Combinatorial gene therapy renders increased survival in cirrhotic rats

Abstract

Background: Liver fibrosis ranks as the second cause of death in México's productive-age population. This pathology is characterized by accumulation of fibrillar proteins in hepatic parenchyma causing synthetic and metabolic disfunction. Remotion of excessive fibrous proteins might result in benefit for subjects increasing survival index. The goal of this work was to find whether the already known therapeutical effect of human urokinase Plasminogen Activator and human Matrix Metalloprotease 8 extends survival index in cirrhotic animals.

Methods: Wistar rats (80 g) underwent chronic intoxication with CCl4: mineral oil for 8 weeks. Cirrhotic animals were injected with a combined dose of Ad-delta-huPA plus Ad-MMP8 (3 x 10(11) and 1.5 x 10(11) vp/Kg, respectively) or with Ad-beta-Gal (4.5 x 10(11)) and were killed after 2, 4, 6, 8 and 10 days. Then, liver and serum were collected. An additional set of cirrhotic animals injected with combined gene therapy was also monitored for their probability of survival.

Results: Only the cirrhotic animals treated with therapeutical genes (Ad-delta-huPA+Ad-MMP-8) showed improvement in liver fibrosis. These results correlated with hydroxyproline determinations. A significant decrement in alpha-SMA and TGF-beta1 gene expression was also observed. Cirrhotic rats treated with Ad-delta-huPA plus Ad-MMP8 had a higher probability of survival at 60 days with respect to Ad-beta-Gal-injected animals.

Conclusion: A single administration of Ad-delta-huPA plus Ad-MMP-8 is efficient to induce fibrosis regression and increase survival in experimental liver fibrosis.

Figure 1

Experimental design. Rats were made cirrhotic with different CCl4: mineral oil dilutions during seven weeks. After the animals were treated with the combined gene therapy, they were continuosly injected (three times a week) with the hepatoxin. When indicated, serum and liver extracts were obtained for the determinations specified in Materials and Methods.

Figure 2

Macroscopic aspect of liver and functional hepatic tests. A, a) a representative image a normal rat liver, b and c shows a recovered hepatic smooth texture in animals treated with the combinatorial gene therapy at 8 and 10 days, respectively, as compared with irrelevant gene therapy (e and f), d) shows a control fibrotic liver injected with saline. Histograms in B, show a significative tendency to normal values of hepatic functional tests (ALT and AST).

Figure 3

Fibrosis analysis: histologic and molecular. A, shows a decrement in collagen content in cirrhotic animals treated with uPA plus MMP-8. B, liver sections Masson's stained in each experimental group, a) a representative histological image of a normal liver rat, in b-d cirrhotic animals treated with combinatorial gene therapy at 6, 8 and 10 days respectively. The histological images of f-h are representative of cirrhotic animals treated with irrelevant vector (Ad-β-Gal) at 6, 8 and 10 days, respectively, e) shows a control fibrotic liver injected with saline. C, Morphometric analysis indicates that animals treated with therapeutically gene therapy has a major recuperation at 6 to 10 days post-treatment (inducing a 55% of regression). D, TGF-β, evaluated by semicuantitative RT-PCR, showed a major decrement in mRNA at 8 days after treatment with Ad-uPA plus Ad-MMP-8 as compared with irrelevant control. N, normal; F, fibrotic.

Figure 4

Human transgenes detection and fibrinolytic activities. Panels A and B show uPA and MMP-8 proteins in cirrhotic animals, both control and experimental (2^nd through 10^th days) detected by immunohistochemistry, indicating percentage of positive cells. C, transgenes activity, shows major activity of both trangenes (uPA and MMP-8) at 4 days after treatment. D, both enzymes (MM-2 and MM-9) increment their functional activities only in the cirrhotic animals treated with Ad-uPA plus Ad-MMP-8 and the maximum activity was at 4 days post-treatment.

Figure 5

Cellular proliferation and survival. A, PCNA protein (regeneration index) shows increment only with the treatment of combinatorial gene therapy at 2-4 days post treatment. B, gene expression of HGF and c-met determined by semiquantitative RT-PCR that indicate a light increment of both genes on the animals treated with Ad-uPA plus Ad-MMP-8 to respect Ad-β-Gal. C, activated HSC (α-SMA⁺) show significant decrement after 2 days of treatment with therapeutically gene therapy. D, Survival analysis (Kaplan Meier) of cirrhotic animals in each experimental group. Increment of survival (60 days) in the animals receiving the combined gene therapy is noticeable.