8 research outputs found
Transforming growth factor Ī² neutralization ameliorates pre-existing hepatic fibrosis and reduces cholangiocarcinoma in thioacetamide-treated rats.
Considerable evidence has demonstrated that transforming growth factor Ī² (TGF-Ī²) plays a key role in hepatic fibrosis, the final common pathway for a variety of chronic liver diseases leading to liver insufficiency. Although a few studies have reported that blocking TGF-Ī² with soluble receptors or siRNA can prevent the progression of hepatic fibrosis, as yet no evidence has been provided that TGF-Ī² antagonism can improve pre-existing hepatic fibrosis. The aim of this study was to examine the effects of a murine neutralizing TGF-Ī² monoclonal antibody (1D11), in a rat model of thioacetamide (TAA)-induced hepatic fibrosis. TAA administration for 8 weeks induced extensive hepatic fibrosis, whereupon 1D11 dosing was initiated and maintained for 8 additional weeks. Comparing the extent of fibrosis at two time points, pre- and post-1D11 dosing, we observed a profound regression of tissue injury and fibrosis upon treatment, as reflected by a reduction of collagen deposition to a level significantly less than that observed before 1D11 dosing. Hepatic TGF-Ī²1 mRNA, tissue hydroxyproline, and plasminogen activator inhibitor 1 (PAI-1) levels were significantly elevated at the end of the 8 week TAA treatment. Vehicle and antibody control groups demonstrated progressive injury through 16 weeks, whereas those animals treated for 8 weeks with 1D11 showed striking improvement in histologic and molecular endpoints. During the course of tissue injury, TAA also induced cholangiocarcinomas. At the end of study, the number and area of cholangiocarcinomas were significantly diminished in rats receiving 1D11 as compared to control groups, presumably by the marked reduction of supporting fibrosis/stroma. The present study demonstrates that 1D11 can reverse pre-existing hepatic fibrosis induced by extended dosing of TAA. The regression of fibrosis was accompanied by a marked reduction in concomitantly developed cholangiocarcinomas. These data provide evidence that therapeutic dosing of a TGF-Ī² antagonist can diminish and potentially reverse hepatic fibrosis and also reduce the number and size of attendant cholangiocarcinomas
1D11 reversed pre-existing hepatic fibrosis.
<p><b>A</b>. Representative photomicrographs of picrosirius red stained liver sections following treatment with TAA for 8 weeks (ābaselineā; TAA-8W), or an additional 8 weeks with 13C4 (13C4ā16W) or 1D11 (1D11ā16W) after cessation of TAA administration. A normal rat liver is also shown (Normal). Livers of rats dosed with TAA for 8 weeks (TAA-8W) had substantial lesions with widely spread fibrous bands (septa), originating from portal areas and extending into the parenchyma. 8 weeks after stopping TAA dosing, fibrotic areas were further expanded, with extensive architectural disorganization and more fibrosis covering a greater percentage of the livers (13C4ā16W). Treatment with 1D11 for 8 weeks significantly ameliorated the TAA-mediated histopathological lesions, as shown by an overall improvement in hepatic morphology (1D11ā16W). Liver collagen deposition in rats treated with 1D11 was noticeably much reduced than the pre-established fibrosis seen at baseline (TAA-W8), shown in rats that were examined at baseline. Magnification: 40x. <b>B</b>. Morphometric analysis of picrosirius red stained sections. Significantly less collagen was deposited in livers treated with 1D11 (ā“) for 4 weeks or 8 weeks, when compared to the PBS (ā¢, * p<0.01) or 13C4 (āŖ, * p<0.01) treatment groups. Moreover, comparison of 1D11 treated livers to those at week 8 before therapeutic treatment started, showed significantly less collagen deposition (treatment for 4 weeks: 9.61%; 8 weeks: 7.53%, * p<0.05) than that harvested at baseline (week 8ā¶12.7%). Morphometric quantification further supports the histology of a reversal of pre-existing hepatic fibrosis when TGF-Ī² was neutralized by 1D11. Values represent the percentage of collagen deposition in the total liver section and are expressed as mean Ā± SE (nā=ā8, except nā=ā4 for normal controls at week 0).</p
1D11decreased TGF-Ī²1 mRNA.
