| Abstract |
INTRODUCTION:
Liver fibrosis is a dynamic phenomenon, the progression of which depends upon the activation of hepatic stellate cells. The production of extracellular matrix protein is regulated by many cytokines while clinical evaluation of patients with chronic liver disease demands a regular evaluation of the fibrotic process. Until now liver biopsy remains the gold standard, but it is an invasive procedure with some degree of morbidity.
Moreover, therapeutic substances might negatively influence the development of fibrosis with detrimental results.
Octreotide, a synthetic somatostatin, is widely used for the control of variceal bleeding and the therapy of HCC. The effect of Octreotide in the process of fibrosis has not been studied.
AIM:
To study the effect of Octreotide on serum parameters related to fibrosis of patients with chronic liver disease.
ΜETHODS:
Serum proteins that have been shown to be associated with the fibrotic process were chosen. Four groups of proteins were studied:
a) Leptin, Laminin, Collagen-IV and Hyaluronan were measured in the peripheral blood of patients with chronic liver disease as well as in the hepatic vein blood after catheterization of cirrhotic patients. Areas Under the Curve (AUC) were created to investigate the possibility of discrimination between patients with chronic viral hepatitis and viral cirrhosis, as well as the possibility of discrimination between patients with early PBC and advanced PBC. The effect of Octreotide on serum levels of these factors was also studied. Measurements were done with commercially available ELISAs.
b) In the same patients, the group of TGFb proteins (TGFb1, TGFb2, TGFb3) and the effect of Octreotide in their levels were also biochemically studied (ELISA). Moreover, in this part we used histochemistry (methods of alkaline phosphatase and immunofluorescence) to localize these proteins in the hepatic tissue. Also, in a limited number of hepatic tissues the mRNA
expression of the protein FoxP3, a protein characteristic of Treg cells was studied , since it could potentially be influenced by the TGFb isoform levels. c) In an other group of patients the protein Activin A (ELISA), which has also been associated with either fibrosis or with regeneration of hepatocytes was evaluated. The effect of Octreotide in the levels of Activin A was also studied. Since few information exist about the origin of Activin A, in this part we studied the localization of the protein in non-parenchymal cells of the rat liver. Kupffer cells and hepatic stellate cells from were isolated and the expression of Activin A and the effect of Octreotide were evaluated using a semiquantitative PCR.
d) Finally, serum levels and the effect of Octreotide on metalloproteases (MMPs) and the metalloprotease inhibitor TIMP1 was also evaluated by ELISA.
RESULTS:
α)) Leptin, Laminin, Collagen-IV and Hyaluronan were measured in the peripheral blood of 62 PBC patients (27 stage III- IV and 35 stage Ι-ΙΙ according to Ludwing), 44 chronic hepatitis C patients, 38 HCC patients before and after 6 months of Octreotide treatment, 34 viral cirrhosis patients and compared to 60 normal controls. They were also measured in the hepatic vein blood of 15 cirrhotic PBC patients and 17 patients with viral cirrhosis before and after 6 months of Octreotide treatment.
All disease groups had significantly increased levels of Hyaluronan and Collagen IV, while Laminin was significantly increased only in viral cirrhosis. Levels of Leptin were not significantly different among groups. Hyaluronan levels were significantly different between early (54.6ng/ml, 95%CI 27.3-426.7) and late PBC (154.5ng/ml 95% CI 55.3-764.4, plt;0.05). AUC was 0.74 for Hyaluronan, 0.63 for Leptin , 0.59 for Laminin and 0.70 for collagen IV. Hyaluronan had high Sensitivity and Negative Prognostic Value (NPV) in identifying late stages of PBC (96% and 90% respectively).
No single measurement was able to differentiate between early and advanced stage liver fibrosis , but Hyaluronan can be used for longitudinal studies of fibrosis progression, in PBC patients. Administration of Octreotide did not influence the values of the fibrosis markers studied..
β) Isoforms of TGFβ were studied in the same group of patients.
