on hydrodynamic gene delivery. The pCMV-Luc plasmid was hydrodynamically delivered to the rats at different time post bile duct ligation (BDL) and luciferase activity in the liver was analyzed 4 hours after gene delivery. The values represent mean ± SD. (All five lobes from five rats in each group, n = 25 samples for each value from 40 rats) *for P < 0.05, ** P < 0.01, *** P < 0.001, and N.S., no statistical significance between the level in LF and LF-MMP13 groups. One-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test.

Figure 5

Histological analysis of luciferase expression in the liver. Immunohistochemical staining with antiluciferase antibody was performed on liver tissues collected 10 weeks after the treatment. Scale bar represents 200 µm (100 ×). (a) Normal, (b) liver fibrosis (LF), and (c) LF-MMP. (d) Quantitative analysis of positively stained cells. Five different liver sections from five lobes of five rats each in three groups were immunohistochemically stained with antiluciferase antibody, and a quantitative analysis was performed using ImageJ software (version 1.6.0_20, national Institutes of Health). The values represent mean ± SD. (n = 125 for each value) (e) The level of MMP13 in 10 weeks after the procedure. Serum concentration of MMP13 was quantified by enzyme-linked immunosorbent assay (ELISA). The values represent mean ± SD. (n = 5 for each group) **P < 0.01, *** P < 0.001, and N.S., no statistical significance. One-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test.

Figure 6

Serum biochemical analysis. The standard serum biochemical analysis was performed on the serum collected 10 weeks after the bile duct ligation and MMP13 gene transfer. The values represent average concentrations of (a) aspartate aminotransferase (AST), (b) alanine aminotransferase (ALT), (c) lactate dehydrogenase (LDH), (d) alkaline phosphatase (ALP), (e) albumin (ALB), (f) hyaluronic acid (HA), and (g) type IV collagen (Type IV Col) in serum. The values represent mean ± SD (n = 5 rats for each value). * P < 0.05, ** P < 0.01, and NS, no statistical significance. One-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test.