<p>mRNA expression of TGF-Ī²1 was analyzed by quantitative real-time RT-PCR for livers harvested at baseline (prior to treatment) and at week 16 (after treatment). TAA caused a sustained overexpression of TGF-Ī²1 (>6 fold) throughout the study and treatment with 1D11 significantly reduced mRNA levels compared to baseline levels and in both PBS and 13C4 groups at end of study (week 16). Data at week 16 with 1D11 treatment suggest a reversal of fibrosis. Values are expressed as means Ā± SE, nā=ā8 per group; * p<0.01 vs. normal controls at week 8; ** p<0.05 vs. PBS or 13C4 group at week 16.</p
Experimental protocol of TAA induced liver fibrosis.
<p>Fischer rats were dosed with TAA intraperitoneally for 8 weeks (300 mg/kg for 6 weeks, three times weekly, then two times weekly for 2 weeks). A cohort of rats received PBS as normal controls. At the end of 8 weeks, TAA was withdrawn and rats were divided into three cohorts that were given PBS, 13C4 or 1D11 for 8 weeks. 13C4 and 1D11 were dosed at a concentration of 5 mg/kg, intraperitoneally, three times per week.</p
Increased PAI-1 protein in fibrotic liver was reduced in rats dosed with 1D11.
<p><b>A</b>. A representative Western blot of pooled samples (nā=ā3) showed marked increase in PAI-1 expression before the start of therapeutic dosing at week 8. Levels were maintained in both PBS and 13C4 groups at the end of study at week 16. Levels of PAI-1 were normalized in livers treated with 1D11. <b>B and C</b>. Hepatic PAI-1 protein was also analyzed by ELISA. At the end of TAA administration, total (B) and active PAI-1 (C) were markedly elevated in fibrotic livers, with a further increase by week 16 in rats treated with PBS or 13C4. After 8 weeks of 1D11 dosing, total PAI-1 and active PAI-1 had returned to normal levels, confirming the immunoblotting data. Values are expressed as mean Ā± SE, nā=ā8; * p<0.01 vs. normal controls at week 8; ** p<0.01 vs. PBS or 13C4 group at week 16.</p
1D11 reduced cholangiocarcinomas induced by TAA.
<p><b>A</b>. TAA induced profound neoplastic changes in biliary ductules (cholangiocarcinoma) at week 16. As visualized by CK staining, these neoplastic cells extended into the parenchyma, as fibrosis evolved (arrow heads). The neoplasia displayed a typical āintestinal metaplasiaā-like appearance (arrows). The number of neoplastic biliary ductules or cells was much lower with 1D11 treatment, compared to the PBS or 13C4 groups. For comparison, a normal liver stained for CK is shown. Magnification: x100 <b>B</b>. CK staining was quantitated. The TAA-induced neoplasia was significantly reduced in rats treated with 1D11 for 8 weeks. In contrast, the neoplasia showed no reduction in rats dosed with PBS or 13C4. Values are expressed as mean Ā± SE of percentage of CK staining of the entire area of liver section. nā=ā8; * p<0.01 vs. normal controls; ** p<0.01 vs. PBS, 13C4 and normal control groups. <b>C</b>. To quantify the areas of cholangiocarcinoma, liver sections from the 16 time point were scanned by ChromaVision Imaging Analysis System. The area of the cholangiocarcinomas defined as neoplastic biliary epithelial cells plus stromal fibrotic tissue and infiltrated cells, was measured and expressed as a percentage of the total area of the liver section. Data revealed a striking reduction with 1D11 treatment, as compared to the PBS and 13C4 groups, reflecting a diminishment of cholangiocarcinoma by TGF-Ī² neutralization. nā=ā8, * p<0.01 vs. normal controls; ** p<0.01 vs. PBS, 13C4 and normal control groups. Values represent mean Ā± SE.</p