TGFβ1 was significantly decreased in all cirrhotic patients (PBC 13.4ng/ml, range 7.4-26.2, HCV cirrhosis 11.6 ng/ml, 5.0-33.8) compared to controls ( 30.9ng/ml, 20.9-37.8).
TGFβ2 was increased in viral cirrhosis but not in PBC and chronic HCV hepatitis.
TGFβ3 (controls values 47.2pg/ml, range 27.0-79.7) was increased in both early and late PBC (94.3pg/ml, 41.5-358.6 and 152.8pg/ml,60.4-361.2 respectively, Plt;0.001 for both groups).
By contrast, TGFβ3 was decreased in viral cirrhosis (37.4pg/ml, 13.3-84.0, Plt;0.05).
The levels of all isoforms in hepatic veins were analogous to the peripheral blood.
Immunohistochemistry identified the presence of all 3 isoforms in portal tract lymphocytes (probably Tregs), sinusoidal cells and cholangiocytes. This last localization has not been previously reported in bibliography.
The study of FoxP3 expression in the liver tissue with the semiquantitave PCR, did not show any difference in the expression between PBC and viral cirrhosis.
ROC curves showed that among the 3 isoforms only TGFb1 deseves a prospective evaluation as a serum fibrosis marker potentially capable to differentiate between early and late PBC but not between chronic viral hepatitis and viral cirrhosis. The other two isoforms were not suitable for use as indicators of fibrosis.
Finally, Octreotide did not influence the levels of all 3 isoforms.
γ)) Τhe results from the Activin A study were totally different.
Activin levels were significantly increased (Plt;0.001) in serum of patients with alcoholic cirrhosis (median 673pg/ml, range 449-3279), compared to either controls (149pg/ml,91-193) or patients with viral cirrhosis (189pg/ml, 81-480), Chronic Hepatitis C ( 142 pg/ml, 65-559) PBC stage I-II (100 pg/ml 59-597) and PBC stage IV ( 104 pg/ml, 81-579). Patients with HCC associated with viral cirrhosis had levels similar to controls, but patients with HCC associated with alcoholic cirrhosis had significantly increased levels ( 131 pg/ml, 42-773 vs 2403 pg/ml,1561-7220pg/ml, respectively plt;0.001). Levels in the hepatic vein were similar to the peripheral blood.
The Activin A was expressed in the stellate and Kupffer cells, a fact that has not been previously described..
Octreotide either used therapeutically in patients or during in vitro incubation of sinusoidal cells did not have any significant effect in either Activin serum levels or in mRNA expression in sinusoidal cells.
d) The levels of total MMPs were similar among all groups of patients with chronic liver diseases and normal controls, either in the peripheral blood or in the hepatic vein blood. By contrast TIMP1 was increased in patients with alcoholic liver disease. Treatment with Octreotide did not influence serum values.
CONCLUSIONS:
1) None of the biochemical markers that were studied can separate with precision early from advanced liver fibrosis. However, hyaluronan and TGFb1 might be used for longitudinal studies of PBC patients. A prospective evaluation is required.
2) Octreotide does not influence the levels of proteins related to fibrosis and hence can be used without problems in chronic liver diseases.
3) Serum profile of TGFβ isoforms is different depending on the cause of liver disease. Increased TGFβ3 is a characteristic of PBC already observed in early disease. It is possible therefore that it might be related to the pathogenesis of the disease.
4) The isoforms of TGFb apart from the already described localizations in the liver (lymphocytes and sinusoidal cells) are also localized in the cells of the small intrahepatic bile ducts.
5) Serum levels of Activin A are characteristically increased in patients with alcoholic cirrhosis with or without HCC and allowed discrimination of alcoholic from either viral cirrhosis or PBC. The origin of Activin A in the peripheral blood seems to be derived almost exclusively from the liver where apart from the already described localizations (hepatocytes and stellate cells ), in the present study was found to be also expressed in the Kupffer cells.
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